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Whole poultry source H5N2 subtype avian influenza recombinant strain, vaccine and application thereof

A technology for avian influenza and epidemic strains, which is applied in the field of reverse genetics technology and animal vaccines, can solve the problems of unguaranteed safety and the infection of humans by recombinant avian influenza vaccines, and achieves good protection effects.

Active Publication Date: 2021-06-25
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In influenza vaccines for human use, recombinant avian influenza vaccines are constructed by choosing to utilize human influenza vaccine backbones (for example: Hoffmann E, Krauss S, Perez D et al. Eight-plasmid system for rapid generation of influenza virus vaccines. Vaccine , 2002, 20:3165- 3170), because it contains human genes, it is easy to cause the risk of human infection by recombinant avian influenza vaccine, and the safety cannot be guaranteed

Method used

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  • Whole poultry source H5N2 subtype avian influenza recombinant strain, vaccine and application thereof
  • Whole poultry source H5N2 subtype avian influenza recombinant strain, vaccine and application thereof
  • Whole poultry source H5N2 subtype avian influenza recombinant strain, vaccine and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 The construction of the reverse genetics operating system of the whole avian source avian influenza vaccine based on the D7 strain

[0046] 1. Construction of reverse genetic eight-plasmid system vector pSMC (such as figure 2 with image 3 )

[0047] (1) Transformation of pCI vector

[0048] The pCI vector is the product of Promega (product number: BR180). In order to place the pCI vector on the plasmid Bsm BI restriction site removal, with restriction endonuclease ear I digest the pCI vector to obtain long fragment A and short fragment B. Design the amplification primer pCI- on the short fragment B ear I-1 and pCI- ear I-2.

[0049] pCI- ear I-1: 5'-TAGCGAGAGGCCGCACG-3';

[0050] pCI- ear I-2: 5'-TCTTCGTTCGGTCACAGCTTCTGTAAG-3';

[0051] And use the short fragment B as a template to amplify to obtain fragment C; recover the above PCR product to obtain fragment C, and use fragment A separately ear After digestion and recovery, ligation, tra...

Embodiment 2

[0064] Example 2 Construction of H5N2 Subtype Avian Influenza Recombinant Strain

[0065] 1. Extraction and reverse transcription of viral RNA

[0066] The total RNA of the allantoic fluid was extracted using a total RNA extraction kit, referring to the manual of M-MLV reverse transcriptase, the reverse primer sequence was: 5'-AGCAAAAGCAGG-3', and cDNA was obtained by reverse transcription.

[0067] 2. Primer design

[0068] Refer to the general primers designed for the 8 fragments of the influenza virus, design the full-length amplification primers for the HA fragment, and then design and modify the segmented primers for the cleavage site of the HA gene according to the HA sequence of the GD123 strain. The specific sequence is as follows, where the underlined part is a restriction endonuclease Bsm Recognition sequence of BI.

[0069] GD123-HA1-F:

[0070] 5’- CTCAGAAATAGTCCTCTAAGAGAAAGAGGACTGTTTGGAGCT-3’

[0071] GD123-HA2-R:

[0072] 5’-AAACAGTCCTCTTTTCTCTAGAGGACTATTT...

Embodiment 3

[0085] Example 3 Preparation of vaccine H51901 by recombinant virus rGD123

[0086] 1. Vaccine preparation

[0087] Large-scale preparation of antigens: Dilute the seedling strain rGD123 with sterile DMEM cell culture medium to 10 -4 , take 9-11 day-old SPF embryos, inoculate the diluted virus liquid into the allantoic cavity of chicken embryos aseptically, 0.2 mL / piece, seal it and put it in a 37°C incubator. After incubation for 60 hours, place it in a biological safety cabinet. The allantoic fluid of chicken embryos was collected and its hemagglutination (HA) titer was determined.

[0088] Antigen inactivation: Inactivate the above collected virus liquid with formaldehyde at a final concentration of 0.1%, seal it, put it in a shaker, and incubate at 37°C for 24 hours; then take the inactivated virus liquid and inoculate it for 9 to 11 days Age SPF chicken embryos, 0.2 mL / piece, cultured at 37°C for 48 h, then detected the hemagglutination titer to verify whether the virus...

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Abstract

The invention discloses a whole poultry source H5N2 subtype avian influenza recombinant strain, a vaccine preparation method, a vaccine composition and application. The recombinant strain is composed of an HA gene modified by a highly pathogenic H5N6 subtype avian influenza epidemic strain and seven internal genes PB2, PB1, PA, NP, NA, M and NS of an H5N2 subtype avian influenza D7 strain. The sequence of the modified HA gene is as shown in SEQ ID NO: 9; Nucleotide sequences of seven internal genes PB2, PB1, PA, NP, NA, M and NS of the D7 strain are sequentially shown as SEQ ID NO: 1-3 and SEQ ID NO: 5-8. The recombinant virus is a recombinant avian influenza virus rescued by using a D7 system as a skeleton, is a non-virulence strain, can maintain the original immunogenicity, can maintain high virus titer in the chick embryo culture process, completely meets the biological safety requirement, and has great application prospect.

Description

technical field [0001] The present invention relates to the field of reverse genetics technology and animal vaccine technology, and more specifically, relates to a recombinant strain of H5N2 subtype avian influenza of whole avian origin, a vaccine preparation method, a vaccine composition and application. Background technique [0002] Avian influenza (Avian influenza, AI) is an infection and / or disease syndrome caused by avian influenza virus (AIV), which seriously affects the development of poultry farming and threatens human health. According to different clinical symptoms, it can be divided into low pathogenic avian influenza (LPAI) and highly pathogenic avian influenza (HPAI) according to the pathogenicity. Since 2003, H5 subtype highly pathogenic avian influenza has been circulating in China and other parts of the world or even outbreaks; since 2016, the number of H5 subtype highly pathogenic avian influenza in my country has increased significantly, and H5N6 has become...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/85C12N15/44A61K39/145A61P31/16C12R1/93
CPCC12N7/00C12N15/85C07K14/005A61K39/12A61P31/16C12N2760/16121C12N2760/16122C12N2760/16152C12N2760/16134C12N2760/16163A61K2039/5252A61K2039/552A61K39/00C12N2760/16162
Inventor 亓文宝廖明陈意群张家豪李波黄锦瑜李华楠
Owner SOUTH CHINA AGRI UNIV
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