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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48 h提取细胞RNA及蛋白,分别以RT-PCR和Western 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928 bp,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K1 mRNA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。
Objective To construct an eukaryotic expression plasmid containing the oncogenic gene K1 of human herpesvirus type 8 (HHV-8), which was then introduced into endothelial cells (ECs) and prostate cancer cells (PC-3) for expression. Methods PCR primers were designed according to the nucleotide sequence of K1 gene registered in GenBank. The Xho I and Xba I restriction sites were introduced into the 5 ’and 3’ end of the PCR product, and the Flag tagged protein sequence was introduced downstream for K1 protein expression The test. The K1 gene was amplified by using the HHV-8 DNA containing K1 gene. The double-digested K1 gene was cloned into the eukaryotic expression vector pCI-neo. The recombinant plasmids were transiently transfected into EC and PC-3 cell lines after being identified by nucleic acid sequencing. RNA and protein were extracted 48 h after transfection. The transcription and expression of K1 gene were detected by RT-PCR and Western blot respectively. Results The recombinant plasmid pCI-K1 containing K1 gene was successfully constructed. Nucleotide sequence analysis showed that the cloned K1 gene was 928 bp in length, which was 100% homologous to the K1 gene registered in GenBank. The introduced Flag sequence was completely correct. K1 mRNA transcription and protein expression were detected in both EC and PC-3 cells transfected with pCI-K1. Conclusion The recombinant plasmid pCI-K1 was successfully constructed and correctly expressed in EC and PC-3 cells.