Prokaryotic Expression Of Ten Genes Of H3N2Subtype Swine Influenza Virus And Establishment Of Hybridoma Cell Lines Secreting Monoclonal Antibody Against Hemagglutinin

Swine influenza virus is a single stranded negative-strand RNA virusesbelongingto the family of Orthomyxoviridae, and is classified as a type Ainfluenzavirus. There are three types of influenza viruses, identified as A、B and C,each of them is able to infect pigs, and cause the infection of swine influenza.Swineinfluenza is an acute respiratory disease of pigs, characterized by high fever, sudden,runny nose, depression, difficulty breathing, weight loss, exhaustion, cough, repeatedepisodes and so on.By far, there are11subtypes of SIV, including H1N1、H1N2、H1N7、H3N2、H2N3、H3N1、H3N6、H5N2、H4N6、H5N1and H9N2, have beenisolated from pigs.According to the study of serology, etiology and epidemiology,subtypes found in China are H9N2, H5N1, H3N2, H1N2and H1N1.Among thesesubtypes, the prevalence and hazards caused by H1N1and H3N2are relatively serious. Many different subtypes of influenza virus long-termcoexistence in herds, which create conditions to produce rearrangement viruses, willbe a potential threat to the pig industry and human public health and so on.Theinternational academic community has generally believed that pigs can produce thevirus strains with pandemic potential in the crowd.Therefore, the impact of swineinfluenza is not only in its implications for veterinary epidemiology, but also in theprofound public health significance.In this study, A/swine/Henan/1/2010(H3N2) swine influenza virus was isolatedand sequenced by our laboratory. Based on those results, HA, NA, NP, M1, M2, NS1,NS2, PA, PB1, and PB2genes were analyzed by DNAStar5.0, according to majorantigenic domain of the regional, we designed11pairs of primers (for M2designed2)by DNAStar5.0and Primer Premier5.0,by PCR (for M2gene, using the OE-PCRmethod) method to clone them, and then connected the pET-28a(+) or pET-32a(+) toconstruct the corresponding recombinant expression vector and expressed in E.colisystem, which laid the foundation for the development of SIV subtypeH3N2subunitvaccine and specific identification of monoclonal antibodies in the next experiment. 6-week-old BALB/c female mices were immunized by purified H3N2swineinfluenza virus, mouse spleen cells were taken and fused with myeloma cells NS0,then coated enzyme-linked reaction plate with whole virus, and screening by indirectELISA method. After three limited dilution cloning, finally got a stable monoclonalantibody secreting anti-H3N2-positive hybridoma cell lines, named1C10.Aftertesting, the antibody titer of mice ascites was1∶51200; Indirect immunofluorescenceindicates that itcould bind with the antigen specifically.The results of cross reactiontest showed that the monoclonal antibody did not cross reactivity with H1、H5、PRRS、PCV2、PPV.With the10proteins of H3N2as antigen to be used to coatenzyme-linked reaction plate, indirect ELISA results showed that the monoclonalantibody specifically recognized the H3N2hemagglutinin (HA) protein，and it showedthat prokaryotic expression ofH3N2HA had a natural activity after refolding, whichlaid the foundation for the development of SIV subtypeH3N2subunit vaccines;The obtainedmonoclonal antibody of1C10laid the foundation fortheestablishment ofSIV rapid diagnostic methods as well as more in-depth study of SIV.