CN108728419A - Express aviadenovirus penton Protein reconstitutions newcastle disease vaccine Candidate Strain rAI4-penton and construction method - Google Patents

Abstract

The invention relates to a recombinant Newcastle disease vaccine candidate strain rAI4-penton expressing avian adenovirus penton protein and a construction method thereof. The Newcastle disease virus strain rAI4-penton has a preservation number of CGMCC No: 15492. The construction method is to use the established reverse genetic operation platform of attenuated Newcastle disease virus strain, insert the penton gene sequence of avian adenovirus into the full-length transcription vector pNDV/rAI4 of the genome of the AI4 strain, and obtain the full-length cDNA of the recombinant Newcastle disease virus genome Cloning pNDV/rAI4-penton, the recombinant virus rAI4-penton obtained by transfection successfully expressed penton protein. The recombinant virus rAI4-penton has a high reproductive titer on chicken embryos, and continuous passage can stabilize the penton protein, which is suitable for large-scale production of vaccines and can be used to manufacture vaccines.

Description

Expression of avian adenovirus penton protein recombinant Newcastle disease vaccine candidate rAI4- Penton and its construction method

technical field

The invention relates to the application of reverse genetic technology to rescue a recombinant virus strain rAI4-penton expressing poultry adenovirus penton protein with Newcastle disease virus as a carrier, and is used for vaccine development.

Background technique

Reverse genetics technology is the process of reverse-transcribing the RNA virus genome into cDNA, and then the cDNA interacts with various auxiliary proteins to assemble infectious RNA [Neumann G, Kawaoka Y. A decade after the generation of a negative-sense RNA virus from cloned cDNA - what have we learned? [J]. TheJournal of general virology, 2002, 83 (Pt 11): 2635-62.], this process is also called "virus rescue". Since the RNA virus finally "rescued" by reverse genetics technology is derived from cDNA clones, various in vitro artificial manipulations can be performed on the genome of RNA viruses at the DNA level, such as gene mutation, gene knockout (deletion), gene The transformation of insertion, gene replacement and gene complementation has solved the difficult problem of RNA virus genome manipulation, which greatly facilitates people's research on the function of virus genes, pathogenic mechanism and viral disease prevention strategies. The research and development of the newcastle provides new ideas [Huang Z, Elankumaran S, Panda A, et al. Recombinant Newcastle disease virus as a vaccine vector. Poultry Science, 2003, 82:899-906.].

Newcastle disease (ND) is an acute, septic, highly contagious infectious disease caused by Newcastle disease virus (NDV) that mainly infects chickens, pigeons, quails, turkeys and other poultry and wild poultry . The genome of Newcastle disease virus is non-segmented, single-stranded negative-strand RNA, and the genome pattern is 3'-leader-NP-P-M-F-HN-L-trailer-5', which encodes six structural proteins in sequence: nucleocapsid protein ( Nucleocapsid protein, NP), phosphoprotein (Phosphoprotein, P), matrix protein (Matrix protein, M), fusion protein (Fusion protein, F), hemagglutinin-neuraminidase protein (Heamagglutinin-Neuraminidase protein, HN) and Large protein (Large protein, L). Among them, F protein has the function of inducing cell fusion and destroying cells. F protein is the main reason for the virulence of different NDVs, and the cleavage site of F protein plays an important role [peeters B P, Deleeuw, Koch G, et al. Rescue of Newcastle disease virus from cloned cDNA: evidence that cleavability of the fusion protein is a major determinant for virulence[J].Journal of virology,1999,73(6):5001-9.].

Fowl adenovirus (Fowl adenovirus, FAV) belongs to the family Adenoviridae, and its nucleocapsid consists of hexon, penton and fiber. Divided into 3 groups according to group-specific antigens: Group I avian adenovirus includes strains of various serotypes isolated from different poultry, with identical group-specific antigens, can be divided into five genes A-E according to RFLP technology According to serum cross neutralization reaction, it can be divided into 12 serotypes (serotypes 1-7, 8a, 8b, 9-11), and its representative strain is chicken embryo lethal orphan virus (CELOV) [Meulemans G, ouvreur B, Decaesstecker M, et al.Phylogenetic analysis of fowl adenoviruses[J C].Avian pathology:journal of the WVPA,2004,33(2):164-70.]. Group II adenoviruses include turkey hemorrhagic enteritis virus (HEV), pheasant marble spleen disease (MSDV) and avian splenomegaly virus (AASV); group III adenoviruses include egg drop syndrome (EDSV). Among them, group I serotype 4 avian adenovirus (FAdV4) can cause inclusion body hepatitis and pericardial effusion syndrome (HPS) [Kim J N, Byun S H, Kim M J, et al. Outbreaks of hydropericardium syndrome and molecular characterization of Korean fowl adenoviral isolates [J]. Avian diseases, 2008, 52(3): 526-30.].

At present, countries around the world mainly use inactivated oil emulsion vaccines to prevent HPS [Du D, Zhang P, Li X, et al. Cell-culture derived fowl adenovirus serotype 4 inactivated vaccine provides complete protection for virus infection on SPF chickens [J]. Virusdisease, 2017, 28 (2):182-8.], but the vaccine produced by this method has high cost, inconvenient use, and the risk of spreading virus due to inactivation. Therefore, new genetically engineered vaccines such as gene recombinant vaccines, subunit vaccines, and nucleic acid vaccines have become the focus of vaccine research in recent years.

One of the most important characteristics of the carrier virus is that it can be used to quickly develop genetically engineered vaccines against emerging highly pathogenic infectious diseases. In addition to this characteristic, Newcastle disease virus does not recombine with homologous viruses and expresses a large number of The source gene will not cause severe weakening of self-replication, and after weakening, it is not pathogenic to humans and animals, and can be used as a safe vector [Bukreyev A, Skiadopoulos M H, Murphy B R, et al.Nonsegmented negative-strandviruses as vaccine vectors[ J]. Journal of Virology, 2006, 80(21): 10293-10306.]. The recombinant virus after inserting foreign genes into the NDV genome can replicate in animals and express foreign proteins stably and continuously. Using this recombinant virus as a vaccine can induce the body to produce double immune protection against foreign proteins and Newcastle disease [Veits J, Wiesner D, Fuchs W, et al. Newcastle disease virus expressing H5hemagglutinin gene protects chickens against Newcastle disease and avian influenza [J]. ProcNatlAcadSci U S A, 2006, 103(21): 8197-8202. Schroer D, Veits J, Grund C,et al.Vaccination with Newcastle disease virus vectored vaccineprotects chickens against highly pathogenic H7avian influenza virus[J].AvianDis,2009,53(2):190-197.], Lamb et al.[Lamb,R.A., Kolakofsky,D., et al. (2001). Paramyxoviridae: the viruses and their replication, p.1305-1340. In D. M. Knipe and P.M. Howley, Fields virology. Lippincott Williams & Wilkins Philadelphia, Pa.] found that the transcription of paramyxoviridae has a polarity effect, the more The gene near the 3' end has more transcripts, and the closer to the 5' end, the lower the amount of transcripts. According to the current research, the expression level of exogenous gene is higher when it is inserted between P and M genes, which is the first choice for exogenous gene insertion. In 2011, Hu Zenglei et al. successfully constructed the full-length clone pNDV/AI4 after mutating the F protein cleavage site of the Ⅶd subtype NDV strain JS5/05 isolated in our laboratory, and obtained attenuated AI4 through reverse genetic manipulation, which has excellent Reproductive performance, the titer of its allantoic fluid can reach 10log2, suitable as a carrier for expressing foreign genes [Hu Z, Hu S, MengC, et al. Generation of a genotype VII Newcastle disease virus vaccine candidate with high yield in embryonated chicken eggs [J].Avian diseases,2011,55(3):391-7.].

The protective antigens of avian adenovirus include: hexon, penton and fibril protein. At present, there is no newcastle disease virus as a carrier to express foreign genes of avian adenovirus to construct a double precedent for vaccines.

Penton protein is an important structural protein of avian adenovirus, which is involved in the process of virus invasion into cells and virion assembly. Studies have shown that anti-penton protein antibodies have significant virus neutralization and can provide animals with a 90% protection rate [Shah M S, Ashraf A, Rahman M, et al. A subunit vaccine against thydropericardium syndrome using adenovirus penton capsid protein [J ]. Vaccine, 2012, 30(50): 7153-6.].

Contents of the invention

The object of the present invention is to use NDV as a vector to express the recombinant virus strain of avian adenovirus penton protein, which is used for making vaccines.

The recombinant virus strain rAI4-penton expressing the avian adenovirus penton gene of the present invention was deposited in the General Microbiology Center of the China Committee for the Collection of Microorganisms on March 20, 2018, and its preservation number is CGMCC No: 15492.

The invention discloses the construction method of the recombinant virus strain rAI4-penton expressing the penton protein of avian adenovirus: using the genome full-length transcription vector pNDV/rAI4 of the attenuated strain AI4 of gene type VII NDV as the backbone, inserting the penton gene of the avian adenovirus Between the P and M genes of the full-length transcription vector pNDV/rAI4 of the AI4 strain genome, the recombinant virus rAI4-penton was obtained by reverse genetics technology.

The present invention utilizes the established reverse genetic operation platform of Newcastle disease virus AI4 strain to insert the penton gene of a strain FAdV4 isolated from the Key Open Laboratory of Livestock and Poultry Infectious Diseases of the Ministry of Agriculture of Yangzhou University into the P of NDV full-length transcription vector pNDV/rAI4 Between the M gene and the recombinant virus strain rAI4-penton, the recombinant virus rAI4-penton has high reproductive performance and is suitable for large-scale production of vaccines.

Method of the present invention specifically comprises the following steps:

1. Construction of full-length clone pNDV/rAI4-penton

1) Construction of the intermediate plasmid pNAI4-blunt (AgeⅠ-BstZ17Ⅰ)

Primers primer1 and primer6 were designed, and pNDV/rAI4 was used as a template for PCR, and the product was ligated into blunt vector to construct plasmid pNAI4-blunt (AgeI-BstZ17I).

Primer1: 5'-AACTCTCACAACCGGTGC-3' (SEQ ID NO.1)

Primer6: 5'-CTGAGACGAGGTGTATACATTGACTG-3' (SEQ ID NO.6)

2) Cloning of the avian adenovirus penton gene

According to the full-length sequence of an avian adenovirus strain CH/JSXZ/2015 published in GenBank, the penton gene was synthesized by GenScript, and primers NAI4-P3 and NAI4-P4 were designed to amplify the penton gene coding region (SEQ ID NO .9), was connected into the plasmid pNAI4-blunt (AgeI-BstZ17I) by the method of homologous recombination, and the plasmid with the correct sequencing result was named pNAI4-penton-blunt (AgeI-BstZ17I).

NAI4-P3:5'-AAAAAATACGGGTAGAAGCACCATGTGGGGGTTGCAGCCGC-3' (SEQ ID NO.3)

NAI4-P4:5'-GATTCCGTGTGACGCCTACTGCAAGGTCGCGGAAC-3' (SEQ ID NO.4)

3) Acquisition of full-length clone pNDV/rAI4-penton

Plasmid pNAI4-penton-Blunt (AgeI-BstZ17I) and plasmid pNDV/rAI4 were digested by AgeI and BstZ17I, and the 3428bp band of pNAI4-penton-Blunt (AgeI-BstZ17I) and pNDV/rAI4 were recovered by agarose gel electrophoresis The large bands were then connected to construct the full-length pNDV/rAI4-penton of the recombinant NDV genome. The specific construction method is shown in Figure 1

2. Rescue of recombinant virus pNDV/rAI4-penton strain

Co-transfect the full-length plasmid pNDV/rAI4-penton with three helper plasmids pCI-NP, pCI-P and pCI-L into BSR-T7/5 cells, inoculate 9-11-day-old SPF chicken embryos 60 hours later, and blindly pass one generation Then the recombinant virus rAI4-penton was obtained.

The present invention uses different Newcastle disease viruses as vectors to insert protective antigens hexon (hexon), penton (penton) and fibril protein (fiber) of different avian adenoviruses respectively, and in the obtained recombinant Newcastle disease virus, finally Only the recombinant virus strain inserted into the penton gene can stimulate animals to produce neutralizing antibodies against avian adenovirus.

The present invention uses pNDV/rAI4 as the backbone to construct a recombinant Newcastle disease virus strain rAI4-penton capable of expressing avian adenovirus penton protein, which successfully stimulates animals to produce antibodies against Newcastle disease virus and avian adenovirus, which can be used to deal with current Newcastle disease and the prevalence of pericardial effusion.

Description of drawings

Fig. 1 Construction model diagram of recombinant plasmid pNDV/rAI4-penton.

Fig. 2 Construction model diagram of recombinant plasmid pNDV/rAI4-fiber2.

Fig. 3 Electropherogram of recombinant virus cDNA RT-PCR identification. M: 1000bp DNA ladder marker, 1: rAI4-penton amplified fragment, 2: rAI4-fiber2 amplified fragment.

Fig. 4 Western blot detection results of exogenous proteins after recombinant plasmids infected DF1 cells. 1: Marker, 2: rAI4-penton, 3: rAI4-fiber2, 4: AI4.

Figure 5 HI antibody levels against NDV virus after immunization.

Fig. 6 Anti-avian adenovirus neutralization titer in immunized chicken serum.

Fig. 7 Construction model diagram of the intermediate plasmid pAI4-2FHN-blunt (AgeI-AflII).

Fig. 8 Construction model diagram of recombinant plasmid pNDV/rAI4-2FHN-hexon.

Fig. 9 RT-PCR results of exogenous gene of recombinant virus. M: 1000bp DNA ladder marker, 1: rAI4-2FHN-hexon amplified fragment.

Figure 10 Western blot analysis of exogenous protein expression. 1: Marker, 2: rAI4-2FHN-hexon, 3: rAI4-2FHN-penton, 4: rAI4-2FHN-fiber2.

Figure 11 HI antibody levels against vector virus after immunization.

Anti-avian adenovirus neutralizing antibody level after Fig. 12 immunization

The recombinant Newcastle disease vaccine candidate strain rAI4-penton constructed by the present invention expressing avian adenovirus penton protein was preserved in the General Microbiology Center of China Microbiological Culture Collection Management Committee (Address: No. 1 Beichen West Road, Chaoyang District, Beijing) on March 20, 2018 No. 3, Institute of Microbiology, Chinese Academy of Sciences), classified as: Avian Paramyxovirus Type I; preservation number CGMCC No.15492.

Detailed ways

Example 1: Using pNDV/rAI4 as a carrier

Step 1: Construction of recombinant Newcastle disease vaccine candidate rAI4-penton full-length expression clone expressing avian adenovirus penton protein

The full-length expression vector pNDV/rAI4 of NDV AI4 strain (Hu Z, Hu S, Meng C, et al. Generation of a Genotype VII Newcastle Disease Virus Vaccine Candidate with High Yield in Embryonated Chicken Eggs. Avian Diseases 2011,55(3):1759- 1769) was constructed and preserved by the Key Open Laboratory of Livestock and Poultry Infectious Diseases of the Ministry of Agriculture of Yangzhou University. Blunt vector: purchased from Transgen; Dephospharylation (BAP) Kit: purchased from Bao Biological Engineering (Dalian) Co., Ltd.; AMV reverse transcriptase, Highfidelity DNA polymerase, T4 DNA ligase, Agarose Gel DNA Extraction Kit were purchased from Roche; transfection Reagents SuperFect and plasmid extraction kit (QIAprep Spin MiniPrep Kit) are products of QIAGEN; other conventional reagents are of domestic analytical grade.

1. Construction of the intermediate plasmid pNAI4-blunt (AgeⅠ-BstZ17Ⅰ)

Primers primer1 and primer6 were designed according to the sequence of plasmid pNDV/rAI4 to amplify the sequence between AgeI (2879)-BstZ17I (4705) restriction sites of plasmid pNDV/rAI4. The primer sequences are as follows:

Primer1: 5'-AACTCTCACAACCGGTGC-3' (SEQ ID NO.1)

Primer6: 5'-CTGAGACGAGGTGTATACATTGACTG-3' (SEQ ID NO.6)

The bold part of the primer sequence is the restriction site sequence, and the italic part is the homologous sequence. The above primers were synthesized by Shanghai Bioengineering Co., Ltd.

The PCR reaction uses the plasmid pNDV/rAI4 as a template to configure the following system:

PCR reaction program: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 2 min, and 15 cycles; denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 2 min, and 15 cycles; Extend at 72°C for 10 minutes, and store at 4°C.

After agarose gel electrophoresis, the 1852bp band was cut, and the PCR product was recovered and stored at 4 degrees Celsius.

After the PCR product was recovered and purified, it was ligated with the blunt vector and transformed into DH5α competent. The extracted plasmid was verified by PCR with Primer1 and Primer6, and the positive plasmid was named pNAI4-blunt (AgeⅠ-BstZ17Ⅰ).

2. Cloning of avian adenovirus penton gene

The penton gene of avian adenovirus strain CH/JSXZ/2015 (GenBank: KU569296.1) was synthesized by GenScript, and primers NAI4-P3 and NAI4-P4 were designed according to the sequence of the penton gene to amplify the coding region of the penton gene . Primers Primer1, Primer2, Primer5 and Primer6 were designed according to the sequence of pNDV/rAI4 for homologous recombination reaction.

In the primer sequence, the bold part is the enzyme cutting site sequence, the underlined dotted line is the kozak sequence, which can improve the expression level of foreign genes, the lower solid line is the GE and GS sequence of Newcastle disease virus, and the italic part is the homonym on the primer. The source sequence and primers were synthesized by Nanjing Jinsirui Company. Use Primer1 and Primer2 as primers to amplify the fragment between 2879 (AgeI) and 3114 (PacI) of pNDV/rAI4, use NAI4-P3 and NAI4-P4 as primers to amplify the ORF fragment of the penton gene, Primer5 and Primer6 In order to amplify the fragment between 3114 (PacI) and 4705 (BstZ17I) of pNDV/rAI4 with primers, the plasmid pNAI4-blunt (AgeI-BstZ17I) was double-digested with AgeI and BstZ17I to expose the homology arm, and recovered Vector fragment, the above three PCR fragments and the vector fragment can be used for homologous recombination reaction to construct the plasmid pNAI4-penton-blunt (AgeⅠ-BstZ17Ⅰ); BstZ17I double enzyme cut and ligated into the full-length plasmid pNDV/rAI4 to obtain the full-length plasmid pNDV/rAI4-penton of the recombinant virus.

PCR reaction system:

PCR reaction program: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30s, annealing at 60°C for 30s, extension at 72°C for 30s, and 15 cycles; denaturation at 94°C for 30s, annealing at 58°C for 30s, extension at 72°C for 30s, and 15 cycles; Extend at 72°C for 10 minutes, and store at 4°C.

The fragments amplified by the pair of primers NAI4-P3 and NAI4-P4 were sequenced to obtain the gene sequence of the penton protein, which is the sequence shown in SEQ ID NO.7:

The PCR products were subjected to agarose gel electrophoresis, and the target bands of 265bp, 1794bp and 1603bp could be obtained respectively, and the target bands were recovered with a gel recovery reagent and placed at 4°C for later use.

The plasmid pNAI4-Blunt (AgeI-BstZ17I) was double-digested with AgeI and BstZ17I, and the 3982bp band was recovered, which was ligated with the 265bp, 1794bp and 1603bp bands with the In Fusion Advantage PCR Cloning Kit kit, and transformed into DH5α Competent cells, the extracted plasmid was sent to Nanjing Jinsirui Company for sequencing, and the plasmid with the correct sequencing result was named pNAI4-penton-blunt (AgeⅠ-BstZ17Ⅰ).

3. Construction of full-length clone pNDV/rAI4-penton

Plasmid pNAI4-penton-blunt (AgeI-BstZ17I) and plasmid pNDV/rAI4 were digested by AgeI and BstZ17I, and the 3428bp band of pNAI4-penton-blunt (AgeI-BstZ17I) and pNDV/rAI4 were recovered by agarose gel electrophoresis The large bands were then ligated and transformed into DH5α competent cells, and the identified positive plasmid was named pNDV/rAI4-penton. (see picture 1)

Step 2: Construction of a recombinant Newcastle disease vaccine candidate rAI4-fiber2 full-length expression clone expressing avian adenovirus fiber2 protein

1. Cloning of fiber2 gene of avian adenovirus

The fiber2 gene of avian adenovirus strain CH/JSXZ/2015 was synthesized by GenScript Company, GenBank sequence number is KU569296.1, and primers NAI4-F3 and NAI4-F4 were designed according to the fiber2 gene sequence to amplify the code of the fiber2 gene Area. Primers Primer1, Primer2, Primer5 and Primer6 were designed according to the sequence of pNDV/rAI4 for homologous recombination reaction.

In the primer sequence, the bold part is the enzyme cutting site sequence, the underlined dotted line is the kozak sequence, which can improve the expression level of foreign genes, and the lower solid line is the GE and GS sequence of Newcastle disease virus. The primers were provided by Shanghai Bioengineering Co., Ltd. synthesis.

PCR reaction system:

PCR reaction program: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30s, annealing at 60°C for 30s, extension at 72°C for 30s, and 15 cycles; denaturation at 94°C for 30s, annealing at 58°C for 30s, extension at 72°C for 30s, and 15 cycles; Extend at 72°C for 10 minutes, and store at 4°C.

Sequence the fragments amplified by the pair of primers NAI4-F3 and NAI4-F4 to obtain the gene sequence of the fiber2 protein, which is the sequence shown in SEQ ID NO.10:

The plasmid pNAI4-blunt (AgeI-BstZ17I) was double-digested with AgeI and BstZ17I to expose the homology arm, and a 3982bp band was recovered. The fiber2 gene was ligated into the vector by homologous recombination in the same way as in step 1 to obtain The plasmid with the correct sequencing result was named pNAI4-fiber2-blunt (AgeI-BstZ17I).

2. Construction of full-length clone pNDV/rAI4-fiber2

Plasmid pNAI4-fiber2-blunt (AgeI-BstZ17I) and plasmid pNDV/rAI4 were digested by AgeI and BstZ17I, and the 3428bp band of pNAI4-fiber2-blunt (AgeI-BstZ17I) and pNDV/rAI4 were recovered by agarose gel electrophoresis The large bands were then ligated and transformed into DH5α competent cells, and the identified positive plasmid was named pNDV/rAI4-fiber2 (see Figure 2).

Step 3: Rescue of recombinant virus rAI4-penton strain and rAI4-fiber2 strain

BSR-T7/5 cells that can stably express T7 RNA polymerase: donated by Researcher Bu Zhigao, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. CEF cells were prepared by the laboratory according to conventional methods.

Eukaryotic expression plasmids pCI-NP, pCI-P, pCI-L (Hu Shunlin, Zhang Yanmei, Sun Qing, Liu Yuliang, etc. Establishment of a rescue system for Newcastle disease virus from goose [J]. Microbiology Bulletin. 2007, 34(3)) : Constructed and preserved by the Key Open Laboratory of Livestock and Poultry Infectious Diseases, Ministry of Agriculture, Yangzhou University. Among them, the eukaryotic expression plasmids pCI-NP and pCI-P expressing NP and P genes are also disclosed in (Liu Yuliang. (2005). Production of infectious ZJ1 strain Newcastle disease from goose origin from cDNA clone); eukaryotic expression plasmid expressing L gene Plasmid pCI-L is also disclosed in (Hu Shunlin. (2007). Establishment and application of the reverse genetic technology platform of goose-derived Newcastle disease virus).

SPF eggs were purchased from Beijing Merial Company and hatched in the Key Open Laboratory of Livestock and Poultry Infectious Diseases, Ministry of Agriculture, Yangzhou University.

1. Rescue of recombinant virus

The plasmids used for transfection (full-length plasmids, pCI-NP, pCI-P and pCI-L) were all extracted with QIAprepSpinMiniPrepKit. Co-transfect the three helper plasmids pCI-NP, pCI-P and pCI-L with the recombinant NDV genome full-length cDNA clones pNDV/rAI4-penton and pNDV/rAI4-fiber2 into BSR-T7/5 cells, after 18-24h Add the final concentration of 10% SPF embryo allantoic fluid, freeze and thaw the transfection sample once after 96 hours, harvest and inoculate 9-11 day-old SPF chicken embryos, collect the chicken embryo allantoic fluid, and measure the hemagglutination effect according to the OIE standard The recombinant Newcastle disease vaccine candidate strains rAI4-penton and rAI4-fiber2 expressing avian adenovirus protein were obtained. The hemagglutination properties of the two recombinant viruses were inhibited by polyclonal antibodies against NDV.

After the rAI4-penton strain was passed on chicken embryos for 5 times, the measured HA titer was 9log ₂ , which was 1 titer lower than that of the mother strain AI4, while the HA titer of the recombinant virus rAI4-fiber2 strain was 7log ₂ , compared with the AI4 strain, the titer decreased by 2, and the insertion of the fiber2 gene more significantly inhibited the replication of Newcastle disease virus.

2. RT-PCR verification of the rescued recombinant virus

Hemagglutination test (hemagglutination, HA) and hemagglutination inhibition test (hemagglutination inhibition, HI) were positive in the allantoic fluid, after the SPF chicken embryo was uploaded for 4 generations, the allantoic fluid was collected to extract the total RNA of the virus, and the total RNA of the virus was extracted with 6nt Random primers were reverse transcribed into cDNA for RT-PCR reaction.

The primers Primer1 and AI4a were used to amplify penton and fiber-2 with lengths of 2044bp and 1906bp respectively. The PCR products were recovered and sequenced. The results showed that the foreign gene was successfully inserted into the Newcastle disease virus vector through reverse genetics. (See Figure 3)

Primer1: 5'-AACTCTCACAACCGGTGC-3' (SEQ ID NO.1)

AI4a: 5'-CCTGGATGAGTCCATTTTGGTG-3' (SEQ ID NO.11)

Step 4: Identification of the biological characteristics of the recombinant virus

1. Determination of MDT

Determination of mean death time (MDT) of chicken embryos: 10-fold (10 ^-6 , 10 ^-7 ... 10 ^-10 ) serial dilutions of the recombinant virus were made with sterilized physiological saline, and 5 plates were inoculated for each dilution. 9-11 day-old SPF chicken embryos, 0.1 mL per embryo, and 5 chicken embryos inoculated with saline as a control. Incubate at 37°C, discard chicken embryos that died within 24 hours, then observe every 12 hours until 120 hours, and calculate the MDT of the virus according to the OIE standard method.

Results: Chicken embryos inoculated with 5 dilutions of the recombinant virus did not die within 120 hours, indicating that the MDT values of the recombinant viruses rAI4-penton and rAI4-fiber2 were greater than 120 hours.

2. Western blot test

The recombinant viruses rAI4-penton and rAI4-fiber2 were inoculated into DF1 cells at a dose of MOI=1. After 24 hours of infection, the cells were lysed to collect cytoplasmic proteins, and chicken embryo fibroblasts not infected with the virus were lysed as a blank control. The treated samples were subjected to SDS-PAGE electrophoresis, and the protein was transferred to a PVDF membrane with a pore size of 0.22um, blocked with 5% skim milk, and the chicken-derived anti-avian adenovirus polyantibody serum was used as the primary antibody, and the HRP-labeled rabbit anti-chicken IgG was Secondary antibody for antigen-antibody binding reaction. The color was developed by ECL luminescence method, and the signal was collected by gel imaging system.

Results: The recombinant viruses rAI4-penton and rAI4-fiber2 successfully expressed penton protein and fiber2 protein respectively (see Figure 4).

Step 5: Immunological potency test of the vaccine carrier strain

The 1-day-old SPF chickens were randomly divided into 10 groups and raised in isolation. They were inoculated with the vector vaccines rAI4-penton and rAI4-fiber2 by intranasal and eye drops, respectively, at a dose of 10 ⁶ EID ₅₀ per bird. At the same time, the AI4 vaccinated group was set as a blank Control, PBS inoculated group served as negative control. The venous blood of each test chicken was collected 3 weeks after immunization, and the seroconversion level against NDV and avian adenovirus was detected.

Results: The immunized chickens did not show any clinical symptoms, which indicated that the recombinant virus strain was not pathogenic to chickens and could be used safely. Recombinant viruses rAI4-penton and rAI4-fiber2 can stimulate animals to produce antibodies against Newcastle disease virus after inoculating animals, and the HI titer is more than 4log ₂ (see Figure 5 for details); rAI4-penton virus strain can be effective in SPF chicken Stimulate the production of antibodies against avian adenoviruses; however, no neutralizing antibodies against avian adenoviruses were detected after the rAI4-fiber2 virus strain immunized animals. It is suspected that the insertion of the fiber2 gene led to a decline in the reproductive performance of the recombinant virus rAI4-fiber2, resulting in its immunity The originality is reduced (see Figure 6 for details).

Example 2: Using pNDV/rAI4-T4FHN as a carrier

The deposit number of the plasmid pNDV/rAI4-T4FHN is CGMCC No: 12987, which has been disclosed by Chinese patent application 201710596136.7.

Step 1: Construction of recombinant Newcastle disease vaccine candidate rAI4-2FHN-hexon full-length expression clone expressing avian adenovirus hexon protein

1. Construction of intermediate plasmid pAI4-2FHN-blunt (AgeⅠ-BstZ17Ⅰ)

Design primers with reference to pNDV/rAI4-T4FHN as follows:

The bold part of the primer sequence is the restriction site sequence, and the italic part is the overlapping sequence, and the primers were synthesized by Jinsirui Company.

Use pNDV/rAI4-T4FHN as template for PCR reaction:

PCR reaction program: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 4 min, and 15 cycles; denaturation at 94°C for 30 s, annealing at 54°C for 30 s, extension at 72°C for 4 min, and 15 cycles; Extend at 72°C for 10 minutes, and store at 4°C.

After electrophoresis, the 3371bp band was cut, the PCR product was recovered and purified, connected to the Blunt Zero vector, and transformed into DH5α competent. The extracted plasmid was verified by PCR with Primer1 and FHa, and the positive plasmid was named pAI4-2FHN-blunt (Age Ⅰ-AflⅡ ). The construction process can be seen in Figure 7.

2. Cloning of avian adenovirus hexon gene

The hexon gene of avian adenovirus strain CH/JSXZ/2015 was synthesized by GenScript Company, GenBank sequence number is KU569296.1, and primers NAI4-H3 and NAI4-H4 were designed according to the hexon gene sequence to amplify the coding of the hexon gene Area. Primers Primer1, Primer2, Primer5 and FHa were designed according to the sequence of pNDV/rAI4-T4FHN for homologous recombination reaction.

In the primer sequence, the bold part is the enzyme cutting site sequence, the underlined dotted line is the kozak sequence, which can improve the expression level of foreign genes, and the lower solid line is the GE and GS sequence of Newcastle disease virus. The primers were provided by Shanghai Bioengineering Co., Ltd. synthesis.

PCR reaction system:

PCR reaction program: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30s, annealing at 60°C for 30s, extension at 72°C for 30s, and 15 cycles; denaturation at 94°C for 30s, annealing at 58°C for 30s, extension at 72°C for 30s, and 15 cycles; Extend at 72°C for 10 minutes, and store at 4°C.

Sequence the fragments amplified by the pair of primers NAI4-H3 and NAI4-H4 to obtain the gene sequence of the hexon protein, which is the sequence shown in SEQ ID NO.16:

The plasmid pAI4-2FHN-blunt (AgeⅠ-AflⅡ) was double-digested with AgeⅠ and AflⅡ, the homology arm was exposed, and the 3982bp band was recovered. The hexon gene was connected into the vector by homologous recombination in the same way as in step 1. The obtained plasmid was sequenced, and the plasmid with correct sequencing result was named pNAI4-2FHN-hexon-blunt (AgeI-AflII).

2. Construction of full-length clone pNDV/rAI4-2FHN-hexon

Plasmid pNAI4-2FHN-hexon-blunt (AgeⅠ-AflⅡ) and plasmid pAI4-2FHN (AgeⅠ-FspAI) were digested by PacⅠ and AflⅡ, and the target fragment was recovered and ligated. The successfully ligated plasmid was named pNAI4-2FHN- hexon (AgeI-FspAI).

Digest pNDV/rAI4 and pNAI4-2FHN-hexon (AgeI-FspAI) by PacI and FspAI double enzymes, recover the target fragments and perform ligation reaction. The plasmid identified as positive by the ligation result is named pNDV/rAI4-2FHN-hexon, the positive plasmid Store at -70°C. The construction process can be seen in Figure 8.

Step 3: Rescue of recombinant virus rAI4-2FHN-hexon

BSR-T7/5 cells that can stably express T7 RNA polymerase: donated by Researcher Bu Zhigao, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. CEF cells were prepared by the laboratory according to conventional methods.

Eukaryotic expression plasmids pCI-NP, pCI-P, pCI-L (Hu Shunlin, Zhang Yanmei, Sun Qing, Liu Yuliang, etc. Establishment of a rescue system for Newcastle disease virus from goose [J]. Microbiology Bulletin. 2007, 34(3)) : Constructed and preserved by the Key Open Laboratory of Livestock and Poultry Infectious Diseases, Ministry of Agriculture, Yangzhou University. Among them, the eukaryotic expression plasmids pCI-NP and pCI-P expressing NP and P genes are also disclosed in (Liu Yuliang. (2005). Production of infectious ZJ1 strain Newcastle disease from goose origin from cDNA clone); eukaryotic expression plasmid expressing L gene Plasmid pCI-L is also disclosed in (Hu Shunlin. (2007). Establishment and application of the reverse genetic technology platform of goose-derived Newcastle disease virus).

SPF eggs were purchased from Beijing Merial Company and hatched in the Key Open Laboratory of Livestock and Poultry Infectious Diseases, Ministry of Agriculture, Yangzhou University.

1. Rescue of recombinant virus

The plasmids used for transfection (full-length clones, pCI-NP, pCI-P and pCI-L) were all extracted with QIAprepSpinMiniPrepKit. Co-transfect BSR-T7/5 cells with the three helper plasmids pCI-NP, pCI-P and pCI-L and the recombinant NDV genome full-length cDNA clone rAI4-2FHN-hexon respectively, and add the final concentration of 10% after 18-24h SPF embryo allantoic fluid, freeze and thaw transfected samples once after 96 hours, harvest and inoculate SPF chicken embryos aged 9-11 days, collect chicken embryo allantoic fluid, measure hemagglutination titer according to OIE standard, and obtain expression poultry gland Recombinant Newcastle disease vaccine candidate rAI4-2FHN-hexon of viral proteins.

2. RT-PCR verification of the rescued recombinant virus

Hemagglutination test (hemagglutination, HA) and hemagglutination inhibition test (hemagglutination inhibition, HI) were positive in the allantoic fluid, after the SPF chicken embryo was uploaded for 4 generations, the allantoic fluid was collected to extract the total RNA of the virus, and the total RNA of the virus was extracted with 6nt Random primers were reverse transcribed into cDNA for RT-PCR reaction.

The hexon was amplified with primers NAI4-H3 and NAI4-H4, with a length of 2852bp. The PCR product was recovered and sequenced, indicating that the foreign gene was successfully inserted into the Newcastle disease virus vector through reverse genetics. (See Figure 9)

Step 3: Identification of the biological characteristics of the recombinant virus

1. Determination of MDT

Determination of mean death time (MDT) of chicken embryos: 10-fold (10 ^-6 , 10 ^-7 ... 10 ^-10 ) serial dilutions of the recombinant virus were made with sterilized physiological saline, and 5 pieces were inoculated for each dilution. 9-11 day-old SPF chicken embryos, 0.1 mL per embryo, and 5 chicken embryos inoculated with saline as a control. Incubate at 37°C, discard chicken embryos that died within 24 hours, then observe every 12 hours until 120 hours, and calculate the MDT of the virus according to the OIE standard method.

Results: Chicken embryos inoculated with 5 dilutions of the recombinant virus did not die within 120 hours, indicating that the MDT value of the recombinant virus rAI4-2FHN-hexon was greater than 120 hours.

2. Western blot test

The recombinant virus rAI4-2FHN-hexon was inoculated into DF1 cells at a dose of MOI=1. After 24 hours of infection, the cells were lysed to collect cytoplasmic proteins, and chicken embryo fibroblasts not infected with the virus were lysed as a blank control. The treated samples were subjected to SDS-PAGE electrophoresis, and the protein was transferred to a PVDF membrane with a pore size of 0.22um, blocked with 5% skim milk, and the chicken-derived anti-avian adenovirus polyantibody serum was used as the primary antibody, and the HRP-labeled rabbit anti-chicken IgG was Secondary antibody for antigen-antibody binding reaction. The color was developed by ECL luminescence method, and the signal was collected by gel imaging system.

Results: Three recombinant viruses were constructed with the same construction strategy: rAI4-2FHN-hexon, rAI4-2FHN-penton, rAI4-2FHN-fiber2. AI4-T4FHN was used as the vector to insert the hexon, penton and fiber2 genes respectively. The recombinant virus rAI4-2FHN-hexon did not express the hexon protein, but the recombinant virus rAI4-2FHN-penton and rAI4-2FHN-fiber2 successfully expressed the penton protein And fiber2 protein, hexon gene length is 2814bp, penton gene and fiber2 gene length are 1578bp and 1440bp respectively, may be because hexon gene is too big, has exceeded the upper limit of exogenous gene expression of Newcastle disease virus vector (see Figure 10).

Step 5: Immunological potency test of the vaccine carrier strain

The 1-day-old SPF chickens were randomly divided into 10 groups, kept in isolation, and were inoculated with carrier vaccines rAI4-2FHN-hexon, rAI4-2FHN-penton, and rAI4-2FHN-hexon by intranasal and eye drops at a dose of 10 ⁶ EID ₅₀ / At the same time, the PBS inoculated group was set as the control. The venous blood of each test chicken was collected 3 weeks after immunization, and the seroconversion level against avian adenovirus was detected.

Results: The immunized chickens did not show any clinical symptoms, which indicated that the recombinant virus strain was not pathogenic to chickens and could be used safely. After inoculating animals with the recombinant virus, HI antibodies against the carrier Newcastle disease virus AI4-T4FHN were detected, indicating that the recombinant virus can replicate effectively in animals (see Figure 11 for details).

The rAI4-2FHN-penton recombinant virus stimulated animals to produce neutralizing antibodies, but the rAI4-2FHN-hexon and rAI4-2FHN-fiber2 immunization groups did not detect neutralizing antibodies against avian adenovirus, we believe that the recombinant virus rAI4-2FHN- Hexon did not express hexon protein, so the neutralizing antibody against avian adenovirus was not produced after immunizing animals; it can be seen from Figure 11 that the antibody level produced by rAI4-2FHN-fiber2 stimulated animals was the lowest three weeks after immunization, so the fiber2 protein The expression reduces the replication ability of the vector virus, so that the rAI4-2FHN-fiber2 virus does not stimulate the animals to produce neutralizing antibodies against the avian adenovirus (see Figure 12 for details).

The experimental results show that only the recombinant viruses rAI4-penton and rAI4-2FHN-penton inserted into the penton gene can stimulate animals to produce neutralizing antibodies against avian adenovirus, because the carrier virus used by rAI4-2FHN-penton is chimeric Newcastle disease virus AI4 -T4FHN, its F and HN proteins are derived from avian paramyxovirus serotype 2, which has serotype differences with Newcastle disease virus of avian paramyxovirus serotype 1, and cannot provide animals with immune protection against Newcastle disease virus; while the recombinant virus The F and HN proteins of rAI4-penton are derived from avian paramyxovirus serotype 1, and the HI titer of Newcastle disease antibody produced after immunizing animals is higher than 4log ₂ , so it can be used to prevent Newcastle disease virus and avian adenovirus infection.

SEQUENCE LISTING

<110> Yangzhou University

<120> Recombinant Newcastle Disease Vaccine Candidate Strain rAI4-penton Expressing Avian Adenovirus Penton Protein and Its Construction Method

<130>

<160> 16

<170> PatentIn version 3.3

<210> 1

<211> 18

<212>DNA

<213> Artificial sequence

<400> 1

aactctcaca accggtgc 18

<210> 2

<211> 49

<212>DNA

<213> Artificial sequence

<400> 2

ctacccgtat tttttcttaa ttaacaggag cctgttatga gttgtgatg 49

<210> 3

<211> 42

<212>DNA

<213> Artificial sequence

<400> 3

aaaaaatacg ggtagaagcc accatgtggg ggttgcagcc gc 42

<210> 4

<211> 35

<212>DNA

<213> Artificial sequence

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gattccgtgt gacgcctact gcaaggtcgc ggaac 35

<210> 5

<211> 15

<212>DNA

<213> Artificial sequence

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gcgtcacacg gaatc 15

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<212>DNA

<213> Artificial sequence

<400> 6

ctgagacgag gtgtatacat tgactg 26

<210> 7

<211> 1578

<212>DNA

<213> Avian adenovirus

<400> 7

atgtgggggt tgcagccgcc gacgtcgatt ccgccgcctc ctccgccgac cgagttaacg 60

ccctcgacct atccggcgat ggtgaacggc tatccgcctc cggccgcgtc cgcgcagagc 120

tgtccctcta gcgacggtca gagcgagctg tatatgcccc ttcagcgggt gatggcccct 180

acggggggac ggaacagcat taagtatcgc gattacacgc cgtgtcgtaa caccaccaag 240

ctgttttacg tagacaacaa ggctagcgat atcgatacgt ataacaaaga cgccaaccat 300

agcaatttcc gcaccacggt gatccataac caggatctgg acgcggacac ggccgccacc 360

gagtccatcc agttggacaa ccgctcctgc tggggcggcg acctaaaaac agccgtgcgc 420

accaactgcc cgaacgtgag cagttttttc cagagtaaca gcgtgcgcgt gcgcatgatg 480

tggaagcgcg acccgccgac tagcacggct cctccgagcg cggtaggcag cggctattcg 540

gtgcccggcg cgcagtacaa gtggtacgac ctgacgatac ccgagggtaa ctacgcgctg 600

tgcgaactga tagacctgct caacgagggc atcgtgcagc tctacctgag cgaggggcgc 660

cagaacaacg tgcaaaaatc ggacatcggg gtcaagttcg acacgcgcaa cttcggcttg 720

ctccgcgacc ccgtgacggg actggtaact ccgggcacgt acgtgtacaa gggttaccac 780

cccgacatcg tgctgctgcc cggatgcgcg atcgacttta cgtacagccg cctgagcctg 840

ctcctgggca tagggaagcg cgagccctac tcgaaggggt tcgttattac ctacgaggat 900

ctgcagggag gggatatccc ggctctgctg gacctcgact ccgtcgacgt gaacgacgct 960

gacggtgaag tgatcgagct cgacaacgct gctccccttt tacatgacag cgcgggcgtg 1020

tcgtataacg tcatatacga ccaggtgacg ggtaaacccg tgacggtgta tcgatcgtgg 1080

atgttggctt acaacgtgcc taactcgccg gccaatcaga cgaccttgct gacggtgccc 1140

gatatggcgg gcgggatcgg ggcgatgtac acgtccctgc ccgatacctt tatcgcgcct 1200

accgggttca aggaagataa cacgaccaac ctttgcccgg tcgtcggcat gaacctgttc 1260

cccacctaca ataaagttta ttaccaggcg gcgtccacgt acgtgcagcg cctggaaaat 1320

tcctgccagt cggccacagc cgccttcaac cgctttcccg aaaacgagat tctgaagcaa 1380

gcgcccccca tgaatgtttc gtccgtgtgc gataaccaac ccgccgtcgt tcagcagggt 1440

gtgttgcctg tgaagacctc gctccccgga ctgcagcgcg tgctgatcac agacgaccag 1500

cgtcgtccga taccctacgt gtataagtct atcgcgacgg ttcagccgac cgttctgagt 1560

tccgcgacct tgcagtag 1578

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<212>DNA

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aaaaaatacg ggtagaagcc accatgctcc gggcccctaa aag 43

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<212>DNA

<213> Artificial sequence

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gattccgtgt gacgcttacg ggagggaggc cgc 33

<210> 10

<211> 1440

<212>DNA

<213> Avian adenovirus

<400> 10

atgctccggg cccctaaaag aagacattcc gaaaacggga agcccgagac cgaagcggga 60

ccttccccgg ctccaatcaa gcgcgccaaa cgcatggtga gagcatccca gcttgacctg 120

gtttatcctt tcgattacgt ggccgacccc gtcggagggc tcaacccgcc ttttttggga 180

ggctcaggac ccctagtgga ccagggcgga cagcttacgc tcaacgtcac cgatcccatc 240

atcatcaaga acagatcggt ggacttggcc cacgacccca gtctcgatgt caacgcccaa 300

ggtcaactgg cggtggccgt tgaccccgaa ggggccctgg acatcacccc cgatggactg 360

gacgtcaagg tcgacggagt gaccgtaatg gtcaacgatg actgggaact ggccgtaaaa 420

gtcgacccgt ccggcggatt ggattccacc gcgggtggac tgggggtcag cgtggacgac 480

accttgctcg tggatcaggg agaactgggc gtacacctca accaacaagg acccatcact 540

gccgatagca gtggtatcga cctcgagatc aatcctaaca tgttcacggt caacacctcg 600

accggaagcg gagtgctgga actcaaccta aaagcgcagg gaggcatcca agccgacagt 660

tcgggagtgg gcgtttccgt ggatgaaagc ctacagattg tcaacaacac tctggaagtg 720

aaaccggatc ccagcggacc gcttacggtc tccgccaatg gcctagggct gaagtacgac 780

actaataccc tagcggtgac cgcgggcgct ttaaccgtgg tcggaggggg gagcgtctcc 840

acacccatcg ctacttttgt ctcgggaagt cccagcctca acacctacaa tgccacgacc 900

gtcaattcca gcgcgaacgc cttctcttgc gcctactacc ttcaacagtg gaacatacag 960

gggctccttg ttacctccct ctacttgaaa ttggacagcg ccaccatggg gaatcgccct 1020

ggggacctca actccgccaa tgccaaatgg ttcacctttt gggtgtccgc ctatctccag 1080

caatgcaacc cctccgggat tcaagcggga acggtcagcc cctccaccgc caccctcacg 1140

gactttgaac ccatggccaa taggagcgtg accagcccat ggacgtactc ggccaatgga 1200

tactatgaac catccatcgg ggaattccaa gtgttcagcc cggtggtaac aggtgcctgg 1260

aacccgggaa acatagggat ccgcgtcctc cccgtgccgg tttcggcctc cggagagcga 1320

tacacccttc tatgctatag tctgcagtgc acgaacgcga gcatttttaa tccaaacaac 1380

agcggaacca tgatcgtggg acccgtgctc tacagctgtc cagcggcctc cctcccgtaa 1440

<210> 11

<211> 22

<212>DNA

<213> Artificial sequence

<400> 11

cctggatgag tccattttgg tg 22

<210> 12

<211> 21

<212>DNA

<213> Artificial sequence

<400> 12

atggactcat ccaggacaat c 21

<210> 13

<211> 31

<212>DNA

<213> Artificial sequence

<400> 13

gtaatttttt cttaagtctt ctacttgaca g 31

<210> 14

<211> 44

<212>DNA

<213> Artificial sequence

<400> 14

aaaaaatacg ggtagaagcc accatggcgg ccctcacgcc cgac 44

<210> 15

<211> 37

<212>DNA

<213> Artificial sequence

<400> 15

gattccgtgt gacgcttatt acacggcgtt gcctgtg 37

<210> 16

<211> 2814

<212>DNA

<213> Avian adenovirus

<400> 16

atggcggccc tcacgcccga cctgactacc gcgactccgc ggctccagta ttttcacatc 60

gcgggccccg ggacgcgcga atacctctct gaggacctcc aacagttcat ttccgccacc 120

ggaagctact ttgacttgaa aaacaagttc agacagacgg tcgtggcgcc cacccgaaat 180

gtcacgacag aaaaggctca acggctgcaa atccgctttt accccatcca aaccgacgac 240

acgtcgacgg gctaccgcgt gcggtacaac atcaatgtgg gcgacggttg ggtcctggac 300

atggggtcga cctatttcga catcaaggga atcctagacc gagggccgtc cttcaagccc 360

tactgcggca cggcttacaa cccgctggct cccaaggagt ccatgtttaa caactggtcg 420

gagacggcac ccgggcagaa cgtgtccgcc tccggtcagc tgtccaacgt ctataccaac 480

acgagcacct ccaaagacac gacggcggcg caggtgacga agatttccgg cgtcttcccc 540

aatcccaacc agggacccgg aagaaatcct ctgcgacggg tacaaaacgc caacaccggc 600

gtgctcggtc gcttcgccaa gtctcagtac aattacgctt acggtgccta cgtcaagccc 660

gtcgccgccg acggttccca gtccctcacg cagaccccct actggatcat ggataacacg 720

ggcaccaatt acctgggagc ggtggccgtc gaggactaca ccaacagcct ctcgtaccca 780

gataccatag tcgtgccgcc tcccgaggac tacgacgatt ataacatagg caccacgcgt 840

gcgctcaggc ccaactacat cgggttcagg gataacttca ttaacctgct gtatcacgac 900

tccggcgtgt gctcgggcac cctcaactcg gagcgttcgg gcatgaacgt ggtggtcgag 960

ctgcccgacc ggaataccga gctcagctac cagtacatgc tggccgacat gatgtcccgc 1020

catcactatt tcgccctgtg gaaccaggcc gtggaccagt acgaccccga ggtgcgagtc 1080

ttctccaatg acggttacga agaaggcgcg cccagctacg ccttcaaccc cgaagcggta 1140

ggcgcgggag aaggctacgg ccccgatctc agtcaaatta aactctacac caacaacacc 1200

gccgcgaacg acaaaaacac cgccgtggct aacgccacta ccaacttcta cttcggcacg 1260

gtaccctcct acgaaatcga tatcagcgct accccagaggc gcaactttat catggccaac 1320

atcgccgagt atctgcccga ccgttacaag tttagcatct ccggcttcga cgccaccagc 1380

gtcgcgccta ccacctacga gtacatgaac aagcgcgtcc ccctcaccaa cgtcgtcgac 1440

atgttcacga acgtgggtgc gcgttggtcc atcgaccaga tggacaacgt caaccccttc 1500

aaccaccaca gaaactgggg gctgaaatac cgctcccagc tgctgggaaa cagtcgctac 1560

gtcaacttcc acatccaagt gccccaaaaa ttcttcgcca tcaaaaacct gctgctgctc 1620

tccggctcgt acacctacga gtgggtgctg cgcaaagacc ccaacatgat cctccaatcc 1680

agtctgggca acgacctgcg cgccgacggc gccagcatca tctacaacga ggtgaacctc 1740

atggccaact tcatgcccat ggatcacaac accagtaacc agctcgagct gatgctgaga 1800

aacgccacca acgatcagac cttcgtggac tacctgggag ccaaaaacgc tctatactcg 1860

gtgcccgcgg gctccaccgc cctcaccatc aacattcccg ctcgcacctg ggaggggatg 1920

cgcgggtggt ccttcactcg catcaaggcg gccgagacgc ctcagctggt cgcccagtac 1980

gacgtcaact tcaagtactc gggcagcatc gcctactcag atggaggctt ctacctctcg 2040

cacaccttcc gtaacatgag catcctcttc gacacgtcca tcaactggcc gggcaacgac 2100

cggttgctca cgcctaacat gttcgagatc aagcgctcgg tggcgctcga caccgagggc 2160

ttcaccatga gccagtgcga catcaccaag gactggtacc tgatccagat ggccacgaac 2220

tacaacttcg tctataacgg ctatcgattc tggcccgatc gtcagtactt ccactacgac 2280

ttcctgcgaa atttcgaccc catgacgcgc cagggaccca acttcgcatt gcccggcctc 2340

ttcgacctcg tgtcttacac ccctaccacg gacaacagcg gacagcagcc tagtcaggaa 2400

gccgtgcgca acaactctgg gtttatcgcc ccccgctcct ggcccgtctg gagcgctcac 2460

cagggcgaga gctggcccgc caactggccg tacccgctct gcggtcagca ggccatccaa 2520

cccgcacagg tcctcagcta caagaagttc ctctgcgaca actacctgtg gaccatcccg 2580

ttcagttccg actttatgta catgggcgaa ctgacagatc tgggccagaa ccccatgtac 2640

accaacaact cgcacagcat ggtcatcaac ttcgagctcg accacatgga tgatcccact 2700

tacgtgtaca tgctctatgg cgtgttcgac accgttaggg tcaaccagcc cgaacgtaac 2760

gtgctagcta tggcttactt ccgtacgcct ttcgccacag gcaacgccgt gtaa 2814

Claims (2)

1. expressing recombinant Newcastle disease vaccine candidate the strain rAI4-penton, preserving number CGMCC of aviadenovirus penton albumen No：15492.

2. expressing the recombinant Newcastle disease vaccine candidate strain rAI4-penton of aviadenovirus penton albumen described in claim 1 Construction method, it is characterised in that：Using newcastle disease attenuated IBDVs as carrier, aviadenovirus penton albumen is expressed.