| 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 |
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| 引用本文： | 闵治超,王子盾,糜远源,程伟,华立新,冯宁翰. 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[J]. 江苏医药, 2011, 37(1) |
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| 作者姓名： | 闵治超 王子盾 糜远源 程伟 华立新 冯宁翰 |
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| 作者单位： | 南京医科大学第一附属医院泌尿外科,210029 |
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| 基金项目： | 中国博士后基金资助(20080441064) |
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| 摘 要： | 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。
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| 关 键 词： | 人类疱疹病毒8型 K1致瘤基因 真核表达 前列腺癌 |
Clone and expression of K1 gene of HHV-8 in EC cells and PC-3 cells |
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| MIN Zhichao,WANG Zidun,MI Yuanyuan,et al.. Clone and expression of K1 gene of HHV-8 in EC cells and PC-3 cells[J]. Jiangsu Medical Journal, 2011, 37(1) |
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| Authors: | MIN Zhichao WANG Zidun MI Yuanyuan et al. |
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| Affiliation: | MIN Zhichao,WANG Zidun,MI Yuanyuan,et al.Department of Urology,First Affiliated Hospital,Nanjing Medical University,Nanjing 210029,CHINA |
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| Abstract: | 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 facilita... |
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| Keywords: | Human herpesvirus 8 K1 oncoprorein Eukaryotic expression Prostate cancer |
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