人巨细胞病毒gB基因真核表达载体的构建与鉴定

Construction and Identification of Eukaryotic Expression Vector of Human Cytomegalovirus gB Gene

目的 克隆人巨细胞病毒 (HCMV)gB基因并构建真核表达载体。方法 根据Genebank中HCMV核苷酸序列设计引物 ,并在引物的 5′端分别加入BamHⅠ、XhoⅠ限制性内切酶位点 ,特异性扩增gB编码基因片段。TA克隆后经双酶切、PCR、测序等鉴定重组质粒 ,再经双酶切、连接构建含HCMVgB编码基因的真核表达载体 ,转染COS7细胞 ,通过间接免疫荧光法 (IFA)检测该重组真核表达载体在COS7细胞中的表达。结果 重组质粒经BamHⅠ、XhoⅠ双酶切成大小为 5 0kb与 2 7kb的片段 ,表明表达载体pcDNA3 1中插入了HCMVgB基因片段 ,测序结果表明读码框正确。重组表达载体pcDNA3 1 gB转染COS7细胞后 ,经IFA检测胞浆中可见黄绿色荧光。结论成功构建了pcDNA3 1 gB真核表达载体 。

Objective To clone human cytomegalovirus (HCMV) gB gene and construct an eukaryotic expression vector.Methods According to the nucleotide sequence of HCMV in Genbank, design and synthesize a pair of primers with BamH I and Xho I sites at 5′-terminal respectively and amplify HCMV gB DNA fragment by PCR. Insert the amplified gB gene fragment into pGEM-T vector and identify the recombinant plasmid by restriction analysis, PCR and sequencing.Subclone the amplified gB gene fragment into an eukaryotic vector pcDNA3.1 by a series of molecular biological methods including restriction digest and ligation, and transfect the recombinant expression plasmid pcDNA 3.1/gB into COS7 cells. Identify the expressed product by IFA.Results Two gene fragments, at the lengths of 5.0 kb and 2.7 kb,were obtained by the digestion of recombinant plasmid pcDNA 3.1 with BamH I and Xho I respectively.It proved that HCMV gB gene fragment was correctly inserted into expression vector pcDNA3.1.Sequencing showed a correct ORF. IFA proved the gB protein was expressed in COS7 cells.Conclusion An eukaryotic expression vector was successfully constructed, and gB protein was expressed in COS7 cells.