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目的构建沙眼衣原体包涵体膜蛋白CT813真核表达载体并观察其在Hela细胞中的表达,为研究其与宿主间的相互作用奠定基础。方法克隆CT813基因,分别对CT813与pcDNA3.1/Myc-HisA空质粒进行双酶切,T4连接酶进行连接,然后进行菌落PCR、酶切及测序鉴定;将构建的重组质粒pcDNA3.1/Myc-HisA-CT813转染Hela细胞后采用Western blot与间接免疫荧光法检测目的蛋白表达。结果 PCR显示,扩增的目的基因片段约795bp,与预期相符;经NotⅠ与kpnⅠ双酶切鉴定重组质粒构建正确,测定其序列与NCBI数据库完全一致;Western blot显示重组质粒转化Hela表达的CT813融合蛋白分子质量单位为29.4kd左右;间接免疫荧光法检测该蛋白位于细胞胞浆中,是衣原体分泌性蛋白。结论成功构建了PcDNA3.1/Myc-His A-CT813真核表达载体并表达了沙眼衣原体包涵体膜蛋白CT813,对研究其生物学功能及与宿主配体间的相互作用具有重要意义。
Objective To construct eukaryotic expression vector of inclusion body of CT813 of Chlamydia trachomatis and observe its expression in Hela cells, which lays the foundation for studying its interaction with host. Methods The CT813 gene was cloned and the CT813 and pcDNA3.1 / Myc-HisA empty plasmids were double digested and ligated with T4 ligase, respectively. Then the colony PCR, restriction enzyme digestion and sequencing were carried out. The constructed recombinant plasmid pcDNA3.1 / Myc -HisA-CT813 transfected Hela cells, Western blot and indirect immunofluorescence method to detect the expression of the target protein. The results of PCR showed that the amplified target gene fragment of about 795bp, in line with expectations; identified by NotⅠ and kpn Ⅰ double digestion of the recombinant plasmid was constructed correctly determined by the NCBI database and the sequence exactly the same; Western blot showed that recombinant plasmid Hela expression of CT813 fusion The molecular mass unit of protein was about 29.4kd. Indirect immunofluorescence assay showed that the protein was located in the cytoplasm of the cell and was a chlamydia secreted protein. Conclusion The pcDNA3.1 / Myc-His A-CT813 eukaryotic expression vector was successfully constructed and CT813 was expressed in the inclusion body of C. trachomatis. It is of great significance to study its biological function and its interaction with host ligands.