人白介素24融合载体的构建、突变纠正和表达优化

Prokaryotic Expression Optimization,Point Mutation Correction and Cloning of Human Interleukin-24

以武汉大学病毒学国家重点实验室保存的白介素24(IL-24)为模板,构建原核表达载体pET28a-IL-24,转化大肠杆菌DH5α,挑取单克隆,测序结果显示:在IL-24第18位A→G,21位C→T,412位G→A,18位和21位的突变没有使该位上的氨基酸发生改变,而412位的突变导致了该位点上的氨基酸发生改变——由丙氨酸A变为苏氨酸T。通过设计突变引物,二次PCR将其纠正,转化大肠杆菌BL21。通过改变诱导剂IPTG的浓度、诱导时间和诱导温度摸索最佳诱导条件,SDS-PAGE、Western blot检测显示有融合蛋白6hisIL-24表达。结果表明:通过二次PCR成功构建了IL-24原核表达载体,表达产物主要以包涵体的形式存在,最佳诱导温度为28℃,最佳诱导剂浓度为1mmol/L,最佳诱导时间是8h。

Taking the interleukin（IL-24）in the lab as the template,recombinant prokaryotic vector of expressing pET28a-IL-24 was constructed,the vector was transferred into E.coli DH5α and the monoclones was picked out.The mutant（G→A in IL-24cDNA）resulted in the change of IL-24protein（A412T）,The mutants（A→G,C→T,in IL-24cDNA）did not change the amino acids on the bit.By designing mutation primers,correction of mutation was performed by a two-step PCR reaction,the correct recombinant vector was transformed into E.coli.BL21.The optimal condition was confirmed by changing the concentration of IPTG and induction time and temperature,the fusion protein 6his-IL-24 was detected by SDS-PAGE and Western blotting.As a result,by two-step PCR,the prokaryotic expression vector was constructed,the expression product was existed in the form of inclusion body,the best induction temperature was 28 ℃,the optimal IPTG concentration was 1 mmol/L and the best induction time was 8h.