H1亚型猪流感病毒抗体间接ELISA检测方法的建立及其应用

Indirect ELISA for detection of antibodies against swine influenza virus (H1N1)

摘要:

为建立H1亚型猪流感病毒抗体检测方法，扩增了H1N1亚型猪流感病毒流行株的血凝素基因HA1部分，构建原核表达载体pET30a-HA1，并转化大肠杆菌BL21表达重组蛋白。对重组蛋白包涵体进行变性、复性和Ni-NTA亲和层析纯化。以纯化后的蛋白作包被抗原，建立间接ELISA检测方法。利用该检测方法检测了2008?2009年采集的猪血清785份，阳性率为15.54％，不同省份的阳性率存在差异 (8％~47％)。以IDEXX相关试剂盒检测结果作为参照，该方法的诊断特异性达到91％，诊断敏感性达到95％。

Abstract:

In order to detect antibody against swine influenza virus (H1N1), HA1region of hemagglutinin gene in epidemic swine influenza virus (H1N1) strain was amplified and subcloned into prokaryotic expression vector pET30a. Then recombinant HA1 protein was expressed by Escherichia coli BL21. The purified recombinant HA1 protein was obtained after the treatment of denaturing, refolding and affinity chromatography with immobilized nickel chelating NTA (Ni-NTA). An indirect enzyme-linked immunosorbent assay (ELISA) method was established using the purified protein as antigen. Then 785 swine serum samples collected during 2008?2009 were detected by this method, and the positive ratio was 15.54%. There were diversities among provinces (8%?47%). The diagnostic specificity and diagnostic sensitivity of this method arrived at 91% and 95% respectively, using the results of IDEXX ELISA kit as reference.