川泽泻鲨烯合酶基因的原核表达研究

Cloning and Prokaryotic Expression of Squalene Synthase( SS) from Alisma orientale

目的:构建川泽泻鲨烯合酶(squalene synthase,SS)基因的原核表达载体。方法:在建泽泻SS基因序列的基础上,采用RT-PCR技术,从川泽泻新鲜叶片中克隆得到SS基因,并在BL21(DE3)细胞中分别用不同的表达载体、IPTG浓度、温度诱导该蛋白表达。结果:该基因在大肠杆菌中表达,但为包涵体,将其克隆到带有GST标签的pGEX-6t-1载体中进行原核表达并未改善蛋白溶解性;30℃诱导蛋白表达最佳;不同IPTG浓度对蛋白表达影响不大。结论:川泽泻的原核表达为后续对该基因的生物学功能及川泽泻品质改良奠定了基础。

Objectives：Construction of expression plasmid of SS and analysis of prokaryotic expression. Methods：SS coding ses- quence was cloned from the fresh leaves of Alisma orientale by RT-PCR, and subsequently inserted into pET28 （ with His tag） and pGEX-6t-1 （with GST tag）, to construct two prokaryotic expression plasmids. Then the expression plasmids were transformed into Esche- richia coli BL21 （DE3）competent cells and recombinant expression was explored with different induction temperatures and IPTG con- centrations. Results：The SS protein was expressed as the inclusion body with the vector of pET28. The GST tag fusion with pGEX-6t-1 did not improve the solubility of SS. In addition, the highest protein expression of SS was observed at the induction temperature of 30 ℃ and there was no significant difference with induction by different concentrations of IPTG, indicating that the optimal induction tempera- ture of SS was 30 ℃and the IPTG concentration had little influence on the expression. Conclusions：The present work will be helpful and lay the foundation for further investigation of SS biological functions and quality improvement of Alisma orientale.