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目的构建肺炎衣原体AR39株Cpn0308基因真核表达重组质粒,体外转染HeLa细胞并检测表达情况,为Cpn核酸疫苗的研制做准备。方法应用PCR技术从CpnAR39株基因组中扩增Cpn0308全长基因,重组入pcDNA3.1/HisA真核表达载体相应位点,进行酶切鉴定和序列分析后,运用脂质体介导重组质粒转染HeLa细胞,间接免疫荧光技术检测目的蛋白在真核细胞中表达情况。结果 PCR扩增得到393bp的特异性Cpn0308基因,筛选鉴定出pcDNA3.1/HisA-Cpn0308真核表达重组体,序列测定证实与GeneBank登录的CpnAR39株Cpn0308基因一致;间接免疫荧光法检测到重组质粒转染的HeLa细胞能够表达目的蛋白。结论成功构建pcDNA3.1/HisA-Cpn0308真核表达重组体,且该重组体能够在体外真核细胞中表达目的蛋白,为进一步研究CpnDNA疫苗以及研究该蛋白的生物学功能提供实验基础。
Objective To construct a recombinant plasmid expressing Cpn0308 gene of Chlamydia pneumoniae AR39 and transfect HeLa cells in vitro and detect the expression of Cpn0308 gene in preparation for Cpn nucleic acid vaccine. Methods Cpn0308 full-length gene was amplified from CpnAR39 genome by PCR and recombined into the corresponding sites of eukaryotic expression vector pcDNA3.1 / HisA. After restriction analysis and sequence analysis, the recombinant plasmid was transfected HeLa cells, indirect immunofluorescence assay to detect the expression of the target protein in eukaryotic cells. Results The 393bp Cpn0308 gene was amplified by PCR. The eukaryotic expression recombinant pcDNA3.1 / HisA-Cpn0308 was identified by PCR. The sequence was confirmed to be consistent with the Cpn0308 gene of CpnAR39 strain registered by GeneBank. The recombinant plasmid was detected by indirect immunofluorescence Dyed HeLa cells express the protein of interest. Conclusion The recombinant eukaryotic expression vector pcDNA3.1 / HisA-Cpn0308 was successfully constructed and expressed in eukaryotic cells in vitro. This study provides an experimental basis for further study of CpnDNA vaccine and its biological function.