H9N2亚型猪流感病毒血凝素基因的表达及LAT诊断方法的研究

根据H9N2亚型猪流感病毒血凝素基因序列设计并合成引物,从本室分离保存的H9N2亚型猪流感病毒中扩增出血凝素基因,并将其克隆进pMD18-T载体,限制性酶切及序列测定后,进一步将其信号肽缺失并亚克隆到pGEX-KG中,使其在E.coli BL21（DE3）中与GST融合表达。SDS-PAGE显示表达的融合蛋白的相对分子质量约为90 000。Western印迹表明表达的蛋白质具有免疫学活性。表达产物位于包涵体中,包涵体经变性、复性处理,利用复性产物作为抗原,经碳二亚胺（EDAC）将表达产物和羧基化的乳胶共价偶联,建立了H9亚型猪流感病毒抗体的乳胶凝集试验的检测方法,结果表明,应用HA重组蛋白作为诊断抗原具有特异性强、敏感性高、重复性好的特点,可用于H9亚型猪流感抗体水平监测、流行病学调查和现地疫病普查。

Expression of hemagglutinin of swine influenza virus(SIV) and the development of research of latex agglutination test

A latex agglutination test(LAT) was developed for quick detection of hemagglutinin serum antibodies of H9 swine influenza virus(SIV).The HA gene of H9N2 SIV was amplified by RT-PCR from a strain of field isolated H9N2 SIV,and its identity was confirmed by sequencing.The HA gene was subcloned into prokaryotic expression vector pGEX-KG with its secretion signal sequence removed.The expressed HA-GST fusion protein in E.coli BL21 was characterized by SDS-PAGE and Western-blotting analysis as a 90 000 protein with immunogenicity.The fusion protein was present primarily in inclusion bodies and was purified via denaturation and renenaturation.The HA-GST fusion protein was covalently linked to carboxylated polyethylene latex beads by EDAC.The sensitisation condition such as ionic strength of the dilutingmixture pH,concentration,antigen,the times of linking were optimized.282 serum were detected by this LAT and HI simultaneously,the result shows that the assay has excellent specificity to H9 SIV,sensitivity and reduplication.It could be used for quick detection of serum antibodies and epidemiological study of H9 SIV.