Expression Of HA2 Subunit Of H7N9 Avian Influenza And Preparation Of Its Monoclonal Antibodies

In February 2013,the H7N9 virus crossed from bird to human.The H7N9 case of Avian influenza was first detected in Shanghai in late March and subsequently appeared in Beijing,Henan,Anhui and Jiangsu.Until the end of 2016,the World Health Organization(WHO)has reported more than 800 cases of infected people.Infected patients often develop fever,cough and other symptoms and rapidly turn to acute pneumonia,resulting in organ exhaustion with a mortality rate of 35%～40%.At present,the first-line drugs against avian influenza,such as neuraminidase inhibitors and ion channel inhibitors(M2 protein blockers),have been shown to be resistant to M2 protein blockers by virus and have been reported to produce 10%of the virus resistant to neuraminidase inhibitors.In recent years,some vaccines and antibody drugs developed by methods of genetic engineering have shown a good application prospect.With the improvement of technology,the deficiency of traditional methods has been constantly made up.H7N9 belongs to Orthomyxoviridae,Influenzavirus A,which genome includes single-stranded negative-sense RNA with 8 segments and encodes a total of 11 proteins.Avian flu can be divided into Low pathogenic AIV(LPAIV)and Highly pathogenic AIV(HPAIV)according to its pathogenicity in the bird.Influenza A virus can be divided into several subtypes.There are 16 forms(HA～H16)of HA and 9 forms(N1～N9)of NA according to the antigenic difference of hemagglutinin(HA)and neuraminidase(NA).Hemagglutinin(HA)is divided into the HA 1 subunit and the HA2 subunit,both of which are linked by a disulfide bond.HA1 plays a key role in the viral recognition of sialic acid glycoprotein receptors on the surface of the membrane.HA2 contains more conservative binding sites than HA1 and plays a major role in the process of entry of the viral nucleic acid into the host cell after fusion of the virus with the membrane.Neutralizing antibody against HA can effectively block the virus infection mechanism in vivo and induce multiple antiviral responses,which lays a good foundation for the prevention and control of avian influenza virus.In this article,HA7 subunit of H7N9 virus was genetically engineered by using the methods established in our laboratory.The eukaryotic expression vector was constructed and introduced into CHO cells.Then we screen out stable expression strain and utilize CHO eukaryotic expression system to highly express H7N9 HA2 antigen protein.The purified recombinant H7N9 HA2 antigen was sufficiently emulsified with Freund’s adjuvant,which was immunized in BALB/c mice.The hybridoma cells obtained by fusing myeloma cells and mouse spleen cells that were detected by enzyme-linked immunosorbent assay(ELISA),screened by multiple dilution-limited cloning,and finally we get the 4 hybridoma cell strains that can stably expressing the anti-H7N9 HA2 monoclonal antibody named as:4C6-1、4C6-3、4F1-2、4F1-3.These three hybridomas were passaged 10 times in succession and resuscitation after cryopreserved,which did not affect the stability of their secreted antibodies.After the H7N9 avian influenza virus infects MDCK cells,the monoclonal antibody prepared in this experiment is used as a primary antibody to incubate.Immunofluorescence results showed that the monoclonal antibody prepared in this experiment can be effectively hybridized with the H7N9 virus.Chicken embryo protection experiments showed that the protective effect of antibodies on chicken embryos was positively correlated.When the amount of antibodies reached 400 μg,100%protection of chick embryos could be achieved.Preparation of monoclonal antibody against H7N9 HA2 antigen provides a basis for the rapid identification of H7N9 virus and the preparation of antibody drugs.