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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 a prokaryotic expression vector encoding the curcin mature protein gene and study on its high expression conditions. Methods: Primers were designed according to the cDNA sequence of Jatropha curcas from GenBank. The gene coding for the mature protein of Jatropha curcas was amplified by PCR from the Jatropha curcas genome and introduced into the prokaryotic expression vector pQE-30, pET-32 And transformed into E. coli to obtain a recombinant strain. Under different inducing agents, different temperatures, different induction time and other conditions induced recombinant protein production and testing comparison. Results: The gene coding for the mature protein of Jatropha curcas was amplified from the Jatropha curcas genome by PCR. The prokaryotic expression vector pQE-R and pET-R were successfully constructed and the recombinant strains PRM and PRB were obtained. PRB had no recombinant protein induced by different inducing agents, different temperatures and different induction time, but PRM expressed curcin in the form of inclusion body. Conclusion: The expression of curcin protein is most efficient when PRM of recombinant bacteria is induced at 6 h, 28 ℃ and IPTG 0.5 mmol·L-1.