CN103525777A - VII type Newcastle disease virus L-gene mutation attenuated vaccine strain and preparation method thereof - Google Patents

Abstract

The invention relates to an attenuated vaccine strain with L gene mutation of Type VII Newcastle disease virus produced by reverse genetic manipulation technology and a preparation method thereof, belonging to the field of biological products and biotechnology, and attenuation of L gene mutation of Type VII Newcastle disease virus The virulence of the vaccine strains is obviously weakened, and at the same time, it has high titer on chicken embryos, stable genetic performance, and high antigen matching with the epidemic virus. It is suitable for large-scale production of vaccines and can be used to manufacture vaccines. Compared with conventional vaccines (gene Ⅰ Compared with genotype Ⅱ), the attenuated vaccine strain with L gene mutation of Ⅶ Newcastle disease virus will show broad application prospects in controlling the incidence and prevalence of Newcastle disease.

Description

Attenuated vaccine strain with L gene mutation of type VII Newcastle disease virus and preparation method thereof

technical field

The invention relates to an attenuated vaccine strain with mutation of the L gene of VII Newcastle disease virus produced by reverse genetic manipulation technology and a preparation method thereof, belonging to the field of biological products and biotechnology.

Background technique

Newcastle disease is caused by Newcastle disease virus (NDV), which affects chickens and turkeys, and can also infect other poultry and birds. It is an acute and highly contagious disease. One of the infectious diseases (the other being highly pathogenic avian influenza). NDV belongs to the genus Mumpsvirus of the Paramyxoviridae family, has an envelope structure, and its genome is a single-strand negative-sense RNA virus that is not segmented. It is composed of 6 structural proteins, namely NP, P, M, F, HN and L. Proteins: M, F and HN proteins are related to the formation of the viral envelope; NP protein is the nucleocapsid protein, P protein is an essential factor for viral RNA synthesis; L protein is the largest protein of the virus, which is among the six structural proteins of NDV It is the most conservative and has biological activities such as RNA polymerase and post-transcriptional modification. It can form a viral replication complex with NP and P proteins. This complex participates in the transcription and replication of the viral genome. In addition to participating in the replication of the viral genome, the L protein, It also has the functions of capping at the 5' end, cap-dependent methyltransferase activity and adding a polyadenylic acid tail at the 3' end.

The virulence of different NDV isolates varies greatly. According to the pathogenicity and virulence to chickens and chicken embryos, NDV can be divided into three categories: strong virulence (rapid type), moderate virulence type (medium virulence) type) and attenuated type (slow type).

Previous studies believed that the infectivity and virulence of NDV were determined by the F gene. The cleavage site of the F protein of the strong strain was RRQKRF, while the cleavage site of the F protein of the weak strain LaSota was GRQGRL. However, with the continuous development of reverse genetics technology and its in-depth application in NDV research, new research progress has been made on the pathogenic mechanism of NDV. Peeters, De Leeuw, and Panda rescued the new recombinant virus after mutating the F gene cleavage site of the attenuated strain to a cleavage site consistent with the strong strain. Experiments showed that although its virulence was significantly improved, it did not reach The level consistent with the virulence of the virulent strain indicates that in addition to the cleavage site of the F gene, there may be other genes that can affect the virulence of the virus in the NDV genome. Recent studies have pointed out that L, HN and V proteins are also related to the virulence and pathogenicity of the virus, and the L protein can enhance the virulence of NDV by increasing the replication level of the virus. Rout et al. replaced the L gene of the NDV BC strain with the L gene of LaSota, and the virulence of the virus increased after being rescued. In addition, we successfully rescued 3 gene VII-97 NDV L mutant strains (K1756A, K1917A and E1954Q amino acid mutations) and 1 gene VII-97 NDV F and L co-mutant strain (E1954Q+F). The index measurement found that the four attenuated vaccine strains with mutations in the L gene of type VII Newcastle disease virus could attenuate the virulence of the virus. We have reason to infer that the NDV L protein plays a crucial role in the virulence of the virus. These experiments fully demonstrate that there is a connection between L protein and the virulence of the virus.

At present, the main vaccine strains for the prevention and control of NDV in the world are two strains, LaSota and B1, both of which are genotype II. However, domestic scholars such as Yan Weiwei reported the existence of genotype VII NDV in mainland China for the first time in 2000; The amino acid sequences of the strains at the cleavage site were all similar to the virulent strains of NDV. Genetic phylogenetic tree analysis showed that 7 of the 8 isolates were genotype Ⅶ NDV. These data fully indicated that the main epidemic genotype Ⅶ NDV in my country. Two attenuated vaccines, LaSota and B1, have been used as vaccine strains for decades. Although the vaccines can provide certain immune protection, they cannot prevent the poultry from shedding the virus and inhibit the ability of the virus to spread. Therefore, Newcastle disease is still a threat to the poultry industry. It is one of the most important epidemic diseases in the development of China, which causes huge economic losses every year in China. Moreover, the current prevailing virus strains in China are mainly genotype VII. With the continuous mutation of the virus, the research and development of the same gene type vaccine for genotype VII has become the main task at present. Good progress has been made in the development of Newcastle disease vaccine for genotype VII in my country. After industrialization, it will surely become a necessary part of the immunization program of large-scale farms. The invention lays a solid foundation for the development of NDV attenuated vaccine and NDV-related basic research.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies in the prior art and invent the attenuated vaccine strain of the L gene mutation of VII type Newcastle disease virus and its preparation method, thereby solving the problem of NDV vaccine strains (gene I and gene II) used in the current market in my country. ) is not consistent with the genotype of the current NDV epidemic strain (gene type VII), resulting in incomplete protection efficiency, which cannot effectively prevent the actual problem of virus transmission.

The present invention is achieved through the following technical solutions:

An attenuated vaccine strain with L gene mutation of Type VII Newcastle disease virus is characterized in that the virulence of the attenuated vaccine strain with L gene mutation of Type VII Newcastle disease virus is obviously weakened, and at the same time, it has high titer and heredity on chicken embryos. Stable performance, high antigen matching with epidemic virus, suitable for large-scale production of vaccines, can be used to manufacture vaccines, the antigenic nucleotide sequence of the attenuated vaccine strain of this type VII Newcastle disease virus L gene mutation is SEQ ID NO: 1 to shown in SEQ ID NO:4. the

A kind of preparation method of the attenuated vaccine strain of described VII type Newcastle disease virus L gene mutation, it is characterized in that:

Five cDNA clone fragments covering the entire genome were constructed, and a complete cDNA clone of 15192 nt was obtained by joining and assembling the transcription vector plasmid pBR322 by using the restriction sites in the overlapping parts of each fragment. The T7 RNA polymerase promoter was prefixed at the 5' end of the full-length cDNA fragment, and the autocatalytic function of hepatitis D virus ribozyme (RiB) and T7 transcription termination signal were added after the cDNA fragment, and the constructed plasmid was named prNDV-97; The 14056th base of the L protein coding region in the genomic cDNA was synonymously mutated from A to G by PCR genome, and the Hind III restriction site at 14056bp was eliminated, and it was used as a molecular marker for virus rescue. The constructed plasmid was named as pmNDV-97; two redundant Gs were introduced at the 5' end of the genomic cDNA in the T7 polymerase promoter, which is helpful for the virus rescue of paramyxovirus reverse genetic manipulation.

Five pairs of primers were designed to amplify the NDV Ⅶ-97 strain genome SEQ ID NO:5, the primer sequences are as follows:

F1: 5'ATGCGGCCGCTAATACGACTCACTATAGGGACCAAACAGAGAATCTG

TGAGGTACGATAA3'

R1: 5'TAGGTACCCTTGCAGTGGCAGACTTAATCAATTCAGAG3'

F2: 5'GGGTGAAATGACGCTCAATAAACTCTCACA3'

R2: 5'ATGGTACCCTCCCTGTTGCAGATGTCCGAAGCACACCA3'

F3: 5'TGCAGAGATCACTCACACTCACATCAATAC3'

R3: 5'ACGGTACCAGGTGACCAGCTTCTGTTTCGGGCATAATC3'

F4: 5'ATTAGTTTCTTTCCAATATGTGCTCGCTGAC3'

R4: 5'GTAGTAATGATACAGACCTGTAGGGTACCA3'

F5: 5'GGAGAGAGAATGCAGAAGAAAGTGACCTGACCTCAGATAAAGCAG3'

R5: 5'AGCCGGCGCCAGCGAGGAGGCTGGGACCATGCCGGCCACCAAACAG

AGATTTGGTGAATGACATC3'

Design 2 pairs of primers for mutating the Hind III restriction site at 14056bp to eliminate this restriction site. The primer sequences are as follows:

AFa: 5' CATGTTTTAAAGTTGTCAAG 3'

F6: 5' GGGTCCAATCAGAGCTTATTAGATC 3'

R6: 5' GATCTAATAAGCTCTGATTGGACCC 3'

ARa: 5' GCG GTCGAC ACCGCTTTCAGGTATTTAG3'

On the basis of the pmNDV-97 whole-genome plasmid, construct the NDV-97 L gene mutation attenuated strain, the specific operation is as follows:

Using the following primers, the 1756, 1917 and 1954 amino acids in the NDV parental L protein domain VI were single-point mutated by the Overlapping PCR method, and the amplified target fragment and pmNDV-97 vector were subjected to Hind Ⅲ and Xba Ⅰ Double digestion with restriction endonucleases, nucleic acid electrophoresis, gel recovery of the target fragment and vector pmNDV-97, and then ligation. The obtained mutant plasmids were named pK1756A, pK1917A and pE1954Q respectively.

Four pairs of primers were designed for site-directed mutagenesis of amino acids in NDV VII-97 L protein. The primer sequences are as follows:

AF1: 5' CATGTTTTAAAGTTGTCAAG 3'

AF2: 5' CTTCTTGGTATGCGGCATCTCATCTTC 3'

AF3: 5' GTAGTGATCATCGCAGTACTGTATGC3'

AF4: 5'GAGGGGATATGCAATGTTACCTGATAT3'

AF5: 5' GCG GTCGAC ACCGCTTTCAGGTATTTAG3'

AF6: 5'GAAGATGAGATGCCGCATACCAAGAAG3 '

AF7: 5'GCATACAGTACTGCGATGATCACTA3 '

AF8: 5'ATATCAGGTAACATTGCATATCCCCT3 '

Among them, AF1+AF6, AF1+AF7 and AF1+AF8 primers were used to amplify the target bands respectively by PCR method, which were named A1, B1 and C1 respectively; AF5+AF2, AF5+AF3 and AF5+AF4 The target bands are named A2, B2 and C2 respectively. On this basis, A1+A2, B1+B2 and C1+C2 were respectively used as templates to amplify target fragments of about 6000 bp (including mutation sites) by using AF1+AF5 primers through Overlapping PCR method, respectively to obtain L mutations Fragments L1756, L1917 and L1954.

A method for constructing an attenuated strain of NDV VII-97 L gene mutation and co-mutation of F and L genes, specifically: performing site-directed mutation on the genomic L gene of the isolate NDV VII-97, and performing a single mutation in the L protein domain VI. Point amino acid mutations were used to obtain highly attenuated NDV Ⅶ-97 L attenuated strains K1756A, K1917A and E1954Q. On the basis of E1954Q, the F protein cleavage site was mutated, and the F protein cleavage site (RRQKRF) of the strong strain was mutated into the F protein cleavage site (GRQGRL) of the LaSota attenuated strain, and the attenuated strain obtained was E1954Q+F.

Primers used to mutate the F protein cleavage site are as follows:

AF9: CTCCGACCAAAACCCCCCACACTCCCTGACCC

AR9: GCAGAGTAGAGAAGAATACCCTCCCTGTTGCAGAT

AF10: TCTGTGTCCACGTCTGGAGGAGGGAGACAGGGGCGCCTTATAGGTGCTGT

TATTGGCAG

AR10: CTGCCAATAACAGCACCTATAAGGCGCCCCTGTCTCCCCTCCTCCAGACGT

GGACACAGA

Among them, 2 pairs of primers AF9+AR10 and AF10+AR9 used the PCR method to amplify the target bands respectively, and after the gel was recovered, the product recovered from the gel was used as a template, and the primer pair AF9+AR9 was used for PCR amplification by the Overlapping method to obtain The fragment containing the F protein mutation is about 5458bp long, and the mutant thus obtained is E1954Q +F.

A method for constructing attenuated vaccine strains K1756A, K1917A, E1954Q and E1954Q+F of type VII Newcastle disease virus L gene mutation, specifically:

1) Construction of full-length cDNA of NDV Ⅶ-97 strain genome;

2) Site-directed mutation of amino acids in domain VI of NDV VII-97 L protein and connection of the full-length cDNA of the mutant;

3) On the basis of the E1954Q mutation, the mutation of the NDV VII-97 F protein cleavage site and the connection of the full-length cDNA of the mutant;

4) Attenuated vaccine strains K1756A, K1917A, E1954Q with mutations in the L gene of Newcastle disease virus type VII

and the salvation of E1954Q +F;

Plasmids pK1756A, pK1917A, pE1954Q and pE1954Q+F were co-transfected with three eukaryotic expression plasmids pBSK-NP, pBSK-P and pBSK-L, respectively, in BHK-21 cells (hamster kidney cells), and the cells were plated 72 hours after transfection Inoculate 9-11 day-old SPF chicken embryos in clear water, inoculate 3 pieces per sample, 0.2mL/piece, and successfully rescue the attenuated vaccine strains K1756A, K1917A and E1954Q with mutations in the L gene of Type VII Newcastle disease virus, as well as F protein and L protein Co-mutated attenuated vaccine strain E1954Q+F.

The present invention has following positive effect:

The present invention uses the established NDV reverse genetic operation platform to construct an attenuated vaccine strain with high virus titer, stable genetic performance, high antigen matching and low pathogenicity to host chickens and other poultry by modifying related genes. This will lay the foundation for the development of high-efficiency vaccine breeding. Compared with conventional vaccines (geno I and genotype II), the attenuated vaccine strain with L gene mutation of type VII Newcastle disease virus will show broad application prospects in controlling the incidence and prevalence of Newcastle disease.

Description of drawings

Figure 1 shows the assembly of full-length NDV cDNA from subgenomic overlapping cDNA fragments generated by high-fidelity RT-PCR. The cDNA fragments were ligated at consensus restriction sites and assembled in transcriptional plasmid pBR322 into which the PBZ and T7 terminator sequences were precloned. Figure 1 represents a schematic diagram of ligation of full-length cDNA.

Figure 2 shows the nucleotide changes introduced by the RT-PCR method and verified by automatic sequencer sequencing. The framed is the Hind III restriction site at 14056 bp after PCR mutation (from A to G ).

Fig. 3 is the RT-PCR identification and the sequencing result of the mutation position of the attenuated vaccine strain of the L gene mutation of type VII Newcastle disease virus. Among them, A is the RT-PCR identification result; B is the K1756A sequencing result; C is the K1917A sequencing result; D is the E1954Q sequencing result; E is the E1954Q+F sequencing result.

Figure 4 shows the genetic stability analysis and sequencing results of the attenuated vaccine strain with mutation in the L gene of Type VII Newcastle disease virus. Among them, A is the K1756A sequencing result; B is the K1917A sequencing result; C is the E1954Q sequencing result; D is the E1954Q+F sequencing result.

Figure 5 is the indirect immunofluorescence analysis of attenuated vaccine strains K1756A, K1917A, E1954Q, L and F co-mutated attenuated strains E1954Q+F and rNDV of type VII Newcastle disease virus L gene mutation. Infect BHK-21 cells with K1756A, K1917A, E1954Q, E1954Q+F and rNDV respectively (set negative control at the same time). The infection dose was 1 MOI. Cells were fixed 24 hours after infection, permeabilized, detected with chicken anti-NDV antiserum, and then incubated with FITC-coupled rabbit anti-chicken IgG antibody. Fluorescence was observed under a Leica fluorescence microscope.

Detailed ways

The present invention combines the accumulation of Newcastle disease virus strains in recent years and the analysis and research on its whole genome sequence, conducts site-directed amino acid mutation and amino acid co-mutation of F and L genes in the L gene of China's Type VII Newcastle disease virus strain, and performs mutation on the L gene. Rescued the full-length NDV genome cDNA co-mutated with the L gene and the F gene, and rescued highly attenuated attenuated vaccine strains K1756A, K1917A, E1954Q and E1954Q+F with mutations in the L gene of Type VII Newcastle disease virus. The attenuated vaccine strain of Newcastle disease virus L gene mutation has significantly weakened virulence, and at the same time, it has high titer on chicken embryos, stable genetic performance, and high antigen matching with epidemic viruses. It is suitable for large-scale production of vaccines and can be used for vaccines The antigenic nucleotide sequence of the attenuated vaccine strain of the type VII Newcastle disease virus L gene mutation is shown in SEQ ID NO: 1 to SEQ ID NO: 4.

The present invention will be further described below in conjunction with specific implementation examples.

Embodiment 1. Construction of transcription vector:

The BHK-21 cells used by the present inventor are hamster kidney cells; the culture medium is containing 10% fetal bovine serum (Gibco); the NDVⅦ-97 strain is preserved by the Poultry Disease Laboratory of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences; SPF chicken embryo and SPF All chicks were purchased from Beijing Meria Verton Biotechnology Co., Ltd.

The reverse genetic operation platform of NDV Ⅶ-97 strain is transformed on the basis of pBR322 plasmid, which contains T7 promoter and T7 ribozyme transcription termination sequence at both ends of the whole NDV genome, and the insertion has the function of self-cutting The hepatitis D virus ribozyme sequence. The DNA fragment cloned between the T7 promoter and the ribozyme can be transcribed under the action of T7 RNA polymerase, and due to the autocatalytic function of the hepatitis D virus ribozyme, it can ensure that the 3' end of the transcript is exactly the same as the cloned DNA fragment unanimous.

Construction of embodiment 2.NDV genome full-length cDNA:

Five cDNA clone fragments covering the whole genome were constructed, and a complete cDNA clone of 15192 nt was obtained by ligation and assembly of the transcription vector plasmid pBR322 by using the restriction sites in the overlapping parts of each fragment. The T7 RNA polymerase promoter is prefixed at the 5' end of the full-length cDNA fragment, and the hepatitis D virus ribozyme with autocatalytic function and T7 transcription termination signal are added after the cDNA fragment. The constructed plasmid was named prNDV. In order to eliminate the Hind III restriction site at 14056bp, the 14056th base in the L protein coding region in the genomic cDNA was synonymously mutated from A to G by PCR genome, and as a molecular marker for virus rescue, we simultaneously added T7 polymerase The promoter introduces two redundant Gs at the 5' end of the genomic cDNA, which may be helpful for virus rescue by reverse genetic manipulation of paramyxoviruses.

Five pairs of primers were designed to amplify the NDV Ⅶ-97 strain genome SEQ ID NO:5, the primer sequences are as follows:

F1: 5'ATGCGGCCGCTAATACGACTCACTATAGGGACCAAACAGAGAATCTG

TGAGGTACGATAA3'

R1: 5'TAGGTACCCTTGCAGTGGCAGACTTAATCAATTCAGAG3'

F2: 5'GGGTGAAATGACGCTCAATAAACTCTCACA3'

R2: 5'ATGGTACCCTCCCTGTTGCAGATGTCCGAAGCACACCA3'

F3: 5'TGCAGAGATCACTCACACTCACATCAATAC3'

R3: 5'ACGGTACCAGGTGACCAGCTTCTGTTTCGGGCATAATC3'

F4: 5'ATTAGTTTCTTTCCAATATGTGCTCGCTGAC3'

R4: 5'GTAGTAATGATACAGACCTGTAGGGTACCA3'

F5: 5'GGAGAGAGAATGCAGAAGAAAGTGACCTGACCTCAGATAAAGCAG3'

R5: 5'AGCCGGCGCCAGCGAGGAGGCTGGGACCATGCCGGCCACCAAACAG

AGATTTGGTGAATGACATC3'

Design 2 pairs of primers for mutating the Hind III restriction site at 14056bp to eliminate this restriction site. The primer sequences are as follows:

AFa: 5' CATGTTTTAAAGTTGTCAAG 3'

F6: 5' GGGTCCAATCAGAGCTTATTAGATC 3'

R6: 5' GATCTAATAAGCTCTGATTGGACCC 3'

ARa: 5' GCG GTCGAC ACCGCTTTCAGGTATTTAG3';

Using the Trizol method to extract the genomic RNA in the allantoic fluid inoculated with NDV Ⅶ-97 chicken embryos, the detailed steps are as follows:

① Take 250uL of chicken embryo allantoic fluid, add 750uL Trizol (Invitrogen, USA), vortex and mix well, and place at room temperature for 5 minutes;

② Add 200uL of chloroform to each tube, cap the centrifuge tube tightly, shake the centrifuge tube vigorously for 15 seconds; place at room temperature for 10 minutes, and centrifuge at 12000rpm for 15 minutes; , centrifuge at 12000rpm for 10 minutes; ④ Discard the supernatant, add 75% ethanol at a ratio of at least 1ml per milliliter of Trizol solution, mix well, and centrifuge at 7500g for 5 minutes at 4°C;

⑤ Discard the supernatant carefully, then dry at room temperature for 5-10 minutes, be careful not to dry too much, otherwise the solubility of RNA will be reduced; finally dissolve RNA in water. After the RNA was reverse-transcribed into cDNA, the PCR method was used to amplify each gene fragment, and the gel was cut to recover the target fragment for later use.

The whole genome was divided into 5 fragments (F1-F5) with partially overlapping ends for PCR amplification, and the cDNA fragment was cloned into the pBR322 eukaryotic expression vector; nucleocapsid protein (NP), phosphoprotein (P) and large polymerase protein (L) The open reading frame (ORF) cDNA of the gene was cloned in the downstream of the T7 promoter of the pBluescript II (+/-) plasmid, respectively, to constitute the transcription assistant plasmids pBSK-NP, pBSK-P and pBSK-L, respectively.

The cloned full-length cDNA of the corresponding genome is the basis for establishing the NDV reverse genetic operating system, and serves as a template for genome negative-strand RNA transcription.

Example 3. NDV Ⅶ-97 strain L gene carries out site-directed amino acid mutation and L mutant full-length cDNA connection

The 1756, 1917 and 1954 amino acids in the L protein domain VI of the NDV VII-97 strain were single-point mutated by using the primers respectively by Overlapping PCR (Table 1), and the amplified target fragment and the prNDV-97 vector were subjected to single-point mutations. Hind Ⅲ and Xba Ⅰ restriction endonucleases were double-digested respectively, followed by nucleic acid electrophoresis, and the target fragment was recovered from the gel and ligated with the vector prNDV-97. The obtained mutant plasmids were named pK1756A, pK1917A and pE1954Q, respectively.

On the basis of pE1954Q, the F protein cleavage site was mutated, and the F protein cleavage site (RRQKRF) of the strong strain was mutated into the F protein cleavage site (GRQGRL) of the attenuated LaSota strain. The F protein mutation position was amplified by the Overlapping PCR method with primers, and the amplified target fragment and pE1954Q vector were double-digested with Sal Ⅰ and Afe Ⅰ restriction endonucleases respectively, followed by nucleic acid electrophoresis, and the target fragment and vector were recovered from the gel Ligation was performed after pE1954Q, and the obtained mutant plasmids were respectively named pE1954Q+F. The primers used to mutate the F protein cleavage site are as follows:

AF9: CTCCGACCAAAACCCCCCACACTCCCTGACCC

AR9: GCAGAGTAGAGAAGAATACCCTCCCTGTTGCAGAT

AF10: TCTGTGTCCACGTCTGGAGGAGGGAGACAGGGGCGCCTTATAGGTGCTGT

TATTGGCAG

AR10: CTGCCAATAACAGCACCTATAAGGCGCCCCTGTCTCCCCTCCTCCAGACGT

GGACACAGA

Among them, 2 pairs of primers AF9+AR10 and AF10+AR9 used the PCR method to amplify the target bands respectively, and after the gel was recovered, the product recovered from the gel was used as a template, and the primer pair AF9+AR9 was used for PCR amplification by the Overlapping method to obtain The fragment containing the F protein mutation is about 5458bp long, and the mutant thus obtained is E1954Q +F.

Table 1. L gene mutation position

Example 4. Rescue of attenuated vaccine strains K1756A, K1917A, E1954Q and E1954Q+F of type VII Newcastle disease virus L gene mutation

Before transfection, BHK-21 cells were infected with poxvirus, washed 2-3 times after 1 hour, and then transfected to rescue the virus. Plasmids pK1756A, pK1917A, pE1954Q, and pE1954Q+F were co-transfected with three eukaryotic expression plasmids pBSK-NP, pBSK-P, and pBSK-L, respectively, in BHK-21 cells. After 72 hours, the cells were gently scraped off and mixed with the cells After the supernatant was fully mixed and filtered, it was inoculated with 9-11-day-old SPF chicken embryos, and each sample was inoculated with 3 eggs, 0.2mL per egg. The inoculated SPF embryos continued to be cultured for 3 days, and 50uL of chicken embryo allantoic fluid was routinely tested for NDV. Hemagglutination (HA) and hemagglutination inhibition (HI) tests were performed to obtain attenuated vaccine strains K1756A, K1917A, E1954Q and E1954Q +F with mutations in the L gene of type VII Newcastle disease virus.

Identification of the attenuated vaccine strain K1756A, K1917A, E1954Q and E1954Q+F of embodiment 5.VII type Newcastle disease virus L gene mutation

5.1 Genetic stability analysis

Experimental method: The attenuated vaccine strains (K1756A, K1917A, E1954Q and E1954Q+F) with mutations in the L gene of type VII Newcastle disease virus were subcultured to the 10th generation using 9-day-old SPF chicken embryos, and the virus RNA of the 10th generation was extracted, and the reverse After transcribing into cDNA, the target fragment is amplified, the gel is cut to recover the target fragment, and the sample is sent for sequencing.

Experimental results: The mutation sites of each mutant strain still exist, and the genetic stability is good (Fig. 4).

indirect immunofluorescence test 

In order to detect the replication of the attenuated strains (K1756A, K1917A, E1954Q and E1954Q+F) in BHK-21 cells, allantoxin was used to infect about 70%-80% of monolayer BHK-21 cells at an MOI of 1. 24 hours after infection, the indirect immunofluorescence test was carried out with the positive serum of NDV high-immune SPF chickens as the detection antibody, and a negative control was set up. Through indirect immunofluorescence test, it was found that all the rescued viruses could proliferate on BHK-21 cells.

The detailed steps are as follows:

The attenuated chicken embryo allantoic virus was diluted with DMEM at an appropriate multiple, and infected BHK-21 cells grown in a six-well plate according to the MOI of 1 and 300uL volume, incubated at 37°C for 1 hour, washed three times with DMEM, and then added complete DMEM to continue Cultivate, after 24 hours, fix the cells with 4% paraformaldehyde for 10 minutes, wash the cells with PBS, block with 5% BSA for 30 minutes, then use NDV high-immune SPF chicken positive serum as the primary antibody, dilute 1:500 times, and act at 37°C After 1 hour, wash 5 times with PBS, add 1:800 dilution of fluorescein (FITC)-labeled secondary antibody (Sigma), act for 45 minutes, and observe the results by fluorescence microscope after washing with PBS (Figure 5).

Embodiment 6. Growth characteristic and pathogenicity characteristic of attenuated strain

The purpose of the experiment: To test the growth characteristics of the attenuated vaccine strain with L gene mutation of type VII Newcastle disease virus rescued by reverse genetic technology on chicken embryos and its pathogenicity to SPF chickens.

Experimental method: the allantoic fluid and rNDV of the F4 generation of attenuated strains K1756A, K1917A, E1954Q and E1954Q+F were inoculated into the allantoic cavity of 9-day-old SPF chicken embryos at a dose of ^{10 6} EID ₅₀ , and no dead embryos were found within 96 hours; 96 hours Afterwards, the chicken embryos were frozen to death, and the allantoic fluid was collected. The HA titers were 2 ⁵ , 2 ⁶ , 2 ⁶ , 2 ⁸ and 2 ⁸ , and the EID ₅₀ per ml of allantoic fluid were 2.15×10 ⁹ , 3.16× 10 ⁹ , 1.78×10 ⁹ , 1.78×10 ⁹ and 1×10 ¹⁰ (Table 2). According to the OIE standard, the virulence index of rNDV and its attenuated strains was measured for the median lethal dose (MDT), intracerebral inoculation pathogenicity index (ICPI) and intravenous inoculation pathogenicity index (IVPI). The results are shown in Table 3 (edited by OIE [translated by Bureau of Animal Husbandry and Veterinary, Ministry of Agriculture]. Mammals, poultry and bees Class A and Class B disease diagnostic test and vaccine standard manual. Beijing: China Agricultural Science and Technology Press, 1996:140-146).

Experimental results: The above data show that the attenuated vaccine strain with L gene mutation of VII Newcastle disease virus rescued by reverse genetic manipulation technology is significantly less virulent than the parental strain, and has higher growth titer and The biological characteristics of significantly weakened virulence to SPF chickens.

Table 2. Detection of HA and EID ₅₀ of attenuated vaccine strains with L gene mutation of type VII Newcastle disease virus

the K1756A K1917A E1954Q E1954Q+F wxya HA 2 ⁵ 2 ⁶ 2 ⁶ 2 ⁸ 2 ⁸ EID ₅₀ 2.15×10 ⁹ 3.16×10 ⁹ 1.78×10 ⁹ 1.78×10 ⁹ 1×10 ¹⁰

Table 3. Determination of virus virulence index of attenuated vaccine strains with L gene mutation of type VII Newcastle disease virus

Inoculum MDT* ICPI** IVPI*** K1756A =120h 0.14 0 K1917A >120h 0.3 0 E1954Q >120h 0.3 0 E1954Q+F >120h 0.14 0 wxya =44h 1.6 2.4

* After the virus is diluted in an appropriate proportion, inoculate 9-day-old SPF chicken embryos (100uL/bird), the maximum dilution of the virus that can kill all chicken embryos is the minimum lethal dose (MLD) of the virus, and MDT is the minimum lethal dose (MLD ) caused the average time of chicken embryo death, using this method to determine MDT.

**The virus seed was inoculated into the brain of 1-day-old SPF chicks (50uL/bird) after 10-fold dilution, and the ICPI was determined.

***Injection into the subwing vein (1×10 ⁶ EID ₅₀ /bird), and determine IVPI.

Example 7. The immune effect of inducing protective antibodies in animals

The purpose of the experiment: To determine the immune protection of 2-week-old SPF chicks by the attenuated vaccine strain with L gene mutation of type VII Newcastle disease virus rescued by reverse genetics operating system.

Experimental method: 10 white Leghorn SPF chicks were artificially immunized with the virus amplified from 9-day-old SPF chicken embryos at a dose of 1×10 ⁶ EID ₅₀ via nasal drops and eye drops. The control group and the non-immune group were reared in air negative pressure filter isolators respectively. After 21 days, the blood was collected from the hindwing vein to separate the serum, and the NDV-specific HI antibody was detected routinely. In addition, 21 days after immunization, they were challenged with the highly toxic NDVⅦ-74 (seed poison for preservation in our laboratory) at a dose of 1×10 ⁶ EID ₅₀ via nasal drops and eye drops, observed for two weeks, and counted the morbidity and death.

Experimental results: White Leghorn SPF chicks were artificially immunized with allantoic virus fluid F4 at a dose of 1×10 ⁶ EID ₅₀ via nasal drops and eye drops, and observed for 3 weeks after immunization. There was no significant difference in growth and development compared with the non-immune control group. Serum NDV-specific HI antibody levels were detected 3 weeks after immunization (Table 4). There was no morbidity or death in the 4 groups of immunized groups after challenge, and they were completely protected (10/10), while the non-immune control group at the same age all suffered from morbidity and death (0/10) (Table 5). The results showed that the mutant attenuated strain can induce a high level of protective antibody response in chicks once immunized, and has a good protective effect.

Table 4. Immunogenicity of NDV L mutant attenuated strains

Vaccination* Number of SPF chickens (pieces) Inoculation age Hemagglutination inhibitor ** K1756A 10 14 2 ⁵ K1917A 10 14 2 ⁴ E1954Q 10 14 2 ⁴ E1954Q+F 10 14 2 ⁴ PBS 10 14 0

* The dosage is 1×10 ⁶ EID ₅₀ per bird.

**3 weeks after immunization, blood was collected from the subwing vein, the HI titer was measured, and the average value was calculated.

Table 5. The challenge protection test of NDV L mutant attenuated strains

group Inoculum* Inoculation dose Hemagglutination inhibitor** Survival rate*** test group K1756A 1×10 ⁶ EID ₅₀ 2 ⁵ 10/10 the K1917A 1×10 ⁶ EID ₅₀ 2 ⁷ 10/10 the E1954Q 1×10 ⁶ EID ₅₀ 2 ⁴ 10/10 the E1954Q+F 1×10 ⁶ EID ₅₀ 2 ⁵ 10/10 control group PBS - 0 0/10

* 2-week-old SPF chicks were inoculated with K1756A, K1917A, E1954Q and E1954Q +F (1×10 ⁶ EID ₅₀ /bird) by intranasal or eye drops.

**Blood was collected from the subwing vein, the HI titer was measured, and the average value was calculated.

*** 21 days after immunization, challenge with NDV Ⅶ-74 virulence through nasal drops and eye drops (1×10 ⁶ EID ₅₀ /rattle).

Example 8. Analysis of detoxification of attenuated strains K1756A, K1917A, E1954Q and E1954Q+F

Experimental purpose: To measure the detoxification of 3 NDV Ⅶ-97 L mutant attenuated strains and 1 L and F co-mutated attenuated strain rescued by reverse genetics operating system after immunization.

Experimental method: In this experiment, 10 2-week-old Leghorn SPF chicks were artificially immunized with the virus amplified from 9-day-old SPF chicken embryos at a dose of 1×10 ⁶ EID ₅₀ via nasal drops and eye drops, and a LaSota control group was set up for 10 Only the immunized group and the non-immunized group were reared in air negative pressure filter isolators respectively. 21 days after immunization, the poisonous NDVⅦ-74 (the seed virus used in our laboratory) was used to challenge the virus at a dose of 1×10 ⁶ EID ₅₀ through nasal drops and eye drops, and throat swabs were collected on the 3rd and 6th days after the challenge. Fetch and cloacal swabs were titrated with SPF chicken embryos to detect virus shedding.

Experimental results: After the immunization group and the control group were challenged with the virus, all the chickens used obtained immune protection (except the blank chicken), but the detoxification was detected after the immunization and challenge, and the detoxification amount of the LaSota throat swab was higher than that of the other 4 groups of L mutation attenuated strains (Table 6).

Table 6. Detoxification detection of NDV L mutation and attenuated strains with F and L co-mutation

Note: "/" means no virus detected

The present invention adopts recombinant poxvirus stably expressing T7 RNA polymerase system to infect BHK-21 cells, transcribes full-length genome negative-strand RNA in transfected cells, and expresses NP, P and L proteins required for initiating virus replication at the same time, and obtains High virus rescue efficiency.

Three NDV VII-97 L mutant attenuated strains K1756A, K1917A and E1954Q and one F protein cleavage site and L co-mutated attenuated strain E1954Q+F that were rescued by reverse genetic manipulation technology could achieve the same level in chicken embryos as The growth titer of the parental rNDV is similar, and the rescued virus was inoculated into the allantoic cavity of 9-day-old SPF chicken embryos at a dose of 10 ⁶ EID ₅₀ , the chicken embryos were not lethal within 96 hours, and the HA were 2 ⁵ , 2 ⁶ , 2 ⁶ and 2 ⁸ ; The EID ₅₀ per milliliter of allantoic fluid was 2.15×10 ⁹ , 3.16×10 ⁹ , 1.78×10 ⁹ , 1.78×10 ⁹ , immunizing 14-day-old SPF chicks could induce a high level of protective antibody response, and 21 days after immunization The antibody titers were 2 ⁵ , 2 ⁴ , 2 ⁴ and 2 ⁴ , respectively, fully showing that the reverse genetic operating system of the present invention rescued 4 attenuated vaccine strains with L gene mutations of type VII Newcastle disease virus and could be used as vaccine strains. The attenuated vaccine strain has good chicken embryo growth characteristics and high virus growth titer, low production cost and convenient use. NDV attenuated vaccine immunization is an essential immunization program for almost all newborn chicks in my country’s poultry industry, and the genotype is consistent with the current epidemic strains in my country. Of course, 4 strains of attenuated vaccines with mutations in the L gene of Newcastle disease virus type VII were rescued strains are the most attractive vaccine candidates.

<110> Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences

<120> Attenuated Vaccine Strain with L Gene Mutation of Type VII Newcastle Disease Virus and Preparation Method thereof

<150> CN2013102702343

<151> 2013-07-01

<160> 5

<170> PatentIn Version 3.3

<210> 1

<211> 15192

<212>DNA

<213> Artificial sequence (SEQ ID NO: 1)

<400> 1

accaaacaga gaatctgtga ggtacgataa aaggcgaaga agcaatcgag atcgtacggg 60

tagaaggtgt gaatcccgag tgcgaggccg aagctcgaac ctgaggggac cttctaccga 120

tatgtcgtct gttttcgacg agtacgagca gctcctcgct gctcagaccc gccctaacgg 180

aactcatgga gggggagaga aagggagcac tttaaaagtt gaggtcccag tattcaccct 240

aaacagtgat gatccagagg atagatggaa ttttgcggta ttctgtcttc ggattgctgt 300

tagcgaggat gccaacaaac cactcaggca aggtgctctt atatccctct tatgctccca 360

ttctcaggtg atgagaaacc atgttgccct tgcagggaaa caaaatgagg ccacactggc 420

tgttcttgag atcgatggtt ttgctaacag tgtgccccag ttcaacaata ggagtggagt 480

gtctgaggaa agagcacaga gattcatggt aatcgcagga tctctccctc gggcgtgcag 540

caacggtact ccgtttgtca cggctggggt tgaagatgat gcaccagaag atatcactga 600

cactctggaa agaatcctat ctatccaagt tcaggtatgg gtcacagtag caaaggccat 660

gactgcatat gagacagcag atgagtcaga aacaagaaga ataaataagt atatgcagca 720

aggtcgagtt cagaagaagt acatccttca tcctgtatgc aggagtgcaa ttcaactcac 780

aatcagacat tctctggcag tccgtatctt cctagttagt gagctcaaga ggggccgcaa 840

tacagcaggt gggagctcta catattacaa cttggtcggg gatgtagact catacatcag 900

gaacaccggg cttactgcat ttttcctaac actcaaatat ggaatcaata ccaagacgtc 960

agccctcgca ctcagcagcc tcacaggtga tatccaaaaa atgaaacagc tcatgcgttt 1020

atatcggatg aaaggtgaaa atgcaccata catgacattg ttaggtgaca gtgaccagat 1080

gagctttgca cctgctgagt atgcacaact ttattctttt gccatgggca tggcatcagt 1140

cttagataag ggaactggca agtaccaatt cgccagggac tttatgagca catcattctg 1200

gagacttgga gtagagtatg ctcaggccca gggaagtagc attaatgagg acatggctgc 1260

tgagctaaaa ctaaccccgg cagcaaggag aggcctggca gctgctgccc aacgagtatc 1320

cgaagaaatc ggcagcatgg aattcccac tcaacaggcg ggagtcctca ccgggctcag 1380

tgatgaaggc ccccgaactc cacagggcag atcaaacaag ccgcaagggc aaccagatgc 1440

tggggatggg gagacccaat tcttggattt tatgagagca gtggcgaacg gcatgcggga 1500

agcgccaaat cctgtacaga gcaccacccca tccagagcct cccccaaccc ctggggcatc 1560

ccaagacaac gacactgact gggggtactg atcgacaaca cccagcctgc cttcacagga 1620

tcacaccaaa ccctccgacc aaaaccctcc cacactccct gacccacaac cccgcacgac 1680

cacaccaaca aaagctcccc cccaccctct cccccactcc cagccaacg atcccgccca 1740

cccgggacaa cacaggcaca gctcagttcg tcgacaatcc gcccagagcc caaggtatta 1800

gaaaaaaata cgggtagaag agagacatcc agagaccagg acgagtcacc aagttctctg 1860

ttctcccttc tacccagtga attagggtga agatggccac ttttacagat gcggagatag 1920

atgacatatt tgagaccagt gggactgtca ttgacagcat aattacggcc cagggcaaat 1980

cagctgagac cgttggaaga agtgcgatcc cgcagggcaa gaccaaagct ctaagcacag 2040

catgggagaa gcacgggagt atccagccac acgccagtca ggacgcccct gaccaacaag 2100

acagaataga aaaacagcca tccacacctg agcaggcgac tctacacaac aatccgccga 2160

tcgcatccac tgaaccgcct cccactcagg ccgcaagcga gaccagcgac acacagctca 2220

agactggggc aagcaactcc cttctgtcca tgctcgacaa actgagtaat aaatcgtcca 2280

atgctaaaaa gggcccatgg tcgggatccc aagaagggca tcaccaacct ccggcccaac 2340

aacatgggaa ccagccgagc tatggaagca accagggaag accgcagcat caggccaagg 2400

ccgtccctgg aaaccggggc acagacgaga acacagcata tcatggacag cggaaggagt 2460

cacaaccatc agctggtgca accccctcatg cgccccagtc agggcagagc caagacaata 2520

ctcctgcacc tgtggatcgt gtccagctac ctgccgactt tgtgcaggcg atgatgtcta 2580

tgatggaggc attatcacag aaggtaagta aagttgatca tcagctggac ctagtcttga 2640

aacagacatc ctccattcct atgatgcgat ctgaaatcca acagctcaag acatctgttg 2700

cgatcatgga agctaactta ggcatgatga aaattctgga ccctggttgt gctaatgttt 2760

catccttaag tgatctccgg gcagtagccc gatcccatcc agtcctagtt tcaggccctg 2820

gagacccatc tccttacgtg acacaagggg gtgaaatgac gctcaataaa ctctcacaac 2880

cggtgcagca cccctctgaa ttgattaagt ctgccactgc aagcgggcct gacatggggag 2940

tggagaagga cactgtccgc gcattaatca cctcgcgccc gatgcatcca agctcctcgg 3000

ctaagctcct gagcaagcta gatgcagcca ggtcaattga agagatcagg aagatcaaac 3060

gccttgcgct gaatggttga tggccatcac aactcataac aggctcccgt cactttagcg 3120

tcacacggaa tccctcgggg gccctccctc gcaaatctat gcttcaacac ccaaaacaac 3180

agccctctct cacccccccc aatccctcga atgaccgcac aactgcaacc aatccagcag 3240

cattagaaat taagaaaaaa tacgggtaga atcaaagtgc cttgattgca ccaaaatgga 3300

ctcatccagg acaattgggc tgtactttga ttctgccctc ccttccagca gcctgttagc 3360

atttccgatt gtcttacaag acacgggaga cgggaagaag caaatcaccc cacaatacag 3420

gatccagcgt cttgattcgt ggacagacag taaggaagac tcggtattca tcaccaccta 3480

cgggttcatc tttcaaattg ggaatgaaga aaccaccgtc ggtgtgatca atgacaatcc 3540

caggcacgag ctactctctt ccgcaatgct ctgcttaggg agtgtcccga acgacggaga 3600

tcttgttgag ctggcgagag cctgcctcac catggtggta acttgcaaaa agagtgcaac 3660

taacactgag agaatagtct tttcagtagt gcaggctcct cgggtgctgc aaagctgtat 3720

ggttgtggca aataggtact catcagtgaa tgcagtgaag catgtgaagg cgccagaaaa 3780

gatccctggg agcggaaccc tagagtataa agtgaatttt gtctctttga ccgtggtgcc 3840

aagaagggat gtctacagga tcccaactgc agtattgaaa gtgtctggct caagcctgta 3900

caatcttgcg ctcaatgtca ctattgatgt ggacgtggat ccgaagagcc cgttagtcaa 3960

atccctttcc aagtccgata gcggatacta tgcgaatctt tttctgcata tcgggcttat 4020

gtccactgta gataagaagg gaaagaaagt gacatttgac aagatagagg aaaagataag 4080

gagactcaat ctatccgtcg ggctcagtga tgtgctcgga ccctctgtgc ttgtgaaggc 4140

gagaggtgca cggactaagc tacttgctcc ttttttctct agcagtggga cagcctgcta 4200

tcctatagca aatgcctctc cccaggttgc caagatactc tggagccaaa ctgcgcacct 4260

gcggagtgtg aaagtcatca ttcaagccgg cactcagcgt gctgtcgcag tgaccgctga 4320

tcatgaggta acctccacta agatagagag gaggcatgcc attgctaaat acaatccttt 4380

caggaaataa gttgcatccc taagactgca gttcacctgc tttcccgaat caccattaca 4440

ccagacaatg atccatctcg actgcttata gttagttcac ctgtctagca aattagaaaa 4500

aacacgggta gaagagtctg gatcccgacc ggcacactca ggacgcaaca tgggctccaa 4560

accttctacc aggatcccag cacctctgat gctggtcacc cggattatgc tgatattggg 4620

ctgtattcgt tcgacaagct ctcttgacgg caggcctctt gcagctgcag gaattgtagt 4680

aacagggat aaggcagtca atgtatacac ctcgtctcag acagggtcaa tcatagtcaa 4740

gttgctcccg aatatgccca gggataagga ggcgtgtgcg aaagccccat tagaggcata 4800

taacagaaca ctgactactt tgctcactcc tcttggcgac tccatccgca agattcaagg 4860

gtctgtgtcc acgtctggag gaaggaggca aaaacgcttt ataggtgctg ttattggcag 4920

tgtagctctt ggggttgcaa cagcggcaca gataacagca gctgcggccc taatacaagc 4980

caacaagaat gctgccaaca tccttcggct taaggagagc attgctgcaa ccaatgaagc 5040

tgtgcatgaa gtcaccgacg gattatcaca actatcagtg gcagttggga agatgcagca 5100

gtttgtcaat gaccagttta ataatacggc gcgagaattg gactgtataa aaatcacaca 5160

acaggttggt gtagaactca acctatacct aactgaattg actacagtat tcgggccaca 5220

gatcacctct cctgcattaa ctcagctgac catccaggca ctttataatt tagctggtgg 5280

caatatggat tacttattaa ctaagttagg tatagggaac aatcaactca gctcattaat 5340

tggtagcggc ctgatcactg gttaccctat actgtatgac tcacagactc aactcttggg 5400

catacaagtg aatttgccct cagtcgggaa cttaaataat atgcgtgcca cctatttgga 5460

gaccttatct gtaagtacaa ccaaaggata tgcctcagca cttgtcccga aagtagtgac 5520

acaagtcggt tctgtgatag aagagcttga cacctcatac tgtatagagt ccgatctgga 5580

tttatattgt actagaatag tgacattccc catgtcccca ggtatttatt cctgtttgag 5640

cggcaacaca tcagcttgca tgtattcaaa gactgaaggc gcactcacta cgccgtatat 5700

ggcccttaaa ggctcagtta ttgccaattg taagataaca acatgtagat gtacagaccc 5760

tcctggtatc atatcgcaaa attatggaga agctgtatcc ctgatagata gacattcgtg 5820

caatgtctta tcattagacg ggataactct gaggctcagc ggagaatttg atgcaactta 5880

tcaaaagaac atctcaatac tagattctca agtcatcgtg acaggcaatc ttgatatatc 5940

aactgaactt ggaaacgtca acaattcaat cagcaatgcc ttggataagt tggcagaaag 6000

caacagtaag atagaaaaag tcaatgtcag attaaccagc acatctgctc tcattaccta 6060

tattgttcta actgtcattt ctctattttt cggtgcactt agtctgggtt tagcgtgtta 6120

cctgatgtac aaacagaagg cacaacaaaa gaccttgcta tggcttggga ataatgccct 6180

tgatcagatg agagccacta caagagcatg aatgcagata agaggtggat atatacccaa 6240

cagcagcctg tgtgtcaatt ccgataatct gtcaagtaga agacttaaga aaaaactact 6300

gggaacaagc aaccaaagag caatacacgg gtagaacggt cagaggagcc acccttcaat 6360

cggaaattag gcttcacaac atccgttcta ccacatcacc atcaacaaga gtcaatcatg 6420

gaccgcgcgg ttaacagagt cgtgctggag aatgaggaaa gagaagcaaa gaacacatgg 6480

cgcctggttt tccggatcgc agtcttactt ttaatggtaa tgactctagc tatctccgca 6540

gctgccctgg catacagtat ggaggccagt acgccgcacg accttgcagg catatcgact 6600

gtgatctcta agacagaaga taaggttacg tctttactca gttcgagtca agatgtgata 6660

gataggatat acaagcaggt ggctcttgaa tccccgctgg cgctactaaa cactgaatct 6720

acaattatga atgcaataac ctctctttct tatcaaatta acggggctgc gaacaatagc 6780

ggatgtgggg cgcctgttca tgacccagat tatatcgggg ggataggcaa agaactcata 6840

gtggacgaca tcagtgatgt cacatcattt tatccttctg catatcaaga acacctgaat 6900

ttcatcccgg cgcctactac aggatccggt tgcactcgga taccctcatt tgacatgagc 6960

accacccatt attgttatac tcacaatgta atactatccg gttgcagaga tcactcacac 7020

tcacatcaat acttagcgct tggtgtgctt cggacatctg caacagggag ggtattcttc 7080

tctactctgc gctccatcaa tttagatgac acccaaaatc ggaagtcctg cagtgtgagt 7140

gcaacccctt taggttgtga tatgctgtgc tctaaggtca cagggactga agaggagat 7200

tacaagtcag ctgccccaac atcaatggtg cacggaaggc tagggtttga cggtcaatac 7260

catgagaagg acttagacac cacggtctta tttaaggatt gggtggcaaa ttaccccagga 7320

gtgggaggag ggtcttttat tgacgaccgt gtatggttcc cagtttacgg agggctcaaa 7380

cccaattcac ccagtgacac tgcacaagaa gggaaatatg taatatacaa gcgccataac 7440

aacacatgcc ccgatggaca agattaccaa attcggatgg ctaaatcttc atataaaccc 7500

gggcgatttg gtggaaagcg cgtacagcaa gccatattat ccatcaaagt gtcaacatcc 7560

ttgggtaagg acccggtgct gactattcca cctaatacaa tcacactcat gggagccgaa 7620

ggcagaatcc tcacagtagg gacatctcac ttcttgtacc aacgagggtc ttcatatttc 7680

tcccctgcct tattatatcc catgacagta aataacaaaa cggctacact ccatagtcct 7740

tatacgttta atgctttcac tcggccaggt agcgtccctt gccaggcatc agcaagatgc 7800

cccaactcat gcatcactgg ggtctatact gatccatatc ccttaatctt ccataggaat 7860

catactctac gaggggtctt tgggacgatg cttgatgatg aacaagcgag acttaaccccc 7920

gtatctgcag tattcgacaa catatcccgc agtcgtgtca cccgggtgag ttcaagcagc 7980

accaaggcag catacacgac atcgacatgt tttaaagttg tcaagaccaa taaagcttat 8040

tgtcttagta ttgcagagat atccaatacc ctattcgggg aatttaggat cgttccctta 8100

ttagttgaga tcctcaagga tgatagagtt taagaagcta gacttggccg attgagccaa 8160

tcataggatg gttgggaaga cgacaccaca tcaatcatct cccacaatgc ttagagtcaa 8220

tctgaatatt aacataagcc aggatcccat gttgttgggc agccacaatc agacaatact 8280

gacatgatca ttctgagtcc tgcccactat caccttatta agaaaaaata caaaaagcat 8340

tgagatataa gggaaacaa ccaacaagag ggaacacggg taggacatgg cgggctccgg 8400

tcccgaaagg gcagagcacc agatcatcct accagagtca catctatcct ctccatggt 8460

caagcacaaa ttgctatact actggaaatt gactgggcta ccgcttcctg atgaatgcga 8520

ctttgatcat ctcattatca gcaggcaatg gaagagaata ctggagtcgg ccactcctga 8580

cacagagaga atgataaaac tcgggcgggc agtgcaccag actctcaacc acaactccaa 8640

gataaccgga gtgctccatc ccaggtgttt agaagaactg gctagtattg aggtcccaga 8700

ttcaactaac aaattccgga agattgaaaa gaagatccag attcacaaca caaggtatgg 8760

agacctgttc acaaagctgt gcacgcatgt tgagaagaaa ttgctaggat catcccggtc 8820

taataatgtc ccacgatcag aggaattcag tagcatccgt acagatccgg cattctggtt 8880

tcactcaaaa tggtccagag ctaagttcgc gtggctccat ataaaacaag tccaaaggca 8940

tctgattgta gcagcaagga caaggtctgc agtcaacaag ttagtaacat taagtcataa 9000

gataggccac gtctttgtta ctcctgagct tgtcattgtg acacatacag acgagaacaa 9060

gttcacatgc ctcacccagg aacttgtatt gatgtatgcg gatatgatgg aaggcaggga 9120

catggtcaat ataatatctt ctacagcagc acatctcaga aacctatccg agaaaattga 9180

cgatattctg cggttagtag atgccctggc aaaggactta ggtaatcaag tctatgacgt 9240

tgtagcatta atggagggat tcgcatacgg tgctgttcag ctgcttgagc catcaggtac 9300

atttgcagga gatttctttg catttaacct acaggagctc aaagacactt taatcgaact 9360

tctcccaaat aatatagcgg aatcagtaac tcacgctatt gccgctgtat tctccggttt 9420

agaacagaat caagcagctg agatgttgtg cttgctacgt ttgtggggtc atcctttgct 9480

tgagtctcgt agtgcagcaa gagcagtcag gagccagatg tgtgcaccga agatggtaga 9540

cttcgatatg atcctccagg tactatcttt ctttaaagga acaatcatca atggatacag 9600

aaagaagaat tcaggtgtgt ggccgcgtgt caaagtagat acaatatacg ggaatatcat 9660

tgggcagctg tatgctgatt cagcagagat ctcacatgat gtcatgttga aggagtacaa 9720

gagtttatct gcttttgaat ttgagccatg tatagactat gaccctgtta ccaatctaag 9780

catgttccta aaagacaagg caatcgcaca tcctagtgat aattggctcg cctcatttag 9840

gcggaaccta ctctctgagg accagaagaa acagataaaa gaggcaactt caactaaccg 9900

cctcttgata gagttcttag aatcaaatga ttttgatcca tataaagaaa tggaatacct 9960

gacaaccctc gagtacctaa gaggtgacag tgtggcagta tcgtactcac tcaaagagaa 10020

agaggtaaaa gtgaatgggc ggatttttgc taagttaaca aagaaactaa ggaactgcca 10080

ggtaatggca gaaggaattc tagctgacca gattgcacct ttctttcagg gaaatggggt 10140

cattcaagat agcatatcct tgacaaagag tatgttagcg atgagtcaac tgtcctttaa 10200

cagcaataag aaacgtatca ctgactgcaa agagagggtt tcctcgaacc gcaatcatga 10260

tcagaagagc aagaatcgta gaagagttgc cacttttatc acgactgacc tacaaaagta 10320

ttgtcttaac tggagatatc agacagtcaa actattcgcc catgctatca atcagctgat 10380

gggcctacct catttcttcg agtggattca tcttaggctg atggacacta caatgtttgt 10440

aggggatcct ttcaatcctc caagtgaccc gactgactgt gatctatcaa gagtcccaaa 10500

cgatgacata tatattgtca gtgctagagg gggcattgag ggactctgcc agaagctatg 10560

gacgatgatc tcaattgctg caatccaact tgctgcagca agatctcatt gtcgagttgc 10620

ctgcatggta caaggtgaca atcaagtaat ggctgtaacg agagaggtaa gatcagatga 10680

ttccccggat atggtgttga cgcagttgca tcaagctagt gataatttct tcaaggaatt 10740

gattcatgtc aatcatttga ttggccataa tctgaaggat cgtgaaacca ttagatcaga 10800

cacattcttc atatacagca aacgaatatt caaggatgga gcaatactca gtcaggtcct 10860

caaaaattca tctaaattgg tgctaatatc aggcgacctt agcgaaaaca ctgtaatgtc 10920

ctgtgccaac attgcatcca ctgtagcacg actatgtgag aatgggcttc caaaggattt 10980

ctgttactat ttgaactacc taatgagttg tgtgcagaca tactttgatt cggagttctc 11040

tattacccac agctcgcaat cagattccaa ccagtcctgg atcgaggata tctctttcgt 11100

acactcatac gtgttaaccc ctgcccagct ggggggactg agcaaccttc aatactcaag 11160

gctctacaca aggaatattg gtgacccagg gaccactgct ttcgcagagg tcaagcgact 11220

ggaagcagtg gggttgctga gtcccagcat catgactaac atcttaacca ggccacctgg 11280

caatggagac tgggccagcc tatgcaacga cccatactct tttaattttg aaactgttgc 11340

aagcccaaat attgtcctca agaaacatac acagaaagtc ctatttgaga catgttcaaa 11400

ccccttatta tccggggtac atacagagga caatgaggca gaggagaaag cattggctga 11460

attcttactc aatcaagaag tgattcaccc acgtgtcgca catgctatca tggaagcaag 11520

ctctgtgggt aggagaaagc aaattcaagg gcttgttgac acaacgaaca ctgtgattaa 11580

gattgcactg actaggaggc ccctcggtat caaaaggctg atgcggataa tcaattactc 11640

gagcatgcat gcaatgttgt tcagagatga tattttctta tccaaagat ccaaccaccc 11700

attagtttct tccaatatgt gctcgctgac gctagcagat tatgcccgaa acagaagctg 11760

gtcacctctg acaggggggca ggaaaatact gggtgtatcc aaccctgata ccatagaact 11820

tgtggaggga gagattctca gcgtcagtgg agggtgcaca aaatgtgaca gcggagatga 11880

gcagtttact tggttccatc ttccaagcaa tatagagctg actgatgaca ccagaaaaaa 11940

tcccccgatg agagtgccat atcttgggtc gaagactcaa gagaggagag ctgcctcgct 12000

tgcgaaaata gcccacatgt caccacatgt gaaagcagca ctaagggcat catccgtgtt 12060

aatctgggct tatggggaca acgaagtgaa ctggactgct gctcttaata ttgcaaggtc 12120

tcgatgcaac ataagctcag agtaccttcg gctattgtca cccctgccca cggctgggaa 12180

tctccaacat agattggatg atggcataac ccagatgaca tttacccctg catctctcta 12240

cagagtgtcg ccttacgttc acatatccaa tgattctcaa aggctattca ccgaagaagg 12300

ggtcaaagag ggaaacgtgg tttaccaaca aattatgctc ttgggtttat ccctaattga 12360

atcactcttc ccaatgacag caaccagaac atatgatgag atcacattac acctccacag 12420

taaatttagc tgctgtatcc gagaagcgcc tgttgcggtt cccttcgagc tcctcgggct 12480

ggcaccggaa ttaagaatgg taacctcaaa taagttcatg tatgatccta gccctatatc 12540

agagagagat ttcgcgagac ttgacttagc tatcttcaag agttacgagc ttaatttgga 12600

atcatattcc acgctggagc taatgaacat tctttcaata tctagcggga agttgattgg 12660

ccaatctgtg gtttcttatg atgaagacac ctctataaag aatgatgcta taatagtgta 12720

tgacaacaca cgaaattgga ttagtgaggc gcagaactca gatgtggtcc gcttgtttga 12780

gtatgcagca ctcgaagtgc ttcttgactg tgcttatcaa ctctactatc tgagggtaag 12840

gggtctaaac aacatcgtcc tatacatgaa tgacttatat aagaacatgc cagggatcct 12900

actctccaat attgcggcca cgatatccca ccccatcatt cactcaaggt tgaatgcagt 12960

aggcctaatt aaccatgacg ggtcacacca gctcgcagat atagacttcg tcgaggtgtc 13020

tgcaaaattg ttagtctctt gcactcgacg cgtggtctca ggcttatatg cagggaataa 13080

gtacgatctg ctgttcccat ctgtcttaga tgataacctg aatgagaaga tgctccaact 13140

gatttcccgg ttatgctgtc tgtacacagt gctctttgct acaacaagag aaatcccaaa 13200

aataaggggc ctatcagcag aagagaaatg ctcaatactc actgagtatc tactgtcaga 13260

tgctgtaaaa ccattgctta ggcccgaaca agtgaattct atcatgtctc ccaacataat 13320

cacgttccca gccaatctat attacatgtc taggaagagc cttaatttga tcagagaacg 13380

agaggacaga gatactatct tgtcattgtt gttccctcag gaaccactgc ttgagcttcg 13440

cccagtacgg gacattggtg ctcgagtgaa agacccgttt acccggcaac ccgcatcatt 13500

catacaagag ctagatctga gtgccccagc aaggtacgac gcatttacac tgagtaaggc 13560

ctgcttcgag catacattac cgaacccaaa ggaggattac ctagtacggt acttgttcag 13620

aggaataggg actgcttcat cttcttggta tgcggcatct catcttctat ccgtacctga 13680

ggtcaggtgt gcaagacatg ggaactcctt atacttagcg gaaggaagcg gagccatcat 13740

gagtcttctt gaattgcata taccacatga gactatctat tacaatacac ttttctcgaa 13800

tgagatgaac cctccacagc gacatttcgg acctacacca acacagtttc taaactcggt 13860

cgtttatagg aatctacaag cggaagtgcc atgtaaagat ggatatgtcc aggagttctg 13920

cccattatgg agagagaatg cagaagaaag tgacctgacc tcagataaag cagttggata 13980

tatcacatcc gtggtaccct acaggtctgt atcattacta cattgtgaca ttgagattcc 14040

tccagggtcc aatcaaagct tattagatca actggctact aatttatccc tgattgccat 14100

gcattctgtg aggggagggcg gggtagtgat catcaaagta ctgtatgcaa tggggtacta 14160

cttccatta ctcatgaatt tattcactcc atgttccaca aaaggatata tactctccaa 14220

tggctatgcc tgtagagggg atatggagtg ttacctgata ttcgttatgg gctacttggg 14280

cgggcccacc ttcgtgcacg aagtggtaag gatggcaaaa actctaatac aacgacacgg 14340

tacactccta tctaaatcag atgaaattac attgactaag ctatttacct cacagcagcg 14400

tcatgtaaca gatatcctat ccagtccttt accgaagcta atgaagctct tgagagaaaa 14460

tattgatgcc gcactaattg aagctggggg acagcccgtc cgtccattct gtgcggaaag 14520

tttggtgagc acactaacag atatgactca gacagctcag atcattgcca gccacattga 14580

cacagtcatt cggtctgtaa tttacatgga ggctgagggt gacctcgccg acagtgtt 14640

cttatttact ccttacaatc tatccacaga cggtaaaagg agaacatcac ttaagcagtg 14700

caccaaacag atcttggaag tcacaatact gggtctcaga gccaaagata tcaataaagt 14760

aggtgatgta atcagtttag tactcagagg tgcggtttct ctagaggacc tcatcccatt 14820

aaggacatac ctgaagcgca gtacctgcac taaatacctg aaagcggtcc taggtattac 14880

taaactcaaa gaaatgttca cagatacctc attackgtac ttgactcgtg ctcaacaaaa 14940

attctacatg aaaaccatag gtaatgctgc caagggatat tactgtaata atgactctta 15000

aaggcaatcg tacgccaatc agttatcttc ctaactgatg actccctcat tgactcaatt 15060

ataccagatt agaaaaaagt taaattccga ctctttggaa ctcgtattcg gattcagtta 15120

gttaacttta agcaagagtg cgcaaagtca tccctaatta tagtgatgtc attcaccaaa 15180

tctctgtttg gt 15192

the

<210> 2

<211> 15192

<212>DNA

<213> Artificial sequence (SEQ ID NO: 2)

<400> 2

accaaacaga gaatctgtga ggtacgataa aaggcgaaga agcaatcgag atcgtacggg 60

tagaaggtgt gaatcccgag tgcgaggccg aagctcgaac ctgaggggac cttctaccga 120

tatgtcgtct gttttcgacg agtacgagca gctcctcgct gctcagaccc gccctaacgg 180

aactcatgga gggggagaga aagggagcac tttaaaagtt gaggtcccag tattcaccct 240

aaacagtgat gatccagagg atagatggaa ttttgcggta ttctgtcttc ggattgctgt 300

tagcgaggat gccaacaaac cactcaggca aggtgctctt atatccctct tatgctccca 360

ttctcaggtg atgagaaacc atgttgccct tgcagggaaa caaaatgagg ccacactggc 420

tgttcttgag atcgatggtt ttgctaacag tgtgccccag ttcaacaata ggagtggagt 480

gtctgaggaa agagcacaga gattcatggt aatcgcagga tctctccctc gggcgtgcag 540

caacggtact ccgtttgtca cggctggggt tgaagatgat gcaccagaag atatcactga 600

cactctggaa agaatcctat ctatccaagt tcaggtatgg gtcacagtag caaaggccat 660

gactgcatat gagacagcag atgagtcaga aacaagaaga ataaataagt atatgcagca 720

aggtcgagtt cagaagaagt acatccttca tcctgtatgc aggagtgcaa ttcaactcac 780

aatcagacat tctctggcag tccgtatctt cctagttagt gagctcaaga ggggccgcaa 840

tacagcaggt gggagctcta catattacaa cttggtcggg gatgtagact catacatcag 900

gaacaccggg cttactgcat ttttcctaac actcaaatat ggaatcaata ccaagacgtc 960

agccctcgca ctcagcagcc tcacaggtga tatccaaaaa atgaaacagc tcatgcgttt 1020

atatcggatg aaaggtgaaa atgcaccata catgacattg ttaggtgaca gtgaccagat 1080

gagctttgca cctgctgagt atgcacaact ttattctttt gccatgggca tggcatcagt 1140

cttagataag ggaactggca agtaccaatt cgccagggac tttatgagca catcattctg 1200

gagacttgga gtagagtatg ctcaggccca gggaagtagc attaatgagg acatggctgc 1260

tgagctaaaa ctaaccccgg cagcaaggag aggcctggca gctgctgccc aacgagtatc 1320

cgaagaaatc ggcagcatgg aattcccac tcaacaggcg ggagtcctca ccgggctcag 1380

tgatgaaggc ccccgaactc cacagggcag atcaaacaag ccgcaagggc aaccagatgc 1440

tggggatggg gagacccaat tcttggattt tatgagagca gtggcgaacg gcatgcggga 1500

agcgccaaat cctgtacaga gcaccacccca tccagagcct cccccaaccc ctggggcatc 1560

ccaagacaac gacactgact gggggtactg atcgacaaca cccagcctgc cttcacagga 1620

tcacaccaaa ccctccgacc aaaaccctcc cacactccct gacccacaac cccgcacgac 1680

cacaccaaca aaagctcccc cccaccctct cccccactcc cagccaacg atcccgccca 1740

cccgggacaa cacaggcaca gctcagttcg tcgacaatcc gcccagagcc caaggtatta 1800

gaaaaaaata cgggtagaag agagacatcc agagaccagg acgagtcacc aagttctctg 1860

ttctcccttc tacccagtga attagggtga agatggccac ttttacagat gcggagatag 1920

atgacatatt tgagaccagt gggactgtca ttgacagcat aattacggcc cagggcaaat 1980

cagctgagac cgttggaaga agtgcgatcc cgcagggcaa gaccaaagct ctaagcacag 2040

catgggagaa gcacgggagt atccagccac acgccagtca ggacgcccct gaccaacaag 2100

acagaataga aaaacagcca tccacacctg agcaggcgac tctacacaac aatccgccga 2160

tcgcatccac tgaaccgcct cccactcagg ccgcaagcga gaccagcgac acacagctca 2220

agactggggc aagcaactcc cttctgtcca tgctcgacaa actgagtaat aaatcgtcca 2280

atgctaaaaa gggcccatgg tcgggatccc aagaagggca tcaccaacct ccggcccaac 2340

aacatgggaa ccagccgagc tatggaagca accagggaag accgcagcat caggccaagg 2400

ccgtccctgg aaaccggggc acagacgaga acacagcata tcatggacag cggaaggagt 2460

cacaaccatc agctggtgca accccctcatg cgccccagtc agggcagagc caagacaata 2520

ctcctgcacc tgtggatcgt gtccagctac ctgccgactt tgtgcaggcg atgatgtcta 2580

tgatggaggc attatcacag aaggtaagta aagttgatca tcagctggac ctagtcttga 2640

aacagacatc ctccattcct atgatgcgat ctgaaatcca acagctcaag acatctgttg 2700

cgatcatgga agctaactta ggcatgatga aaattctgga ccctggttgt gctaatgttt 2760

catccttaag tgatctccgg gcagtagccc gatcccatcc agtcctagtt tcaggccctg 2820

gagacccatc tccttacgtg acacaagggg gtgaaatgac gctcaataaa ctctcacaac 2880

cggtgcagca cccctctgaa ttgattaagt ctgccactgc aagcgggcct gacatgggag 2940

tggagaagga cactgtccgc gcattaatca cctcgcgccc gatgcatcca agctcctcgg 3000

ctaagctcct gagcaagcta gatgcagcca ggtcaattga agagatcagg aagatcaaac 3060

gccttgcgct gaatggttga tggccatcac aactcataac aggctcccgt cactttagcg 3120

tcacacggaa tccctcgggg gccctccctc gcaaatctat gcttcaacac ccaaaacaac 3180

agccctctct cacccccccc aatccctcga atgaccgcac aactgcaacc aatccagcag 3240

cattagaaat taagaaaaaa tacgggtaga atcaaagtgc cttgattgca ccaaaatgga 3300

ctcatccagg acaattgggc tgtactttga ttctgccctc ccttccagca gcctgttagc 3360

atttccgatt gtcttacaag acacgggaga cgggaagaag caaatcaccc cacaatacag 3420

gatccagcgt cttgattcgt ggacagacag taaggaagac tcggtattca tcaccaccta 3480

cgggttcatc tttcaaattg ggaatgaaga aaccaccgtc ggtgtgatca atgacaatcc 3540

caggcacgag ctactctctt ccgcaatgct ctgcttaggg agtgtcccga acgacggaga 3600

tcttgttgag ctggcgagag cctgcctcac catggtggta acttgcaaaa agagtgcaac 3660

taacactgag agaatagtct tttcagtagt gcaggctcct cgggtgctgc aaagctgtat 3720

ggttgtggca aataggtact catcagtgaa tgcagtgaag catgtgaagg cgccagaaaa 3780

gatccctggg agcggaaccc tagagtataa agtgaatttt gtctctttga ccgtggtgcc 3840

aagaagggat gtctacagga tcccaactgc agtattgaaa gtgtctggct caagcctgta 3900

caatcttgcg ctcaatgtca ctattgatgt ggacgtggat ccgaagagcc cgttagtcaa 3960

atccctttcc aagtccgata gcggatacta tgcgaatctt tttctgcata tcgggcttat 4020

gtccactgta gataagaagg gaaagaaagt gacatttgac aagatagagg aaaagataag 4080

gagactcaat ctatccgtcg ggctcagtga tgtgctcgga ccctctgtgc ttgtgaaggc 4140

gagaggtgca cggactaagc tacttgctcc ttttttctct agcagtggga cagcctgcta 4200

tcctatagca aatgcctctc cccaggttgc caagatactc tggagccaaa ctgcgcacct 4260

gcggagtgtg aaagtcatca ttcaagccgg cactcagcgt gctgtcgcag tgaccgctga 4320

tcatgaggta acctccacta agatagagag gaggcatgcc attgctaaat acaatccttt 4380

caggaaataa gttgcatccc taagactgca gttcacctgc tttcccgaat caccattaca 4440

ccagacaatg atccatctcg actgcttata gttagttcac ctgtctagca aattagaaaa 4500

aacacgggta gaagagtctg gatcccgacc ggcacactca ggacgcaaca tgggctccaa 4560

accttctacc aggatcccag cacctctgat gctggtcacc cggattatgc tgatattggg 4620

ctgtattcgt tcgacaagct ctcttgacgg caggcctctt gcagctgcag gaattgtagt 4680

aacagggat aaggcagtca atgtatacac ctcgtctcag acagggtcaa tcatagtcaa 4740

gttgctcccg aatatgccca gggataagga ggcgtgtgcg aaagccccat tagaggcata 4800

taacagaaca ctgactactt tgctcactcc tcttggcgac tccatccgca agattcaagg 4860

gtctgtgtcc acgtctggag gaaggaggca aaaacgcttt ataggtgctg ttattggcag 4920

tgtagctctt ggggttgcaa cagcggcaca gataacagca gctgcggccc taatacaagc 4980

caacaagaat gctgccaaca tccttcggct taaggagagc attgctgcaa ccaatgaagc 5040

tgtgcatgaa gtcaccgacg gattatcaca actatcagtg gcagttggga agatgcagca 5100

gtttgtcaat gaccagttta ataatacggc gcgagaattg gactgtataa aaatcacaca 5160

acaggttggt gtagaactca acctatacct aactgaattg actacagtat tcgggccaca 5220

gatcacctct cctgcattaa ctcagctgac catccaggca ctttataatt tagctggtgg 5280

caatatggat tacttattaa ctaagttagg tatagggaac aatcaactca gctcattaat 5340

tggtagcggc ctgatcactg gttaccctat actgtatgac tcacagactc aactcttggg 5400

catacaagtg aatttgccct cagtcgggaa cttaaataat atgcgtgcca cctatttgga 5460

gaccttatct gtaagtacaa ccaaaggata tgcctcagca cttgtcccga aagtagtgac 5520

acaagtcggt tctgtgatag aagagcttga cacctcatac tgtatagagt ccgatctgga 5580

tttatattgt actagaatag tgacattccc catgtcccca ggtatttatt cctgtttgag 5640

cggcaacaca tcagcttgca tgtattcaaa gactgaaggc gcactcacta cgccgtatat 5700

ggcccttaaa ggctcagtta ttgccaattg taagataaca acatgtagat gtacagaccc 5760

tcctggtatc atatcgcaaa attatggaga agctgtatcc ctgatagata gacattcgtg 5820

caatgtctta tcattagacg ggataactct gaggctcagc ggagaatttg atgcaactta 5880

tcaaaagaac atctcaatac tagattctca agtcatcgtg acaggcaatc ttgatatatc 5940

aactgaactt ggaaacgtca acaattcaat cagcaatgcc ttggataagt tggcagaaag 6000

caacagtaag atagaaaaag tcaatgtcag attaaccagc acatctgctc tcattaccta 6060

tattgttcta actgtcattt ctctattttt cggtgcactt agtctgggtt tagcgtgtta 6120

cctgatgtac aaacagaagg cacaacaaaa gaccttgcta tggcttggga ataatgccct 6180

tgatcagatg agagccacta caagagcatg aatgcagata agaggtggat atatacccaa 6240

cagcagcctg tgtgtcaatt ccgataatct gtcaagtaga agacttaaga aaaaactact 6300

gggaacaagc aaccaaagag caatacacgg gtagaacggt cagaggagcc acccttcaat 6360

cggaaattag gcttcacaac atccgttcta ccacatcacc atcaacaaga gtcaatcatg 6420

gaccgcgcgg ttaacagagt cgtgctggag aatgaggaaa gagaagcaaa gaacacatgg 6480

cgcctggttt tccggatcgc agtcttactt ttaatggtaa tgactctagc tatctccgca 6540

gctgccctgg catacagtat ggaggccagt acgccgcacg accttgcagg catatcgact 6600

gtgatctcta agacagaaga taaggttacg tctttactca gttcgagtca agatgtgata 6660

gataggatat acaagcaggt ggctcttgaa tccccgctgg cgctactaaa cactgaatct 6720

acaattatga atgcaataac ctctctttct tatcaaatta acggggctgc gaacaatagc 6780

ggatgtgggg cgcctgttca tgacccagat tatatcgggg ggataggcaa agaactcata 6840

gtggacgaca tcagtgatgt cacatcattt tatccttctg catatcaaga acacctgaat 6900

ttcatcccgg cgcctactac aggatccggt tgcactcgga taccctcatt tgacatgagc 6960

accacccatt attgttatac tcacaatgta atactatccg gttgcagaga tcactcacac 7020

tcacatcaat acttagcgct tggtgtgctt cggacatctg caacagggag ggtattcttc 7080

tctactctgc gctccatcaa tttagatgac acccaaaatc ggaagtcctg cagtgtgagt 7140

gcaacccctt taggttgtga tatgctgtgc tctaaggtca cagggactga agaggagat 7200

tacaagtcag ctgccccaac atcaatggtg cacggaaggc tagggtttga cggtcaatac 7260

catgagaagg acttagacac cacggtctta tttaaggatt gggtggcaaa ttaccccagga 7320

gtgggaggag ggtcttttat tgacgaccgt gtatggttcc cagtttacgg agggctcaaa 7380

cccaattcac ccagtgacac tgcacaagaa gggaaatatg taatatacaa gcgccataac 7440

aacacatgcc ccgatggaca agattaccaa attcggatgg ctaaatcttc atataaaccc 7500

gggcgatttg gtggaaagcg cgtacagcaa gccatattat ccatcaaagt gtcaacatcc 7560

ttgggtaagg acccggtgct gactattcca cctaatacaa tcacactcat gggagccgaa 7620

ggcagaatcc tcacagtagg gacatctcac ttcttgtacc aacgagggtc ttcatatttc 7680

tcccctgcct tattatatcc catgacagta aataacaaaa cggctacact ccatagtcct 7740

tatacgttta atgctttcac tcggccaggt agcgtccctt gccaggcatc agcaagatgc 7800

cccaactcat gcatcactgg ggtctatact gatccatatc ccttaatctt ccataggaat 7860

catactctac gaggggtctt tgggacgatg cttgatgatg aacaagcgag acttaaccccc 7920

gtatctgcag tattcgacaa catatcccgc agtcgtgtca cccgggtgag ttcaagcagc 7980

accaaggcag catacacgac atcgacatgt tttaaagttg tcaagaccaa taaagcttat 8040

tgtcttagta ttgcagagat atccaatacc ctattcgggg aatttaggat cgttccctta 8100

ttagttgaga tcctcaagga tgatagagtt taagaagcta gacttggccg attgagccaa 8160

tcataggatg gttgggaaga cgacaccaca tcaatcatct cccacaatgc ttagagtcaa 8220

tctgaatatt aacataagcc aggatcccat gttgttgggc agccacaatc agacaatact 8280

gacatgatca ttctgagtcc tgcccactat caccttatta agaaaaaata caaaaagcat 8340

tgagatataa ggggaaacaa ccaacaagag ggaacacggg taggacatgg cgggctccgg 8400

tcccgaaagg gcagagcacc agatcatcct accagagtca catctatcct ctccatggt 8460

caagcacaaa ttgctatact actggaaatt gactgggcta ccgcttcctg atgaatgcga 8520

ctttgatcat ctcattatca gcaggcaatg gaagagaata ctggagtcgg ccactcctga 8580

cacagagaga atgataaaac tcgggcgggc agtgcaccag actctcaacc acaactccaa 8640

gataaccgga gtgctccatc ccaggtgttt agaagaactg gctagtattg aggtcccaga 8700

ttcaactaac aaattccgga agattgaaaa gaagatccag attcacaaca caaggtatgg 8760

agacctgttc acaaagctgt gcacgcatgt tgagaagaaa ttgctaggat catcccggtc 8820

taataatgtc ccacgatcag aggaattcag tagcatccgt acagatccgg cattctggtt 8880

tcactcaaaa tggtccagag ctaagttcgc gtggctccat ataaaacaag tccaaaggca 8940

tctgattgta gcagcaagga caaggtctgc agtcaacaag ttagtaacat taagtcataa 9000

gataggccac gtctttgtta ctcctgagct tgtcattgtg acacatacag acgagaacaa 9060

gttcacatgc ctcacccagg aacttgtatt gatgtatgcg gatatgatgg aaggcaggga 9120

catggtcaat ataatatctt ctacagcagc acatctcaga aacctatccg agaaaattga 9180

cgatattctg cggttagtag atgccctggc aaaggactta ggtaatcaag tctatgacgt 9240

tgtagcatta atggagggat tcgcatacgg tgctgttcag ctgcttgagc catcaggtac 9300

atttgcagga gatttctttg catttaacct acaggagctc aaagacactt taatcgaact 9360

tctcccaaat aatatagcgg aatcagtaac tcacgctatt gccgctgtat tctccggttt 9420

agaacagaat caagcagctg agatgttgtg cttgctacgt ttgtggggtc atcctttgct 9480

tgagtctcgt agtgcagcaa gagcagtcag gagccagatg tgtgcaccga agatggtaga 9540

cttcgatatg atcctccagg tactatcttt ctttaaagga acaatcatca atggatacag 9600

aaagaagaat tcaggtgtgt ggccgcgtgt caaagtagat acaatatacg ggaatatcat 9660

tgggcagctg tatgctgatt cagcagagat ctcacatgat gtcatgttga aggagtacaa 9720

gagtttatct gcttttgaat ttgagccatg tatagactat gaccctgtta ccaatctaag 9780

catgttccta aaagacaagg caatcgcaca tcctagtgat aattggctcg cctcatttag 9840

gcggaaccta ctctctgagg accagaagaa acagataaaa gaggcaactt caactaaccg 9900

cctcttgata gagttcttag aatcaaatga ttttgatcca tataaagaaa tggaatacct 9960

gacaaccctc gagtacctaa gaggtgacag tgtggcagta tcgtactcac tcaaagagaa 10020

agaggtaaaa gtgaatgggc ggatttttgc taagttaaca aagaaactaa ggaactgcca 10080

ggtaatggca gaaggaattc tagctgacca gattgcacct ttctttcagg gaaatggggt 10140

cattcaagat agcatatcct tgacaaagag tatgttagcg atgagtcaac tgtcctttaa 10200

cagcaataag aaacgtatca ctgactgcaa agagagggtt tcctcgaacc gcaatcatga 10260

tcagaagagc aagaatcgta gaagagttgc cacttttatc acgactgacc tacaaaagta 10320

ttgtcttaac tggagatatc agacagtcaa actattcgcc catgctatca atcagctgat 10380

gggcctacct catttcttcg agtggattca tcttaggctg atggacacta caatgtttgt 10440

aggggatcct ttcaatcctc caagtgaccc gactgactgt gatctatcaa gagtcccaaa 10500

cgatgacata tatattgtca gtgctagagg gggcattgag ggactctgcc agaagctatg 10560

gacgatgatc tcaattgctg caatccaact tgctgcagca agatctcatt gtcgagttgc 10620

ctgcatggta caaggtgaca atcaagtaat ggctgtaacg agagaggtaa gatcagatga 10680

ttccccggat atggtgttga cgcagttgca tcaagctagt gataatttct tcaaggaatt 10740

gattcatgtc aatcatttga ttggccataa tctgaaggat cgtgaaacca ttagatcaga 10800

cacattcttc atatacagca aacgaatatt caaggatgga gcaatactca gtcaggtcct 10860

caaaaattca tctaaattgg tgctaatatc aggcgacctt agcgaaaaca ctgtaatgtc 10920

ctgtgccaac attgcatcca ctgtagcacg actatgtgag aatgggcttc caaaggattt 10980

ctgttactat ttgaactacc taatgagttg tgtgcagaca tactttgatt cggagttctc 11040

tattacccac agctcgcaat cagattccaa ccagtcctgg atcgaggata tctctttcgt 11100

acactcatac gtgttaaccc ctgcccagct ggggggactg agcaaccttc aatactcaag 11160

gctctacaca aggaatattg gtgacccagg gaccactgct ttcgcagagg tcaagcgact 11220

ggaagcagtg gggttgctga gtcccagcat catgactaac atcttaacca ggccacctgg 11280

caatggagac tgggccagcc tatgcaacga cccatactct tttaattttg aaactgttgc 11340

aagcccaaat attgtcctca agaaacatac acagaaagtc ctatttgaga catgttcaaa 11400

ccccttatta tccggggtac atacagagga caatgaggca gaggagaaag cattggctga 11460

attcttactc aatcaagaag tgattcaccc acgtgtcgca catgctatca tggaagcaag 11520

ctctgtgggt aggagaaagc aaattcaagg gcttgttgac acaacgaaca ctgtgattaa 11580

gattgcactg actaggaggc ccctcggtat caaaaggctg atgcggataa tcaattactc 11640

gagcatgcat gcaatgttgt tcagagatga tattttctta tccaaagat ccaaccaccc 11700

attagtttct tccaatatgt gctcgctgac gctagcagat tatgcccgaa acagaagctg 11760

gtcacctctg acaggggggca ggaaaatact gggtgtatcc aaccctgata ccatagaact 11820

tgtggaggga gagattctca gcgtcagtgg agggtgcaca aaatgtgaca gcggagatga 11880

gcagtttact tggttccatc ttccaagcaa tatagagctg actgatgaca ccagaaaaaa 11940

tcccccgatg agagtgccat atcttgggtc gaagactcaa gagaggagag ctgcctcgct 12000

tgcgaaaata gcccacatgt caccacatgt gaaagcagca ctaagggcat catccgtgtt 12060

aatctgggct tatggggaca acgaagtgaa ctggactgct gctcttaata ttgcaaggtc 12120

tcgatgcaac ataagctcag agtaccttcg gctattgtca cccctgccca cggctgggaa 12180

tctccaacat agattggatg atggcataac ccagatgaca tttacccctg catctctcta 12240

cagagtgtcg ccttacgttc acatatccaa tgattctcaa aggctattca ccgaagaagg 12300

ggtcaaagag ggaaacgtgg tttaccaaca aattatgctc ttgggtttat ccctaattga 12360

atcactcttc ccaatgacag caaccagaac atatgatgag atcacattac acctccacag 12420

taaatttagc tgctgtatcc gagaagcgcc tgttgcggtt cccttcgagc tcctcgggct 12480

ggcaccggaa ttaagaatgg taacctcaaa taagttcatg tatgatccta gccctatatc 12540

agagagagat ttcgcgagac ttgacttagc tatcttcaag agttacgagc ttaatttgga 12600

atcatattcc acgctggagc taatgaacat tctttcaata tctagcggga agttgattgg 12660

ccaatctgtg gtttcttatg atgaagacac ctctataaag aatgatgcta taatagtgta 12720

tgacaacaca cgaaattgga ttagtgaggc gcagaactca gatgtggtcc gcttgtttga 12780

gtatgcagca ctcgaagtgc ttcttgactg tgcttatcaa ctctactatc tgagggtaag 12840

gggtctaaac aacatcgtcc tatacatgaa tgacttatat aagaacatgc cagggatcct 12900

actctccaat attgcggcca cgatatccca ccccatcatt cactcaaggt tgaatgcagt 12960

aggcctaatt aaccatgacg ggtcacacca gctcgcagat atagacttcg tcgaggtgtc 13020

tgcaaaattg ttagtctctt gcactcgacg cgtggtctca ggcttatatg cagggaataa 13080

gtacgatctg ctgttcccat ctgtcttaga tgataacctg aatgagaaga tgctccaact 13140

gatttcccgg ttatgctgtc tgtacacagt gctctttgct acaacaagag aaatcccaaa 13200

aataaggggc ctatcagcag aagagaaatg ctcaatactc actgagtatc tactgtcaga 13260

tgctgtaaaa ccattgctta ggcccgaaca agtgaattct atcatgtctc ccaacataat 13320

cacgttccca gccaatctat attacatgtc taggaagagc cttaatttga tcagagaacg 13380

agaggacaga gatactatct tgtcattgtt gttccctcag gaaccactgc ttgagcttcg 13440

cccagtacgg gacattggtg ctcgagtgaa agacccgttt acccggcaac ccgcatcatt 13500

catacaagag ctagatctga gtgccccagc aaggtacgac gcatttacac tgagtaaggc 13560

ctgcttcgag catacattac cgaacccaaa ggaggattac ctagtacggt acttgttcag 13620

aggaataggg actgcttcat cttcttggta taaggcatct catcttctat ccgtacctga 13680

ggtcaggtgt gcaagacatg ggaactcctt atacttagcg gaaggaagcg gagccatcat 13740

gagtcttctt gaattgcata taccacatga gactatctat tacaatacac ttttctcgaa 13800

tgagatgaac cctccacagc gacatttcgg acctacacca acacagtttc taaactcggt 13860

cgtttatagg aatctacaag cggaagtgcc atgtaaagat ggatatgtcc aggagttctg 13920

cccattatgg agagagaatg cagaagaaag tgacctgacc tcagataaag cagttggata 13980

tatcacatcc gtggtaccct acaggtctgt atcattacta cattgtgaca ttgagattcc 14040

tccagggtcc aatcaaagct tattagatca actggctact aatttatccc tgattgccat 14100

gcattctgtg aggggagggcg gggtagtgat catcgcagta ctgtatgcaa tggggtacta 14160

cttccatta ctcatgaatt tattcactcc atgttccaca aaaggatata tactctccaa 14220

tggctatgcc tgtagagggg atatggagtg ttacctgata ttcgttatgg gctacttggg 14280

cgggcccacc ttcgtgcacg aagtggtaag gatggcaaaa actctaatac aacgacacgg 14340

tacactccta tctaaatcag atgaaattac attgactaag ctatttacct cacagcagcg 14400

tcatgtaaca gatatcctat ccagtccttt accgaagcta atgaagctct tgagagaaaa 14460

tattgatgcc gcactaattg aagctggggg acagcccgtc cgtccattct gtgcggaaag 14520

tttggtgagc acactaacag atatgactca gacagctcag atcattgcca gccacattga 14580

cacagtcatt cggtctgtaa tttacatgga ggctgagggt gacctcgccg acagtgtt 14640

cttatttact ccttacaatc tatccacaga cggtaaaagg agaacatcac ttaagcagtg 14700

caccaaacag atcttggaag tcacaatact gggtctcaga gccaaagata tcaataaagt 14760

aggtgatgta atcagtttag tactcagagg tgcggtttct ctagaggacc tcatcccatt 14820

aaggacatac ctgaagcgca gtacctgcac taaatacctg aaagcggtcc taggtattac 14880

taaactcaaa gaaatgttca cagatacctc attackgtac ttgactcgtg ctcaacaaaa 14940

attctacatg aaaaccatag gtaatgctgc caagggatat tactgtaata atgactctta 15000

aaggcaatcg tacgccaatc agttatcttc ctaactgatg actccctcat tgactcaatt 15060

ataccagatt agaaaaaagt taaattccga ctctttggaa ctcgtattcg gattcagtta 15120

gttaacttta agcaagagtg cgcaaagtca tccctaatta tagtgatgtc attcaccaaa 15180

tctctgtttg gt 15192

the

<210> 3

<211> 15192

<212>DNA

<213> Artificial sequence (SEQ ID NO: 3)

<400> 3

accaaacaga gaatctgtga ggtacgataa aaggcgaaga agcaatcgag atcgtacggg 60

tagaaggtgt gaatcccgag tgcgaggccg aagctcgaac ctgaggggac cttctaccga 120

tatgtcgtct gttttcgacg agtacgagca gctcctcgct gctcagaccc gccctaacgg 180

aactcatgga gggggagaga aagggagcac tttaaaagtt gaggtcccag tattcaccct 240

aaacagtgat gatccagagg atagatggaa ttttgcggta ttctgtcttc ggattgctgt 300

tagcgaggat gccaacaaac cactcaggca aggtgctctt atatccctct tatgctccca 360

ttctcaggtg atgagaaacc atgttgccct tgcagggaaa caaaatgagg ccacactggc 420

tgttcttgag atcgatggtt ttgctaacag tgtgccccag ttcaacaata ggagtggagt 480

gtctgaggaa agagcacaga gattcatggt aatcgcagga tctctccctc gggcgtgcag 540

caacggtact ccgtttgtca cggctggggt tgaagatgat gcaccagaag atatcactga 600

cactctggaa agaatcctat ctatccaagt tcaggtatgg gtcacagtag caaaggccat 660

gactgcatat gagacagcag atgagtcaga aacaagaaga ataaataagt atatgcagca 720

aggtcgagtt cagaagaagt acatccttca tcctgtatgc aggagtgcaa ttcaactcac 780

aatcagacat tctctggcag tccgtatctt cctagttagt gagctcaaga ggggccgcaa 840

tacagcaggt gggagctcta catattacaa cttggtcggg gatgtagact catacatcag 900

gaacaccggg cttactgcat ttttcctaac actcaaatat ggaatcaata ccaagacgtc 960

agccctcgca ctcagcagcc tcacaggtga tatccaaaaa atgaaacagc tcatgcgttt 1020

atatcggatg aaaggtgaaa atgcaccata catgacattg ttaggtgaca gtgaccagat 1080

gagctttgca cctgctgagt atgcacaact ttattctttt gccatgggca tggcatcagt 1140

cttagataag ggaactggca agtaccaatt cgccagggac tttatgagca catcattctg 1200

gagacttgga gtagagtatg ctcaggccca gggaagtagc attaatgagg acatggctgc 1260

tgagctaaaa ctaaccccgg cagcaaggag aggcctggca gctgctgccc aacgagtatc 1320

cgaagaaatc ggcagcatgg aattcccac tcaacaggcg ggagtcctca ccgggctcag 1380

tgatgaaggc ccccgaactc cacagggcag atcaaacaag ccgcaagggc aaccagatgc 1440

tggggatggg gagacccaat tcttggattt tatgagagca gtggcgaacg gcatgcggga 1500

agcgccaaat cctgtacaga gcaccacccca tccagagcct cccccaaccc ctggggcatc 1560

ccaagacaac gacactgact gggggtactg atcgacaaca cccagcctgc cttcacagga 1620

tcacaccaaa ccctccgacc aaaaccctcc cacactccct gacccacaac cccgcacgac 1680

cacaccaaca aaagctcccc cccaccctct cccccactcc cagccaacg atcccgccca 1740

cccgggacaa cacaggcaca gctcagttcg tcgacaatcc gcccagagcc caaggtatta 1800

gaaaaaaata cgggtagaag agagacatcc agagaccagg acgagtcacc aagttctctg 1860

ttctcccttc tacccagtga attagggtga agatggccac ttttacagat gcggagatag 1920

atgacatatt tgagaccagt gggactgtca ttgacagcat aattacggcc cagggcaaat 1980

cagctgagac cgttggaaga agtgcgatcc cgcagggcaa gaccaaagct ctaagcacag 2040

catgggagaa gcacgggagt atccagccac acgccagtca ggacgcccct gaccaacaag 2100

acagaataga aaaacagcca tccacacctg agcaggcgac tctacacaac aatccgccga 2160

tcgcatccac tgaaccgcct cccactcagg ccgcaagcga gaccagcgac acacagctca 2220

agactggggc aagcaactcc cttctgtcca tgctcgacaa actgagtaat aaatcgtcca 2280

atgctaaaaa gggcccatgg tcgggatccc aagaagggca tcaccaacct ccggcccaac 2340

aacatgggaa ccagccgagc tatggaagca accagggaag accgcagcat caggccaagg 2400

ccgtccctgg aaaccggggc acagacgaga acacagcata tcatggacag cggaaggagt 2460

cacaaccatc agctggtgca accccctcatg cgccccagtc agggcagagc caagacaata 2520

ctcctgcacc tgtggatcgt gtccagctac ctgccgactt tgtgcaggcg atgatgtcta 2580

tgatggaggc attatcacag aaggtaagta aagttgatca tcagctggac ctagtcttga 2640

aacagacatc ctccattcct atgatgcgat ctgaaatcca acagctcaag acatctgttg 2700

cgatcatgga agctaactta ggcatgatga aaattctgga ccctggttgt gctaatgttt 2760

catccttaag tgatctccgg gcagtagccc gatcccatcc agtcctagtt tcaggccctg 2820

gagacccatc tccttacgtg acacaagggg gtgaaatgac gctcaataaa ctctcacaac 2880

cggtgcagca cccctctgaa ttgattaagt ctgccactgc aagcgggcct gacatgggag 2940

tggagaagga cactgtccgc gcattaatca cctcgcgccc gatgcatcca agctcctcgg 3000

ctaagctcct gagcaagcta gatgcagcca ggtcaattga agagatcagg aagatcaaac 3060

gccttgcgct gaatggttga tggccatcac aactcataac aggctcccgt cactttagcg 3120

tcacacggaa tccctcgggg gccctccctc gcaaatctat gcttcaacac ccaaaacaac 3180

agccctctct cacccccccc aatccctcga atgaccgcac aactgcaacc aatccagcag 3240

cattagaaat taagaaaaaa tacgggtaga atcaaagtgc cttgattgca ccaaaatgga 3300

ctcatccagg acaattgggc tgtactttga ttctgccctc ccttccagca gcctgttagc 3360

atttccgatt gtcttacaag acacgggaga cgggaagaag caaatcaccc cacaatacag 3420

gatccagcgt cttgattcgt ggacagacag taaggaagac tcggtattca tcaccaccta 3480

cgggttcatc tttcaaattg ggaatgaaga aaccaccgtc ggtgtgatca atgacaatcc 3540

caggcacgag ctactctctt ccgcaatgct ctgcttaggg agtgtcccga acgacggaga 3600

tcttgttgag ctggcgagag cctgcctcac catggtggta acttgcaaaa agagtgcaac 3660

taacactgag agaatagtct tttcagtagt gcaggctcct cgggtgctgc aaagctgtat 3720

ggttgtggca aataggtact catcagtgaa tgcagtgaag catgtgaagg cgccagaaaa 3780

gatccctggg agcggaaccc tagagtataa agtgaatttt gtctctttga ccgtggtgcc 3840

aagaagggat gtctacagga tcccaactgc agtattgaaa gtgtctggct caagcctgta 3900

caatcttgcg ctcaatgtca ctattgatgt ggacgtggat ccgaagagcc cgttagtcaa 3960

atccctttcc aagtccgata gcggatacta tgcgaatctt tttctgcata tcgggcttat 4020

gtccactgta gataagaagg gaaagaaagt gacatttgac aagatagagg aaaagataag 4080

gagactcaat ctatccgtcg ggctcagtga tgtgctcgga ccctctgtgc ttgtgaaggc 4140

gagaggtgca cggactaagc tacttgctcc ttttttctct agcagtggga cagcctgcta 4200

tcctatagca aatgcctctc cccaggttgc caagatactc tggagccaaa ctgcgcacct 4260

gcggagtgtg aaagtcatca ttcaagccgg cactcagcgt gctgtcgcag tgaccgctga 4320

tcatgaggta acctccacta agatagagag gaggcatgcc attgctaaat acaatccttt 4380

caggaaataa gttgcatccc taagactgca gttcacctgc tttcccgaat caccattaca 4440

ccagacaatg atccatctcg actgcttata gttagttcac ctgtctagca aattagaaaa 4500

aacacgggta gaagagtctg gatcccgacc ggcacactca ggacgcaaca tgggctccaa 4560

accttctacc aggatcccag cacctctgat gctggtcacc cggattatgc tgatattggg 4620

ctgtattcgt tcgacaagct ctcttgacgg caggcctctt gcagctgcag gaattgtagt 4680

aacagggat aaggcagtca atgtatacac ctcgtctcag acagggtcaa tcatagtcaa 4740

gttgctcccg aatatgccca gggataagga ggcgtgtgcg aaagccccat tagaggcata 4800

taacagaaca ctgactactt tgctcactcc tcttggcgac tccatccgca agattcaagg 4860

gtctgtgtcc acgtctggag gaaggaggca aaaacgcttt ataggtgctg ttattggcag 4920

tgtagctctt ggggttgcaa cagcggcaca gataacagca gctgcggccc taatacaagc 4980

caacaagaat gctgccaaca tccttcggct taaggagagc attgctgcaa ccaatgaagc 5040

tgtgcatgaa gtcaccgacg gattatcaca actatcagtg gcagttggga agatgcagca 5100

gtttgtcaat gaccagttta ataatacggc gcgagaattg gactgtataa aaatcacaca 5160

acaggttggt gtagaactca acctatacct aactgaattg actacagtat tcgggccaca 5220

gatcacctct cctgcattaa ctcagctgac catccaggca ctttataatt tagctggtgg 5280

caatatggat tacttattaa ctaagttagg tatagggaac aatcaactca gctcattaat 5340

tggtagcggc ctgatcactg gttaccctat actgtatgac tcacagactc aactcttggg 5400

catacaagtg aatttgccct cagtcgggaa cttaaataat atgcgtgcca cctatttgga 5460

gaccttatct gtaagtacaa ccaaaggata tgcctcagca cttgtcccga aagtagtgac 5520

acaagtcggt tctgtgatag aagagcttga cacctcatac tgtatagagt ccgatctgga 5580

tttatattgt actagaatag tgacattccc catgtcccca ggtatttatt cctgtttgag 5640

cggcaacaca tcagcttgca tgtattcaaa gactgaaggc gcactcacta cgccgtatat 5700

ggcccttaaa ggctcagtta ttgccaattg taagataaca acatgtagat gtacagaccc 5760

tcctggtatc atatcgcaaa attatggaga agctgtatcc ctgatagata gacattcgtg 5820

caatgtctta tcattagacg ggataactct gaggctcagc ggagaatttg atgcaactta 5880

tcaaaagaac atctcaatac tagattctca agtcatcgtg acaggcaatc ttgatatatc 5940

aactgaactt ggaaacgtca acaattcaat cagcaatgcc ttggataagt tggcagaaag 6000

caacagtaag atagaaaaag tcaatgtcag attaaccagc acatctgctc tcattaccta 6060

tattgttcta actgtcattt ctctattttt cggtgcactt agtctgggtt tagcgtgtta 6120

cctgatgtac aaacagaagg cacaacaaaa gaccttgcta tggcttggga ataatgccct 6180

tgatcagatg agagccacta caagagcatg aatgcagata agaggtggat atatacccaa 6240

cagcagcctg tgtgtcaatt ccgataatct gtcaagtaga agacttaaga aaaaactact 6300

gggaacaagc aaccaaagag caatacacgg gtagaacggt cagaggagcc acccttcaat 6360

cggaaattag gcttcacaac atccgttcta ccacatcacc atcaacaaga gtcaatcatg 6420

gaccgcgcgg ttaacagagt cgtgctggag aatgaggaaa gagaagcaaa gaacacatgg 6480

cgcctggttt tccggatcgc agtcttactt ttaatggtaa tgactctagc tatctccgca 6540

gctgccctgg catacagtat ggaggccagt acgccgcacg accttgcagg catatcgact 6600

gtgatctcta agacagaaga taaggttacg tctttactca gttcgagtca agatgtgata 6660

gataggatat acaagcaggt ggctcttgaa tccccgctgg cgctactaaa cactgaatct 6720

acaattatga atgcaataac ctctctttct tatcaaatta acggggctgc gaacaatagc 6780

ggatgtgggg cgcctgttca tgacccagat tatatcgggg ggataggcaa agaactcata 6840

gtggacgaca tcagtgatgt cacatcattt tatccttctg catatcaaga acacctgaat 6900

ttcatcccgg cgcctactac aggatccggt tgcactcgga taccctcatt tgacatgagc 6960

accacccatt attgttatac tcacaatgta atactatccg gttgcagaga tcactcacac 7020

tcacatcaat acttagcgct tggtgtgctt cggacatctg caacagggag ggtattcttc 7080

tctactctgc gctccatcaa tttagatgac acccaaaatc ggaagtcctg cagtgtgagt 7140

gcaacccctt taggttgtga tatgctgtgc tctaaggtca cagggactga agaggagat 7200

tacaagtcag ctgccccaac atcaatggtg cacggaaggc tagggtttga cggtcaatac 7260

catgagaagg acttagacac cacggtctta tttaaggatt gggtggcaaa ttaccccagga 7320

gtgggaggag ggtcttttat tgacgaccgt gtatggttcc cagtttacgg agggctcaaa 7380

cccaattcac ccagtgacac tgcacaagaa gggaaatatg taatatacaa gcgccataac 7440

aacacatgcc ccgatggaca agattaccaa attcggatgg ctaaatcttc atataaaccc 7500

gggcgatttg gtggaaagcg cgtacagcaa gccatattat ccatcaaagt gtcaacatcc 7560

ttgggtaagg acccggtgct gactattcca cctaatacaa tcacactcat gggagccgaa 7620

ggcagaatcc tcacagtagg gacatctcac ttcttgtacc aacgagggtc ttcatatttc 7680

tcccctgcct tattatatcc catgacagta aataacaaaa cggctacact ccatagtcct 7740

tatacgttta atgctttcac tcggccaggt agcgtccctt gccaggcatc agcaagatgc 7800

cccaactcat gcatcactgg ggtctatact gatccatatc ccttaatctt ccataggaat 7860

catactctac gaggggtctt tgggacgatg cttgatgatg aacaagcgag acttaaccccc 7920

gtatctgcag tattcgacaa catatcccgc agtcgtgtca cccgggtgag ttcaagcagc 7980

accaaggcag catacacgac atcgacatgt tttaaagttg tcaagaccaa taaagcttat 8040

tgtcttagta ttgcagagat atccaatacc ctattcgggg aatttaggat cgttccctta 8100

ttagttgaga tcctcaagga tgatagagtt taagaagcta gacttggccg attgagccaa 8160

tcataggatg gttgggaaga cgacaccaca tcaatcatct cccacaatgc ttagagtcaa 8220

tctgaatatt aacataagcc aggatcccat gttgttgggc agccacaatc agacaatact 8280

gacatgatca ttctgagtcc tgcccactat caccttatta agaaaaaata caaaaagcat 8340

tgagatataa ggggaaacaa ccaacaagag ggaacacggg taggacatgg cgggctccgg 8400

tcccgaaagg gcagagcacc agatcatcct accagagtca catctatcct ctccatggt 8460

caagcacaaa ttgctatact actggaaatt gactgggcta ccgcttcctg atgaatgcga 8520

ctttgatcat ctcattatca gcaggcaatg gaagagaata ctggagtcgg ccactcctga 8580

cacagagaga atgataaaac tcgggcgggc agtgcaccag actctcaacc acaactccaa 8640

gataaccgga gtgctccatc ccaggtgttt agaagaactg gctagtattg aggtcccaga 8700

ttcaactaac aaattccgga agattgaaaa gaagatccag attcacaaca caaggtatgg 8760

agacctgttc acaaagctgt gcacgcatgt tgagaagaaa ttgctaggat catcccggtc 8820

taataatgtc ccacgatcag aggaattcag tagcatccgt acagatccgg cattctggtt 8880

tcactcaaaa tggtccagag ctaagttcgc gtggctccat ataaaacaag tccaaaggca 8940

tctgattgta gcagcaagga caaggtctgc agtcaacaag ttagtaacat taagtcataa 9000

gataggccac gtctttgtta ctcctgagct tgtcattgtg acacatacag acgagaacaa 9060

gttcacatgc ctcacccagg aacttgtatt gatgtatgcg gatatgatgg aaggcaggga 9120

catggtcaat ataatatctt ctacagcagc acatctcaga aacctatccg agaaaattga 9180

cgatattctg cggttagtag atgccctggc aaaggactta ggtaatcaag tctatgacgt 9240

tgtagcatta atggagggat tcgcatacgg tgctgttcag ctgcttgagc catcaggtac 9300

atttgcagga gatttctttg catttaacct acaggagctc aaagacactt taatcgaact 9360

tctcccaaat aatatagcgg aatcagtaac tcacgctatt gccgctgtat tctccggttt 9420

agaacagaat caagcagctg agatgttgtg cttgctacgt ttgtggggtc atcctttgct 9480

tgagtctcgt agtgcagcaa gagcagtcag gagccagatg tgtgcaccga agatggtaga 9540

cttcgatatg atcctccagg tactatcttt ctttaaagga acaatcatca atggatacag 9600

aaagaagaat tcaggtgtgt ggccgcgtgt caaagtagat acaatatacg ggaatatcat 9660

tgggcagctg tatgctgatt cagcagagat ctcacatgat gtcatgttga aggagtacaa 9720

gagtttatct gcttttgaat ttgagccatg tatagactat gaccctgtta ccaatctaag 9780

catgttccta aaagacaagg caatcgcaca tcctagtgat aattggctcg cctcatttag 9840

gcggaaccta ctctctgagg accagaagaa acagataaaa gaggcaactt caactaaccg 9900

cctcttgata gagttcttag aatcaaatga ttttgatcca tataaagaaa tggaatacct 9960

gacaaccctc gagtacctaa gaggtgacag tgtggcagta tcgtactcac tcaaagagaa 10020

agaggtaaaa gtgaatgggc ggatttttgc taagttaaca aagaaactaa ggaactgcca 10080

ggtaatggca gaaggaattc tagctgacca gattgcacct ttctttcagg gaaatggggt 10140

cattcaagat agcatatcct tgacaaagag tatgttagcg atgagtcaac tgtcctttaa 10200

cagcaataag aaacgtatca ctgactgcaa agagagggtt tcctcgaacc gcaatcatga 10260

tcagaagagc aagaatcgta gaagagttgc cacttttatc acgactgacc tacaaaagta 10320

ttgtcttaac tggagatatc agacagtcaa actattcgcc catgctatca atcagctgat 10380

gggcctacct catttcttcg agtggattca tcttaggctg atggacacta caatgtttgt 10440

aggggatcct ttcaatcctc caagtgaccc gactgactgt gatctatcaa gagtcccaaa 10500

cgatgacata tatattgtca gtgctagagg gggcattgag ggactctgcc agaagctatg 10560

gacgatgatc tcaattgctg caatccaact tgctgcagca agatctcatt gtcgagttgc 10620

ctgcatggta caaggtgaca atcaagtaat ggctgtaacg agagaggtaa gatcagatga 10680

ttccccggat atggtgttga cgcagttgca tcaagctagt gataatttct tcaaggaatt 10740

gattcatgtc aatcatttga ttggccataa tctgaaggat cgtgaaacca ttagatcaga 10800

cacattcttc atatacagca aacgaatatt caaggatgga gcaatactca gtcaggtcct 10860

caaaaattca tctaaattgg tgctaatatc aggcgacctt agcgaaaaca ctgtaatgtc 10920

ctgtgccaac attgcatcca ctgtagcacg actatgtgag aatgggcttc caaaggattt 10980

ctgttactat ttgaactacc taatgagttg tgtgcagaca tactttgatt cggagttctc 11040

tattacccac agctcgcaat cagattccaa ccagtcctgg atcgaggata tctctttcgt 11100

acactcatac gtgttaaccc ctgcccagct ggggggactg agcaaccttc aatactcaag 11160

gctctacaca aggaatattg gtgacccagg gaccactgct ttcgcagagg tcaagcgact 11220

ggaagcagtg gggttgctga gtcccagcat catgactaac atcttaacca ggccacctgg 11280

caatggagac tgggccagcc tatgcaacga cccatactct tttaattttg aaactgttgc 11340

aagcccaaat attgtcctca agaaacatac acagaaagtc ctatttgaga catgttcaaa 11400

ccccttatta tccggggtac atacagagga caatgaggca gaggagaaag cattggctga 11460

attcttactc aatcaagaag tgattcaccc acgtgtcgca catgctatca tggaagcaag 11520

ctctgtgggt aggagaaagc aaattcaagg gcttgttgac acaacgaaca ctgtgattaa 11580

gattgcactg actaggaggc ccctcggtat caaaaggctg atgcggataa tcaattactc 11640

gagcatgcat gcaatgttgt tcagagatga tattttctta tccaaagat ccaaccaccc 11700

attagtttct tccaatatgt gctcgctgac gctagcagat tatgcccgaa acagaagctg 11760

gtcacctctg acaggggggca ggaaaatact gggtgtatcc aaccctgata ccatagaact 11820

tgtggaggga gagattctca gcgtcagtgg agggtgcaca aaatgtgaca gcggagatga 11880

gcagtttact tggttccatc ttccaagcaa tatagagctg actgatgaca ccagaaaaaa 11940

tcccccgatg agagtgccat atcttgggtc gaagactcaa gagaggagag ctgcctcgct 12000

tgcgaaaata gcccacatgt caccacatgt gaaagcagca ctaagggcat catccgtgtt 12060

aatctgggct tatggggaca acgaagtgaa ctggactgct gctcttaata ttgcaaggtc 12120

tcgatgcaac ataagctcag agtaccttcg gctattgtca cccctgccca cggctgggaa 12180

tctccaacat agattggatg atggcataac ccagatgaca tttacccctg catctctcta 12240

cagagtgtcg ccttacgttc acatatccaa tgattctcaa aggctattca ccgaagaagg 12300

ggtcaaagag ggaaacgtgg tttaccaaca aattatgctc ttgggtttat ccctaattga 12360

atcactcttc ccaatgacag caaccagaac atatgatgag atcacattac acctccacag 12420

taaatttagc tgctgtatcc gagaagcgcc tgttgcggtt cccttcgagc tcctcgggct 12480

ggcaccggaa ttaagaatgg taacctcaaa taagttcatg tatgatccta gccctatatc 12540

agagagagat ttcgcgagac ttgacttagc tatcttcaag agttacgagc ttaatttgga 12600

atcatattcc acgctggagc taatgaacat tctttcaata tctagcggga agttgattgg 12660

ccaatctgtg gtttcttatg atgaagacac ctctataaag aatgatgcta taatagtgta 12720

tgacaacaca cgaaattgga ttagtgaggc gcagaactca gatgtggtcc gcttgtttga 12780

gtatgcagca ctcgaagtgc ttcttgactg tgcttatcaa ctctactatc tgagggtaag 12840

gggtctaaac aacatcgtcc tatacatgaa tgacttatat aagaacatgc cagggatcct 12900

actctccaat attgcggcca cgatatccca ccccatcatt cactcaaggt tgaatgcagt 12960

aggcctaatt aaccatgacg ggtcacacca gctcgcagat atagacttcg tcgaggtgtc 13020

tgcaaaattg ttagtctctt gcactcgacg cgtggtctca ggcttatatg cagggaataa 13080

gtacgatctg ctgttcccat ctgtcttaga tgataacctg aatgagaaga tgctccaact 13140

gatttcccgg ttatgctgtc tgtacacagt gctctttgct acaacaagag aaatcccaaa 13200

aataaggggc ctatcagcag aagagaaatg ctcaatactc actgagtatc tactgtcaga 13260

tgctgtaaaa ccattgctta ggcccgaaca agtgaattct atcatgtctc ccaacataat 13320

cacgttccca gccaatctat attacatgtc taggaagagc cttaatttga tcagagaacg 13380

agaggacaga gatactatct tgtcattgtt gttccctcag gaaccactgc ttgagcttcg 13440

cccagtacgg gacattggtg ctcgagtgaa agacccgttt acccggcaac ccgcatcatt 13500

catacaagag ctagatctga gtgccccagc aaggtacgac gcatttacac tgagtaaggc 13560

ctgcttcgag catacattac cgaacccaaa ggaggattac ctagtacggt acttgttcag 13620

aggaataggg actgcttcat cttcttggta taaggcatct catcttctat ccgtacctga 13680

ggtcaggtgt gcaagacatg ggaactcctt atacttagcg gaaggaagcg gagccatcat 13740

gagtcttctt gaattgcata taccacatga gactatctat tacaatacac ttttctcgaa 13800

tgagatgaac cctccacagc gacatttcgg acctacacca acacagtttc taaactcggt 13860

cgtttatagg aatctacaag cggaagtgcc atgtaaagat ggatatgtcc aggagttctg 13920

cccattatgg agagagaatg cagaagaaag tgacctgacc tcagataaag cagttggata 13980

tatcacatcc gtggtaccct acaggtctgt atcattacta cattgtgaca ttgagattcc 14040

tccagggtcc aatcaaagct tattagatca actggctact aatttatccc tgattgccat 14100

gcattctgtg aggggagggcg gggtagtgat catcaaagta ctgtatgcaa tggggtacta 14160

cttccatta ctcatgaatt tattcactcc atgttccaca aaaggatata tactctccaa 14220

tggctatgcc tgtagagggg atatgcaatg ttacctgata ttcgttatgg gctacttggg 14280

cgggcccacc ttcgtgcacg aagtggtaag gatggcaaaa actctaatac aacgacacgg 14340

tacactccta tctaaatcag atgaaattac attgactaag ctatttacct cacagcagcg 14400

tcatgtaaca gatatcctat ccagtccttt accgaagcta atgaagctct tgagagaaaa 14460

tattgatgcc gcactaattg aagctggggg acagcccgtc cgtccattct gtgcggaaag 14520

tttggtgagc acactaacag atatgactca gacagctcag atcattgcca gccacattga 14580

cacagtcatt cggtctgtaa tttacatgga ggctgagggt gacctcgccg acagtgtt 14640

cttatttact ccttacaatc tatccacaga cggtaaaagg agaacatcac ttaagcagtg 14700

caccaaacag atcttggaag tcacaatact gggtctcaga gccaaagata tcaataaagt 14760

aggtgatgta atcagtttag tactcagagg tgcggtttct ctagaggacc tcatcccatt 14820

aaggacatac ctgaagcgca gtacctgcac taaatacctg aaagcggtcc taggtattac 14880

taaactcaaa gaaatgttca cagatacctc attackgtac ttgactcgtg ctcaacaaaa 14940

attctacatg aaaaccatag gtaatgctgc caagggatat tactgtaata atgactctta 15000

aaggcaatcg tacgccaatc agttatcttc ctaactgatg actccctcat tgactcaatt 15060

ataccagatt agaaaaaagt taaattccga ctctttggaa ctcgtattcg gattcagtta 15120

gttaacttta agcaagagtg cgcaaagtca tccctaatta tagtgatgtc attcaccaaa 15180

tctctgtttg gt 15192

the

<210> 4

<211> 15192

<212> DNA

<213> Artificial sequence (SEQ ID NO: 4)

<400> 4

accaaacaga gaatctgtga ggtacgataa aaggcgaaga agcaatcgag atcgtacggg 60

tagaaggtgt gaatcccgag tgcgaggccg aagctcgaac ctgaggggac cttctaccga 120

tatgtcgtct gttttcgacg agtacgagca gctcctcgct gctcagaccc gccctaacgg 180

aactcatgga gggggagaga aagggagcac tttaaaagtt gaggtcccag tattcaccct 240

aaacagtgat gatccagagg atagatggaa ttttgcggta ttctgtcttc ggattgctgt 300

tagcgaggat gccaacaaac cactcaggca aggtgctctt atatccctct tatgctccca 360

ttctcaggtg atgagaaacc atgttgccct tgcagggaaa caaaatgagg ccacactggc 420

tgttcttgag atcgatggtt ttgctaacag tgtgccccag ttcaacaata ggagtggagt 480

gtctgaggaa agagcacaga gattcatggt aatcgcagga tctctccctc gggcgtgcag 540

caacggtact ccgtttgtca cggctggggt tgaagatgat gcaccagaag atatcactga 600

cactctggaa agaatcctat ctatccaagt tcaggtatgg gtcacagtag caaaggccat 660

gactgcatat gagacagcag atgagtcaga aacaagaaga ataaataagt atatgcagca 720

aggtcgagtt cagaagaagt acatccttca tcctgtatgc aggagtgcaa ttcaactcac 780

aatcagacat tctctggcag tccgtatctt cctagttagt gagctcaaga ggggccgcaa 840

tacagcaggt gggagctcta catattacaa cttggtcggg gatgtagact catacatcag 900

gaacaccggg cttactgcat ttttcctaac actcaaatat ggaatcaata ccaagacgtc 960

agccctcgca ctcagcagcc tcacaggtga tatccaaaaa atgaaacagc tcatgcgttt 1020

atatcggatg aaaggtgaaa atgcaccata catgacattg ttaggtgaca gtgaccagat 1080

gagctttgca cctgctgagt atgcacaact ttattctttt gccatgggca tggcatcagt 1140

cttagataag ggaactggca agtaccaatt cgccagggac tttatgagca catcattctg 1200

gagacttgga gtagagtatg ctcaggccca gggaagtagc attaatgagg acatggctgc 1260

tgagctaaaa ctaaccccgg cagcaaggag aggcctggca gctgctgccc aacgagtatc 1320

cgaagaaatc ggcagcatgg aattcccac tcaacaggcg ggagtcctca ccgggctcag 1380

tgatgaaggc ccccgaactc cacagggcag atcaaacaag ccgcaagggc aaccagatgc 1440

tggggatggg gagacccaat tcttggattt tatgagagca gtggcgaacg gcatgcggga 1500

agcgccaaat cctgtacaga gcaccacccca tccagagcct cccccaaccc ctggggcatc 1560

ccaagacaac gacactgact gggggtactg atcgacaaca cccagcctgc cttcacagga 1620

tcacaccaaa ccctccgacc aaaaccctcc cacactccct gacccacaac cccgcacgac 1680

cacaccaaca aaagctcccc cccaccctct cccccactcc cagccaacg atcccgccca 1740

cccgggacaa cacaggcaca gctcagttcg tcgacaatcc gcccagagcc caaggtatta 1800

gaaaaaaata cgggtagaag agagacatcc agagaccagg acgagtcacc aagttctctg 1860

ttctcccttc tacccagtga attagggtga agatggccac ttttacagat gcggagatag 1920

atgacatatt tgagaccagt gggactgtca ttgacagcat aattacggcc cagggcaaat 1980

cagctgagac cgttggaaga agtgcgatcc cgcagggcaa gaccaaagct ctaagcacag 2040

catgggagaa gcacgggagt atccagccac acgccagtca ggacgcccct gaccaacaag 2100

acagaataga aaaacagcca tccacacctg agcaggcgac tctacacaac aatccgccga 2160

tcgcatccac tgaaccgcct cccactcagg ccgcaagcga gaccagcgac acacagctca 2220

agactggggc aagcaactcc cttctgtcca tgctcgacaa actgagtaat aaatcgtcca 2280

atgctaaaaa gggcccatgg tcgggatccc aagaagggca tcaccaacct ccggcccaac 2340

aacatgggaa ccagccgagc tatggaagca accagggaag accgcagcat caggccaagg 2400

ccgtccctgg aaaccggggc acagacgaga acacagcata tcatggacag cggaaggagt 2460

cacaaccatc agctggtgca accccctcatg cgccccagtc agggcagagc caagacaata 2520

ctcctgcacc tgtggatcgt gtccagctac ctgccgactt tgtgcaggcg atgatgtcta 2580

tgatggaggc attatcacag aaggtaagta aagttgatca tcagctggac ctagtcttga 2640

aacagacatc ctccattcct atgatgcgat ctgaaatcca acagctcaag acatctgttg 2700

cgatcatgga agctaactta ggcatgatga aaattctgga ccctggttgt gctaatgttt 2760

catccttaag tgatctccgg gcagtagccc gatcccatcc agtcctagtt tcaggccctg 2820

gagacccatc tccttacgtg acacaagggg gtgaaatgac gctcaataaa ctctcacaac 2880

cggtgcagca cccctctgaa ttgattaagt ctgccactgc aagcgggcct gacatggggag 2940

tggagaagga cactgtccgc gcattaatca cctcgcgccc gatgcatcca agctcctcgg 3000

ctaagctcct gagcaagcta gatgcagcca ggtcaattga agagatcagg aagatcaaac 3060

gccttgcgct gaatggttga tggccatcac aactcataac aggctcccgt cactttagcg 3120

tcacacggaa tccctcgggg gccctccctc gcaaatctat gcttcaacac ccaaaacaac 3180

agccctctct cacccccccc aatccctcga atgaccgcac aactgcaacc aatccagcag 3240

cattagaaat taagaaaaaa tacgggtaga atcaaagtgc cttgattgca ccaaaatgga 3300

ctcatccagg acaattgggc tgtactttga ttctgccctc ccttccagca gcctgttagc 3360

atttccgatt gtcttacaag acacgggaga cgggaagaag caaatcaccc cacaatacag 3420

gatccagcgt cttgattcgt ggacagacag taaggaagac tcggtattca tcaccaccta 3480

cgggttcatc tttcaaattg ggaatgaaga aaccaccgtc ggtgtgatca atgacaatcc 3540

caggcacgag ctactctctt ccgcaatgct ctgcttaggg agtgtcccga acgacggaga 3600

tcttgttgag ctggcgagag cctgcctcac catggtggta acttgcaaaa agagtgcaac 3660

taacactgag agaatagtct tttcagtagt gcaggctcct cgggtgctgc aaagctgtat 3720

ggttgtggca aataggtact catcagtgaa tgcagtgaag catgtgaagg cgccagaaaa 3780

gatccctggg agcggaaccc tagagtataa agtgaatttt gtctctttga ccgtggtgcc 3840

aagaagggat gtctacagga tcccaactgc agtattgaaa gtgtctggct caagcctgta 3900

caatcttgcg ctcaatgtca ctattgatgt ggacgtggat ccgaagagcc cgttagtcaa 3960

atccctttcc aagtccgata gcggatacta tgcgaatctt tttctgcata tcgggcttat 4020

gtccactgta gataagaagg gaaagaaagt gacatttgac aagatagagg aaaagataag 4080

gagactcaat ctatccgtcg ggctcagtga tgtgctcgga ccctctgtgc ttgtgaaggc 4140

gagaggtgca cggactaagc tacttgctcc ttttttctct agcagtggga cagcctgcta 4200

tcctatagca aatgcctctc cccaggttgc caagatactc tggagccaaa ctgcgcacct 4260

gcggagtgtg aaagtcatca ttcaagccgg cactcagcgt gctgtcgcag tgaccgctga 4320

tcatgaggta acctccacta agatagagag gaggcatgcc attgctaaat acaatccttt 4380

caggaaataa gttgcatccc taagactgca gttcacctgc tttcccgaat caccattaca 4440

ccagacaatg atccatctcg actgcttata gttagttcac ctgtctagca aattagaaaa 4500

aacacgggta gaagagtctg gatcccgacc ggcacactca ggacgcaaca tgggctccaa 4560

accttctacc aggatcccag cacctctgat gctggtcacc cggattatgc tgatattggg 4620

ctgtattcgt tcgacaagct ctcttgacgg caggcctctt gcagctgcag gaattgtagt 4680

aacagggat aaggcagtca atgtatacac ctcgtctcag acagggtcaa tcatagtcaa 4740

gttgctcccg aatatgccca gggataagga ggcgtgtgcg aaagccccat tagaggcata 4800

taacagaaca ctgactactt tgctcactcc tcttggcgac tccatccgca agattcaagg 4860

gtctgtgtcc acgtctggag gagggagaca ggggcgcctt ataggtgctg ttattggcag 4920

tgtagctctt ggggttgcaa cagcggcaca gataacagca gctgcggccc taatacaagc 4980

caacaagaat gctgccaaca tccttcggct taaggagagc attgctgcaa ccaatgaagc 5040

tgtgcatgaa gtcaccgacg gattatcaca actatcagtg gcagttggga agatgcagca 5100

gtttgtcaat gaccagttta ataatacggc gcgagaattg gactgtataa aaatcacaca 5160

acaggttggt gtagaactca acctatacct aactgaattg actacagtat tcgggccaca 5220

gatcacctct cctgcattaa ctcagctgac catccaggca ctttataatt tagctggtgg 5280

caatatggat tacttattaa ctaagttagg tatagggaac aatcaactca gctcattaat 5340

tggtagcggc ctgatcactg gttaccctat actgtatgac tcacagactc aactcttggg 5400

catacaagtg aatttgccct cagtcgggaa cttaaataat atgcgtgcca cctatttgga 5460

gaccttatct gtaagtacaa ccaaaggata tgcctcagca cttgtcccga aagtagtgac 5520

acaagtcggt tctgtgatag aagagcttga cacctcatac tgtatagagt ccgatctgga 5580

tttatattgt actagaatag tgacattccc catgtcccca ggtatttatt cctgtttgag 5640

cggcaacaca tcagcttgca tgtattcaaa gactgaaggc gcactcacta cgccgtatat 5700

ggcccttaaa ggctcagtta ttgccaattg taagataaca acatgtagat gtacagaccc 5760

tcctggtatc atatcgcaaa attatggaga agctgtatcc ctgatagata gacattcgtg 5820

caatgtctta tcattagacg ggataactct gaggctcagc ggagaatttg atgcaactta 5880

tcaaaagaac atctcaatac tagattctca agtcatcgtg acaggcaatc ttgatatatc 5940

aactgaactt ggaaacgtca acaattcaat cagcaatgcc ttggataagt tggcagaaag 6000

caacagtaag atagaaaaag tcaatgtcag attaaccagc acatctgctc tcattaccta 6060

tattgttcta actgtcattt ctctattttt cggtgcactt agtctgggtt tagcgtgtta 6120

cctgatgtac aaacagaagg cacaacaaaa gaccttgcta tggcttggga ataatgccct 6180

tgatcagatg agagccacta caagagcatg aatgcagata agaggtggat atatacccaa 6240

cagcagcctg tgtgtcaatt ccgataatct gtcaagtaga agacttaaga aaaaactact 6300

gggaacaagc aaccaaagag caatacacgg gtagaacggt cagaggagcc acccttcaat 6360

cggaaattag gcttcacaac atccgttcta ccacatcacc atcaacaaga gtcaatcatg 6420

gaccgcgcgg ttaacagagt cgtgctggag aatgaggaaa gagaagcaaa gaacacatgg 6480

cgcctggttt tccggatcgc agtcttactt ttaatggtaa tgactctagc tatctccgca 6540

gctgccctgg catacagtat ggaggccagt acgccgcacg accttgcagg catatcgact 6600

gtgatctcta agacagaaga taaggttacg tctttactca gttcgagtca agatgtgata 6660

gataggatat acaagcaggt ggctcttgaa tccccgctgg cgctactaaa cactgaatct 6720

acaattatga atgcaataac ctctctttct tatcaaatta acggggctgc gaacaatagc 6780

ggatgtgggg cgcctgttca tgacccagat tatatcgggg ggataggcaa agaactcata 6840

gtggacgaca tcagtgatgt cacatcattt tatccttctg catatcaaga acacctgaat 6900

ttcatcccgg cgcctactac aggatccggt tgcactcgga taccctcatt tgacatgagc 6960

accacccatt attgttatac tcacaatgta atactatccg gttgcagaga tcactcacac 7020

tcacatcaat acttagcgct tggtgtgctt cggacatctg caacagggag ggtattcttc 7080

tctactctgc gctccatcaa tttagatgac acccaaaatc ggaagtcctg cagtgtgagt 7140

gcaacccctt taggttgtga tatgctgtgc tctaaggtca cagggactga agaggagat 7200

tacaagtcag ctgccccaac atcaatggtg cacggaaggc tagggtttga cggtcaatac 7260

catgagaagg acttagacac cacggtctta tttaaggatt gggtggcaaa ttaccccagga 7320

gtgggaggag ggtcttttat tgacgaccgt gtatggttcc cagtttacgg agggctcaaa 7380

cccaattcac ccagtgacac tgcacaagaa gggaaatatg taatatacaa gcgccataac 7440

aacacatgcc ccgatggaca agattaccaa attcggatgg ctaaatcttc atataaaccc 7500

gggcgatttg gtggaaagcg cgtacagcaa gccatattat ccatcaaagt gtcaacatcc 7560

ttgggtaagg acccggtgct gactattcca cctaatacaa tcacactcat gggagccgaa 7620

ggcagaatcc tcacagtagg gacatctcac ttcttgtacc aacgagggtc ttcatatttc 7680

tcccctgcct tattatatcc catgacagta aataacaaaa cggctacact ccatagtcct 7740

tatacgttta atgctttcac tcggccaggt agcgtccctt gccaggcatc agcaagatgc 7800

cccaactcat gcatcactgg ggtctatact gatccatatc ccttaatctt ccataggaat 7860

catactctac gaggggtctt tgggacgatg cttgatgatg aacaagcgag acttaaccccc 7920

gtatctgcag tattcgacaa catatcccgc agtcgtgtca cccgggtgag ttcaagcagc 7980

accaaggcag catacacgac atcgacatgt tttaaagttg tcaagaccaa taaagcttat 8040

tgtcttagta ttgcagagat atccaatacc ctattcgggg aatttaggat cgttccctta 8100

ttagttgaga tcctcaagga tgatagagtt taagaagcta gacttggccg attgagccaa 8160

tcataggatg gttgggaaga cgacaccaca tcaatcatct cccacaatgc ttagagtcaa 8220

tctgaatatt aacataagcc aggatcccat gttgttgggc agccacaatc agacaatact 8280

gacatgatca ttctgagtcc tgcccactat caccttatta agaaaaaata caaaaagcat 8340

tgagatataa ggggaaacaa ccaacaagag ggaacacggg taggacatgg cgggctccgg 8400

tcccgaaagg gcagagcacc agatcatcct accagagtca catctatcct ctccatggt 8460

caagcacaaa ttgctatact actggaaatt gactgggcta ccgcttcctg atgaatgcga 8520

ctttgatcat ctcattatca gcaggcaatg gaagagaata ctggagtcgg ccactcctga 8580

cacagagaga atgataaaac tcgggcgggc agtgcaccag actctcaacc acaactccaa 8640

gataaccgga gtgctccatc ccaggtgttt agaagaactg gctagtattg aggtcccaga 8700

ttcaactaac aaattccgga agattgaaaa gaagatccag attcacaaca caaggtatgg 8760

agacctgttc acaaagctgt gcacgcatgt tgagaagaaa ttgctaggat catcccggtc 8820

taataatgtc ccacgatcag aggaattcag tagcatccgt acagatccgg cattctggtt 8880

tcactcaaaa tggtccagag ctaagttcgc gtggctccat ataaaacaag tccaaaggca 8940

tctgattgta gcagcaagga caaggtctgc agtcaacaag ttagtaacat taagtcataa 9000

gataggccac gtctttgtta ctcctgagct tgtcattgtg acacatacag acgagaacaa 9060

gttcacatgc ctcacccagg aacttgtatt gatgtatgcg gatatgatgg aaggcaggga 9120

catggtcaat ataatatctt ctacagcagc acatctcaga aacctatccg agaaaattga 9180

cgatattctg cggttagtag atgccctggc aaaggactta ggtaatcaag tctatgacgt 9240

tgtagcatta atggagggat tcgcatacgg tgctgttcag ctgcttgagc catcaggtac 9300

atttgcagga gatttctttg catttaacct acaggagctc aaagacactt taatcgaact 9360

tctcccaaat aatatagcgg aatcagtaac tcacgctatt gccgctgtat tctccggttt 9420

agaacagaat caagcagctg agatgttgtg cttgctacgt ttgtggggtc atcctttgct 9480

tgagtctcgt agtgcagcaa gagcagtcag gagccagatg tgtgcaccga agatggtaga 9540

cttcgatatg atcctccagg tactatcttt ctttaaagga acaatcatca atggatacag 9600

aaagaagaat tcaggtgtgt ggccgcgtgt caaagtagat acaatatacg ggaatatcat 9660

tgggcagctg tatgctgatt cagcagagat ctcacatgat gtcatgttga aggagtacaa 9720

gagtttatct gcttttgaat ttgagccatg tatagactat gaccctgtta ccaatctaag 9780

catgttccta aaagacaagg caatcgcaca tcctagtgat aattggctcg cctcatttag 9840

gcggaaccta ctctctgagg accagaagaa acagataaaa gaggcaactt caactaaccg 9900

cctcttgata gagttcttag aatcaaatga ttttgatcca tataaagaaa tggaatacct 9960

gacaaccctc gagtacctaa gaggtgacag tgtggcagta tcgtactcac tcaaagagaa 10020

agaggtaaaa gtgaatgggc ggatttttgc taagttaaca aagaaactaa ggaactgcca 10080

ggtaatggca gaaggaattc tagctgacca gattgcacct ttctttcagg gaaatggggt 10140

cattcaagat agcatatcct tgacaaagag tatgttagcg atgagtcaac tgtcctttaa 10200

cagcaataag aaacgtatca ctgactgcaa agagagggtt tcctcgaacc gcaatcatga 10260

tcagaagagc aagaatcgta gaagagttgc cacttttatc acgactgacc tacaaaagta 10320

ttgtcttaac tggagatatc agacagtcaa actattcgcc catgctatca atcagctgat 10380

gggcctacct catttcttcg agtggattca tcttaggctg atggacacta caatgtttgt 10440

aggggatcct ttcaatcctc caagtgaccc gactgactgt gatctatcaa gagtcccaaa 10500

cgatgacata tatattgtca gtgctagagg gggcattgag ggactctgcc agaagctatg 10560

gacgatgatc tcaattgctg caatccaact tgctgcagca agatctcatt gtcgagttgc 10620

ctgcatggta caaggtgaca atcaagtaat ggctgtaacg agagaggtaa gatcagatga 10680

ttccccggat atggtgttga cgcagttgca tcaagctagt gataatttct tcaaggaatt 10740

gattcatgtc aatcatttga ttggccataa tctgaaggat cgtgaaacca ttagatcaga 10800

cacattcttc atatacagca aacgaatatt caaggatgga gcaatactca gtcaggtcct 10860

caaaaattca tctaaattgg tgctaatatc aggcgacctt agcgaaaaca ctgtaatgtc 10920

ctgtgccaac attgcatcca ctgtagcacg actatgtgag aatgggcttc caaaggattt 10980

ctgttactat ttgaactacc taatgagttg tgtgcagaca tactttgatt cggagttctc 11040

tattacccac agctcgcaat cagattccaa ccagtcctgg atcgaggata tctctttcgt 11100

acactcatac gtgttaaccc ctgcccagct ggggggactg agcaaccttc aatactcaag 11160

gctctacaca aggaatattg gtgacccagg gaccactgct ttcgcagagg tcaagcgact 11220

ggaagcagtg gggttgctga gtcccagcat catgactaac atcttaacca ggccacctgg 11280

caatggagac tgggccagcc tatgcaacga cccatactct tttaattttg aaactgttgc 11340

aagcccaaat attgtcctca agaaacatac acagaaagtc ctatttgaga catgttcaaa 11400

ccccttatta tccggggtac atacagagga caatgaggca gaggagaaag cattggctga 11460

attcttactc aatcaagaag tgattcaccc acgtgtcgca catgctatca tggaagcaag 11520

ctctgtgggt aggagaaagc aaattcaagg gcttgttgac acaacgaaca ctgtgattaa 11580

gattgcactg actaggaggc ccctcggtat caaaaggctg atgcggataa tcaattactc 11640

gagcatgcat gcaatgttgt tcagagatga tattttctta tccaaagat ccaaccaccc 11700

attagtttct tccaatatgt gctcgctgac gctagcagat tatgcccgaa acagaagctg 11760

gtcacctctg acaggggggca ggaaaatact gggtgtatcc aaccctgata ccatagaact 11820

tgtggaggga gagattctca gcgtcagtgg agggtgcaca aaatgtgaca gcggagatga 11880

gcagtttact tggttccatc ttccaagcaa tatagagctg actgatgaca ccagaaaaaa 11940

tcccccgatg agagtgccat atcttgggtc gaagactcaa gagaggagag ctgcctcgct 12000

tgcgaaaata gcccacatgt caccacatgt gaaagcagca ctaagggcat catccgtgtt 12060

aatctgggct tatggggaca acgaagtgaa ctggactgct gctcttaata ttgcaaggtc 12120

tcgatgcaac ataagctcag agtaccttcg gctattgtca cccctgccca cggctgggaa 12180

tctccaacat agattggatg atggcataac ccagatgaca tttacccctg catctctcta 12240

cagagtgtcg ccttacgttc acatatccaa tgattctcaa aggctattca ccgaagaagg 12300

ggtcaaagag ggaaacgtgg tttaccaaca aattatgctc ttgggtttat ccctaattga 12360

atcactcttc ccaatgacag caaccagaac atatgatgag atcacattac acctccacag 12420

taaatttagc tgctgtatcc gagaagcgcc tgttgcggtt cccttcgagc tcctcgggct 12480

ggcaccggaa ttaagaatgg taacctcaaa taagttcatg tatgatccta gccctatatc 12540

agagagagat ttcgcgagac ttgacttagc tatcttcaag agttacgagc ttaatttgga 12600

atcatattcc acgctggagc taatgaacat tctttcaata tctagcggga agttgattgg 12660

ccaatctgtg gtttcttatg atgaagacac ctctataaag aatgatgcta taatagtgta 12720

tgacaacaca cgaaattgga ttagtgaggc gcagaactca gatgtggtcc gcttgtttga 12780

gtatgcagca ctcgaagtgc ttcttgactg tgcttatcaa ctctactatc tgagggtaag 12840

gggtctaaac aacatcgtcc tatacatgaa tgacttatat aagaacatgc cagggatcct 12900

actctccaat attgcggcca cgatatccca ccccatcatt cactcaaggt tgaatgcagt 12960

aggcctaatt aaccatgacg ggtcacacca gctcgcagat atagacttcg tcgaggtgtc 13020

tgcaaaattg ttagtctctt gcactcgacg cgtggtctca ggcttatatg cagggaataa 13080

gtacgatctg ctgttcccat ctgtcttaga tgataacctg aatgagaaga tgctccaact 13140

gatttcccgg ttatgctgtc tgtacacagt gctctttgct acaacaagag aaatcccaaa 13200

aataaggggc ctatcagcag aagagaaatg ctcaatactc actgagtatc tactgtcaga 13260

tgctgtaaaa ccattgctta ggcccgaaca agtgaattct atcatgtctc ccaacataat 13320

cacgttccca gccaatctat attacatgtc taggaagagc cttaatttga tcagagaacg 13380

agaggacaga gatactatct tgtcattgtt gttccctcag gaaccactgc ttgagcttcg 13440

cccagtacgg gacattggtg ctcgagtgaa agacccgttt acccggcaac ccgcatcatt 13500

catacaagag ctagatctga gtgccccagc aaggtacgac gcatttacac tgagtaaggc 13560

ctgcttcgag catacattac cgaacccaaa ggaggattac ctagtacggt acttgttcag 13620

aggaataggg actgcttcat cttcttggta taaggcatct catcttctat ccgtacctga 13680

ggtcaggtgt gcaagacatg ggaactcctt atacttagcg gaaggaagcg gagccatcat 13740

gagtcttctt gaattgcata taccacatga gactatctat tacaatacac ttttctcgaa 13800

tgagatgaac cctccacagc gacatttcgg acctacacca acacagtttc taaactcggt 13860

cgtttatagg aatctacaag cggaagtgcc atgtaaagat ggatatgtcc aggagttctg 13920

cccattatgg agagagaatg cagaagaaag tgacctgacc tcagataaag cagttggata 13980

tatcacatcc gtggtaccct acaggtctgt atcattacta cattgtgaca ttgagattcc 14040

tccagggtcc aatcaaagct tattagatca actggctact aatttatccc tgattgccat 14100

gcattctgtg aggggagggcg gggtagtgat catcaaagta ctgtatgcaa tggggtacta 14160

cttccatta ctcatgaatt tattcactcc atgttccaca aaaggatata tactctccaa 14220

tggctatgcc tgtagagggg atatgcaatg ttacctgata ttcgttatgg gctacttggg 14280

cgggcccacc ttcgtgcacg aagtggtaag gatggcaaaa actctaatac aacgacacgg 14340

tacactccta tctaaatcag atgaaattac attgactaag ctatttacct cacagcagcg 14400

tcatgtaaca gatatcctat ccagtccttt accgaagcta atgaagctct tgagagaaaa 14460

tattgatgcc gcactaattg aagctggggg acagcccgtc cgtccattct gtgcggaaag 14520

tttggtgagc acactaacag atatgactca gacagctcag atcattgcca gccacattga 14580

cacagtcatt cggtctgtaa tttacatgga ggctgagggt gacctcgccg acagtgtt 14640

cttatttact ccttacaatc tatccacaga cggtaaaagg agaacatcac ttaagcagtg 14700

caccaaacag atcttggaag tcacaatact gggtctcaga gccaaagata tcaataaagt 14760

aggtgatgta atcagtttag tactcagagg tgcggtttct ctagaggacc tcatcccatt 14820

aaggacatac ctgaagcgca gtacctgcac taaatacctg aaagcggtcc taggtattac 14880

taaactcaaa gaaatgttca cagatacctc attackgtac ttgactcgtg ctcaacaaaa 14940

attctacatg aaaaccatag gtaatgctgc caagggatat tactgtaata atgactctta 15000

aaggcaatcg tacgccaatc agttatcttc ctaactgatg actccctcat tgactcaatt 15060

ataccagatt agaaaaaagt taaattccga ctctttggaa ctcgtattcg gattcagtta 15120

gttaacttta agcaagagtg cgcaaagtca tccctaatta tagtgatgtc attcaccaaa 15180

tctctgtttg gt 15192

the

<210> 5

<211> 15192

<212> DNA

<213> Newcastle disease virus (SEQ ID NO: 5)

<400> 5

accaaacaga gaatctgtga ggtacgataa aaggcgaaga agcaatcgag atcgtacggg 60

tagaaggtgt gaatcccgag tgcgaggccg aagctcgaac ctgaggggac cttctaccga 120

tatgtcgtct gttttcgacg agtacgagca gctcctcgct gctcagaccc gccctaacgg 180

aactcatgga gggggagaga aagggagcac tttaaaagtt gaggtcccag tattcaccct 240

aaacagtgat gatccagagg atagatggaa ttttgcggta ttctgtcttc ggattgctgt 300

tagcgaggat gccaacaaac cactcaggca aggtgctctt atatccctct tatgctccca 360

ttctcaggtg atgagaaacc atgttgccct tgcagggaaa caaaatgagg ccacactggc 420

tgttcttgag atcgatggtt ttgctaacag tgtgccccag ttcaacaata ggagtggagt 480

gtctgaggaa agagcacaga gattcatggt aatcgcagga tctctccctc gggcgtgcag 540

caacggtact ccgtttgtca cggctggggt tgaagatgat gcaccagaag atatcactga 600

cactctggaa agaatcctat ctatccaagt tcaggtatgg gtcacagtag caaaggccat 660

gactgcatat gagacagcag atgagtcaga aacaagaaga ataaataagt atatgcagca 720

aggtcgagtt cagaagaagt acatccttca tcctgtatgc aggagtgcaa ttcaactcac 780

aatcagacat tctctggcag tccgtatctt cctagttagt gagctcaaga ggggccgcaa 840

tacagcaggt gggagctcta catattacaa cttggtcggg gatgtagact catacatcag 900

gaacaccggg cttactgcat ttttcctaac actcaaatat ggaatcaata ccaagacgtc 960

agccctcgca ctcagcagcc tcacaggtga tatccaaaaa atgaaacagc tcatgcgttt 1020

atatcggatg aaaggtgaaa atgcaccata catgacattg ttaggtgaca gtgaccagat 1080

gagctttgca cctgctgagt atgcacaact ttattctttt gccatgggca tggcatcagt 1140

cttagataag ggaactggca agtaccaatt cgccagggac tttatgagca catcattctg 1200

gagacttgga gtagagtatg ctcaggccca gggaagtagc attaatgagg acatggctgc 1260

tgagctaaaa ctaaccccgg cagcaaggag aggcctggca gctgctgccc aacgagtatc 1320

cgaagaaatc ggcagcatgg aattcccac tcaacaggcg ggagtcctca ccgggctcag 1380

tgatgaaggc ccccgaactc cacagggcag atcaaacaag ccgcaagggc aaccagatgc 1440

tggggatggg gagacccaat tcttggattt tatgagagca gtggcgaacg gcatgcggga 1500

agcgccaaat cctgtacaga gcaccacccca tccagagcct cccccaaccc ctggggcatc 1560

ccaagacaac gacactgact gggggtactg atcgacaaca cccagcctgc cttcacagga 1620

tcacaccaaa ccctccgacc aaaaccctcc cacactccct gacccacaac cccgcacgac 1680

cacaccaaca aaagctcccc cccaccctct cccccactcc cagccaacg atcccgccca 1740

cccgggacaa cacaggcaca gctcagttcg tcgacaatcc gcccagagcc caaggtatta 1800

gaaaaaaata cgggtagaag agagacatcc agagaccagg acgagtcacc aagttctctg 1860

ttctcccttc tacccagtga attagggtga agatggccac ttttacagat gcggagatag 1920

atgacatatt tgagaccagt gggactgtca ttgacagcat aattacggcc cagggcaaat 1980

cagctgagac cgttggaaga agtgcgatcc cgcagggcaa gaccaaagct ctaagcacag 2040

catgggagaa gcacgggagt atccagccac acgccagtca ggacgcccct gaccaacaag 2100

acagaataga aaaacagcca tccacacctg agcaggcgac tctacacaac aatccgccga 2160

tcgcatccac tgaaccgcct cccactcagg ccgcaagcga gaccagcgac acacagctca 2220

agactggggc aagcaactcc cttctgtcca tgctcgacaa actgagtaat aaatcgtcca 2280

atgctaaaaa gggcccatgg tcgggatccc aagaagggca tcaccaacct ccggcccaac 2340

aacatgggaa ccagccgagc tatggaagca accagggaag accgcagcat caggccaagg 2400

ccgtccctgg aaaccggggc acagacgaga acacagcata tcatggacag cggaaggagt 2460

cacaaccatc agctggtgca accccctcatg cgccccagtc agggcagagc caagacaata 2520

ctcctgcacc tgtggatcgt gtccagctac ctgccgactt tgtgcaggcg atgatgtcta 2580

tgatggaggc attatcacag aaggtaagta aagttgatca tcagctggac ctagtcttga 2640

aacagacatc ctccattcct atgatgcgat ctgaaatcca acagctcaag acatctgttg 2700

cgatcatgga agctaactta ggcatgatga aaattctgga ccctggttgt gctaatgttt 2760

catccttaag tgatctccgg gcagtagccc gatcccatcc agtcctagtt tcaggccctg 2820

gagacccatc tccttacgtg acacaagggg gtgaaatgac gctcaataaa ctctcacaac 2880

cggtgcagca cccctctgaa ttgattaagt ctgccactgc aagcgggcct gacatgggag 2940

tggagaagga cactgtccgc gcattaatca cctcgcgccc gatgcatcca agctcctcgg 3000

ctaagctcct gagcaagcta gatgcagcca ggtcaattga agagatcagg aagatcaaac 3060

gccttgcgct gaatggttga tggccatcac aactcataac aggctcccgt cactttagcg 3120

tcacacggaa tccctcgggg gccctccctc gcaaatctat gcttcaacac ccaaaacaac 3180

agccctctct cacccccccc aatccctcga atgaccgcac aactgcaacc aatccagcag 3240

cattagaaat taagaaaaaa tacgggtaga atcaaagtgc cttgattgca ccaaaatgga 3300

ctcatccagg acaattgggc tgtactttga ttctgccctc ccttccagca gcctgttagc 3360

atttccgatt gtcttacaag acacgggaga cgggaagaag caaatcaccc cacaatacag 3420

gatccagcgt cttgattcgt ggacagacag taaggaagac tcggtattca tcaccaccta 3480

cgggttcatc tttcaaattg ggaatgaaga aaccaccgtc ggtgtgatca atgacaatcc 3540

caggcacgag ctactctctt ccgcaatgct ctgcttaggg agtgtcccga acgacggaga 3600

tcttgttgag ctggcgagag cctgcctcac catggtggta acttgcaaaa agagtgcaac 3660

taacactgag agaatagtct tttcagtagt gcaggctcct cgggtgctgc aaagctgtat 3720

ggttgtggca aataggtact catcagtgaa tgcagtgaag catgtgaagg cgccagaaaa 3780

gatccctggg agcggaaccc tagagtataa agtgaatttt gtctctttga ccgtggtgcc 3840

aagaagggat gtctacagga tcccaactgc agtattgaaa gtgtctggct caagcctgta 3900

caatcttgcg ctcaatgtca ctattgatgt ggacgtggat ccgaagagcc cgttagtcaa 3960

atccctttcc aagtccgata gcggatacta tgcgaatctt tttctgcata tcgggcttat 4020

gtccactgta gataagaagg gaaagaaagt gacatttgac aagatagagg aaaagataag 4080

gagactcaat ctatccgtcg ggctcagtga tgtgctcgga ccctctgtgc ttgtgaaggc 4140

gagaggtgca cggactaagc tacttgctcc ttttttctct agcagtggga cagcctgcta 4200

tcctatagca aatgcctctc cccaggttgc caagatactc tggagccaaa ctgcgcacct 4260

gcggagtgtg aaagtcatca ttcaagccgg cactcagcgt gctgtcgcag tgaccgctga 4320

tcatgaggta acctccacta agatagagag gaggcatgcc attgctaaat acaatccttt 4380

caggaaataa gttgcatccc taagactgca gttcacctgc tttcccgaat caccattaca 4440

ccagacaatg atccatctcg actgcttata gttagttcac ctgtctagca aattagaaaa 4500

aacacgggta gaagagtctg gatcccgacc ggcacactca ggacgcaaca tgggctccaa 4560

accttctacc aggatcccag cacctctgat gctggtcacc cggattatgc tgatattggg 4620

ctgtattcgt tcgacaagct ctcttgacgg caggcctctt gcagctgcag gaattgtagt 4680

aacagggat aaggcagtca atgtatacac ctcgtctcag acagggtcaa tcatagtcaa 4740

gttgctcccg aatatgccca gggataagga ggcgtgtgcg aaagccccat tagaggcata 4800

taacagaaca ctgactactt tgctcactcc tcttggcgac tccatccgca agattcaagg 4860

gtctgtgtcc acgtctggag gaaggaggca aaaacgcttt ataggtgctg ttattggcag 4920

tgtagctctt ggggttgcaa cagcggcaca gataacagca gctgcggccc taatacaagc 4980

caacaagaat gctgccaaca tccttcggct taaggagagc attgctgcaa ccaatgaagc 5040

tgtgcatgaa gtcaccgacg gattatcaca actatcagtg gcagttggga agatgcagca 5100

gtttgtcaat gaccagttta ataatacggc gcgagaattg gactgtataa aaatcacaca 5160

acaggttggt gtagaactca acctatacct aactgaattg actacagtat tcgggccaca 5220

gatcacctct cctgcattaa ctcagctgac catccaggca ctttataatt tagctggtgg 5280

caatatggat tacttattaa ctaagttagg tatagggaac aatcaactca gctcattaat 5340

tggtagcggc ctgatcactg gttaccctat actgtatgac tcacagactc aactcttggg 5400

catacaagtg aatttgccct cagtcgggaa cttaaataat atgcgtgcca cctatttgga 5460

gaccttatct gtaagtacaa ccaaaggata tgcctcagca cttgtcccga aagtagtgac 5520

acaagtcggt tctgtgatag aagagcttga cacctcatac tgtatagagt ccgatctgga 5580

tttatattgt actagaatag tgacattccc catgtcccca ggtatttatt cctgtttgag 5640

cggcaacaca tcagcttgca tgtattcaaa gactgaaggc gcactcacta cgccgtatat 5700

ggcccttaaa ggctcagtta ttgccaattg taagataaca acatgtagat gtacagaccc 5760

tcctggtatc atatcgcaaa attatggaga agctgtatcc ctgatagata gacattcgtg 5820

caatgtctta tcattagacg ggataactct gaggctcagc ggagaatttg atgcaactta 5880

tcaaaagaac atctcaatac tagattctca agtcatcgtg acaggcaatc ttgatatatc 5940

aactgaactt ggaaacgtca acaattcaat cagcaatgcc ttggataagt tggcagaaag 6000

caacagtaag atagaaaaag tcaatgtcag attaaccagc acatctgctc tcattaccta 6060

tattgttcta actgtcattt ctctattttt cggtgcactt agtctgggtt tagcgtgtta 6120

cctgatgtac aaacagaagg cacaacaaaa gaccttgcta tggcttggga ataatgccct 6180

tgatcagatg agagccacta caagagcatg aatgcagata agaggtggat atatacccaa 6240

cagcagcctg tgtgtcaatt ccgataatct gtcaagtaga agacttaaga aaaaactact 6300

gggaacaagc aaccaaagag caatacacgg gtagaacggt cagaggagcc acccttcaat 6360

cggaaattag gcttcacaac atccgttcta ccacatcacc atcaacaaga gtcaatcatg 6420

gaccgcgcgg ttaacagagt cgtgctggag aatgaggaaa gagaagcaaa gaacacatgg 6480

cgcctggttt tccggatcgc agtcttactt ttaatggtaa tgactctagc tatctccgca 6540

gctgccctgg catacagtat ggaggccagt acgccgcacg accttgcagg catatcgact 6600

gtgatctcta agacagaaga taaggttacg tctttactca gttcgagtca agatgtgata 6660

gataggatat acaagcaggt ggctcttgaa tccccgctgg cgctactaaa cactgaatct 6720

acaattatga atgcaataac ctctctttct tatcaaatta acggggctgc gaacaatagc 6780

ggatgtgggg cgcctgttca tgacccagat tatatcgggg ggataggcaa agaactcata 6840

gtggacgaca tcagtgatgt cacatcattt tatccttctg catatcaaga acacctgaat 6900

ttcatcccgg cgcctactac aggatccggt tgcactcgga taccctcatt tgacatgagc 6960

accacccatt attgttatac tcacaatgta atactatccg gttgcagaga tcactcacac 7020

tcacatcaat acttagcgct tggtgtgctt cggacatctg caacagggag ggtattcttc 7080

tctactctgc gctccatcaa tttagatgac acccaaaatc ggaagtcctg cagtgtgagt 7140

gcaacccctt taggttgtga tatgctgtgc tctaaggtca cagggactga agaggagat 7200

tacaagtcag ctgccccaac atcaatggtg cacggaaggc tagggtttga cggtcaatac 7260

catgagaagg acttagacac cacggtctta tttaaggatt gggtggcaaa ttaccccagga 7320

gtgggaggag ggtcttttat tgacgaccgt gtatggttcc cagtttacgg agggctcaaa 7380

cccaattcac ccagtgacac tgcacaagaa gggaaatatg taatatacaa gcgccataac 7440

aacacatgcc ccgatggaca agattaccaa attcggatgg ctaaatcttc atataaaccc 7500

gggcgatttg gtggaaagcg cgtacagcaa gccatattat ccatcaaagt gtcaacatcc 7560

ttgggtaagg acccggtgct gactattcca cctaatacaa tcacactcat gggagccgaa 7620

ggcagaatcc tcacagtagg gacatctcac ttcttgtacc aacgagggtc ttcatatttc 7680

tcccctgcct tattatatcc catgacagta aataacaaaa cggctacact ccatagtcct 7740

tatacgttta atgctttcac tcggccaggt agcgtccctt gccaggcatc agcaagatgc 7800

cccaactcat gcatcactgg ggtctatact gatccatatc ccttaatctt ccataggaat 7860

catactctac gaggggtctt tgggacgatg cttgatgatg aacaagcgag acttaaccccc 7920

gtatctgcag tattcgacaa catatcccgc agtcgtgtca cccgggtgag ttcaagcagc 7980

accaaggcag catacacgac atcgacatgt tttaaagttg tcaagaccaa taaagcttat 8040

tgtcttagta ttgcagagat atccaatacc ctattcgggg aatttaggat cgttccctta 8100

ttagttgaga tcctcaagga tgatagagtt taagaagcta gacttggccg attgagccaa 8160

tcataggatg gttgggaaga cgacaccaca tcaatcatct cccacaatgc ttagagtcaa 8220

tctgaatatt aacataagcc aggatcccat gttgttgggc agccacaatc agacaatact 8280

gacatgatca ttctgagtcc tgcccactat caccttatta agaaaaaata caaaaagcat 8340

tgagatataa ggggaaacaa ccaacaagag ggaacacggg taggacatgg cgggctccgg 8400

tcccgaaagg gcagagcacc agatcatcct accagagtca catctatcct ctccatggt 8460

caagcacaaa ttgctatact actggaaatt gactgggcta ccgcttcctg atgaatgcga 8520

ctttgatcat ctcattatca gcaggcaatg gaagagaata ctggagtcgg ccactcctga 8580

cacagagaga atgataaaac tcgggcgggc agtgcaccag actctcaacc acaactccaa 8640

gataaccgga gtgctccatc ccaggtgttt agaagaactg gctagtattg aggtcccaga 8700

ttcaactaac aaattccgga agattgaaaa gaagatccag attcacaaca caaggtatgg 8760

agacctgttc acaaagctgt gcacgcatgt tgagaagaaa ttgctaggat catcccggtc 8820

taataatgtc ccacgatcag aggaattcag tagcatccgt acagatccgg cattctggtt 8880

tcactcaaaa tggtccagag ctaagttcgc gtggctccat ataaaacaag tccaaaggca 8940

tctgattgta gcagcaagga caaggtctgc agtcaacaag ttagtaacat taagtcataa 9000

gataggccac gtctttgtta ctcctgagct tgtcattgtg acacatacag acgagaacaa 9060

gttcacatgc ctcacccagg aacttgtatt gatgtatgcg gatatgatgg aaggcaggga 9120

catggtcaat ataatatctt ctacagcagc acatctcaga aacctatccg agaaaattga 9180

cgatattctg cggttagtag atgccctggc aaaggactta ggtaatcaag tctatgacgt 9240

tgtagcatta atggagggat tcgcatacgg tgctgttcag ctgcttgagc catcaggtac 9300

atttgcagga gatttctttg catttaacct acaggagctc aaagacactt taatcgaact 9360

tctcccaaat aatatagcgg aatcagtaac tcacgctatt gccgctgtat tctccggttt 9420

agaacagaat caagcagctg agatgttgtg cttgctacgt ttgtggggtc atcctttgct 9480

tgagtctcgt agtgcagcaa gagcagtcag gagccagatg tgtgcaccga agatggtaga 9540

cttcgatatg atcctccagg tactatcttt ctttaaagga acaatcatca atggatacag 9600

aaagaagaat tcaggtgtgt ggccgcgtgt caaagtagat acaatatacg ggaatatcat 9660

tgggcagctg tatgctgatt cagcagagat ctcacatgat gtcatgttga aggagtacaa 9720

gagtttatct gcttttgaat ttgagccatg tatagactat gaccctgtta ccaatctaag 9780

catgttccta aaagacaagg caatcgcaca tcctagtgat aattggctcg cctcatttag 9840

gcggaaccta ctctctgagg accagaagaa acagataaaa gaggcaactt caactaaccg 9900

cctcttgata gagttcttag aatcaaatga ttttgatcca tataaagaaa tggaatacct 9960

gacaaccctc gagtacctaa gaggtgacag tgtggcagta tcgtactcac tcaaagagaa 10020

agaggtaaaa gtgaatgggc ggatttttgc taagttaaca aagaaactaa ggaactgcca 10080

ggtaatggca gaaggaattc tagctgacca gattgcacct ttctttcagg gaaatggggt 10140

cattcaagat agcatatcct tgacaaagag tatgttagcg atgagtcaac tgtcctttaa 10200

cagcaataag aaacgtatca ctgactgcaa agagagggtt tcctcgaacc gcaatcatga 10260

tcagaagagc aagaatcgta gaagagttgc cacttttatc acgactgacc tacaaaagta 10320

ttgtcttaac tggagatatc agacagtcaa actattcgcc catgctatca atcagctgat 10380

gggcctacct catttcttcg agtggattca tcttaggctg atggacacta caatgtttgt 10440

aggggatcct ttcaatcctc caagtgaccc gactgactgt gatctatcaa gagtcccaaa 10500

cgatgacata tatattgtca gtgctagagg gggcattgag ggactctgcc agaagctatg 10560

gacgatgatc tcaattgctg caatccaact tgctgcagca agatctcatt gtcgagttgc 10620

ctgcatggta caaggtgaca atcaagtaat ggctgtaacg agagaggtaa gatcagatga 10680

ttccccggat atggtgttga cgcagttgca tcaagctagt gataatttct tcaaggaatt 10740

gattcatgtc aatcatttga ttggccataa tctgaaggat cgtgaaacca ttagatcaga 10800

cacattcttc atatacagca aacgaatatt caaggatgga gcaatactca gtcaggtcct 10860

caaaaattca tctaaattgg tgctaatatc aggcgacctt agcgaaaaca ctgtaatgtc 10920

ctgtgccaac attgcatcca ctgtagcacg actatgtgag aatgggcttc caaaggattt 10980

ctgttactat ttgaactacc taatgagttg tgtgcagaca tactttgatt cggagttctc 11040

tattacccac agctcgcaat cagattccaa ccagtcctgg atcgaggata tctctttcgt 11100

acactcatac gtgttaaccc ctgcccagct ggggggactg agcaaccttc aatactcaag 11160

gctctacaca aggaatattg gtgacccagg gaccactgct ttcgcagagg tcaagcgact 11220

ggaagcagtg gggttgctga gtcccagcat catgactaac atcttaacca ggccacctgg 11280

caatggagac tgggccagcc tatgcaacga cccatactct tttaattttg aaactgttgc 11340

aagcccaaat attgtcctca agaaacatac acagaaagtc ctatttgaga catgttcaaa 11400

ccccttatta tccggggtac atacagagga caatgaggca gaggagaaag cattggctga 11460

attcttactc aatcaagaag tgattcaccc acgtgtcgca catgctatca tggaagcaag 11520

ctctgtgggt aggagaaagc aaattcaagg gcttgttgac acaacgaaca ctgtgattaa 11580

gattgcactg actaggaggc ccctcggtat caaaaggctg atgcggataa tcaattactc 11640

gagcatgcat gcaatgttgt tcagagatga tattttctta tccaaagat ccaaccaccc 11700

attagtttct tccaatatgt gctcgctgac gctagcagat tatgcccgaa acagaagctg 11760

gtcacctctg acaggggggca ggaaaatact gggtgtatcc aaccctgata ccatagaact 11820

tgtggaggga gagattctca gcgtcagtgg agggtgcaca aaatgtgaca gcggagatga 11880

gcagtttact tggttccatc ttccaagcaa tatagagctg actgatgaca ccagaaaaaa 11940

tcccccgatg agagtgccat atcttgggtc gaagactcaa gagaggagag ctgcctcgct 12000

tgcgaaaata gcccacatgt caccacatgt gaaagcagca ctaagggcat catccgtgtt 12060

aatctgggct tatggggaca acgaagtgaa ctggactgct gctcttaata ttgcaaggtc 12120

tcgatgcaac ataagctcag agtaccttcg gctattgtca cccctgccca cggctgggaa 12180

tctccaacat agattggatg atggcataac ccagatgaca tttacccctg catctctcta 12240

cagagtgtcg ccttacgttc acatatccaa tgattctcaa aggctattca ccgaagaagg 12300

ggtcaaagag ggaaacgtgg tttaccaaca aattatgctc ttgggtttat ccctaattga 12360

atcactcttc ccaatgacag caaccagaac atatgatgag atcacattac acctccacag 12420

taaatttagc tgctgtatcc gagaagcgcc tgttgcggtt cccttcgagc tcctcgggct 12480

ggcaccggaa ttaagaatgg taacctcaaa taagttcatg tatgatccta gccctatatc 12540

agagagagat ttcgcgagac ttgacttagc tatcttcaag agttacgagc ttaatttgga 12600

atcatattcc acgctggagc taatgaacat tctttcaata tctagcggga agttgattgg 12660

ccaatctgtg gtttcttatg atgaagacac ctctataaag aatgatgcta taatagtgta 12720

tgacaacaca cgaaattgga ttagtgaggc gcagaactca gatgtggtcc gcttgtttga 12780

gtatgcagca ctcgaagtgc ttcttgactg tgcttatcaa ctctactatc tgagggtaag 12840

gggtctaaac aacatcgtcc tatacatgaa tgacttatat aagaacatgc cagggatcct 12900

actctccaat attgcggcca cgatatccca ccccatcatt cactcaaggt tgaatgcagt 12960

aggcctaatt aaccatgacg ggtcacacca gctcgcagat atagacttcg tcgaggtgtc 13020

tgcaaaattg ttagtctctt gcactcgacg cgtggtctca ggcttatatg cagggaataa 13080

gtacgatctg ctgttcccat ctgtcttaga tgataacctg aatgagaaga tgctccaact 13140

gatttcccgg ttatgctgtc tgtacacagt gctctttgct acaacaagag aaatcccaaa 13200

aataaggggc ctatcagcag aagagaaatg ctcaatactc actgagtatc tactgtcaga 13260

tgctgtaaaa ccattgctta ggcccgaaca agtgaattct atcatgtctc ccaacataat 13320

cacgttccca gccaatctat attacatgtc taggaagagc cttaatttga tcagagaacg 13380

agaggacaga gatactatct tgtcattgtt gttccctcag gaaccactgc ttgagcttcg 13440

cccagtacgg gacattggtg ctcgagtgaa agacccgttt acccggcaac ccgcatcatt 13500

catacaagag ctagatctga gtgccccagc aaggtacgac gcatttacac tgagtaaggc 13560

ctgcttcgag catacattac cgaacccaaa ggaggattac ctagtacggt acttgttcag 13620

aggaataggg actgcttcat cttcttggta taaggcatct catcttctat ccgtacctga 13680

ggtcaggtgt gcaagacatg ggaactcctt atacttagcg gaaggaagcg gagccatcat 13740

gagtcttctt gaattgcata taccacatga gactatctat tacaatacac ttttctcgaa 13800

tgagatgaac cctccacagc gacatttcgg acctacacca acacagtttc taaactcggt 13860

cgtttatagg aatctacaag cggaagtgcc atgtaaagat ggatatgtcc aggagttctg 13920

cccattatgg agagagaatg cagaagaaag tgacctgacc tcagataaag cagttggata 13980

tatcacatcc gtggtaccct acaggtctgt atcattacta cattgtgaca ttgagattcc 14040

tccagggtcc aatcaaagct tattagatca actggctact aatttatccc tgattgccat 14100

gcattctgtg aggggagggcg gggtagtgat catcaaagta ctgtatgcaa tggggtacta 14160

cttccatta ctcatgaatt tattcactcc atgttccaca aaaggatata tactctccaa 14220

tggctatgcc tgtagagggg atatggagtg ttacctgata ttcgttatgg gctacttggg 14280

cgggcccacc ttcgtgcacg aagtggtaag gatggcaaaa actctaatac aacgacacgg 14340

tacactccta tctaaatcag atgaaattac attgactaag ctatttacct cacagcagcg 14400

tcatgtaaca gatatcctat ccagtccttt accgaagcta atgaagctct tgagagaaaa 14460

tattgatgcc gcactaattg aagctggggg acagcccgtc cgtccattct gtgcggaaag 14520

tttggtgagc acactaacag atatgactca gacagctcag atcattgcca gccacattga 14580

cacagtcatt cggtctgtaa tttacatgga ggctgagggt gacctcgccg acagtgtt 14640

cttatttact ccttacaatc tatccacaga cggtaaaagg agaacatcac ttaagcagtg 14700

caccaaacag atcttggaag tcacaatact gggtctcaga gccaaagata tcaataaagt 14760

aggtgatgta atcagtttag tactcagagg tgcggtttct ctagaggacc tcatcccatt 14820

aaggacatac ctgaagcgca gtacctgcac taaatacctg aaagcggtcc taggtattac 14880

taaactcaaa gaaatgttca cagatacctc attackgtac ttgactcgtg ctcaacaaaa 14940

attctacatg aaaaccatag gtaatgctgc caagggatat tactgtaata atgactctta 15000

aaggcaatcg tacgccaatc agttatcttc ctaactgatg actccctcat tgactcaatt 15060

ataccagatt agaaaaaagt taaattccga ctctttggaa ctcgtattcg gattcagtta 15120

gttaacttta agcaagagtg cgcaaagtca tccctaatta tagtgatgtc attcaccaaa 15180

tctctgtttg gt 15192

Claims (4)

1. the attenuated vaccine strain of VII type Avian pneumo-encephalitis virus L transgenation, it is characterized in that: the attenuated vaccine strain virulence of this VII type Avian pneumo-encephalitis virus L transgenation obviously weakens, while has high titre on chicken embryo, heritability is stable, high with the antigen matching of popular virus, be suitable for the scale operation of vaccine, can be used for manufacturing vaccine, the antigen nucleotides sequence of the attenuated vaccine strain of this VII type Avian pneumo-encephalitis virus L transgenation is classified as shown in SEQ ID NO:1 to SEQ ID NO:4.

2. the preparation method of the attenuated vaccine strain of VII type Avian pneumo-encephalitis virus L transgenation, it is characterized in that: built and covered whole genomic five cDNA cloned sequences, utilize the restriction enzyme site of the intersegmental lap of each sheet, at transcription vector plasmid pBR322, assemble the global cDNA clone who has obtained 15192nt; 5 ' the end prefix t7 rna polymerase promotor in full-length cDNA fragment adds fourth hepatovirus ribozyme (RiB) and the T7 transcription termination signal with self-catalysis, the plasmid called after prNDV-97 building after cDNA fragment; By pcr gene group, by the 14056th bit base of the L protein-coding region in genome cDNA, by A same sense mutation, be G, eliminate the Hind III restriction enzyme site at 14056bp place, and as the viral molecule marker of rescue, the plasmid called after pmNDV-97 having built; At T7 polymerase promoter, in 5 ' end of genome cDNA, introduce two unnecessary G simultaneously, contribute to the virus rescue of paramyxovirus reverse genetic manipulation;

Design 5 pairs of primers NDV VII-97 pnca gene group SEQ ID NO:5 that is used for increasing, primer sequence is as follows:

F1：5'ATGCGGCCGCTAATACGACTCACTATAGGGACCAAACAGAGAATCTG

TGAGGTACGATAA3'

R1：5'TAGGTACCCTTGCAGTGGCAGACTTAATCAATTCAGAG3'

F2：5'GGGTGAAATGACGCTCAATAAACTCTCACA3'

R2：5'ATGGTACCCTCCCTGTTGCAGATGTCCGAAGCACACCA3'

F3：5'TGCAGAGATCACTCACACTCACATCAATAC3'

R3：5'ACGGTACCAGGTGACCAGCTTCTGTTTCGGGCATAATC3'

F4：5'ATTAGTTTCTTCCAATATGTGCTCGCTGAC3'

R4：5'GTAGTAATGATACAGACCTGTAGGGTACCA3'

F5：5'GGAGAGAGAATGCAGAAGAAAGTGACCTGACCTCAGATAAAGCAG3'

R5：5'AGCCGGCGCCAGCGAGGAGGCTGGGACCATGCCGGCCACCAAACAG

AGATTTGGTGAATGACATC3'

Design 2 pairs for the 14056bp place that suddenlys change hindiII restriction enzyme site primer, eliminates this restriction enzyme site, and primer sequence is as follows:

AFa：5' CATGTTTTAAAGTTGTCAAG 3'

F6：5' GGGTCCAATCAGAGCTTATTAGATC 3'

R6：5' GATCTAATAAGCTCTGATTGGACCC 3'

ARa：5' GCG GTCGAC ACCGCTTTCAGGTATTTAG3'；

On the basis of the full geneome plasmid of pmNDV-97, build NDV-97 L transgenation attenuated strain, concrete operations are:

By following primer, respectively through Overlapping PCR method, by 1756,1917 and 1954 amino acids difference simple point mutations in NDV parent poison L protein structure domain VI, by object fragment and pmNDV-97 carrier warp after amplification hindiII and xbai restriction enzyme is double digestion respectively, carries out nucleic acid electrophoresis, and glue reclaims object fragment and is connected with after carrier pmNDV-97, and the mutant plasmid obtaining is called after pK1756A, pK1917A and pE1954Q respectively;

Design 4 pairs of primers for NDV VII-97 L Argine Monohydrochloride rite-directed mutagenesis, primer sequence is as follows:

AF1：5' CATGTTTTAAAGTTGTCAAG 3'

AF2：5' CTTCTTGGTATGCGGCATCTCATCTTC 3'

AF3：5' GTAGTGATCATCGCAGTACTGTATGC3'

AF4：5' GAGGGGATATGCAATGTTACCTGATAT3'

AF5：5' GCG GTCGAC ACCGCTTTCAGGTATTTAG3'

AF6： 5'GAAGATGAGATGCCGCATACCAAGAAG3'

AF7： 5'GCATACAGTACTGCGATGATCACTA3'

AF8： 5'ATATCAGGTAACATTGCATATCCCCT3'

Wherein, AF1+AF6, AF1+AF7 and AF1+AF8 primer utilize PCR method to expand respectively object band, respectively called after A1, B1 and C1; AF5+AF2, AF5+AF3 and AF5+AF4 primer utilize PCR method to expand respectively object band, respectively called after A2, B2 and C2; On this basis, utilize AF1+AF5 primer respectively through Overlapping PCR method, with A1+A2, B1+B2 and C1+C2, be respectively template, the about 6000bp(that increases is containing mutational site) object fragment, obtain respectively L sudden change fragment L1756, L1917 and L1954.

3. the preparation method of the attenuated vaccine strain of VII type Avian pneumo-encephalitis virus L as claimed in claim 2 transgenation, is characterized in that:

Genome L gene with strain isolated NDV VII-97 carries out rite-directed mutagenesis, to carrying out single-point amino acid mutation in L protein structure domain VI, obtains highly causing weak NDV VII-97 L attenuated strain K1756A, K1917A and E1954Q; On the basis of E1954Q, F protein cleavage site is suddenlyd change, the F protein cleavage site (RRQKRF) of virulent strain is sported to the F protein cleavage site (GRQGRL) of LaSota low virulent strain, the attenuated strain obtaining is E1954Q+F;

Primer for the F protein cleavage site that suddenlys change is as follows:

AF9：CTCCGACCAAAACCCTCCCACACTCCCTGACCC

AR9：GCAGAGTAGAGAAGAATACCCTCCCTGTTGCAGAT

AF10：TCTGTGTCCACGTCTGGAGGAGGGAGACAGGGGCGCCTTATAGGTGCTGT

TATTGGCAG

AR10：CTGCCAATAACAGCACCTATAAGGCGCCCCTGTCTCCCTCCTCCAGACGT

GGACACAGA

Wherein, 2 pairs of primers of AF9+AR10 and AF10+AR9 utilize PCR method to expand respectively object band, it is template that the glue of take again after glue recovery reclaims product, with this 1 pair of primer of AF9+AR9, utilize Overlapping method to carry out pcr amplification, the fragment that acquisition contains F protein mutation is about 5458bp, and take this mutant obtaining is E1954Q+F.

4. the preparation method of the attenuated vaccine strain of VII type Avian pneumo-encephalitis virus L transgenation according to claim 3, is characterized in that:

1) structure of NDV VII-97 pnca gene group full-length cDNA;

2) connection of Amino Acid-Induced Site-Directed Mutation and mutant full-length cDNA in NDV VII-97 L protein structure domain VI;

3) on the basis of E1954Q sudden change, the sudden change in F protein cleavage site, NDV VII-97 and the connection of mutant full-length cDNA;

4) attenuated vaccine strain K1756A, K1917A, the E1954Q of VII type Avian pneumo-encephalitis virus L transgenation

And the rescue of E1954Q+F;

By plasmid pK1756A, pK1917A, pE1954Q and pE1954Q+F respectively with pBSK-NP, pBSK-P and tri-eukaryon expression plasmid cotransfection BHK-21 cells of pBSK-L, after transfection 72h, cell conditioned medium is inoculated to 9-11 age in days SPF chicken embryo, 3 pieces of every duplicate samples inoculations, 0.2mL/ piece, successfully rescues and obtains NDV VII-97 L transgenation attenuated strain K1756A, K1917A and E1954Q and F albumen and the L albumen attenuated vaccine strain E1954Q+F that suddenlys change altogether.

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