Recombinant turkey herpesvirus expressing HA gene and application thereof

A technology of turkey herpes virus and gene, which is applied to the recombinant turkey herpes virus expressing HA gene and its application field, which can solve the problems of poor protection, large interference, and inability to cross-protect inactivated vaccines, and achieve safe use and immunity The effect of good originality and reduced screening steps

Pending Publication Date: 2019-09-06
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Live vector vaccines, subunit vaccines or nucleic acid vaccines based on the HA gene can all produce nearly 100% protection against the challenge of the same HA subtype virus, but due to the extremely high mutation rate of HA, the protective effect of multiple vaccines based on this gene It is subtype-specific, that is, it is only limited to the same subtype as the expressed HA, and has poor protection against other subtypes of virus attack
[0005] At present, the use of H9N2 subtype AIV inactivated vaccines has played an important role in the prevention and control of the disease, but inactivated vaccines cannot produce cross-protection against mutant strains of the same subtype or different subtypes. It cannot effectively induce cellular immunity and mucosal immune response, and is greatly interfered by maternal antibodies during use

Method used

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  • Recombinant turkey herpesvirus expressing HA gene and application thereof
  • Recombinant turkey herpesvirus expressing HA gene and application thereof
  • Recombinant turkey herpesvirus expressing HA gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] Construction of Bacterial Artificial Chromosome Plasmid pHVT-BAC of Turkey Herpesvirus

[0090] 1) Primer design and synthesis

[0091] According to the complete genome sequence (AF291866) of the HVT Fc-126 strain published in GenBank, the pBeloBAC11 vector sequence and the pGE-gpt plasmid (see Figure 15 ) sequence, designed three pairs of PCR amplification primers as shown in Table 1, and the primers were synthesized by Huada Gene Company.

[0092] Table 1 Recombinant turkey herpes virus transfer vector primers

[0093]

[0094] 2) Extraction of total DNA from HVT-infected cells

[0095] SPF chicken embryos aged 9-11 days were taken to make primary chicken embryo fibroblasts (chicken embryo fiborblast, CEF). Take out the frozen HVT virus and inoculate the CEF cells that have confluent monolayer. The cells were cultured at 37° C., and when 70% of the cells had lesions (plaques), the virus-infected cells were collected to extract DNA. The total DNA of diseased c...

Embodiment 2

[0151] Construction of recombinant HVT expressing H9N2 AIV HA gene

[0152] 1) Primers used for RED / ET recombination and HA gene amplification

[0153] According to the full-length genome sequence of HVT Fc-126 strain (Accession NO.AF291866), pcDNA3.1(-) sequence and avian influenza virus A / goose / Gongdong / 3 / 96 strain (H9N2) HA gene sequence published on GenBank, the design Primers as shown in Table 7:

[0154] Table 7: Primers required for RED / ET recombination

[0155]

[0156] The RNA of avian influenza virus A / goose / Gongdong / 3 / 96 was extracted according to the instruction of RNeasy plus Mini Kit. Reverse Transcription.

[0157] The above RNA was taken and reverse-transcribed according to the instructions of the reverse transcription kit to obtain cDNA, which was used as a template for HA gene amplification. Reaction system: cDNA 2μL, 10×LA Taq Buffer 10μL, 2.5mmol dNTP 4μL, 20pmol HA gene upstream and downstream primers 1μL, LA Taq DNA polymerase 0.5μL, sterilized dd ...

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Abstract

The invention provides a recombinant turkey herpesvirus expressing HA gene and an application thereof. Based on a viral vector HVT- BAC as a platform, combined with a RED / ET recombination technology and a ccdB reverse screening technology, and the recombinant turkey herpesvirus expressing HA gene is prepared by a two-step recombination method, wherein, a nucleotide sequence of the HA gene is shownas SEQ ID No. 1. The beneficial effect is that the HA gene used in the present invention is derived from a H9N2 toxic strain widely prevalent in recent years in China, and has broad immunogenicity tobirds. A preparation method of the invention realizes rapid and accurate recombination of the exogenous gene, and a used screening marker is beneficial to reduce a screening step, and the obtained recombinant virus strain can be stably passaged. A vaccine prepared by the recombinant virus strain can stimulate the latent infection of the chicken body, causes lifelong immunity, provides technical support for effectively preventing and controlling H9N2 avian influenza and Marek's disease, and has important production guiding significance.

Description

technical field [0001] The invention belongs to the technical field of animal genetic engineering vaccines, and more specifically relates to a recombinant turkey herpes virus expressing HA gene and application thereof. Background technique [0002] Although the H9N2 subtype avian influenza virus (AIV) whole-virus inactivated vaccine widely used in poultry production has a good immune effect, this type of vaccine cannot induce effective cellular and mucosal immune responses. Highly interfered by maternal antibodies. [0003] AIV is prone to antigenic drift, and generally causes small and medium epidemics of the virus, but antigenic variation can easily cause a worldwide pandemic. Therefore, one of the difficulties in the prevention and control of avian influenza lies in the antigenic variation of AIV. Currently commonly used diagnostic methods are virus isolation, hemagglutination test and hemagglutination inhibition test, virus neutralization test, fluorescent immunoassay,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/869C12N15/44A61K39/145A61P31/16C12R1/93
CPCC12N7/00C12N15/86C07K14/005A61K39/12A61P31/16C12N2710/16021C12N2710/16043C12N2710/16052C12N2760/16122C12N2760/16134A61K2039/5256A61K2039/552
Inventor 谢青梅封柯宇申晓晨蔺文成张新珩李鸿鑫
Owner SOUTH CHINA AGRI UNIV
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