PRRSV Nsp2高变区基因的表达及免疫学特性鉴定

Expression of Hypervariable Region in PRRSV Nsp2 Gene and Identification of Its Immunogenicity Characteristics

本研究分别扩增从"高热病"猪体内分离株(TA-12)及美洲经典株ATTC-VR2332 PRRSV Nsp2基因的高变区,亚克隆至表达载体pET-28a中,转化至E.coliBL21中,在IPTG的诱导下进行表达。用SDS-PAGE、Western-blot对表达产物进行鉴定。表达产物纯化后免疫BALB/c小鼠,对其免疫原性进行研究。建立了以纯化的重组蛋白作为包被抗原的间接ELISA,确定了临界值,用建立的ELISA方法并对400份猪血清进行了检测和应用。结果表明,成功的表达了分离株TA-12及ATTC-VR2332 Nsp2基因的高变区。表达蛋白纯化分别免疫小鼠后,获得多克隆抗体,其抗体效价均为1∶100000。优化的ELISA条件为:包被缓冲液为磷酸盐缓冲液,800 ng/孔,血清样品稀释浓度为1∶100。临界值为0.4。血清检测结果表明以两种蛋白为包被抗原均为阳性的血清最多,占到总数的58.5%,前者阳性后者阴性的血清阳性率28%,前者阴性后者阳性的血清阳性率8.75%,二者均为阴性的占到4.75%。为建立区别两种毒株的鉴别诊断间接ELISA试剂盒奠定基础。

Hypervariable region in Nsp2 gene of isolate（TA-12） and ATTC-VR2332 were cloned and subcloned into pET-28a prokaryotic expression system to get recombinant protein after induction of IPTG.The recombinant proteins were identified by SDS-PAGE and Western-blot.BALB/c mice were immunized to analyze their immunogenicity.An indirect ELISA was developed coating with recombinant protein.400 sera were detected by the developed ELISA.The results showed hypervariable region in Nsp2 gene of isolate and ATTC-VR2332 were over expressed in pET-28a vector.High antibody levels were both acquired after immunized with purified products.The optimized conditions for ELISA were phosphates balanced solution as coating buffer,800 ng/well with purified products,sample area diluted as 1：100,the cutoff value is 0.4.The positive sera occupied 58.8 % detecting with both recombinant protein.Sera positive with recombinant protein from isolate and negative form ATTC-VR2332 was 28 %.Sera negative with recombinant protein from isolate and positive form ATTC-VR2332 was 8.75 %,both negative was 4.75 %.Two recombinant proteins could discriminate different sera.It provide the important foundation for development of an ELISA to discriminate two type virus in serology level.