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目的在原核系统表达并初步鉴定细粒棘球绦虫硫氧还蛋白过氧化物酶(EgTPx)基因重组蛋白的抗原性。方法对已构建的重组质粒pMD-18T-EgTPx进行限制性酶切,获取目的基因片段后连接到表达质粒载体pET30a,构建重组表达质粒pET30a-EgTPx,转化大肠杆菌DH5α。筛选阳性克隆,经限制性酶切分析、PCR及测序鉴定后,转化大肠杆菌BL21(DE3),以异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析表达产物,对表达产物纯化后用Western-blot方法和ELISA鉴定重组EgTPx蛋白的抗原性。结果构建的重组表达质粒pET30a-EgTPx阳性克隆经PCR与双酶切鉴定,与预期结果一致,测序鉴定无基因突变,开放阅读框正确;含有pET30a-EgTPx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显示表达产物分子量约为27 kDa,而且主要以包涵体形式存在;Western-blot和ELISA结果表明EgTPx重组抗原可以被棘球蚴感染羊阳性血清特异性识别。结论成功构建了pET30a-EgTPx表达质粒,EgTPx重组蛋白得到了高效表达,表达产物具有良好的抗原性。
Objective To express and preliminarily identify the antigenicity of the recombinant protein of EgTPx gene of Echinococcus granulosus in prokaryotic system. Methods The constructed recombinant plasmid pMD-18T-EgTPx was digested with restriction enzyme. The target gene fragment was obtained and ligated into the expression vector pET30a. The recombinant plasmid pET30a-EgTPx was constructed and transformed into E. coli DH5α. The positive clones were screened and identified by PCR, restriction endonuclease analysis and sequencing. The recombinant plasmids were transformed into E. coli BL21 (DE3) and induced by isopropyl-β-D-thiogalactoside (IPTG). The expressed product was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The expressed product was purified and the antigenicity of the recombinant EgTPx protein was identified by Western-blot and ELISA. Results The recombinant plasmid pET30a-EgTPx was identified by PCR and double enzyme digestion. The results were consistent with the expected results. Genomic DNA was identified by sequencing. The correct ORF was constructed. E. coli BL21 (DE3) containing pET30a-EgTPx recombinant plasmid was induced SDS-PAGE showed that the molecular weight of the expressed product was about 27 kDa, and mainly existed in the form of inclusion bodies. Western-blot and ELISA showed that EgTPx recombinant antigen was specifically recognized by the positive serum of sheep infected with hydatid cysts. Conclusion The pET30a-EgTPx expression plasmid was constructed successfully. EgTPx recombinant protein was highly expressed and the expressed product had good antigenicity.