nsp7的原核表达、纯化与免疫原性鉴定  

Prokaryotic expression,purification and characterization identification of nsp7

作　　者：刘利娅[1] 李淑慧[2] 严峻 胡川闽[2] 余晓东[1] 

机构地区：[1]重庆师范大学生命科学学院,401331 [2]第三军医大学医学检验系临床生物化学教研室,重庆400038 [3]重庆业为基生物科技有限公司,400039

出　　处：《国际检验医学杂志》2012年第9期1025-1027,共3页International Journal of Laboratory Medicine

基　　金：2011年重庆市科技攻关项目

摘　　要：目的获得高纯度和高免疫原性的猪繁殖与呼吸综合征(PRRS)病毒非结构蛋白7(nsp7)。方法 nsp7编码DNA片段是PRRS病毒基因组裂解产物,酶切后连接至克隆质粒pET-28a构建为表达质粒pET-28a-nsp7,转化至大肠杆菌BL21中,IPTG诱导蛋白表达,采用Ni2+-NTA树脂纯化、Q阴离子交换层析、镍柱浓缩3个连续的过程进行纯化和用SDS-PAGE、Western blot、ELISA法进行鉴定。在此基础上通过间接ELISA法对135份血清样品进行检测,并与进口LSI-ELISA试剂盒进行比较。结果 SDS-PAGE分析表明表达产物的相对分子质量约为34×103,与理论值相一致;该蛋白产量高、可溶性好,纯度达95%;Western blot和间接ELISA法鉴定结果显示该重组蛋白能够与PRRS病毒的阳性血清反应,与进口LSI-ELISA试剂盒相比其符合率达91%。结论成功构建了nsp7蛋白的表达与纯化系统,获得了高纯度和高免疫原性的nsp7蛋白,为PRRS血清学诊断奠定了基础。Objective To obtain nonstructural protein 7（nsp7） with high purity and high immunogenicity.Methods DNA fragment coding nsp7,which was cleaved product of the genome of porcine reproductive and respiratory syndrome virus（PRRSV）,was cloned into pET-28a vector to construct expression plasmid pET-28a-nsp7,that was then transformed into Escherichia coli（E.coli） BL21 strain.Recombinant protein of nsp7 was expressed by induction with IPTG,purified by nickel-metal chelate chromatography,Q anion exchange chromatography and nickel concentrate and identified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis（SDS-PAGE）,Western blotting and enzyme linked immunosorbent assay（ELISA）.Indirect ELISA was used to detect the immunogenicity of nsp7 through compared with the LSI-ELISA kit.Results SDS-PAGE showed that the size of the nsp7 protein was 34×10^3,being consistent with theoretical data.Recombinant protein of nsp7 was easily expressed,with good solubility and with purity 95%.Western blot and indirect ELISA indicated that nsp7 recombinant protein was able to react with serum samples positive with PRRSV,and the detected results were high correlated with those detected by LSI-ELISA.Conclusion An expression and purification system of nsp7 was successfully constructed,and recombinant nsp7 protein with high purity and high immunogenicity was obtained,which lay a foundation for further study of serological diagnosis of PRRS.

关 键 词：猪呼吸与生殖综合征病毒 病毒非结构蛋白质类 原核细胞 基因表达 酶联免疫吸附测定 纯化 

分 类 号：S858.28[农业科学—临床兽医学]