鸭病毒性肝炎病毒VP1基因表达及其抗体检测ELISA方法的建立

Expression of VP1 gene and ELISA detection of antibodies against duck hepatitis virus

摘要:

【目的】研究DHV VP1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ELISA方法。【方法】采用RT-PCR技术，扩增VP1基因，与pMD18-T载体进行连接，构建DHV VP1基因克隆重组质粒。然后定向插入到pET-32a（+）表达载体，筛选原核表达载体pET-32a-VP1，进行ITPG诱导表达分析。以纯化的重组蛋白为抗原建立间接ELISA方法并初步应用于临床。【结果】DHV VP1基因可在大肠杆菌中稳定、高效地表达。Western blot检测表明，表达的重组蛋白能与鸭肝炎阳性血清发生特异性反应。确定间接ELISA方法的抗原最佳包被浓度为5 mg/孔， 血清最佳稀释度为1∶100，临界值为OD450值≥0.302，建立的ELISA方法具有较好的敏感性、特异性和重复性。通过对80份血清样品的检测表明，该方法与中和试验的符合率为97.5%，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DHV VP1重组蛋白为抗原建立的间接ELISA方法可用于鸭病毒性肝炎抗体的检测。

Abstract:

[objective] We developed an indirect enzyme-linked immunosorbent assay(ELISA) by the recombinant VP1protein of duck hepatitis virus (DHV) expressed in Escherichia coli to detect antibodies against DHV. [Methods] The VP1 gene of DHV was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and cloned into pMD18-T and pET-32a vectors to get a prokaryotic expression vector pET-32a -VP1. DHV VP1 gene was expressed and analyzed. A method of enzyme-linked immunosorbent assay (ELISA) was developed and studied. [Results] We obtained the recombinant plasmid pET-32a-VP1 with correct sequence and orientation. DHV VP1 gene was expressed in E. coli BL21(DE3) at a high level and had good immunoreactivity by SDS-PAGE and western blot. The optimum working concentration of antigen was 5.0 mg in 100 mL per well, the working concentration of serum samples was 1∶100 dilution and the critical value was OD450≥0.302 for the ELISA assay. The rate of coincidence of ELISA and virus neutralization test(VN) was 97.5% by detecting 80 serum samples. The method was specific, sensitive and could be applied for antibody detection of maternal antibody and the rule of antibody growth and decline in immunizing ducklings. [Conclusion] The ELISA method developed by the purified recombinant protein could be used to detect antibodies against DHV.