Sj28GST基因克隆和高效表达产物的纯化

Gene cloning of Sj28GST and purification of its highly expressed product

目的 从日本血吸虫中国大陆株成虫cDNA文库中克隆 2 8kDa谷胱甘肽 -S -转移酶 (Sj2 8GST)基因 ,获得用于疫苗和T细胞表位研究的高纯度重组融合蛋白 2 8GST -TRX。方法 应用引物特异性PCR技术 ,从日本血吸虫中国大陆株成虫cDNA文库中扩增Sj2 8GST基因 ,经琼脂糖凝胶电泳结合碱基序列分析鉴定后 ,采用定向克隆技术构建Sj2 8GST -TRX融合蛋白重组表达载体并诱导其在大肠杆菌BL2 1(DE3)的表达 ,用Western -blotting测定以融合蛋白方式表达的Sj2 8GST的免疫原性 ,通过包涵体纯化结合Ni+柱亲和层析获得高纯度重组融合蛋白。结果 Sj2 8GSTPCR产物为约 6 4 0bp ,其碱基序列 (6 33bp)和推导的氨基酸序列 (2 11aa)与GenBank报道一致 ;获得了Sj2 8GST -TRX重组质粒 ;2 8GST -TRX融合蛋白在大肠杆菌以包涵体形式高效表达 ;在分子量为约 4 3kDa处有一能与羊抗Sj2 8GST抗体产生特异性反应的含 2 8GST融合蛋白条带 ;包涵体纯化法结合Ni+ 亲和层析可获得高纯度的重组融合蛋白。结论 获得到了日本血吸虫中国大陆株 2 8GST分子的全长基因和高纯度重组 2 8GST -TRX融合蛋白 ,该融合蛋白具有天然Sj2 8GST免疫原性。

Objective To clone Schistosom japonicum 28kDa glutathione S-transferase(Sj28GST) gene from adult worm cDNA library and obtain expression product of the gene for selection of T cell epitopes and further vaccine study.Methods Sj28GST gene was amplified from adult worm cDNA library using specific primers PCR.The PCR product was purified and identified by agarose gel electrophoresis and DNA sequencing,and then inserted plasmid pET32c,and expressed in E.coli BL21(DE3) by IPTG induction.The Sj28GST which was expressed as a fusion protein was identified by SDS-PAGE and Western-blotting for immunogenicity,and then purified by washing its inclusion body and nickel affinity chromotograph.Results Sj28GST gene was 633bp in length,coding for 211 amino acids,The result was the same as that in GenBank.The recombinant plasmid pET32c/Sj28GST was constructed successfully,and highly expressed the fusion protein 28GST-TRX with 6×His in inclusion body,which was recognized on Western-blotting by the specific antibodies of sheep serum immunized with recombinant 28GST from the Chinese strain of Schistosoma japonicum.The fusion protein was purified,and identified by SDS-PAGE as the single band shown.Conclusion A Schistosoma japonicum cDNA coding for a full length S.japonicum 28kDaGST and the higher purity of recombinant fusion protein 28GST-TRX has been obtained,which can be used for T cell epitope selection and vaccine studies.