PEDV流行株N基因主要抗原表位原核表达及ELISA方法的建立

Prokaryotic expression of N gene major antigen region of PEDV and development of an indirect ELISA based on expressed protein

为建立检测猪流行性腹泻病毒（PEDV）N蛋白抗体的ELISA方法，应用RT-PCR从腹泻仔猪样本中扩增获得了1段PEDV河南流行毒株N基因全长序列，根据对其氨基酸序列的抗原性分析结果，将N基因上2个大的抗原表位区（112～321位氨基酸）克隆至原核表达载体pET-32u，构建pET-32a-N重组质粒并转化至宿主菌BL21（DE3）中。将重组菌在37℃条件下加入IPTG进行诱导使该蛋白获得了高效表达，表达产物为融合蛋白，相对分子质量约41300。Western-blot分析表明，所表达的蛋白可与抗PEDV全病毒小鼠阳性血清发生反应。以纯化的重组N蛋白作为包被抗原建立了检测PEDV抗体的间接ELISA方法，抗原最佳包被量0．226ug／μL，血清最佳稀释度为1：100，二抗最佳稀释度为1：2000，待检血清n450值≥0．28判定为阳性。国内首次用该方法对临床猪血清样本进行了检测，母猪血清PEDV抗体阳性率为84．26％（166／197），仔猪血清阳性率为27．93％（31／111）。结果表明，建立的间接ELISA方法特异性、灵敏性和可重复性良好，可用于-1盏床上PEDV抗体水平的监测和PED流行病学调查。

In order to develope an indirect ELISA to detect anti-PEDV antibody, the complete N genes of PEDV isolated from Henan province were cloned, sequenced and analyzed. Two major an- tigen region of N gene was amplified by RT-PCR and inserted into the expression vector pET- 32a,and the recombinant plasmid was named as pET-32a-N. Fusion protein was expressed in BL21 （DE3） with transfected pET-32a-N and induced by IPTG. The molecular weight of the re- combinant protein was about 41 300 analyzed by SDS-PAGE, and the immunoreaction activity of the recombinant protein was confirmed by Western-blot when mouse positive serum against PEDV was used. An indirect ELISA for detecting antibody against PEDV was developed by using expressed N protein as coating antigen. Results showed that the optimal coating concentration of N protein is 0. 226 μg/well,and the optimal dilution of the serum is 1 - 100 and that of the HRP- SPA is 1 ：2 000. The positive criterion for this ELISA assay is D450 ≥0. 28. When field serum samples were tested with developed the ELISA,the positive rate was 84.26o/00 （166/197） for sow serum,and 27.93o//00 （31/111） for piglets serum. The results indicated that developed ELISA was specific,sensitive,reproducible and suitable as a rapid serology detection method for monitoring anti-PEDV antibody and epidemiologic survey of PED.