内源性血管生成抑制因子Arresten基因原核表达条件的优化

目的 优化内源性血管生成抑制因子Arresten基因的原核表达条件。方法从健康产妇的胎盘组织中提取总RNA,RT-PCR扩增Arresten基因,构建重组表达质粒pBV220-Arr,分别转化感受态E.coli JM109、DH5α、BL21、BL21(DE3),诱导表达,SDS-PAGE分析表达产物,筛选出最优表达菌种,并对其菌体培养温度和时间、诱导温度和时间及溶解氧量进行优化。结果重组表达质粒pBV220-Arr经酶切及测序证明构建正确,重组表达质粒在E.coli JM109、DH5α、BL21、BL21(DE3)4种菌中诱导2和4 h,均能表达出Arresten蛋白,诱导表达4 h,E.coli BL21(DE3)目的 蛋白表达量最高,湿菌重也明显大于其他菌种。E.coli BL21(DE3)/pBV220-Arr的最佳表达条件为:在500 ml培养瓶中加入100 ml LB氨苄阳性培养基,菌体于30℃培养4 h后,再42℃诱导表达4 h。结论优化了Arresten基因工程菌的表达条件,为重组Arresten蛋白的大量表达提供了参考。

Optimization of Condition for Prokaryotic Expression of Endogenetic Angiogenesis Inhibitor Arresten Gene

Objective To optimize the condition for prokaryotic expression of endogenetic angiogenesis inhibitor Arresten gene.Methods Total RNAs were extracted from the placenta tissues of healthy puerperal women,from which Arresten gene was amplified by RT-PCR and inserted into vector pBV220.The constructed recombinant plasmid pBV220-Arr was transformed to E.coli JM109,DH5α,BL21 and BL21(DE3)respectively for expression.The expressed product was analyzed by SDS-PAGE.The optimal bacterial strain for expression was screened,of which the temperatures and times for culture and induction as well as dissolved oxygen content were optimized.Results Both restriction analysis and sequencing proved that recombinant plasmid pBV220-Arr was constructed correctly.Arresten protein was expressed in all the four E.coli strains 2 and 4 h after induction,of which the expression level in E.coli BL21(DE3)4 h after induction was the highest,and the wet weight of bacteria was significantly higher than those of the other three strains.The condition for expression of E.coli BL21(DE3)/ pBV220-Arr was optimized as follows:add 100 ml of ampicillin-positive LB medium into a 500 ml flask and culture at 30℃ for 4 h then induce at 42℃ for 4 h.Conclusion The condition for expression of Arresten gene was optimized,which provided a reference for expression of recombinant Arresten protein in a large quantity.