猪瘟病毒NS3蛋白的原核表达纯化和抗体制备

Expression of the Nonstructural Protein 3(NS3) of Classical Swine Fever Virus(CSFV) in E.coli and Production of Polyclonal Antibody against NS3

以猪瘟病毒(CSFV)石门株全长cDNA克隆pT7SM为模板,PCR扩增CSFVns3基因N末端截短的cDNA片段.PCR产物经限制性内切酶BglⅡ和SalⅠ双酶切消化后,克隆到经BamHⅠ和SalⅠ双酶切消化的pET-28a表达载体,构建重组质粒pET-ΔNS3,转化E.coliBL21-codonPlus(DE3),IPTG诱导重组蛋白表达.采用SDS-PAGE分离纯化非结构蛋白3(NS3)重组蛋白,免疫家兔,制备多克隆抗体.ELISA法及间接免疫荧光检测结果显示,该多克隆抗体效价在1×105以上,能分别与在大肠杆菌中表达的NS3重组蛋白或CSFV感染细胞中表达的NS3天然蛋白发生特异性结合反应.制备的NS3重组蛋白可用作建立NS3抗体ELISA检测方法的包被抗原;NS3抗体可用于检测CSFV病毒感染和NS3功能研究中的蛋白质鉴定.

In order to express and purify the recombinant nonstructural protein 3 （NS3） of classical swine fever virus Shimen strain, a truncated ns3 gene fragment was amplified by PCR with the full-length classical swine fever virus （CSFV） eDNA clone pT7SM as template. After digestion with Bgl Ⅱ and Sal Ⅰ , the PCR product was cloned into pET-28a vector digested with BamH I and Sal Ⅰ. The recombinant plasmid pET-△NS3 was analyzed by restriction enzyme digestion and sequencing,and then was transformed into E. coli BL21-CondonPlus （DE3）-RIL. A truncated NS3 protein fused with the polyhistidine tag was expressed by the induction of IPTG and identified by SDS-PAGE analysis. Polyclonal antibody against NS3 was raised after injecting rabbits with the purified recombinant NS3 protein. The results from ELISA and indirect immunofluoreseence indicated that the antibody titer was higher than 1×10^5 , and showed excellent specificity for CSFV NS3 protein. These results might be contributive to detect CSFV infection and to develop an indirect ELISA for the detection of antibodies against CSFV NS3.