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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。
Objective: To construct prokaryotic expression vector of squalene synthase (SS) gene from Chuanze. Methods: Based on the sequence of SS gene in Alisma orientale, SS gene was cloned by RT-PCR from fresh leaves of Chuanzai. The expression of SS gene in BL21 (DE3) cells was detected by different expression vectors, IPTG concentration, Induce this protein expression. Results: The gene was expressed in E. coli. However, it was an inclusion body, and cloned into pGEX-6t-1 vector with GST tag for prokaryotic expression without improving protein solubility. The protein expression was best at 30 ℃. IPTG concentration has little effect on protein expression. CONCLUSION: The prokaryotic expression of Chuanzai Xie laid the foundation for the biological function of this gene and the improvement of its quality.