A recombinant turkey herpes virus expressing h9 protein of h9n2 subtype avian influenza virus

A technology for turkey herpes virus and avian influenza virus, applied in the direction of double-stranded DNA virus, antisense single-stranded RNA virus, virus, etc., can solve the problem of reducing chicken excretion, failing to effectively control the prevalence of H9N2 subtype AIV, and virus antigenic variation. And other issues

Active Publication Date: 2021-04-27
CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, in the past 20 years, inactivated vaccines can alleviate the clinical symptoms of infected chickens and reduce chicken shedding, but they cannot produce complete protection. At the same time, due to the variation of virus antigenicity, the epidemic of H9N2 subtype AIV has not been effectively controlled.
A survey of the prevalence of H9N2 subtype AIV in immunized chickens in recent years found that the isolation rate of H9N2 subtype AIV increased year by year, from 22.08% in 2010 to 47.08% in 2013, indicating that the traditional inactivated vaccine immunization Cannot effectively prevent and control H9N2 subtype AIV infection, so the development of a new vaccine is imminent

Method used

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  • A recombinant turkey herpes virus expressing h9 protein of h9n2 subtype avian influenza virus
  • A recombinant turkey herpes virus expressing h9 protein of h9n2 subtype avian influenza virus
  • A recombinant turkey herpes virus expressing h9 protein of h9n2 subtype avian influenza virus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1, A / Chicken / Beijing / 0701 / 2015 (H9N2) (hereinafter referred to as A / Chicken / Beijing / 0701 / 2015, BJ / 15 virus strain or CK / BJ / 1 / 15) acquisition and identification

[0055] 1. Isolation and identification of H9N2 influenza virus

[0056] The inventor of the present invention collected throat swab samples of chickens from Beijing, Tianjin, Hebei, Shandong and other places in my country between 2014 and 2015, and then referred to the literature (Pu juan et al.Evolution of the H9N2influenzagenotype that facilitated the genesis of the novel The method described in H7N9virus.Proc Natl AcadSci US A.2015.), the relevant virus of sample is isolated and identified. Among them, 16 strains of H9N2 avian influenza virus were isolated. According to the general nomenclature of influenza viruses, the 16 isolated strains of H9N2 avian influenza viruses were named, and the specific information is shown in Table 1-1.

[0057] Table 1-1. Basic information of H9N2 avian influenza viru...

Embodiment 2

[0091] Embodiment 2, the preparation of the recombinant turkey herpes virus (i.e. recombinant virus rHVT-H9) expressing H9N2 subtype avian influenza virus H9 protein

[0092] 1. Construction of recombinant plasmid pBAC-GFP-US

[0093] 1. The nucleotide sequence of the GFP gene (NCBI sequence number: LC336974.1) was artificially synthesized and inserted into the pUC19 vector to construct the plasmid pUC-GFP. The plasmid pUC-GFP was used as a template, and a primer pair consisting of GFP-F and GFP-R was used for PCR amplification to obtain a DNA fragment of about 720 bp.

[0094] 2. Take the DNA fragment obtained in step 1, perform double digestion with restriction endonucleases NheI and XbaI, and recover the digested fragment.

[0095] 3. Take the pcDNA3.1+ vector, perform double digestion with restriction endonucleases NheI and XbaI, and recover a vector skeleton of about 5.4 kb.

[0096] 4. Ligate the restriction fragment obtained in step 2 with the vector backbone obtained...

Embodiment 3

[0151] Embodiment 3, identification of recombinant virus rHVT-H9

[0152] 1. Gene level identification

[0153] Genomic DNA of the recombinant virus rHVT-H9 was extracted and used as a template to perform PCR amplification with a primer pair composed of H9-F and H9-R to obtain PCR amplification products.

[0154] The results showed that the PCR amplification product contained a DNA fragment of about 1683bp. It can be seen that the H9 gene has been completely inserted into the HVT genome.

[0155] 2. Indirect immunofluorescence identification

[0156] 1. Take a 24-well cell culture plate and add 500 μL of culture medium containing CEF cells (about 2.25×10 5 CEF cells / well), 37°C, 5% CO 2 Culture until CEF cells grow into a monolayer.

[0157] 2. After completing step 1, inoculate the recombinant virus rHVT-H9 (the inoculation dose is 100PFU / well), at 37°C, 5% CO 2 Cultivate for 72h.

[0158] 3. After completing step 2, discard the medium, wash once with PBS buffer soluti...

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Abstract

The invention discloses a recombinant turkey herpes virus expressing H9N2 subtype avian influenza virus H9 protein. The recombinant turkey herpes virus is obtained by carrying out the following transformation on the genome of the turkey herpes virus: the DNA molecule containing the coding gene of the H9N2 subtype avian influenza virus H9 protein is added; the amino acid of the H9N2 subtype avian influenza virus H9 protein The sequence is shown as sequence 2 in the sequence listing. The experiment proved that on the first day of the experiment, the recombinant turkey herpes virus was subcutaneously inoculated into the neck of 1-day-old SPF chickens; on the 35th day of the experiment, the BJ / 15 virus strain was inoculated into the nasal cavity; Neither the cloaca nor the cloaca are detoxified, and the BJ / 15 virus strain cannot efficiently replicate in the organs (such as internal organs, trachea, lung, spleen and kidney) of SPF chickens. It can be seen that the recombinant turkey herpes virus has excellent immune protection effect on H9N2 subtype AIV. The invention has important application value.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a recombinant turkey herpes virus expressing the H9 protein of H9N2 subtype avian influenza virus. Background technique [0002] In 1994, my country first reported chickens infected with H9N2 subtype avian influenza virus (avian influenza virus, AIV). Since then, H9N2 subtype AIV has been widely prevalent in poultry in my country. Secondary infection brings huge economic losses to the breeding industry. H9N2 subtype AIV is prevalent in terrestrial poultry and has evolved into different genotypes. At present, the dominant strain of H9N2 subtype AIV in chickens in my country is the G57 genotype. This genotype first appeared in 2007 and became dominant after 2010. Genotype: The H9N2 subtype AIV of this genotype not only seriously threatens the healthy development of the aquaculture industry, but also plays an important role in the production of new influenza viruses such as H7N9 and H10N...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/01C12N15/44C07K14/11A61K39/145A61P31/16
CPCA61K39/12A61K2039/5256A61K2039/552A61P31/16C07K14/005C12N7/00C12N2710/16021C12N2760/16122C12N2760/16134
Inventor 刘金华刘立涛孙洪磊
Owner CHINA AGRI UNIV
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