人类疱疹病毒8型K1基因的克隆及其在内皮细胞和前列腺癌细胞中的表达

摘要

Objective To isolate and clone K1 gene of HHV-8 and investigate its expression both in endothelial(EC) cells and prostate cancer(PC-3) cells.Methods A pair of PCR primers for k1 gene including Xho Ⅰ and Xba Ⅰ restriction enzyme cut sites was designed according to the sequence registered in GenBank.Then the genome of HHV-8 was taken as template and K1 gene was amplified with PCR.Subsequently, amplified gene fragments were digested with the two enzymes mentioned above.A Flag sequence was introduced to facilitate the detection of K1 protein after identification with nucleotide sequences analysis,and then cloned into pCI-neo vector to generate recombinant eukaryotic expression plasmid designated as pCI-K1, recombinant pCI-K1 was transient transfected into EC and PC-3 cells.The expressions of pCI-K1 mRNA and protein in the two cell lines were detected by RTPCR and Western blot.Results Nucleotide sequences analysis indicated that the isolated and cloned pCI-K1 sequence length was 928 bp.The isolated K1 sequence was 100％ homology with K1 gene registered in GenBank.The specific bands were both detected at the expectant place by RT-PCR and Western blot.Conclusion K1 gene could be correctly expressed in EC and PC-3 cells.%目的 构建含人类疱疹病毒8型(HHV-8)致瘤基因K1的真核表达质粒,并将其导入内皮细胞(EC)和前列腺癌细胞(PC-3)中进行表达.方法 根据GenBank中登记的K1基因核酸序列设计PCR引物,在其5'端及3'端分别引入Xho Ⅰ和Xba Ⅰ酶切位点,并在其下游引入Flag标签蛋白序列用于后期K1蛋白表达的检测.以含有K1基因的HHV-8 DNA为模板扩增K1基因,经双酶切纯化后将其克隆入真核表达载体pCI-neo中.重组质粒经核酸测序鉴定后分别瞬时转染EC和PC-3细胞系,于转染后48h提取细胞RNA及蛋白,分别以RT-PCR和Westrn blot检测K1基因的转录和表达情况.结果 成功构建了含K1基因的重组质粒pCI-K1.核酸序列分析显示,克隆的K1基因全长928bp,与GenBank中登记的K1基因100%同源,引入的Flag序列完全正确.经pCI-K1转染后的EC及PC-3细胞中均可检测到K1mRNA转录和蛋白表达.结论 重组质粒pCI-K1构建成功,并在EC和PC-3细胞中正确表达.