Construction and application of recombinant turkey herpesvirus expressing H7N9 subtype highly pathogenic avian influenza virus HA protein

A technology of turkey herpes virus and avian influenza virus, applied in the fields of molecular biology and genetic engineering, can solve the problems of inaccurate distinction between wild virus infection and vaccine immunity, increasing the difficulty of bird flu prevention and control, and interference of vaccine maternal antibodies , to achieve the effect of shortening the screening time

Active Publication Date: 2019-09-10
SOUTH CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although whole-virus inactivated vaccines can protect poultry and reduce the risk of bird flu outbreaks, the defects of whole-virus inactivated vaccines increase the difficulty of bird flu prevention and control [17,18]
First of all, this type of vaccine is susceptible to interference from maternal antibodies, requiring multiple immunizations in large doses, and the cost is high
Secondly, wild virus infection and vaccine immunity cannot be accurately distinguished, making it more difficult for avian influenza epidemiological monitoring
Moreover, since this type of vaccine mainly produces humoral immunity, the vaccine strain and the circulating strain must be highly homologous, otherwise the immune effect will not be ideal

Method used

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  • Construction and application of recombinant turkey herpesvirus expressing H7N9 subtype highly pathogenic avian influenza virus HA protein
  • Construction and application of recombinant turkey herpesvirus expressing H7N9 subtype highly pathogenic avian influenza virus HA protein
  • Construction and application of recombinant turkey herpesvirus expressing H7N9 subtype highly pathogenic avian influenza virus HA protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Embodiment 1, the extraction of the proliferation of herpesvirus turkey (HVT) and DNA

[0056] Primary and secondary chicken embryo fibroblasts (CEF) were prepared and cultured according to conventional methods using 9-10-day-old SPF chicken embryos (Xingxing Dahuanong Poultry Co., Ltd.), and the cells grew into a single layer after 24 hours. The turkey herpesvirus (HVT) FC126 strain infected the secondary CEF, cultured for 3 to 5 days (days), typical cytopathy appeared, and when the cytopathy reached 80%, the cells containing the virus were digested with trypsin (GIBCO company) and added to frozen cells. Storage solution, liquid nitrogen storage. Extract the total DNA of cells and viruses (for the extraction of total DNA of cells and viruses, refer to the literature [20] mentioned).

Embodiment 2

[0057] Embodiment 2, the construction of transfer plasmid pG-65 / 66H7HA ( figure 1 , 2 )

[0058] 2.1 Transformation of HA gene

[0059] H7N9 subtype avian influenza virus strain (A / Chicken / Huizhou / HZ-3 / 2016 [15] ; GISAID website sequence number is EPI_ISL_248796) is a highly pathogenic avian influenza virus, lethal to chickens, extract the RNA of the virus, reverse transcription to obtain cDNA, use the cDNA as a template with primers (HA-F and HA1-R; HA1-R respectively; -F and HA-R) for PCR amplification to obtain HA1 and HA2 fragments. HA1 and HA2 were fused with primers (HA-F and HA-R) to obtain a modified H7HA fragment (SEQ ID NO: 1), that is, the HA fragment of the highly pathogenic H7N9 subtype avian influenza virus was cleaved into place by PCR mutation technology. The deletion of basic amino acids near the point changed from high pathogenicity to low pathogenicity. The H7HA fragment size is about 1.7kb. The primer sequences are as follows (5'-3'):

[0060] HA-F: ...

Embodiment 3

[0080] Example 3, construction and purification of recombinant turkey herpesvirus rHVT-H7HA

[0081] 3.1 Construction and enrichment of recombinant turkey herpesvirus rHVT-H7HA

[0082] CEFs infected with turkey herpesvirus (HVT) FC126 strain (i.e. virus-containing cells obtained in Example 1) were inoculated with fresh primary CEFs (prepared in Example 1), incubated at 37°C for 4 hours, digested with trypsin, and infected 8×10 after 6 1 primary CEF cells were suspended in PBS buffer, 40 μg of transfer plasmid pG-65 / 66-H7HA linearized with restriction endonuclease BsmBI (NEB company) was added, the total volume was 0.7ml, mixed evenly, and transferred to In the electroporation cup, the electrotransfection conditions are: 1.5kV / cm, pulse time 0.5ms, in the electroporation instrument (Gemini X 2 model) for electrotransfection. After the electroporation was completed, the cells were transferred to a 60mm culture dish (dish), and 10% (v / v) serum (Biological Industries fetal bov...

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Abstract

The invention discloses construction and application of a recombinant turkey herpesvirus expressing H7N9 subtype highly pathogenic avian influenza virus HA protein. The recombinant turkey herpesvirusis obtained by inserting an exogenous gene expression box between the HVT-065 and HVT-066 genes in a non-essential replication area of a coding sequence of a turkey herpesvirus (HVT), wherein the exogenous gene expression box is formed by connecting an MCMV promoter, an exogenous gene and SV40 poly A in sequence; the exogenous gene is the H7HA gene, and the nucleotide sequence of the exogenous gene is shown as SEQ ID NO:1. The construction method of the recombinant turkey herpesvirus is also provided. An ultrasonic cracking and breaking method is adopted for purification, the screening time isshortened, and the obtained recombinant turkey herpesvirus rHVT-H7HA provides good protection for the highly pathogenic H7N9 subtype avian influenza virus and can be used for subsequent development of new avian influenza virus vaccines.

Description

technical field [0001] The invention belongs to the fields of molecular biology and genetic engineering, and particularly relates to the construction and application of a recombinant turkey herpes virus expressing the HA protein of H7N9 subtype highly pathogenic avian influenza virus. Background technique [0002] Turkey herpesvirus (HVT) strain Fc-126 is antigenically related to Marek's disease virus (MDV), and is widely used as a live vaccine against Marek's disease (MD), which is non-pathogenic and has excellent protective effect [1] . Compared with other viral vectors, HVT has its unique advantages [2-7] , such as: HVT itself is a vaccine candidate strain, and it can infect hosts including chickens and waterfowl, but it does not cause disease. The huge genome can provide multiple insertion sites for foreign genes, and has the potential to become a multivalent vaccine; HVT is a cell Conjugated viruses can overcome the influence of maternal antibodies and induce strong c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/66C12N15/869C07K14/11A61K39/245A61P31/22
CPCA61K39/12A61K2039/5252A61K2039/54A61K2039/552A61P31/22C07K14/005C12N7/00C12N15/86C12N2710/16021C12N2710/16034C12N2710/16043C12N2760/16122
Inventor 亓文宝苏冠铭廖明张旭陈意群马凯雄
Owner SOUTH CHINA AGRI UNIV
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