CN100352513C - Novel mucous membrane vaccine for newcastle disease and its construction method - Google Patents

Abstract

The Newcastle disease mucosal DNA vaccine and its construction method relate to a Newcastle disease vaccine and its construction method. Firstly, the fusion protein F gene of Newcastle disease virus is amplified by reverse transcription-polymerase chain RT-PCR reaction; and then the F gene is amplified. The gene was ligated with the plasmid pVAX1 to construct the DNA vaccine pVAX1-F; finally, the plasmid pVAX1-F was transformed into the attenuated Salmonella typhimurium SL7207, and finally the Newcastle disease virus DNA vaccine with a DNA length of 4647bp was obtained. The invention uses the attenuated Salmonella typhimurium to deliver the Newcastle disease virus DNA vaccine to construct a new type of Newcastle disease mucosal vaccine SL7207 (pVAX1-F). Directly immunize poultry (chickens, geese, pigeons, etc.) through oral administration (drinking water or ingredients), which is safer than the traditional Newcastle disease attenuated vaccine, and is more convenient to use than the traditional Newcastle disease oil emulsion inactivated vaccine, which can overcome the maternal source Antibody interference and inactivated oil-emulsion vaccines failed to produce defects in the mucosal immune response and provided protection against virulent challenge with Newcastle disease virus.

Description

Newcastle disease mucosal DNA vaccine and its construction method

technical field

The invention relates to a Newcastle disease vaccine and its construction method.

current technology

Newcastle disease is one of the main infectious diseases that endanger the poultry industry, and is listed as a class A severe infectious disease by the International Office of Epizootics (OIE). At present, the prevention and control of Newcastle disease in my country mainly uses traditional vaccines such as attenuated vaccines of different virulence and inactivated oil emulsion vaccines for immune prevention. However, some attenuated vaccines retain some toxicity, and still have a certain lethality to chicks, and attenuated vaccines can also cause mild lesions in the respiratory tract of chickens, enhancing the susceptibility to other pathogens. Although inactivated vaccines can produce high levels of circulating antibodies, they cannot stimulate the body to produce cellular and mucosal immunity, and require injections for immunization, which is time-consuming and laborious. In addition, the production cost of conventional vaccines is high, and immunization will be interfered by maternal antibodies. Therefore, it is of great practical significance to seek a cheap, safer, more effective and easy-to-use vaccine for the comprehensive prevention and control of Newcastle disease.

Contents of the invention

The purpose of the present invention is to invent a new type of Newcastle disease mucosal vaccine which can be vaccinated through the mucosal route.

The DNA length of the Newcastle disease virus DNA vaccine is 4647bp, and the nucleotide sequence of its DNA vaccine is:

gactcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta 60

atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata 120

acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat 180

aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga 240

ctatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc 300

ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt 360

atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat 420

gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag 480

tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc 540

aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga 600

ggtctatataagcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga 660

aattaatacg actcactata gggagaccca agctggctag cgtttaaact taagcttcaa 720

tatgggctcc agaccttcta ccaggatccc agcacctctg atgctgatca cccggattat 780

gctgatgttg ggctgtatcc gtccgacaag ctctcttgac ggcaggcctc ttgcagctgc 840

aggaattgta gtaacaggag ataaggcagt caatgtatac acctcgtctc agacagggtc 900

aatcatagtc aagttgctcc cgaatatgcc cagggataag gaagcgtgtg cgaaagcccc 960

attagaggca tataacagaa cactgactac tttgctcact cctcttggcg actccatccg 1020

caagatccaa gggtctgtgt ccacgtctgg aggaaggaga caaaaacgct ttataggtgc 1080

tattattggc agtgtagctc ttggggttgc aacagcggca cagataacag cagctgcggc 1140

cctaatacaa gccaaccaga atgctgccaa catcctccgg cttaaggaga gcattgctgc 1200

aaccaatgaa gctgtgcatg aagtcaccga cggattatca caactatcag tggcagttgg 1260

gaagatgcag cagtttgtca atgaccagtt taataataca gcgcgagaat tggactgtat 1320

aaaaatcaca caacaggttg gtgtagaact caacctatac ctaactgaat tgactacagt 1380

attcgggcca cagatcacct cccctgcatt gactcagctg accatccagg cactttataa 1440

tttagctggt ggcaatatgg attacktatt aactaagtta ggtataggga acaatcaact 1500

cagctcatta attggtagcg gcctgatcac tggttaccct attctgtacg actcacagac 1560

tcaactcttg ggcatacaag tgaatttgcc ctcagtcggg aacttaaata atatgcgtgc 1620

cacctatttg gagaccttat ctgtaagtac aaccaaagga tatgcctcag cacttgtccc 1680

gaaagtagtg acacaagtcg gttctgtgat agaagagctt gacacctcat actgtataga 1740

gtccgatctg gatttatatt gtactagaat agtgacattc cccatgtccc caggtattta 1800

ttcctgtttg agcggcaaca catcagcttg catgtattca aagactgaag gcgcactcac 1860

cacgccgtat atggccctta aaggctcagt tattgccaat tgtaagataa caacatgtag 1920

atgtacagac cctcctggta tcatatcgca aaattatgga gaagctgtat ccctgataga 1980

tagacattcg tgcaatgtct tatcattaga tgggataact ctgaggctca gtggggaatt 2040

tgatgcaact tatcaaaaga acatctcaat actagattct caagtcatcg tgacaggcaa 2100

tcttgatata tcaactgaac ttggaaacgt caacaattca atcagcaatg ccttggataa 2160

gttggcggaa agcaacagca agctagaaaa agtcaatgtc agactaacca gcacatctgc 2220

tctcattacc tatattgttc taactgtcat ttctctagtt ttcggtgcac ttagtctggg 2280

tttagcgtgt tacctgatga tcaaacagaa ggcacaacaa aagaccttgc tatggcttgg 2340

gaataatacc ctcgatcaga tgagagccac tacaagagca tgaatgcaga taagaggtgg 2400

gaattctgca gatatccagc acagtggcgg ccgctcgagt ctagagggcc cgtttaaacc 2460

cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc 2520

gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa 2580

attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac 2640

agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg 2700

gcttctactg ggcggtttta tggacagcaa gcgaaccgga attgccagct ggggcgccct 2760

ctggtaaggt tgggaagccc tgcaaagtaa actggatggc tttctcgccg ccaaggatct 2820

gatggcgcag gggatcaagc tctgatcaag agacaggatg aggatcgttt cgcatgattg 2880

aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg 2940

actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg 3000

ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg 3060

aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg 3120

ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc 3160

tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc 3240

tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc 3300

gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc 3360

aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg 3420

atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct 3480

tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt 3540

tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc 3600

tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt 3660

tcttctgaat tattaacgct tacaatttcc tgatgcggta ttttctcctt acgcatctgt 3720

gcggtatttc acaccgcata caggmggcac ttttcgggga aatgtgcgcg gaacccctat 3780

ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata 3840

aatgcttcaa taatagcacg tgctaaaact tcatttttaa tttaaaagga tctaggtgaa 3900

gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc 3960

gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat 4020

ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga 4080

gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt 4140

ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata 4200

cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac 4260

cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg 4320

ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg 4380

tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag 4440

cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct 4500

ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc 4560

aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctgggctt 4620

ttgctggcct tttgctcaca tgttctt.

In addition, the present invention also provides a construction method for the new-type Newcastle disease mucosal vaccine with the above-mentioned characteristics, that is, through reverse transcription-polymerase chain RT-PCR reaction, the Newcastle disease virus fusion protein F gene is amplified; A ligation reaction was carried out with the plasmid pVAX1 to construct the DNA vaccine pVAX1-F; finally, the plasmid pVAX1-F was transformed into attenuated Salmonella typhimurium SL7207. The amplification primer of above-mentioned Newcastle disease virus fusion protein F gene is:

Upstream primer: 5′taagcttcaatatgggctccagacct 3′

Downstream primer: 5′cgaattctccaccctcttatctgcattcatgc 3′.

The invention uses the attenuated Salmonella typhimurium to deliver the Newcastle disease virus DNA vaccine to construct a new type of Newcastle disease mucosal vaccine SL7207 (pVAX1-F). Directly immunize poultry (chickens, geese, pigeons, etc.) through oral administration (drinking water or ingredients), which is safer than the traditional Newcastle disease attenuated vaccine, and is more convenient to use than the traditional Newcastle disease oil emulsion inactivated vaccine, which can overcome the maternal source Antibody interference and inactivated oil-emulsion vaccines failed to produce defects in the mucosal immune response and provided protection against virulent challenge with Newcastle disease virus.

The above-mentioned Newcastle disease virus fusion protein F gene is 1662bp in length, and its DNA sequence is:

atgggctcca gaccttctac caggatccca gcacctctga tgctgatcac ccggattatg 60

ctgatgttgg gctgtatccg tccgacaagc tctcttgacg gcaggcctct tgcagctgca 120

ggaattgtag taacaggaga taaggcagtc aatgtataca cctcgtctca gacagggtca 180

atcatagtca agttgctccc gaatatgccc agggataagg aagcgtgtgc gaaagcccca 240

ttagaggcat ataacagaac actgactact ttgctcactc ctcttggcga ctccatccgc 300

aagatccaag ggtctgtgtc cacgtctgga ggaaggagac aaaaacgctt tataggtgct 360

attattggca gtgtagctct tggggttgca acagcggcac agataacagc agctgcggcc 420

ctaatacaag ccaaccagaa tgctgccaac atcctccggc ttaaggagag cattgctgca 480

accaatgaag ctgtgcatga agtcaccgac ggattatcac aactatcagt ggcagttggg 540

aagatgcagc agtttgtcaa tgaccagttt aataatacag cgcgagaatt ggactgtata 600

aaaatcacac aacaggttgg tgtagaactc aacctatacc taactgaatt gactacagta 660

ttcgggccac agatcacctc ccctgcattg actcagctga ccatccaggc actttataat 720

ttagctggtg gcaatatgga ttacttatta actaagttag gtatagggaa caatcaactc 780

agctcattaa ttggtagcgg cctgatcact ggttacccta ttctgtacga ctcacagact 840

caactcttgg gcatacaagt gaatttgccc tcagtcggga acttaaataa tatgcgtgcc 900

acctatttgg agaccttatc tgtaagtaca accaaaggat atgcctcagc acttgtcccg 960

aaagtagtga cacaagtcgg ttctgtgata gaagagcttg acacctcata ctgtatagag 1020

tccgatctgg atttatattg tactagaata gtgacattcc ccatgtcccc aggtatttat 1080

tcctgtttga gcggcaacac atcagcttgc atgtattcaa agactgaagg cgcactcacc 1140

acgccgtata tggcccttaa aggctcagtt attgccaatt gtaagataac aacatgtaga 1200

tgtacagacc ctcctggtat catatcgcaa aattatggag aagctgtatc cctgatagat 1260

agacattcgt gcaatgtctt atcattagat gggataactc tgaggctcag tggggaattt 1320

gatgcaactt atcaaaagaa catctcaata ctagattctc aagtcatcgt gacaggcaat 1380

cttgatatat caactgaact tggaaacgtc aacaattcaa tcagcaatgc cttggataag 1440

ttggcggaaa gcaacagcaa gctagaaaaa gtcaatgtca gactaaccag cacatctgct 1500

ctcattacct atattgttct aactgtcatt tctctagttt tcggtgcact tagtctgggt 1560

ttagcgtgtt acctgatgat caaacagaag gcacaacaaa agaccttgct atggcttggg 1620

aataataccc tcgatcagat gagagccact acaagagcat ga

specific embodiment

1. Amplification of Newcastle disease virus fusion protein (F) gene:

1. Primers

Upstream primer: 5′t aagctt caatatgg gctccagacct 3′

Downstream primer: 5′c gaattc tccacctcttatctgcattcatgc 3′

The upstream and downstream primers contain HindIII and EcoRI restriction sites (underlined), respectively.

2. Preparation of Newcastle Disease Virus Genomic RNA:

1.5ml of virulent Newcastle disease virus JS5 (separated in this laboratory) allantoic fluid precipitate was suspended in 0.4ml of phosphate buffered saline (PBS), transferred to a DEPC-treated finger tube, and 50 μl of DEPC-treated 10% Mix the SDS upside down, then add 500μl saturated phenol and mix, put it in an ice-water bath for 5min, centrifuge at 4000rpm for 5min, absorb the supernatant and place it in a DEPC-treated finger tube, add 5% volume of 2M sodium acetate, 5% The volume of 4M lithium chloride and 2.5 times the volume of absolute ethanol were mixed and placed at -20°C for more than 1 hour, centrifuged at 12000-14000 rpm for 15 minutes, the supernatant was discarded, and the finger tube was placed in an ultra-clean bench to volatilize for 1 hour.

3. Reverse transcription

Dissolve the above volatilized and dried RNA in 13 μl of DEPC-treated ultrapure water, react in a water bath at 65°C for 5 minutes, and quickly place it in an ice-water bath for 5 minutes, then add 5 μl of AMV RT 5×Buffer, 3 μl of 10 mmol/L dNTP, and upstream of 50 pmol/L Primer 1μl, 25U/μl AMV reverse transcriptase 2μl, 40U/μl RNasin 1μl, mix well and briefly centrifuge, put in 42℃ water bath for 1h, and carry out PCR.

4. PCR

The PCR reaction system is 50 μL:

Reverse transcription product: 6 μL

10× buffer: 5 μL

Upstream primer (50pmol/L): 1μL

Downstream primer (50pmol/L): 1μL

High-fidelity DNA polymerase (2.5U/μL): 0.5 μL

4 kinds of dNTP mixture (2.5mmol/L): 2μL

Ultrapure water: 34.5μL

The PCR reaction conditions were: 94°C 45S, 46°C 45S, 72°C 120S, a total of 30 cycles. After the end, it was separated by electrophoresis at a concentration of 0.8% agarose, and the F gene with a length of 1696bp was recovered.

2. Construction of DNA vaccine pVAX1-F:

The recovered F gene was double-digested with HindIII and EcoRI, and the plasmid pVAX1 was double-digested with HindIII and EcoRI at the same time, and then the F gene was ligated with the vector. The system is as follows:

F gene (HindIII-EcoRI): 3 μL

pVAX1 (HindIII-EcoRI): 1 μL

Ligation buffer: 1 μL

T4 DNA Ligase (1U/μL): 1μL

Ultrapure water: 4μL

Connect at 4°C for 16 hours, then transform Escherichia coli DH5α, smear the LB agar plate containing kanamycin, and cultivate at 37°C for 24 hours, pick out the growing colony and culture it in LB liquid, and identify the plasmid.

The plasmid was first identified by enzyme digestion, double enzyme digestion with HindIII and EcoRI, and the target band of about 1.7kb was observed by 0.8% agarose electrophoresis. Then the plasmid was sent to Shanghai Sangon Bioengineering Technology Service Co., Ltd. for sequence determination. The results showed that the cloned F gene was 1696bp in full length, encoding a precursor protein F ₀ consisting of 553 amino acids, which was consistent with the characteristics of the F gene of the virulent NDV strain reported. It was confirmed that the DNA vaccine pVAX1-F was constructed correctly.

3. Construction of Newcastle Disease New Mucosal Vaccine:

The DNA vaccine pVAX1-F was transformed into attenuated Salmonella typhimurium SL7207 (gifted by Dr. Stocker, Stanford University, USA), and spread on LB agar plates containing kanamycin. After culturing at 37°C for 24 hours, the growing colony was picked for LB liquid culture, and the plasmid was identified. The plasmid was identified by enzyme digestion, double enzyme digestion with HindIII and EcoRI, and the target band of about 1.7kb was observed by 0.8% agarose electrophoresis, which confirmed that the DNA vaccine pVAX1-F had been successfully introduced into the attenuated Salmonella typhimurium SL7207, constituting the following The Newcastle disease virus DNA vaccine delivered by attenuated Salmonella typhimurium has a DNA length of 4647bp.

Application experiment:

1. The safety test results of SL7207(pVAX1-F) on chicks

SL7207(pVAX1-F) was orally inoculated at 10 ⁸ CFU, 10 ⁹ CFU, and 10 ¹⁰ CFU, respectively, in 1-day-old commercial Isa brown layer hens, 10 in each dose group, and 10 in the oral PBS control group. Observe the health status of the test chickens in each group, and kill all the test chickens after 2 weeks to check whether there are visual lesions.

The experimental results showed that during the observation period, all vaccinated chickens had no adverse reactions. They were slaughtered after 2 weeks, and no visual lesions were found in real organs such as liver and spleen during autopsy.

2. The results of the immune efficacy test of SL7207(pVAX1-F) on chicks

The 1-day-old commercial Isa brown layer hens were divided into 4 groups, namely the recombinant bacteria SL7207 (pVAX1-F) immunization group (10 ⁹ CFU/feather, 30 birds), SL7207 (pVAX1) immunization group (10 ⁹ CFU/ feather, 25 feathers), oil emulsion inactivated vaccine immunization group (25 feathers), blank control group (25 feathers). Bacterial immunization was carried out orally three times, the first time was 1 day old, the second time was 2 weeks old, the third time was 4 weeks old, and the oil emulsion inactivated vaccine (product of Yangzhou University Biological Products Research and Development Center) was given on the 1st day. Inoculate 0.2ml/feather subcutaneously in the neck of the young age. On the 14th, 28th, and 35th days after the first immunization group, blood was collected through the wing vein respectively, and 10 chickens were collected from each group, and the serum was separated at room temperature. For excess fat, dissect the intestinal tract with scissors in 5ml PBS (containing 100μg/ml PMSF), cut it into 1cm segments, shake vigorously in a 100ml bottle for 5min, centrifuge at 8000rpm at 4°C for 20min, and take the supernatant to prepare the intestinal tract For mucosal samples, F protein-specific serum and mucosal antibody titers were determined by indirect ELISA. Weigh the body weight of all the chickens in each test group on the 1st day and the 35th day respectively, and observe their weight gain. Ten days after the third immunization, the chickens were challenged with the standard virulent strain of Newcastle disease F ₄₈ E ₈ by intranasal route. The challenge dose was 8×10 ⁴ EID ₅₀ . On the 14th day, the number of survivors in each group was counted, and the immune protection rate was calculated. On the 14th day after the challenge, all surviving chickens were culled, and the general lesions of the test chickens were observed after the challenge; the intestines, trachea, bursa, thymus and other organs of the test chickens were collected, and treated with 10% neutral formaldehyde Forests were fixed, and paraffin tissue sections of the above-mentioned organs were prepared, and some tissues were stained with HE to observe the corresponding pathological changes.

The experimental results showed that immunization with the vaccine SL7207 (pVAX1-F) did not affect the weight gain of chickens. Vaccine SL7207(pVAX1-F) immunization can stimulate chickens to produce both serum antibodies and local mucosal antibodies, while chickens immunized with oil emulsion inactivated vaccine did not produce local mucosal antibodies. Vaccine SL7207(pVAX1-F) immunity can resist the interference of chicken maternal antibody, and provide chickens with good immune protection against virulent Newcastle disease virus challenge, and the immune protection rate is equivalent to that of the oil emulsion inactivated vaccine test group. There were no gross and histological lesions in the glandular gizzard, intestinal tract, cecal tonsil, spleen, bursa of Fabricius, thymus and trachea of chickens challenged with SL7207(pVAX1-F) immunization group, while those challenged with SL7207(pVAX1) empty vector Obvious gross lesions and histological lesions can be seen in the above viscera of poisoned chickens.

Attached Table 1 The effect of vaccine SL7207 (pVAX1-F) immunization on chick weight gain

Group Number of chickens Average body weight at 5 weeks old (kg) SL7207(pVAX1-F) blank control SL7207(pVAX1) seedlings   22161718 0.413±0.0470.438±0.0410.427±0.0440.403±0.055

Attached Table 2 The results of the immunogenicity test of the vaccine SL7207 (pVAX1-F) on commercial Isa brown layer hens

Group Average ELISA antibody titer Serum samples   Small intestinal mucosa sample 2 weeks after the first waiver 1 week after the third exemption   2 weeks after the first waiver 1 week after the third exemption SL7207(pVAX1-F) SL7207(pVAX1) seedling blank 320.00±195.9596.00±35.771024.00±350.5496.00±35.77 576.00±143.1040.00896.00±350.5436.00±35.77  128.0032.0048.00±22.6332.00  192.00±90.5132.0048.00±22.6332.00

Attached Table 3 Vaccine SL7207 (pVAX1-F) to the immune protection results of commercial Isa brown layer hens

Group Number of chickens Survival Deaths Protection rate (%) SL7207(pVAX1-F) SL7207(pVAX1) seedling blank   22171816   175152 512314 77.2729.4283.2212.50

From the above experiments, it is concluded that:

1. The Newcastle disease vaccine SL7207 (pVAX1-F) is easy to prepare, easy to immunize, and suitable for veterinary clinical application.

2. In vivo experiments show that Newcastle disease vaccine SL7207 (pVAX1-F) has good safety for chickens and does not affect the weight gain of chickens; it has the ability to stimulate the body to generate systemic immune response and local mucosal immune response; it can provide Newcastle disease The good immune protection against the virulence of the epidemic virus has laid a solid foundation for the popularization and use of the vaccine.

Claims (3)

1, newcastle mucosa DNA vaccine is characterized in that the nucleotide sequence of its dna vaccination is:

gactcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta 60

atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata 120

acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat 180

aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga 240

ctatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc 300

ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt 360

atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat 420

gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag 480

tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc 540

aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga 600

ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga 660

aattaatacg actcactata gggagaccca agctggctag cgtttaaact taagcttcaa 720

tatgggctcc agaccttcta ccaggatccc agcacctctg atgctgatca cccggattat 780

gctgatgttg ggctgtatcc gtccgacaag ctctcttgac ggcaggcctc ttgcagctgc 840

aggaattgta gtaacaggag ataaggcagt caatgtatac acctcgtctc agacagggtc 900

aatcatagtc aagttgctcc cgaatatgcc cagggataag gaagcgtgtg cgaaagcccc 960

attagaggca tataacagaa cactgactac tttgctcact cctcttggcg actccatccg 1020

caagatccaa gggtctgtgt ccacgtctgg aggaaggaga caagaacgct ttataggtgc 1080

tattattggc agtgtagctc ttggggttgc aacagcggca cagataacag cagctgcggc 1140

cctaatacaa gccaaccaga atgctgccaa catcctccgg cttaaggaga gcattgctgc 1200

aaccaatgaa gctgtgcatg aagtcaccga cggattatca caactatcag tggcagttgg 1260

gaagatgcag cagtttgtca atgaccagtt taataataca gcgcgagaat tggactgtat 1320

aaaaatcaca caacaggttg gtgtagaact caacctatac ctaactgaat tgactacagt 1380

attcgggcca cagatcacct cccctgcatt gactcagctg accatccagg cactttataa 1440

tttagctggt ggcaatatgg attacttatt aactaagtta ggtataggga acaatcaact 1500

cagctcatta attggtagcg gcctgatcac tggttaccct attctgtacg actcacagac 1560

tcaactcttg ggcatacaag tgaatttgcc ctcagtcggg aacttaaata atatgcgtgc 1620

cacctatttg gagaccttat ctgtaagtac aaccaaagga tatgcctcag cacttgtccc 1680

gaaagtagtg acacaagtcg gttctgtgat agaagagctt gacacctcat actgtataga 1740

gtccgatctg gatttatatt gtactagaat agtgacattc cccatgtccc caggtattta 1800

ttcctgtttg agcggcaaca catcagcttg catgtattca aagactgaag gcgcactcac 1860

cacgccgtat atggccctta aaggctcagt tattgccaat tgtaagataa caacatgtag 1920

atgtacagac cctcctggta tcatatcgca aaattatgga gaagctgtat ccctgataga 1980

tagacattcg tgcaatgtct tatcattaga tgggataact ctgaggctca gtggggaatt 2040

tgatgcaact tatcaaaaga acatctcaat actagattct caagtcatcg tgacaggcaa 2100

tcttgatata tcaactgaac ttggaaacgt caacaattca atcagcaatg ccttggataa 2160

gttggcggaa agcaacagca agctagaaaa agtcaatgtc agactaacca gcacatctgc 2220

tctcattacc tatattgttc taactgtcat ttctctagtt ttcggtgcac ttagtctggg 2280

tttagcgtgt tacctgatga tcaaacagaa ggcacaacaa aagaccttgc tatggcttgg 2340

gaataatacc ctcgatcaga tgagagccac tacaagagca tgaatgcaga taagaggtgg 2400

gaattctgca gatatccagc acagtggcgg ccgctcgagt ctagagggcc cgtttaaacc 2460

cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc 2520

gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa 2580

attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac 2640

agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg 2700

gcttctactg ggcggtttta tggacagcaa gcgaaccgga attgccagct ggggcgccct 2760

ctggtaaggt tgggaagccc tgcaaagtaa actggatggc tttctcgccg ccaaggatct 2820

gatggcgcag gggatcaagc tctgatcaag agacaggatg aggatcgttt cgcatgattg 2880

aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg 2940

actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg 3000

ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg 3060

aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg 3120

ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc 3160

tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc 3240

tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc 3300

gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc 3360

aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg 3420

atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct 3480

tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt 3540

tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc 3600

tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt 3660

tcttctgaat tattaacgct tacaatttcc tgatgcggta ttttctcctt acgcatctgt 3720

gcggtatttc acaccgcata caggtggcac ttttcgggga aatgtgcgcg gaacccctat 3780

ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata 3840

aatgcttcaa taatagcacg tgctaaaact tcatttttaa tttaaaagga tctaggtgaa 3900

gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc 3960

gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat 4020

ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga 4080

gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt 4140

ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata 4200

cctcgctctg ctaatcctgt taccagtgg ctgctgccagt ggcgataagt cgtgtcttac 4260

cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg 4320

ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg 4380

tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag 4440

cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct 4500

ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc 4560

aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctgggctt 4620

ttgctggcct tttgctcaca tgttctt。

2, a kind of construction method of the mucosa DNA of newcastle according to claim 1 vaccine is characterized in that: by reverse transcription-polymerase chain RT-PCR reaction, amplify the Avian pneumo-encephalitis virus fusion protein F gene earlier; Again F gene and plasmid pVAX1 are carried out coupled reaction, constructed dna vaccine pVAX1-F; At last plasmid pVAXt-F is transformed among the attenuated salmonella typhimurium SL7207; The amplimer of above-mentioned Avian pneumo-encephalitis virus fusion protein F gene is:

Forward primer: 5 ' taagcttcaatatgg gctccagacct 3 '

Downstream primer: 5 ' cgaattctccacctcttatctgcattcatgc 3 '.

3, according to the construction method of the described newcastle mucosa DNA of claim 2 vaccine, it is characterized in that Avian pneumo-encephalitis virus fusion protein F gene length is 1662bp, its DNA sequence is:

atgggctcca gaccttctac caggatccca gcacctctga tgctgatcac ccggattatg 60

ctgatgttgg gctgtatccg tccgacaagc tctcttgacg gcaggcctct tgcagctgca 120

ggaattgtag taacaggaga taaggcagtc aatgtataca cctcgtctca gacagggtca 180

atcatagtca agttgctccc gaatatgccc agggataagg aagcgtgtgc gaaagcccca 240

ttagaggcat ataacagaac actgactact ttgctcactc ctcttggcga ctccatccgc 300

aagatccaag ggtctgtgtc cacgtctgga ggaaggagac aaaaacgctt tataggtgct 360

attattggca gtgtagctct tggggttgca acagcggcac agataacagc agctgcggcc 420

ctaatacaag ccaaccagaa tgctgccaac atcctccggc ttaaggagag cattgctgca 480

accaatgaag ctgtgcatga agtcaccgac ggattatcac aactatcagt ggcagttggg 540

aagatgcagc agtttgtcaa tgaccagttt aataatacag cgcgagaatt ggactgtata 600

aaaatcacac aacaggttgg tgtagaactc aacctatacc taactgaatt gactacagta 660

ttcgggccac agatcacctc ccctgcattg actcagctga ccatccaggc actttataat 720

ttagctggtg gcaatatgga ttacttatta actaagttag gtatagggaa caatcaactc 780

agctcattaa ttggtagcgg cctgatcact ggttacccta ttctgtacga ctcacagact 840

caactcttgg gcatacaagt gaatttgccc tcagtcggga acttaaataa tatgcgtgcc 900

acctatttgg agaccttatc tgtaagtaca accaaagggat atgcctcagc acttgtcccg 960

aaagtagtga cacaagtcgg ttctgtgata gaagagcttg acacctcata ctgtatagag 1020

tccgatctgg atttatattg tactagaata gtgacattcc ccatgtcccc aggtatttat 1080

tcctgtttga gcggcaacac atcagcttgc atgtattcaa agactgaagg cgcactcacc 1140

acgccgtata tggcccttaa aggctcagtt attgccaatt gtaagataac aacatgtaga 1200

tgtacagacc ctcctggtat catatcgcaa aattatggag aagctgtatc cctgatagat 1260

agacattcgt gcaatgtctt atcattagat gggataactc tgaggctcag tggggaattt 1320

gatgcaactt atcaaaagaa catctcaata ctagattctc aagtcatcgt gacaggcaat 1380

cttgatatat caactgaact tggaaacgtc aacaattcaa tcagcaatgc cttggataag 1440

ttggcggaaa gcaacagcaa gctagaaaaa gtcaatgtca gactaaccag cacatctgct 1500

ctcattacct atattgttct aactgtcatt tctctagttt tcggtgcact tagtctgggt 1560

ttagcgtgtt acctgatgat caaacagaag gcacaacaaa agaccttgct atggcttggg 1620

aataataccc tcgatcagat gagagccact acaagagcat ga。