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pEGFP-C2/LAT-ORF3真核表达载体的构建及其在Vero细胞中的表达

Construction and Identification of Eukaryotic Expression Plasmid pEGFP-C2/LAT-ORF3 and Its Expression in Vero Cells
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摘要 克隆单纯疱疹病毒Ⅱ型(herpes simplex virusⅡ,HSV-2)潜伏相关转录体(latency associated transcript,LAT)第3个开放阅读框(open reading frame3,ORF3)的DNA序列,构建pEGFP-C2/LAT-ORF3真核表达载体,并观察其在非洲绿猴肾细胞(Vero)中的表达,为进一步研究LAT基因及ORF3的生物学功能奠定基础。用HSV-2333株感染Vero细胞,从Vero细胞培养液中收集HSV-2并提取HSV-2基因组。以HSV-2基因组为模板,PCR扩增得到HSV-2LAT ORF3DAN片段。将ORF3片段连接到pEGFP-C2质粒上,用卡那霉毒筛选阳性克隆,经菌落PCR,双酶切及测序验证pEGFP-C2/LAT-ORF3真核表达载体构建成功。用Xfect转染试剂将重组载体转染入Vero细胞,倒置荧光显微镜观察其在Vero细胞中的表达。双酶切及测序结果验证ORF3片段正确插入pEGFP-C2质粒,荧光显微镜观察到融合蛋白在转染的Vero细胞中的表达。成功构建pEGFP-C2/LAT-ORF3真核表达载体,该重组质粒能在Vero细胞中成功表达。 The study was to construct the eukaryotic expression plasmid containing the open reading frame（ORF）3 of the herpes simplex virus 2（HSV-2）latency associated transcript（LAT）gene,and detect its expression in Vero cells in order to lay a foundation for further research on the biological function of LAT gene and ORF3.The Vero cells were infected with HSV-2 333 strain,and HSV-2 genome was extracted from HSV-2 which were collected from cell culture fluid.The HSV-2 LAT ORF3 gene fragment was amplified by PCR,and the amplified products were inserted into pEGFP-C2 to conduct a recombinant plasmid which was selected by kanamycin,then sequenced and identified by restrictive endonuclease digestion.The constructed recombinant plasmid was finally transfected into Vero cells with Xfect transfection reagent and the expression of ORF3 was observed by inverted fluorescence microscope.Results showed that the successful construction of the recombinant plasmid pEGFP-C2/LAT-ORF3 was verified by restrictive endonuclease assay and sequence analysis with LAT ORF3.The expression of fussion protein in Vero cells was observed by fluorescence microscope.The eukaryotic expression plasmid for LAT ORF3 was successfully constructed and its expression in Vero cells was confirmed.
作者 薛礼长 杨慧兰
机构地区 华南理工大学生物科学与工程学院 中国人民解放军广州军区广州总医院皮肤科
出处 《生物技术通报》 CAS CSCD 北大核心 2010年第11期157-161,共5页 Biotechnology Bulletin
基金 国家自然科学基金资助项目(30972666) 全军医学科学技术研究"十一五"计划资助项目(06J008)
关键词 单纯疱疹病毒Ⅱ型 pEGFP-C2质粒 LAT ORF3 真核表达载体 VERO细胞 Herpes simplex virus Ⅱ pEGFP-C2 plasmid LAT ORF3 Eukaryotic expressing vector Vero cells
分类号 Q78 [生物学—分子生物学]
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参考文献16

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生物技术通报
2010年 第11期

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