马铃薯C-8,7甾醇异构酶基因原核表达载体的构建和表达

Construction of prokaryotic expression vectors for StSI1 and its induced expression

为研究甾醇异构酶的功能以及制备抗体,分别构建了马铃薯C-8,7甾醇异构酶基因StSI1携带和去除信号肽序列的原核表达载体pGEX-StSI1b和pGEX-StSI1c,并在大肠杆菌BL21(DE3)工程菌株中优化了融合蛋白GST-StSI1b和GST-StSI1c的诱导表达条件.结果表明,在0.1,0.5,1.0 mmol.L-1的IPTG诱导下,2种融合蛋白GST-StSI1b和GST-StSI1c均能有效表达,并以1.0 mmol.L-1IPTG诱导的效果最好;从诱导表达的时间来看,3种浓度IPTG诱导3 h后融合蛋白均开始表达,且表达量随着诱导时间的延长而逐渐增加,但在诱导9 h后的表达量增加幅度不大,因此确定诱导融合蛋白GST-pStSI1表达的最佳IPTG浓度为1.0 mmol.L-1,诱导时间为9 h.

Two prokaryotic expression vectors pGEX-StSIlb and pGEX-StSI1 c for the potato StSI1 （C- 8,7 sterol isomerase） gene cDNA StSH both with and without signal peptide were constructed and the induced expression conditions for the fusion protein GST-StSI1 b and GST-StSI1 c were optimized in the engineered Escherichia coli BL21 （DE3）. The results showed that the fused GST-StSIlb and GST- StSIlc proteins could be effectively expressed under different concentrations of IPTG, including 0.1, 0.5 and 1.0 mmol · L^-1, and the most suitable concentration of IPTG was 1.0 mmol · L^-1. As to the induction time, the fusion protein began to express after 3 h of induction under the 3 different IPTG concentrations, and its expression abundance increased along with the induction time and reached to the highest point at the 9 h of induction. In general, the most suitable induction condition for the fu- sion protein was 9 h induction under 1.0 mmol · L^-1 IPTG.