亚洲带绦虫膜联蛋白B2基因的表达、纯化及免疫学分析

The expression and purification of the Annexins B2 gene in Taenia saginata asiatica and the immunologic analysis of the recombinant proteins

目的对亚洲带绦虫膜联蛋白B2(Annexins B2)进行克隆表达及免疫学初步研究。方法利用在线生物信息学工具从亚洲带绦虫成虫cDNA文库中筛选出Annexins B2基因,并将该基因克隆到原核表达质粒pET-28a(+)中,在大肠埃希菌BL21/DE3中诱导表达,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,重组后的蛋白用His-镍蛋白纯化柱纯化,纯化的重组蛋白用Western blotting进行免疫学分析。结果 PCR、双酶切及重组质粒DNA测序均显示重组体构建成功,并得到高纯度的蛋白,且该重组蛋白可被亚洲带绦虫、猪带绦虫及牛带绦虫患者的血清识别。结论亚洲带绦虫成虫Annexins B2基因可在原核表达系统中获得具有免疫活性的高效表达。

Objective To clone and express the gene named as Annexins B2 of Taenia saginata asitica so as to analyze the immunogenicity of its recombinant protein.Methods By online analysis of the gene at bioinfomatics websites to identify the gene from the Taenia saginata asiatica full-length cDNA plasmid library,the gene was cloned into prokaryotic expression vector pET-28a（＋） and then transformed to E.coli BL21 with IPTG induction.Thereafter,the gene product was analyzed by SDS-PAGE and purified by Ni-NTA affinity chromatography.In addition,the immunoreactivity of the purified recombinant proteins was analyzed by Western blotting.Results As demonstrated by PCR,double enzyme digestion and DNA sequencing indicated that the recombinant plasmid was successfully constructed and highly expressed.The recombinant protein tested with Western blotting analysis could react with Taenia asiatica and Taeniarhynchus saginatus infected patient serum.Conclusion The Annexins B2 of Taenia saginata asitica has been cloned and expressed in the prokaryotic expression system and the purified protein has been confirmed with immunogenicity.