Abstract:

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 pET41a to construct a recombinant plasmid SjTGR-pET41a. Then the recombinant plasmid SjTGR-pET41a was co-transformed into E. coli BL21 with plasmid pSU ABC. The SjTGR protein was expressed by inducing with IPTG. The recombinant SjTGR was purified from expression products by affinity chromatography with an adenosine 2’, 5’- diphosphate agarose column. The polyclonal antiserum against recombinant SjTGR was obtained by immunizing mice with purified SjTGR. The native TGR in S. japonicum was evidenced by using Western blotting. Thiorendoxin reductase (TrxR) activity, glutathione reductase (GR) activity and gluaredoxin (Grx) activity of recombinant TGR were analyzed according to the biochemical method. Results The chimeric gene of SjTGR with a bacterial-type selenosysteine insertion sequence (SECIS) element was constructed successfully. The bacteria containing the recombinant plasmid SjTGR-pET41a could express the soluble SjTGR by inducing with IPTG at static growth stage for 24 h at 24 ℃. The expressed products of plasmid pSU ABC could promote the integration of selenocysteine and increase the yield of selenoprotein. The result of Western blotting showed that the polyclonal antiserum against recombinant SjTGR could recognize the native TGR in S. japonicum adult worms. The enzymatic assay indicated that SjTGR was a multifunctional enzyme with activities of TrxR, GR and Grx. Conclusion The recombinant SjTGR with biological activity is expressed successfully, which lays the foundation for further study on the function and applied values of SjTGR.

Key words: Schistosoma japonicum, Thiorendoxin glutathione reductase, Expression, Purification, Enzyme activity

摘要：

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杆菌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pET41a中形成重组表达质粒SjTGR-pET41a。将重组表达质粒SjTGR-pET41a 和质粒pSU ABC共转化到大肠杆菌BL21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的SjTGR蛋白。以重组的SjTGR免疫小鼠制备TGR的多克隆血清，免疫印迹实验分析日本血吸虫体内是否存在天然的TGR。采用生物化学方法分析重组SjTGR的硫氧还蛋白还原酶、谷胱甘肽还原酶、谷氧还蛋白的酶活性。结果 含有大肠杆菌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-pET41a的转化子细菌在静止生长期，经IPTG诱导，24 ℃培养24 h可表达出可溶性SjTGR蛋白，质粒pSU ABC表达产物可促进硒代半胱氨酸整合，增加硒蛋白的产量。免疫印迹实验结果显示，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。

关键词: 日本血吸虫, 硫氧还蛋白谷胱甘肽还原酶, 表达, 纯化, 酶活性