摘要
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。
Objectives To construct a eukaryotic expression vector for expression of CT813 and to examine its expression in eukaryotic cells in order to lay the foundation for the study of the interaction between Chlamydia trachomatis and the host. Methods The CT813 gene was obtained using PCR.The CT813 gene and a pcDNA3.1/Myc-HisA null plasmid were digested with two enzymes and joined with T4 ligase.After enzyme digestion,amplification with PCR,and DNA sequencing,the target gene was cloned into the eukaryotic expression vector pcDNA3.1/Myc-HisA.The recombinant plasmid was transfected into Hela cells,and its expression was examined with Western blotting and an indirect immunofluorescence assay. Results Colony PCR indicated that the fragment of the amplified target gene was about 795 bp in length.NotI and kpnI double enzyme digestion revealed that the target gene was correct.Sequencing results were consistent with sequence registered in the NCBI database.Western blotting indicated that the fusion protein CT813 had a relative molecular weight of about 29.4kd.The indirect immunofluorescence assay indicated that the fusion protein was expressed in the cytoplasm of cells. Conclusion The successful construction of the pcDNA3.1/Myc-HisA eukaryotic expression vector is crucial to further study of the biological function of CT813 and interaction between Chlamydia trachomatis and the host.
出处
《中国病原生物学杂志》
CSCD
北大核心
2016年第9期799-801,807,共4页
Journal of Pathogen Biology
基金
河北省自然科学基金项目(No.C2014405041)
