肺炎衣原体0308基因真核表达载体构建及表达检测

Construction of eukaryotic expression vector for Cpn0308 gene and its expression in eukaryotic cells

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 the recombinant plasmid containing Cpn0308 gene from Chlamydia pneumoniae AR39,and transfect it into HeLa cells to express the protein,in preparation for the development of DNA vaccine.Methods Cpn0308 gene was amplified from the genomic DNA of CpnAR39 by polymerase chain reaction（PCR）,the gene was inserted into appropriate site of pcDNA3.1/His A vector.After identification by restrictive enzymes digestion and sequencing,the recombinant plasmid was transfected into HeLa cells using Liposome.The expressed protein was identified by indirect immunofluorescence assay.Results The 393bp specific gene Cpn0308 was obtained,The DNA sequence of Cpn0308 gene was consistent with the nucleotide sequence that had been published on Genebank.Indirect immunofluorescence assay showed that Cpn0308 protein expressed in HeLa cells.Conclusion Successfully constructed the eukaryotic expression recombinant pcDNA3.1/His A-Cpn0308,and the recombinant plasmid can express Cpn0308 protein in eukaryotic system which lay the foundation for studying the biological activities and the development of the chlamydia pneumoniae vaccine.