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目的构建肺炎衣原体Cpn 0147基因真核表达重组质粒,为进一步研究其与宿主相互作用的分子机制打下基础。方法以Cpn AR39株基因组为模板,以Cpn0147全长编码特异性引物进行PCR扩增;将Cpn0147基因插入至pcDNA3.1+/His/Myc载体,构建pcDNA3.1+/His/Myc-Cpn0147重组质粒,经双酶切及测序鉴定后转染至HeLa细胞中,采用RT-PCR检测重组质粒转染情况,采用免疫荧光法及Western blot检测Cpn0147蛋白在细胞中的表达。试验设pcDNA3.1+/His/Myc载体为阴性对照。结果从CpnAR39株基因组DNA中扩增出Cpn0147基因片段,大小466bp,经双酶切、连接、转化、筛选,得到pcDNA3.1+/His/Myc-Cpn0147重组质粒,序列测定证实与GenBank Cpn AR39株Cpn0147基因序列一致;RT-PCR扩增出Cpn0147基因,与理论大小一致;免疫荧光检测重组质粒转染后HeLa细胞胞浆观察到红色荧光,对照组无荧光;Western blot检测到特异反应条带位于16kd,对照组无此条带。结论成功构建pcDNA3.1+/His/Myc-Cpn0147重组质粒并在真核细胞内表达Cpn0147蛋白,为进一步研究其分子生物学功能奠定了基础。
Objective To construct an eukaryotic expression recombinant plasmid of Cpn 0147 gene of Chlamydia pneumoniae, which laid the foundation for further study of the molecular mechanism of its interaction with host. Methods Cpn AR39 strain was used as a template to amplify the Cpn0147 full length coding specific primers. Cpn0147 gene was inserted into pcDNA3.1 + / His / Myc vector to construct pcDNA3.1 + / His / Myc-Cpn0147 recombinant plasmid The recombinant plasmids were transfected into HeLa cells by double enzyme digestion and sequencing. The transfection of recombinant plasmids was detected by RT-PCR. The expression of Cpn0147 protein in cells was detected by immunofluorescence and Western blot. Test set pcDNA3.1 + / His / Myc vector as a negative control. Results Cpn0147 gene fragment was amplified from genomic DNA of CpnAR39 strain and its size was 466 bp. The recombinant plasmid pcDNA3.1 + / His / Myc-Cpn0147 was double digested, ligated, transformed and screened. Cpn0147 gene sequences were identical; Cpn0147 gene was amplified by RT-PCR, which was consistent with the theoretical size; Fluorescent staining of HeLa cells by immunofluorescence showed red fluorescence in the cytoplasm of HeLa cells and no fluorescence in the control group; 16kd, the control group without this band. Conclusion The recombinant plasmid pcDNA3.1 + / His / Myc-Cpn0147 was successfully constructed and expressed Cpn0147 protein in eukaryotic cells, which laid the foundation for further study of its molecular biological functions.