H3N2亚型猪流感病毒重组HA1蛋白间接ELISA诊断方法的建立

Establishment of ELISA Based HA1 Protein for Detecting Antibodies against H3N2 Subtype Swine Influenza Virus

以pMD18-HA质粒为模板,PCR扩增HA1基因片段并与pET30a连接后,转化宿主菌BL21(DE3),IPTG诱导表达HA1蛋白,对表达蛋白进行SDS-PAGE及Western blotting检测。结果表明,表达的重组HA1蛋白具有良好的反应原性,分子量约为45 ku。表达产物经过纯化作为包被抗原建立了检测H3亚型猪流感抗体的间接ELISA方法。该方法具有较高的特异性和敏感性,重复性良好,通过检测40份血清,与HI检测结果相比较,其符合率达86.5%。该方法为检测H3亚型猪流感抗体提供了一种快速、准确、简便的技术手段。

The HA1 gene of H3N2 subtype swine influenza virus （SIV） was cloned into expression plasmid pET-30a. And the recombinant plasmid DNA was named as pET-HA1. Then it was transformed into E. coli BL21 （DE3）, the recombinant protein was expressed in E. coli BL21 （DE3）by induction of IPTG. The expressed HA protein was identified by SDS-PAGE and Western blotting analysis. The results showed that HA1 protein was a 45 ku protein with immunogenieity. The purified HA protein was used to establish the indirect ELISA for detection of the antibodies against H3 subtype of SIV. The assay has excellent specificity, high sensitivity and excellent reproducibility. The total of 40 serum samples were randomly collected from field and evaluated by ELISA with recombinant HA1 and HI test, the coincidental rate between the two tests is 86.5%. These results showed that recombinant HA1 based ELISA is specific, sensitive and easy to perform for serologically diagnosis of SIV infection.