旋毛虫丝氨酸蛋白酶抑制因子基因的克隆与原核表达

Cloning and prokaryotic expression of serine proteinase inhibitor gene of Trichinella spiralis

根据GenBank中旋毛虫丝氨酸蛋白酶抑制因子(Tsserpin)基因序列设计1对引物,从旋毛虫肌幼虫提取总RNA,应用RT-PCR扩增获得目的基因片段,进行限制性酶切后连接到表达质粒载体pET30a,构建重组表达质粒pET30a-Tsserpin,转化大肠杆菌DH5α,筛选阳性克隆,经PCR、限制性酶切分析及测序鉴定后,转化大肠杆菌BL21(DE3),以IPTG进行了诱导表达。SDS-PAGE分析表达产物,表达产物纯化后用Western-blotting鉴定Tsserpin重组蛋白的抗原性。PCR与酶切鉴定结果显示,构建的重组表达质粒pET30a-Tsserpin与预期结果一致。测序结果表明,插入片段为1122bp,编码373个氨基酸,与GenBank中旋毛虫Tsserpin基因的相似性为99%。含有pET30a-Tsserpin重组表达质粒的大肠杆菌BL21(DE3)诱导后得到了高效表达,SDS-PAGE分析结果显示,表达产物的分子质量约为48.5ku,而且主要以包涵体形式存在。Western-blot分析结果表明Tsserpin重组蛋白可以被旋毛虫感染猪阳性血清特异性识别,提示其具有良好的抗原性,可以作为猪旋毛虫病免疫诊断的候选抗原。

A pair of specific primers derived from Trichinella spiralis serine proteinase inhibitor gene（Tsserpin） in GenBank was designed and used to amplify Tsserpin gene by RT-PCR from total RNA extracted from T.spiralis muscle larvae.The RT-PCR product was purified,digested and ligated into the expression vector pET30a and identified by PCR,restriction digestion and sequencing after transformation into Escherichia coli DH5α.pET30a-Tsserpin plasmid was transformed into E.coli BL21（DE3） and induced by IPTG.The expressed product was analyzed by SDS-PAGE and the antigenicity of the purified recombinant protein was identified by Western-blot with T.spiralis-infected swine serum.The EcoRⅠ＋XhoⅠ digestion,PCR amplification and sequencing analysis demonstrated that the constructed pET30a-Tsserpin contained a insert of 1122bp encoding 373 amino acids,which shared 99% identity to Tsserpin gene in GenBank.The recombinant protein was highly expressed in the form of inclusion bodies with the molecular weight of about 48.5ku.Western-blot analysis indicated that the purified recombinant protein could be specifically recognized by T.spiralis-infected swine serum with a good antigenicity and might be a candidate antigen for the immunodiagnosis of swine trichinellosis.