论文部分内容阅读
根据已克隆得到的刺五加P450基因的cDNA序列设计引物,构建刺五加P450基因的原核表达载体pET30a-P450,并对重组菌株BL21/pET30a-P450的原核表达条件进行了筛选优化。实验结果显示,pET30a-P450转化大肠杆菌BL21后可实现P450基因的原核表达,SDS-PAGE分析显示其在53 kDa处有显著诱导条带;表达体系的最佳诱导温度为27~37℃,最佳IPTG浓度为1 mmol·L~(-1),最佳培养基为LB液体培养基,最佳诱导时间为24 h。因此,刺五加P450基因能够通过表达载体BL21/pET30a-P450实现该基因的原核表达,该体系的诱导温度、IPTG浓度、培养基种类以及诱导时间均可影响目的蛋白的表达量,但影响强度不同。
The prokaryotic expression vector pET30a-P450 of Acanthopanax senticosus P450 gene was constructed based on the cloned cDNA sequence of Acanthopanax senticosus P450. The prokaryotic expression conditions of the recombinant strain BL21 / pET30a-P450 were optimized. The results showed that prokaryotic expression of P450 gene was achieved after pET30a-P450 was transformed into E.coli BL21. SDS-PAGE analysis showed that the P450 gene was induced significantly at 53 kDa. The optimum induction temperature of the expression system was 27 ~ 37 ℃ The optimal concentration of IPTG was 1 mmol·L -1, and the best culture medium was LB liquid medium, the best induction time was 24 h. Therefore, Acanthopanax senticosus P450 gene can express prokaryotic expression vector BL21 / pET30a-P450. The induction temperature, IPTG concentration, medium type and induction time can affect the expression of target protein, different.