鸭甲肝病毒1型和3型间接ELISA方法的建立与应用

Indirect ELISA for simultaneous detection of antibodies against duck hepatitis A type 1 and 3 viruses

摘要:

摘要:【目的】研究鸭甲肝病毒(Duck hepatitis A virus，DHAV)1型VP3和3型VP1串联基因在大肠杆菌中的表达，并以纯化的重组蛋白为抗原建立鸭病毒性肝炎抗体检测的间接ELISA方法。【方法】设计2对特异性引物，提取鸭甲肝病毒1型(DHAV-1)和3型(DHAV-3)的RNA，分别RT-PCR扩增得到714bp的DHAV-1 VP3和720bp的DHAV-3 VP1基因，并克隆至pMD18-T载体中，然后依次将DHAV-1 VP3和DHAV-3 VP1定向插入pET-32a (+)表达载体，获得重组表达载体pET-1VP3-3VP1，进行IPTG (Isopropyl-beta-D-1- thiogalactopyranoside)诱导表达分析，以纯化的重组蛋白为抗原建立间接ELISA方法并应用于临床。【结果】经IPTG诱导表达，在大肠杆菌中可稳定、高效地表达DHAV-1VP3-3VP1蛋白。Western blot检测结果表明，表达的重组蛋白与DHAV-1和DHAV-3阳性血清均能发生特异性反应。确定间接ELISA方法的抗原最佳包被浓度为1.0μg/孔，血清最佳稀释度为1∶200，临界值为OD6 50值≥0.38，建立的间接ELISA方法具有较好的敏感性、特异性和重复性。该方法与中和试验分别检测DHAV-3阳性血清，两种方法的符合率各为96.3%和96.7%;初步临床应用结果表明该方法可用于雏鸭母源抗体和免疫后抗体的消长变化的检测。【结论】以大肠杆菌表达的DHAV-1VP3-3VP1重组蛋白建立的间接ELISA方法可用于DHAV-1和DHAV-3抗体的检测。

Abstract:

Abstract:［Objective］ To simultaneously detect antibodies against Duck hepatitis A type 1 (DHAV-1) and type 3 (DHAV-3) viruses，we developed an indirect enzyme-linked immunosobent assay (ELISA) with bacterially expressed recombinant viral protein as antigen in Escherichia coli． ［Methods］We amplified the full-length VP3 gene of DHAV-1 and the full-length VP1 gene of DHAV-3 through reverse transcription-polymerase chain reaction (RT-PCR) and then cloned them into pET-32a expression vector，designated as pET-1VP3- 3VP1．The fusion protein DHAV- 1VP3-3VP1 expressed correctly and was subsequently used to develop an indirect ELISA assay．［Results］DHAV-1VP3-3VP1 fusion protein expressed in BL21 ( DE3) cells following induction by Isopropyl-beta-D-1-thiogalactopyranoside (IPTG)．The expressed protein was very antigenic and reactive to virus-specific antibodies in western blot assay．The optimal working concentration for coating antigen was 1.0 μg per well and the working concentration of serum samples was 1:200 dilution and the cut-off value that distinguished the positive from negative serum samples was OD650≥0.38．［Conclusion］The ELISA method based on the prokaryotic expression of VP3 (DHAV-1) and VP1 proteins (DHAV-3) can be used effectively for the clinical detection antibodies against DHAV-1 and DHAV-3.