麻疯树核糖体失活蛋白curcin在大肠杆菌中高效表达条件的研究

Study on expression of curcin gene cloned from Jatropha curcas in Escherichia coli

目的:编码麻疯树毒蛋白(curcin)成熟蛋白的基因原核表达载体的构建及其高效表达条件的研究。方法:根据GenBank上麻疯树的cDNA序列设计引物,用PCR方法从麻疯树基因组中扩增得到编码麻疯树毒蛋白成熟蛋白的基因;将其导入原核表达载体pQE-30,pET-32中,并将其转入大肠杆菌获得重组菌株。在不同诱导剂、不同温度、不同诱导时间等条件下诱导其产生重组蛋白并进行检测比较。结果:用PCR方法从麻疯树基因组中扩增得到编码麻疯树毒蛋白成熟蛋白的基因,成功构建了原核表达载体pQE-R,pET-R,并获得重组菌株PRM,PRB。在不同诱导剂、不同温度、不同诱导时间等条件诱导下,PRB均无重组蛋白产生,而PRM却能以包涵体的形式表达curcin。结论:重组菌PRM在诱导6 h,28℃,IPTG 0.5 mmol.L-1时表达curcin蛋白的效率最高。

Objective:To construct mature protein curcin gene prokaryotic expression vectors in Escherichia coli and choose the optimal inducing condition of the recombinant strains.Method:The gene encoding of curcin was amplified from the genome of Jatropha curcas seeds by PCR and cloned into the expression vectors pQE-30 and pET-32 obtaining recombinant vectors pQE-R and pET-R respectively.The two vectors were transferred into E.coli BL21(DE3) and the recombinant strains PRM and PRB were attained respectively.PRM and...