旋毛虫成虫丝氨酸蛋白酶抑制剂的原核表达及免疫原性研究

Prokaryotic expression of TsAdSPI gene from Trichinella spiralis adult and immunogenicity study of TsAdSPI

以旋毛虫成虫的总RNA为模板,应用RT-PCR扩增获得旋毛虫成虫丝氨酸蛋白酶抑制剂(TsAdSPI)基因。经克隆鉴定正确后,将TsAdSPI基因连接到pET-28a表达载体,转化至大肠杆菌BL21(DE3)中进行IPTG诱导表达,并将纯化后的目的蛋白免疫新西兰大白兔,制备多克隆抗体。应用Western-blot及ELISA方法对重组蛋白的免疫原性和抗体效价进行分析。SDS-PAGE结果显示,目的蛋白分子质量为44 k Da,以包涵体形式表达。经Western-blot和ELISA方法分析,制备的多克隆抗体效价可高达1∶51200,表明该蛋白具有较好免疫原性。表明TsAdSPI蛋白可作为旋毛虫感染早期血清学诊断方法的候选抗原,为新型的抗旋毛虫及相关寄生虫药物研发打下结实基础。

In this study, we used total RNA from adult Trichinella as a template, using RT-PCR amplified TsAdSPI gene and then cloned on to the pMD18--T vector. The target fragment was ligated to the expression vector pET-28a after being identified cor- rectly. Expression vector pET-28a was transformed into E.coli BL21 （DE3）and induced by IPTG and then used to immunize New Zealand rabbits with purified protein to make polyelonal antibodies. Western-blot and EIlSA were oppeied to analyse the reactionogenicity of recombinant protein and valence of antibody. SDS-PAGE showed that the molecular mass of protein is 44 kDa, expressed in the form of inclusion body. By Western-blot and ELISA analysis, TsAdSPI protein showed good reaetionogenicity, valence of antibody of polyclonal antibody reached up to 1 ： 51200, indicating that the protein had good immunogenicity. As the research results showed, TsAdSPI protein could be used as an early candidate antigen of Triehinella serological diagnostic methods, which laid solid foundations for a new type of anti-parasite Trichinella and related drug development.