【摘要】 将大连蛇岛蝮蛇毒腺中的类凝血酶基因gloshedobin克隆到pET-32a(+)上,构建了T7启动子控制的大肠杆菌表达质粒,并考察了质粒的稳定性. 采用IPTG诱导,以融合蛋白的形式进行表达,得到以包涵体形式存在的类凝血酶. 通过实验对诱导时间、诱导温度及诱导剂浓度进行了优化,并采用正交实验考察了金属离子对表达的影响,结果表明Fe3+, Co2+对类凝血酶的表达有促进作用.更多还原

【Abstract】 The gene of gloshedobin was cloned into pET-32a(+) and expressed in Escherichia coli under T7 promoter with a fusion partner of a Thx.Tag and a 6xHis.Tag. The stability of the plasmid was confirmed according to pET protocol. The effects of induction time, induction temperature and IPTG concentration on the expression, particularly the expression of target protein over impurities, were examined. The maximum expression in the inclusion body was obtained at induction time of 2~3 h, induction temperature of 37oC and IPTG concentration of 0.02 mmol/L. Effects of metal ions on the expression were examined by orthogonal design. Fe3+, Co2+ showed positive stimulation on the expression of insoluble target protein, while the effect of Ca2+ has minor promotion. In contrast, decreased expression was observed as Mg2+ was added in culture media.更多还原