藤黄微球菌Rpf基因的克隆表达及纯化鉴定

Cloning,purification and identification of Micrococcus luteus Rpf gene

目的:克隆藤黄微球菌Rpf基因,序列测定正确后进行融合表达纯化.方法:采用聚合酶链反应(PCR)方法,从藤黄微球菌基因组中扩增出藤黄微球菌Rpf基因,用限制性内切酶消化后插入到pUC-19克隆载体中,测序正确后再亚克隆到融合表达载体pPro-EXHT中,转化大肠杆菌DH5α,目的基因经IPTG诱导,由T7启动子调控表达了氨基端带6个连续组氨酸残基的Rpf蛋白,在变性条件下对目的蛋白进行纯化.结果:获得藤黄微球菌Rpf基因,得到融合6个组氨酸残基的藤黄微球菌Rpf蛋白,可溶性分析发现融合蛋白主要在包涵体中存在,最后在变性条件下,用Ni2+-NTA亲和色谱柱纯化目的融合蛋白,纯化获得的融合蛋白纯度大于90%,Western-Blot证实获得的蛋白为所需要的目的蛋白.结论:构建了藤黄微球菌Rpf基因的重组表达载体,并获得了高纯度的融合表达蛋白,为以后的深入研究奠定了基础.

AIM： To clone Micrococcus luteus Rpf gene and purify its fusion protein. METHODS： The gene encoding Micrococcus luteus Rpf was amplified by polymerase chain reaction （PCR） from genome of Micrococcus luteus , and first inserted into cloning vector pUC-19. After sequenced, Micrococcus luteus Rpf gene was then subcloned into expression vector pPro-EXHT, and transfected into the host strain E. coli DH5α. After induction with IPTG, the strain controlled by T7 promoter expressed the fused Micrococcus luteus Rpf protein with a hexahistidine tail in its Nterminal. The target protein was purified under denaturing condition and confirmed by western blot. RESULTS： SDS-PAGE analysis showed that the fusion protein mainly existed in inclusion bodies. The expressed proteins could be purified to more than 90% v/a Ni^2＋ -NTA system kit in denaturing condition and confirmed by western blot. CONCLUSION： The recombinant expression plasmid is successfully constructed and purified to a higher level, which lays a basis for further study of the Micrococcus luteus Rpf protein.