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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 a 28 kDa glutathione S-transferase (Sj2 8GST) gene from the cDNA library of adult Schistosoma japonicum Chinese mainland strains and obtain the recombinant fusion protein 2 8GST-TRX for vaccine and T cell epitope research. Methods Sj2 8GST gene was amplified from the adult cDNA library of Schistosoma japonicum by primer-specific PCR. After identification by agarose gel electrophoresis and nucleotide sequence analysis, the recombinant Sj2 8GST-TRX fusion protein was constructed by directional cloning Expression vector and induced its expression in E. coli BL21 (DE3), the immunogenicity of Sj2 8GST expressed by fusion protein was determined by Western-blotting, and high purity recombinant fusion was obtained by inclusion body purification combined with Ni + column affinity chromatography protein. Results The Sj2 8GSTPCR product was about 640 bp. The nucleotide sequence (6 33 bp) and deduced amino acid sequence (2 11aa) of Sj2 8GSTPCR were identical with that reported in GenBank. Sj2 8GST-TRX recombinant plasmid was obtained. The 28GST-TRX fusion protein was expressed in Escherichia coli Which is highly expressed in the form of inclusion bodies. A band of 28 GST fusion protein that specifically reacts with goat anti-Sj2 8GST antibody at a molecular weight of about 43 kDa was obtained. Inclusion body purification combined with Ni + affinity chromatography yielded high-purity Recombinant fusion protein. Conclusion The full-length gene of 2 8GST molecule of Schistosoma japonicum in China and the highly purified recombinant 2 8GST-TRX fusion protein were obtained. The fusion protein has the immunogenicity of natural Sj2 8GST.