可溶性重组日本血吸虫26 kDa GST的优化表达

目的探讨在大肠杆菌中表达可溶性重组日本血吸虫谷胱甘肽S-转移酶(rSj26 GST)的优化条件。方法用含有Sj26基因的原核表达载体pGEX-3X转化大肠杆菌BL21(DE3),将影响可溶性rSj26 GST表达的4个因素,即温度、诱导剂IPTG浓度、诱导时细菌的密度和诱导时间进行正交设计,确定其优化表达条件并大量表达,采用谷胱甘肽亲和层析法纯化表达的蛋白,测定酶活性,通过Western blot鉴定免疫活性。结果正交设计的直观分析和方差分析结果显示:可溶性rSj26GST最佳表达条件为温度25℃,IPTG的浓度1 mmol/L,诱导前细菌的生长密度0.9,诱导表达时间8 h。可溶性rSj26 GST纯化的产量为16 mg/L,Western blot证实rSj26 GST具有良好免疫原性和免疫反应性。结论通过正交设计确定了可溶性rSj26 GST在大肠杆菌中表达的优化条件。

Optimizing expression of recombinant 26-kilodalton glutathione S-transferases of Schistosoma japonicum in Escherchia coli

To explore the best expressing condition of the recombinant 26-kilodalton glutathione S-transferases of S.japonicum(rSj26 GST) in Escherchia coli(E.coli) and to analyse its characterization.E.coli BL21(DE3) was transformed with plasmid pGEX-3X.The best expressing condition of rSj26 GST was explored through orthogonal design which took growth temperature,IPTG concentration,culture density,incubation time as indices.The expressed products(rSj26 GST) proteins were purified from supernatant bacterial lysates by affinity chromatography using reduced glutathione resin.It can be detected using functional assays and immunological methods.The pure rSj26 GST with the activity of glutathione S-transferase,and it could be recognized by the sera of rabbit and mouse infected with S.japonica.The rabbit was vaccinated with rSj26 GST and the highest level of the anti-rSj26 GST antibody was 1∶256 000.Western blot analysis showed that the anti-rSj26 GST antibody could recognize the antigen of adult worm of Schistosoma japonica.The best expressing condition of the recombinant 26-kilodalton glutathione S-transferases of S.japonicum(rSj26 GST) in E.coli was determined through orthogonal design.