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High-pathogenicity H7N9 avian influenza virus antigen with low receptor binding activity and preparation method of antigen

An avian influenza virus, highly pathogenic technology, applied in the field of virus genetic engineering, can solve the problems of the influence of receptor affinity results, low receptor binding activity, etc., and achieve the effect of improving biological safety

Inactive Publication Date: 2019-08-09
GUANGZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The second object of the present invention is to provide a HPAI H7N9 influenza virus antigen with low receptor binding activity, so as to solve the problem of the influence of the change of receptor affinity on the result in the HI test

Method used

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  • High-pathogenicity H7N9 avian influenza virus antigen with low receptor binding activity and preparation method of antigen
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  • High-pathogenicity H7N9 avian influenza virus antigen with low receptor binding activity and preparation method of antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Method for Reducing HPAI H7N9 Influenza Virus Receptor Binding Affinity

[0037] The first step is to synthesize the HA gene: first, according to the HA gene sequence of the HPAI H7N9 influenza vaccine strain A / Guangdong / 17SF003 / 2016 (H7N9) recommended by WHO, synthesize the HA gene with multiple basic amino acid deletions at the cleavage site, the cleavage site The HA gene (called H7 / GD16 / WT) with multiple basic amino acid deletions was completed by GenScript. Existing studies have shown that the cleavage site with multiple basic amino acids is a sign of highly pathogenic avian influenza virus, and the deletion of multiple basic amino acids can reduce its pathogenicity, so that the recombinant virus can be tested in the biosafety level two experiment. The operation is carried out in the room without affecting the immunogenicity of the HA protein. For the sake of safety, the cleavage site in the HA gene of the HPAI H7N9 influenza vaccine strain, which has the character...

Embodiment 2

[0052] The difference between Example 2 and Example 1 is that the NA fragment in Example 2 is selected from N9NA gene with non-oseltamivir resistance mutation. The NA gene containing the oseltamivir resistance mutation has the effect of resistance to oseltamivir, and the HPAI H7N9 influenza virus prepared by using this mutated NA gene has the risk of drug-resistant gene proliferation. By using non-oseltamivir The drug-resistant mutated NA gene can prevent the oseltamivir drug-resistant mutated NA gene from spreading influenza virus widely. The synthesis of HA gene of H7 / GD16 / WT, the construction of pM-H7 / GD16 / WT recombinant plasmid and the site-directed mutation of HA gene in Example 2 are all the same as in Example 1 above.

[0053] In the first step, the NA gene of the LPAI H7N9 vaccine strain A / Anhui / 1 / 2013 (H7N9) (GISAIDID: EPI439509, called N9 / AH13) was selected as the NA gene. N9 / AH13 was completed by GenScript, and N9 / AH13 The sequence of is shown in SEQ ID NO.7.

[0...

Embodiment 3

[0066] A method for the preparation of HPAI H7N9 influenza virus with low receptor binding affinity comprising an inactivation step.

[0067] The first step, inactivation of influenza virus

[0068] The recombinant influenza virus prepared in the above-mentioned Example 2 was inactivated, and the specific inactivation method was as follows. Formaldehyde solution was used for inactivation, and formaldehyde solution with a final concentration of 0.1% was added to the virus allantoic fluid, and inactivated at 37° C. for 16 hours. The inactivated influenza virus was continuously passaged in chicken embryos for three times, and it was determined that the inactivation was successful if no hemagglutination titer was detected after passage.

[0069] The inactivated and purified H7N9 / GD16 / WT and H7N9 / GD16 / R220G recombinant viruses were recorded as two HPAI H7N9 influenza viruses, which were HPAI H7N9 influenza virus HI detection antigens.

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Abstract

The invention discloses a high-pathogenicity H7N9 avian influenza virus antigen with low receptor binding activity and a preparation method of the antigen. The method comprises the following steps ofpreparation of a mutant HA gene, wherein a gene sequence of mutant high-pathogenicity H7N9 avian influenza virus full-length HA protein is prepared, and the R220G mutant HA gene is obtained, and the sequence of the R220G mutant HA gene is shown in SEQ ID NO.2; virus rescue, wherein the R220G mutant HA gene is adopted for rescuing a recombinant influenza virus. According to the recombinant high-pathogenicity H7N9 avian influenza virus prepared by means of the method, arginine located at the 220 locus in a receptor binding area is mutated into glycine, and the receptor binding affinity of the HA protein is reduced.

Description

technical field [0001] The invention relates to the field of virus genetic engineering, in particular to a highly pathogenic H7N9 avian influenza virus antigen with low receptor binding activity and a preparation method thereof. Background technique [0002] Influenza A virus (influenza A virus) is a representative species of Orthomyxoviridae. According to the objects infected by influenza viruses, viruses can be divided into human influenza viruses, swine influenza viruses, equine influenza viruses, and avian influenza viruses. According to the difference of hemagglutinin protein (HA protein) and neuraminidase (NA), influenza A virus can be further divided into different HA subtypes (H1-H18) and NA subtypes (N1-N11). When people or animals are infected with the virus, it may cause influenza pandemic, and even cause death of people or animals in severe cases. [0003] In March 2013, a new type of H7N9 subtype influenza virus that first appeared in the world caused an epide...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/44C07K14/11C12N15/66C12N15/85
CPCC07K14/005C12N15/66C12N15/85C12N2760/16022
Inventor 王洋陈凌潘蔚绮吕云华董记
Owner GUANGZHOU MEDICAL UNIV
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