蛇毒类凝血酶基因在大肠埃希菌中诱导表达及影响因素

Expression of Thrombin-like Enzyme Gene in E.coli and Its Influence Factors

将蛇毒类凝血酶基因(TLE)亚克隆到原核表达载体pMAL-p2X上,对重组表达质粒鉴定正确后,转化大肠埃希菌BL21进行诱导表达;同时采用不同的OD值、诱导时间I、PTG浓度和诱导温度对尖吻蝮蛇蛇毒类凝血酶基因重组表达质粒pMAL-p2X进行了条件优化。研究发现,不同的诱导时间、IPTG诱导浓度和诱导温度与融合蛋白表达量有很大关系。SDS-PAGE结果显示,当OD值为0.5,IPTG终浓度为0.3 mmol/L,诱导温度为37℃时,诱导3 h后蛋白表达量最高,超出此范围可使表达量显著减少。在优化诱导条件后,融合蛋白的表达量可达全菌总蛋白量的66%。

TLE gene was subcloned into prokaryotic expression vector pMAL-p2X and transformed into E. coli BL21 after identification of the recombinant vector. The expression of recombinant vector pMakL-p2X which contained TLE gene of Agkistrodon acutus in E. coli BL21 was induced at a variety of OD value, temperature, density of IPTG, and induction time. The results showed that the expression level was correlated with OD value, temperature, density of IPTG and induction time. SDS-PAGE results also confirmed that the expression level was highest in the conditions of OD value 0.5, 0.3 mmol/LPTG,under 37℃ and 3 hours of induction time. The amount of recombinant gene expression products could reach as high as 66% total proteins of the host.