胞内劳森菌LsaA蛋白的原核表达及间接ELISA抗体检测方法的建立

Prokaryotic Expression of LsaA Protein of Lawsonia Intracellularis and Development of a LsaA-based Indirect ELISA for Antibody Detection

为建立胞内劳森菌特异性抗体检测方法,将密码子优化合成的胞内劳森菌表面蛋白LsaA的编码基因克隆于pET32a(+)载体,在大肠杆菌BL21(DE3)中诱导表达。用纯化的重组LsaA蛋白作为包被抗原,通过一系列条件优化,建立了检测胞内劳森菌抗体的间接ELISA方法。结果显示:该方法可以特异地检测抗胞内劳森菌抗体,与大肠杆菌、猪霍乱沙门菌、猪流行性腹泻病毒和猪传染性胃肠炎病毒等常见猪腹泻病原的抗血清均无交叉反应。来自PCR检测粪便呈胞内劳森菌阳性猪的血清,用所建立ELISA检测均为胞内劳森菌抗体阳性。该方法具有较好的敏感性和重复性,阳性血清经过1∶800稀释检测结果仍为阳性,批内和批间重复试验的变异系数分别为0.352%~2.752%和0.877%~3.000%。应用建立的ELISA方法对河南省部分地区猪场胞内劳森菌的感染情况进行了血清流行病学调查,结果显示被检地区猪场均存在不同程度的胞内劳森菌抗体阳性,阳性率在13.3%~57.1%,平均阳性率为30.6%。结果表明,本研究所建立的ELISA方法可以用于胞内劳森菌抗体的检测,为胞内劳森菌感染的监测和流行病学调查提供了方法。

In order to establish a method for detecting specific antibody against Lawsonia intracellularis,the codon-optimized LsaAgene encoding surface protein LsaA of L.intracellularis was cloned into pET32a（＋）vector,and the expression of LsaA protein was induced in E.coli host BL21（DE3）.After optimization of a series of conditions,an indirect ELISA for detecting antibodies against L.intracellularis was established by using purified LsaA protein as coating antigen.The results showed that the anti-L.intracellularis antibodies were detected specifically by the established ELISA,and there were no cross-reactions with the antibodies against common diarrhea-causing pathogens such as E.coli,Salmonella choleraesuis,porcine epidemic diarrhea virus and transmissible gastroenteritis virus.Serum samples from fecal L.intracellularis-positive pigs which were identified by PCR,were also positive for anti-L.intracellularis antibodies detected by the established ELISA.The ELISA has good sensitivity and repeatability.Anti-L.in-tracellularis positive serum with 1：800 dilution was still ELISA positive.The intra-and interassay coefficient of variation was 0.352%-2.752% and 0.877%-3.000%,respectively.Seroepidemiological prevalence of L.intracellularis infection in partial pig farms in Henan province was investigated by using the established ELISA.The results indicated that all the tested pig farms were anti-L.intracellularis antibody positive with positive rate of 13.3%to 57.1%,with average positive rate of 30.6%.The results showed that the established ELISA in this study can be used for the detection of antibodies against L.intracellularis,and provides a method for the monitoring and epidemiological investigation of L.intracellularis.