日本血吸虫硫氧还蛋白谷胱甘肽还原酶的表达及特性分析

目的制备具有生物活性的重组日本血吸虫硫氧还蛋白谷胱甘肽还原酶（SjTGR）硒蛋白。方法采用PCR法,在SjTGR基因开放阅读框的末端融合大肠埃希菌（E.coli）硒蛋白转译的硒代半胱氨酸插入序列以构建一个嵌合基因,将此嵌合基因亚克隆到表达载体pET-41a中形成重组表达质粒SjTGR-pET-41a。将重组表达质粒SjTGR-pET-41a和质粒pSUABC共转化到E.coliBL21中,用异丙基-β-D-硫代吡喃半乳糖苷（IPTG）诱导表达SjTGR蛋白。用阿糖胞苷-2＇,5＇-二磷酸琼脂糖凝胶通过亲和层析从表达产物中纯化重组SjTGR蛋白。以重组SjTGR免疫小鼠制备TGR多克隆血清,免疫印迹试验分析日本血吸虫体内是否存在天然TGR。采用生物化学方法分析重组SjTGR硫氧还蛋白还原酶、谷胱甘肽还原酶、谷氧还蛋白的酶活性。结果含有E.coli硒代半胱氨酸插入序列的SjTGR嵌合基因构建成功。含SjTGR基因的重组质粒SjTGR-pET-41a的转化子细菌在静止生长期经IPTG诱导,24℃培养24h可表达出可溶性SjTGR蛋白,质粒pSUABC表达产物可促进硒代半胱氨酸整合,增加硒蛋白的产量。免疫印迹试验结果显示,纯化重组SjTGR多克隆抗体可以识别血吸虫成虫体内天然的TGR。生化分析结果显示,SjTGR是一种具有硫氧还蛋白还原酶、谷胱甘肽还原酶、谷氧还蛋白活性的多功能酶。结论具有生物活性的SjTGR已被成功表达,为进一步研究其功能与应用价值奠定了基础。

Expression and characterization of thioredoxin glutathione reductase of Schistosoma japonicum

Objective To prepare the recombinant thioredoxin glutathione reductase of Schistosoma japonicum(SjTGR) with biological activity.Methods The open reading frame DNA sequence of SjTGR was fused with a bacterial type selenosysteine insertion sequence(SECIS) element by PCR to form a chimeric gene.The chimeric gene was subcloned into expression plasmid pET-41a to construct a recombinant plasmid SjTGR pET 41a.Then the recombinant plasmid SjTGR pET 41a was co transformed into E.coli BL21 with plasmid pSU ABC.The Sj...