单纯疱疹病毒Ⅱ型ICP22真核表达载体的构建及其对Vero细胞活性的影响  

Construction of eukaryotic expression vector of herpes simplex virus type Ⅱ ICP22 and the effects on the activities of Vero cells

作　　者：张强[1] 薄惠[1] 邢建华[1] 王高峰[1] 

机构地区：[1]黄河科技学院医学院实验中心,河南郑州450063

出　　处：《现代预防医学》2014年第22期4117-4120,共4页Modern Preventive Medicine

摘　　要：目的构建单纯疱疹病毒Ⅱ型(HSV-2)感染细胞蛋白22(ICP22)真核表达载体p EGFP-C2/ICP22并研究其对非洲绿猴肾细胞(Vero)细胞活性的影响。方法经双酶切实验和测序验证,以Xfect为介导将构建的真核表达载体p EGFP-ICP22转染至Vero细胞,并通过反转录PCR(RT-PCR)和绿色荧光检验其在细胞中的表达,最后以MTT法检测细胞活性。结果 48 h后观察,发现转染重组质粒组细胞经RT-PCR验证有目的基因的转录,并通过荧光显微镜观察到融合蛋白表达。而空白质粒组只检测到荧光蛋白但未检测到目的基因转录本,且对照组未检测到荧光蛋白表达。转染重组质粒的细胞相对活性值为(82.39±6.44)%。结论成功构建了p EGFP-ICP22真核表达载体,实现了其在Vero中的表达,融合蛋白GFP-ICP22明显降低了细胞活性。为进一步探讨HSV-ⅡICP22的功能和HSV-Ⅱ潜伏复发机制奠定了实验基础。Objective The aim of this study was to construct pEGFP-C2/HSV-2 ICP22 vector and analyze the effects on the activities of Vero cells in vitro. Methods The HSV-2 ICP22 eukaryotic expression vector pEGFP-ICP22 was constructed and verified by double enzymes digestion and sequencing. Then the recombinant plasmid was transfected into Vero cells mediated by Xfect. The expression of the recombination plasmid in the cells was observed by fluorescence microscopy and the expression of ICP22 was verified by RT-PCR. The cell activity was analyzed by MTF. Results The fragment of the target gene was observed by agarose electrophoresis and the recombinant plasmid pEGFP-C2/ICP22 was verified by restrictive endonuclease assay and sequencing analysis. The expression of fusion protein in Vero cells was observed by fluorescence microscope and its mRNA expression levels were examined by reverse transcription PCR. The relative activity of Vero cells was 82.39 ± 6.44. Conclusion The eukaryotic expression plasmid for ICP22 was successfully constructed and its was expressed effectively in Vero cells, while the expression of fusion protein GFP-ICP22 significantly reduced the activity of the cells, which could lay a foundation for the further study of the function of HSV- Ⅱ ICP22 and the role in the process of HSV- Ⅱ latency-reactivation.

关 键 词：真核表达载体 感染细胞蛋白22 单纯疱疹病毒Ⅱ型 

分 类 号：R319[医药卫生—基础医学]