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目的构建亚洲牛带绦虫NOMO1基因原核重组质粒,进行原核表达、纯化及免疫学研究。方法以亚洲牛带绦虫成虫cDNA文库中含NOMO1基因的质粒作为模板,扩增该基因,将其克隆到原核表达载体pET-28a(+)中,测序鉴定重组质粒后再行诱导表达,表达产物通过十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定,并用蛋白免疫印迹(Western-blotting)分析其免疫反应性。结果成功建构了原核重组质粒pET28a(+)-NOMI1。该基因在大肠埃希菌中以包涵体形式存在,破包涵体后纯化蛋白通过SDS-PAGE鉴定结果表明该基因在大肠埃希菌BL-21/DE3得到高效表达。Western blotting结果显示,该重组蛋白可被亚洲牛带绦虫、牛带绦虫病人血清识别,具有免疫反应性。结论成功克隆亚洲牛带绦虫NOMO1基因、表达和纯化得到了该基因的重组蛋白并且证明该基因具有免疫反应性,为进一步研究该基因的功能提供条件。
Objective To construct prokaryotic recombinant plasmids of NOMO1 gene of Asian Taenia saginata by prokaryotic expression, purification and immunological study. Methods The gene of NOMO1 gene was amplified from the cDNA library of adult Taenia saginata by using the plasmid pET-28a (+) as a template. The recombinant plasmid was identified by sequencing and induced to express. The expression product It was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and its immunoreactivity was analyzed by Western-blotting. Results The prokaryotic recombinant plasmid pET28a (+) - NOMI1 was successfully constructed. The result of SDS-PAGE showed that the gene was highly expressed in Escherichia coli BL-21 / DE3. Western blotting showed that the recombinant protein could be recognized by the serum of Taenia saginata and Taenia saginata as immunoreactive. Conclusion The NOMO1 gene of Taenia asiatica was successfully cloned, and the recombinant protein was obtained by expression and purification. The recombinant protein was proved to be immunoreactive, which provided the conditions for further study on the function of this gene.