Evolution Of H3N2 Subtype Swine Influenza Virus And Establishment Of An Indirect ELISA Diagnostic Method With The Recombinant HA1 Protein

H3N2 swine influenza are recognized as a continuing theat to animal husbandy and human public health. To gain insight into the molecular epidemiology, genetic evolution of Swine Influenza virus (SIV) and spread of SI in China from 2004 to 2005, serological and etiological studies of SI were carried out in some provinces and cities, 1238 samples including nasal swabs and organs were collected from pig flocks. From these samples, 5 strains H3N2 SIV were isolated.Compared with the HA and NA gene sequences of 5 strains of H3N2, They had a close relationship each other. To understand the molecular evolution of the H3N2 virus isolated in China, the entire genome of A/Swine/Gongdong/9/2005 (H3N2) was cloned and sequenced, Phylogenic analysis revealed that the virus originated from recent human. Comparison of amino acid according to HA1 from others reference strains of influenza virus in GenBank, there only some amino acid sites had mutation.The complementary DNA of 1.76kb HA gene was prepared from the viral gene RNA by RT-PCR. The amplified fragment was cloned into the plasmid vector pMD18-T. After identification by PCR, the HA1 fragment was got by PCR, the signal sequence of coding HA protein was deleted. Then the HA1 gene without signal sequence was subcloned into the downstream of the pET30a vector named as pET-HA1. We confirmed that the target gene fragment was inserted into the pET30a vector genome by PCR and restriction enzyme. The recombinant vector was transferred into BL21(DE3) and induced by 1Mm IPTG. SDS-PAGE result showed that the HA gene was expressed in prokaryotic expression system, and the target protein take the proportion of 40% in the whole protein by scanning assay. Additionally, the Western-blot assay proved the HA1 protein of H3N2 subtype swine influenza virus having good antigenic activity.Taking the purified recombinant fusion protein as antigen, We established indirect ELISA method for detecting H3 SIV antibody. To validate the assays, HA1 ELISA was performed with standard positive and negative sera of H3 SIV. Modified indirect reaction condition of ELISA: the antigenic optimal coating concentration 1.5μg/100μl, the dilution of serum sample was 1:100, the working concentration of HRP-labeled rabbit anti-swine IgG was 1:1000. The tests showed no cross-reactivity with antibodies to H1N1 SIV, H5N1 SIV, H9N2 SIV, PPV, PRRSV and PRV. Compared with HI, the diagnostic sensitivity, specificity, and accuracy of HA1 ELISA were more optimization after detecting 96 serum samples collected from different coughing herds in China. The coincidence rate was 86.5% between indirect and HI test. It could be used as an efficient and sensitive method to detect antibodiy against H3 subtype SIV.