FGF20原核表达载体构建及在大肠杆菌中优化表达

Construction of FGF20 prokaryotic expression vector and optimized expression in E.coli

[目的]构建人FGF20原核表达载体并在大肠杆菌(Escherichia coli,E.coli)中优化表达。[方法]从胃癌细胞中经RT-PCR扩增FGF20基因并构建p ET28b-FGF20载体,转化E.coli,IPTG诱导表达。对诱导OD值、IPTG浓度、诱导温度、时间及溶氧量进行优化,同时采用正交试验优化培养基配方,以SDS-PAGE及Western Blot检测蛋白表达,以确定最佳表达条件。[结果]构建了p ET28b-FGF20原核表达载体,实现了FGF20蛋白的表达,确定最佳表达条件为:培养基占锥形瓶容积20%,菌体生长至OD600=0.4,加入终浓度1.0 mmol/L的IPTG,37℃诱导6 h。培养基最佳成分为:蛋白胨11 g/L、酵母提取物5 g/L、氯化钠10 g/L、葡萄糖0 g/L。[结论]实现了FGF20在大肠杆菌中的优化表达,为其基因工程药物生产及药用研究奠定了基础。

[ Objective] To construct the prokaryotic expression vector of human FGF20 and express it in E. coli optimally. [ Methods]The FGF20 genes were amplified from the gastric cancer cell by RT- PCR,then we builded pET28b- FGF20 recombinant plasmid and transferred it into E. coli to induce expression by IPTG. Then expression condition OD600 value, IPTG concentration,induction temperature,induction time and dissolved oxygen were optimized and tested the protein expession by SDS - PAGE and Western Blot to get the best conditions. At the same time, We optimized the cuhure medium formula by orthogonal test to increase production of protein expression. [ Results ] pET28b - FGF20 prokaryotic expression vector was constructed and expressed the FGF20 protein successfully. And the optimal expression condition of FGF20 was： the medium accounted for 20% of taper bottle volume,when bacteria growth to OD600 = 0.4, we added IPTG with 1.0 mmol/L final concentration, then cultivated in 37 ℃ for 6 hours. The best components in culture medium ： peptone 11 g/L, yeast extract 5 g/L, sodium chloride 10 g/L,glucose 0 g/L. [ Conclusion] We had expressed FGF20 protein optimizedly that laid the foundation for the genetic engineering production of drug and its medical research.