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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。
Objective To optimize prokaryotic expression of the endogenous angiogenesis inhibitor Arresten gene. Methods The total RNA was extracted from placenta of healthy maternal and the Arresten gene was amplified by RT-PCR. The recombinant plasmid pBV220-Arr was constructed and transformed into competent E. coli JM109, DH5α, BL21 and BL21 (DE3) -PAGE expression products were screened out the optimal expression strains, and its culture temperature and time, induction temperature and time and dissolved oxygen were optimized. Results The recombinant plasmid pBV220-Arr was constructed and confirmed by restriction enzyme digestion and sequencing. Arresten protein was expressed both in 4 and 4 h after E. coli JM109, DH5α, BL21 and BL21 (DE3) After induced for 4 h, the expression of E.coli BL21 (DE3) protein was the highest, and the wet weight was significantly higher than that of other strains. The optimal conditions for the expression of E.coli BL21 (DE3) / pBV220-Arr were as follows: 100 ml LB ampicillin-positive medium was added to 500 ml culture flask, and the cells were cultured at 30 ° C for 4 h and then induced at 42 ° C for 4 h . Conclusion The expression conditions of Arresten genetically engineered bacteria were optimized, which provided a reference for the mass expression of recombinant Arresten protein.