| 贺庆芝，曾怀才，陆春雪，胡艳群，陈芝喜，王川，吴移谋.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.[J].中南医学科学杂志.,2014,42(3):229-232. |
| 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 |
| Construction and Expression of the Prokaryotic Clone ofRecombinant Protein CPSIT_0018 and Localization in Hela Cells |
| 投稿时间：2013-10-21 |
| DOI： |
| 中文关键词: 鹦鹉热嗜衣原体 CPSIT_0018 克隆表达 细胞定位 |
| 英文关键词:Chlamydophila psittaci CPSIT_0018 cloning and expression cellular localization |
| 基金项目:国家自然科学基金（81202323），湖南省科技厅项目（2012FJ6009），湖南省卫生厅项目（B2012-042），南华大学研究生科研创新项目（2012XCX13）. |
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| 中文摘要: |
| 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。 |
| 英文摘要: |
| 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. |
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