鹦鹉热嗜衣原体CPSIT_0018融合蛋白原核表达载体的构建、表达及其在Hela细胞中的定位

目的构建鹦鹉热嗜衣原体（Cps）CPSIT_0018原核表达载体,表达并纯化该融合蛋白,观察其在感染的Hela细胞中的定位。方法采用PCR技术从Cps 6BC基因组中扩增CPSIT_0018基因,并构建pET30a-CPSIT_0018原核表达载体,然后转化到宿主菌E.coli BL21中,IPTG诱导His-CPSIT_0018融合蛋白表达,进一步用该融合蛋白免疫BALB/c小鼠制备多克隆抗体,间接免疫荧光法分析His-CPSIT_0018蛋白在Hela细胞中的分布特征。结果成功构建了pET30a-CPSIT_0018原核表达载体,并表达和纯化较稳定的重组蛋白;在Cps感染的Hela细胞中CPSIT_0018的分布与主要外膜蛋白MOMP相似,而与包涵体膜蛋白IncA的分布模式不同。结论成功克隆表达了His-CPSIT_0018,该蛋白定位在Cps包涵体内。

Construction and Expression of the Prokaryotic Clone ofRecombinant Protein CPSIT_0018 and Localization in Hela Cells

ObjectiveThe aim is to construct the prokaryotic expression plasmid of CPSIT_0018 gene from Cps 6BC strain;to express and purify the recombinant protein His-CPSIT_0018;and to localize the endogenous CPSIT_0018 protein in Cps- infected Hela cells.MethodsCPSIT_0018 gene was cloned by PCR and inserted into the expression vector pET30a to construct pET30a-CPSIT_0018.The recombinant plasmid was transformed into E.coli BL21,and the recombinant protein was expressed and purified.The purified protein was used to immunize BALB/c mice to produce polyclonal antibody,which were subsequently used to localize the endogenous CPSIT_0018 protein by indirect immunofluorescence assay (IFA).ResultsThe recombinant expression plasmid pET30a-CPSIT_0018 was successfully constructed,and then the recombinant protein was expressed and purified.IFA showed that the distribution pattern of the CPSIT_0018 protein was similar to that of the major outer membrane protein (MOMP),but not to that of inclusion membrane protein A (IncA).ConclusionCPSIT_0018 protein was successfully expressed and purified in prokaryotic expression system;CPSIT_0018 is located on the bacterial organism in Cps-infected Hela cells.