猪繁殖与呼吸综合征病毒重组N蛋白的高效表达及间接ELISA方法的建立  

High-level expression of N protein of PRRSV Hn-1/06 strain and establishment of ELISA diagnosis based on the recombinant fusion protein

作　　者：夏平安[1] 尹彦涛[1] 李素平[1] 党占国[1] 王建举[1] 王中明[1] 李伟娟[1] 崔保安[1] 

机构地区：[1]河南农业大学牧医工程学院,河南郑州450002

出　　处：《中国兽医学报》2009年第5期537-541,共5页Chinese Journal of Veterinary Science

摘　　要：根据GenBank中猪繁殖与呼吸综合征病毒(PRRSV)美洲株ATCC VR2332的N蛋白基因序列,设计合成1对特异性引物,用RT-PCR方法从PRRSV Hn-1/06株中克隆了大小为372bp的N蛋白基因(EU025136)。将N蛋白基因亚克隆到原核表达载体pET-32a中构建了重组表达质粒pET-N,在IPTG的诱导下成功表达了相对分子质量约为33000的可溶性融合蛋白N-His;经SDS-PAGE和凝胶薄层扫描分析,融合蛋白的表达量约占细菌总蛋白量的29%。将融合蛋白加入镍琼脂糖凝胶树脂层析柱,经300mmol/L咪唑磷酸盐缓冲液洗脱,其纯度可达91%。用Western blotting分析纯化后的融合蛋白,显示良好的生物学活性。利用纯化融合蛋白作包被抗原及优化ELISA反应条件,建立了可检测抗PRRSV N蛋白抗体的间接ELISA方法(N-ELISA),并将所建立的N-ELISA与国外进口的PRRS检测试剂盒IDEXX-ELISA对200份临床送检血清进行平行检测,阳性符合率为92.7%。结果表明,本试验建立的N-ELISA与IDEXX-ELISA具有同样高的特异性。A pair of specific primers was designed and synthesized according to the published N gene sequence of PRRSV from GenBank（PRU87392）,and the N gene of PRRSV strain HN-1/06 was amplified by RT-PCR from a template of total PRRSV RNA. The nucleotide sequence of N gene was determined （EU025136）. The N gene was cloned into pET-32a vector to generate the recombinant expressing plasmid pET-N. The PCR-generated plasmids was verified by sequencing of the entire insert. Then pET-N was transformed into the host cell BL21（DE3） and the expression procedure was optimized. The N-His fusion protein was successfully obtained with the induction of 1.0 mmol/ L IPTG. The N-His fusion protein could specifically react to antiserum against PRRSV by Western blotting. The N-His fusion protein was further purified and used as a packaging antigen to establish a novel PRRSV N-ELISA diagnosis assay. The comparison between N-ELISA and kit IDEXX-ELISA showed that the positive results of two methods had 92.7 percent agreement by detecting 200 sera samples, indicating that the indirect N-ELISA was the same specific and sensitive as kit IDEXX-ELISA.

关 键 词：PRRSV N蛋白 原核表达 ELISA 

分 类 号：S852.65[农业科学—基础兽医学]