Construction and application of a genotype VII attenuated Newcastle disease marker vaccine strain rMG7-F-N471A

Abstract

The present invention discloses a genotype VII Newcastle disease attenuated marker vaccine strain rMG7-F-N471A, which has a deposit number of CGMCC No.46293. The full gene nucleotide sequence of the vaccine strain is shown in SEQ ID No: 1; wherein, the 471st amino acid asparagine (N) of the F protein is replaced by alanine (A). The present invention utilizes a Newcastle disease virus reverse genetic operation platform, and on the basis of the Newcastle disease virus MG7 strain, mutates different sites to rescue a genotype VII Newcastle disease virus marker vaccine attenuated strain rMG7-F-N471A with low pathogenicity, good growth characteristics, high virus titer, stable passage, and excellent immunogenicity. The marker vaccine strain has good immunogenicity, can induce a high level of protective antibodies, and achieves complete protection for immunized chickens. It can be used to prevent and control the currently prevalent genotype VII Newcastle disease virus and lay the foundation for distinguishing vaccine immunity from wild virus infection.

Claims

1. A genotype VII Newcastle disease attenuated marker vaccine strain rMG7-F-N471A, characterized in that: Its deposit number is CGMCC No.46293.

2. A method for constructing the genotype VII Newcastle disease attenuated marker vaccine strain rMG7-F-N471A according to claim 1, characterized in that: The construction method comprises the following steps: (a) Using the pCAGGS-MG7-cDNA full genome plasmid as a template, site-directed mutagenesis was performed on the differential amino acid sites of the structural proteins of the MG7 strain to obtain mutant plasmids; (b) The mutant plasmid is subjected to reverse genetic manipulation to construct the genotype VII Newcastle disease attenuated marker vaccine strain rMG7-F-N471A.

3. The construction method according to claim 2, characterized in that In the pCAGGS-MG7-cDNA full genome plasmid, the F protein cleavage site of the MG7 strain has been mutated to the F protein cleavage site of the La Sota strain, and 18 amino acids are missing from positions 443 to 460 of the NP protein of the MG7 strain.

4. The construction method according to claim 2, characterized in that The upstream primer used in the site-directed mutagenesis is 5'- GAACTTGGAAACGTCGCGAATTCAATCAGCAATGCC-3'.

5. The construction method according to claim 2, characterized in that The downstream primer used for the site-directed mutagenesis was 5'-GGCATTGCTGATTGAATTCGCGACGTTTCCAAGTTC-3'.

6. The construction method according to claim 2, characterized in that The site-directed mutagenesis comprises the following steps: (1) PCR amplification: Amplify the DNA fragments upstream and downstream of the mutation site. After gel recovery, the upstream and downstream DNA fragments are mixed in equimolar amounts as templates to amplify the full-length DNA mutation fragment to obtain the amplified product; (2) Ligation: The amplified product and the pCAGGS-MG7-cDNA full genome plasmid were double-digested, gel-recovered to obtain the digestion product, and the digestion product was ligated in a metal bath using T4 DNA ligase to obtain a ligation product; (3) Transformation: The ligation product was transformed into DH5α competent cells, added to LB liquid medium and cultured for 28.5-31.5 min, spread on ampicillin-resistant LB solid medium plates, and cultured for 12 hours; as well as (4) Screening: Identify whether the MG7 mutant plasmid is successfully connected, and screen to obtain the mutant plasmid that is successfully connected.

7. The construction method according to claim 6, characterized in that: The temperature of the metal bath is 15.2-16.8°C.

8. The construction method according to claim 6, characterized in that: The LB solid culture medium is an ampicillin-resistant LB solid culture medium.

9. The construction method according to claim 6, characterized in that: The culture temperature is 35.15-38.85°C.

10. The construction method according to claim 2, characterized in that: The reverse genetic operation comprises the following steps: (i) co-transfecting the successfully linked mutant plasmid and the helper plasmid into BHK-21 cells, culturing, freezing and thawing, and filtering to obtain a cell culture; and (ii) inoculating the cell culture into chicken embryos, incubating the cells, and collecting the chicken embryo allantoic fluid with a positive hemagglutination test result to obtain the genotype VII Newcastle disease attenuated marker vaccine strain rMG7-F-N471A.

11. The construction method according to claim 10, characterized in that: The auxiliary plasmids are pBSK-NP, pBSK-P and pBSK-L.

12. The construction method according to claim 10, characterized in that: The BHK-21 cells are BHK-21 cells pre-infected with the recombinant poxvirus VVT7.

13. The construction method according to claim 12, characterized in that: The multiplicity of infection of the recombinant poxvirus VVT7 is 0.001 to 0.1 MOI.

14. The construction method according to claim 13, characterized in that: The multiplicity of infection of the recombinant poxvirus VVT7 is 0.0095-0.0105 MOI.

15. The construction method according to claim 12, characterized in that: The pre-infection time is 0.5 to 1.5 hours.

16. The construction method according to claim 15, characterized in that: The pre-infection time is 0.95 to 1.05 hours.

17. The construction method according to claim 10, characterized in that: The culture time is 48 to 96 hours.

18. The construction method according to claim 17, characterized in that: The culture time is 68.4 to 75.6 hours.

19. The construction method according to claim 10, characterized in that: The culture temperature is 35.15-38.85°C.

20. The construction method according to claim 10, characterized in that: The culture container is a cell culture box containing 5% CO2.

21. The construction method according to claim 10, characterized in that: The number of freeze-thaw cycles was 1.

22. The construction method according to claim 10, characterized in that: The pore size of the filter is 0.22 μm.

23. The construction method according to claim 10, characterized in that: The chicken embryo is a 9-11 day old SPF chicken embryo.

24. The construction method according to claim 10, characterized in that The incubation time is 2 to 4 days.

25. The construction method according to claim 24, characterized in that: The incubation time is 3 days.

26. Use of the genotype VII Newcastle disease attenuated marker vaccine strain rMG7-F-N471A according to claim 1 in preparing a vaccine for Newcastle disease, characterized in that: The vaccine is a vaccine that has a protective effect on Newcastle disease caused by genotype VII.

27. The use according to claim 26, characterized in that The vaccine is an poultry vaccine.

28. The use according to claim 27, characterized in that The poultry is chicken.

29. The use according to claim 26, characterized in that The protective effect further includes one or more of the following: promoting the replication of the vaccine strain, preventing the excretion of Newcastle disease virus and inhibiting the replication of Newcastle disease virus in organs.

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