【作者】 刘昕；

【导师】 黄孝天；

【作者基本信息】 南昌大学 ， 病原生物学， 2012， 硕士

【摘要】 目的：构建柯萨奇病毒（CVB3）结构蛋白VP1基因的真核质粒pBudCE4.1-VP1，并检测其在HeLa细胞中的表达，研究VP1蛋白对HeLa细胞形态、活性及细胞周期等方面的影响，为探讨CVB3的分子致病机制奠定基础。方法：1.质粒pBudCE4.1-VP1构建：抽提CVB3感染的HeLa细胞的总mRNA，RT-PCR扩增VP1基因，经KpnI和BglII双酶切后，克隆于真核载体pBudCE4.1中，酶切鉴定后进行DNA测序验证；2.质粒pBudCE4.1-VP1表达：将质粒pBudCE4.1-VP1转染HeLa细胞，采用Western blotting和间接免疫荧光技术检测VP1蛋白在HeLa细胞中的表达；3.质粒pBudCE4.1-VP1对细胞形态和活性的影响：质粒转染HeLa细胞后，倒置显微镜连续观察VP1蛋白对细胞形态的影响，通过MTT法检测HeLa细胞的活性变化；4.质粒pBudCE4.1-VP1对高尔基体的影响：采用免疫荧光双标技术分析质粒VP1蛋白对细胞高尔基体基质蛋白GM130结构的影响；5.质粒pBudCE4.1-VP1对细胞周期的影响：以G1/S期为切入点，应用流式细胞仪检测VP1转染后16h、20h、24h、28h、32h等时间点细胞周期的变化。结果：1.限制性内切酶酶切和测序结果表明VP1已成功插入pBudCE4.1载体的多克隆位点，其基因序列与CVB3m株（GI:323432）VP1基因序列完全一致。2. Western blotting和免疫荧光技术结果显示：质粒pBudCE4.1-VP1转染HeLa细胞后能检测到VP1蛋白的表达，且表达量随转染时间的延长而增加；3.质粒pBudCE4.1-VP1转染HeLa细胞后12h，部分细胞开始变圆；随着转染时间的延长，细胞出现较为明显的病变效应，到转染48h、60h后，几乎未见正常细胞。MTT法结果显示：质粒VP1转染的细胞与对照组细胞相比，OD值明显降低（P＜0.05），且转染时间越长，细胞活性越差；4.免疫荧光技术分析结果显示：质粒pBudCE4.1-VP1转染HeLa细胞后，随VP1蛋白表达量的增加，细胞蛋白GM130绿色荧光由带状分布趋于弥散，呈散在分布，且荧光强度减弱；5.细胞周期结果显示：质粒pBudCE4.1-VP1转染HeLa细胞16h、20h、24h、28h、32h后，与空质粒转染对照组G1期细胞相比，VP1转染组G1期细胞分别升高到：58.81%（P＜0.05），57.15%（P＜0.05），54.87%（P＜0.01），55.46%（P＜0.05），56.96%（P＜0.05）。结论：1.成功构建CVB3VP1基因真核表达质粒pBudCE4.1-VP1，并能在HeLa细胞中表达；2. VP1蛋白作用于HeLa细胞后可引起明显的细胞病变效应；3. VP1蛋白可引起高尔基带断裂，VP1蛋白可能是参与CVB3感染引起高尔基带断裂的主要病毒蛋白之一；4. VP1蛋白进入细胞后可将细胞周期阻滞在G1期。更多还原

【Abstract】 Objective:To construct and identify the recombinant eukaryotic plasmid of CoxsackievirusB3VP1gene, and to detect its expression in HeLa cells. Then observe its effects oncell morphous, cell viability and cell cycle with the aim to investigate the molecularmechanism of CVB3infection.Methods:1. Construction of the plasmid pBudCE4.1-VP1: The VP1gene was amplifiedby PCR from a template of total mRNA of CVB3. The amplified DNA wasdirectionally inserted into vector pBudCE4.1. Then the recombinant plasmid wasidentified by restriction endonucleases digestion and sequencing;2. Expression of the plasmid pBudCE4.1-VP1: The expression of VP1genewas observed by Western blotting and immunofluorescently after transfection;3. The effection of the plasmid pBudCE4.1-VP1on HeLa cells was detected byMTT assay and observed the change of cell morphology by inverted microscope;4. To detect the interaction among the CVB3VP1protein and host proteinGM130by confocal microscopy with double immunofluorescent stain aftertransrected;5. The effection of the plasmid pBudCE4.1-VP1on cell cycle:Cell cycledistribution was detected by flow cytometry after transfeced16h,20h,24h,28h and32h.Result:1. The result of restriction endonucleases treatment and sequencing confirmthat VP1gene was successfully inserted into the vector pBudCE4.1in the samesequence to CVB3m type;2. The reusult of Western blotting and immunofluorescent show that the VP1gene expressed obviously in HeLa cells after transfected and the expression increasedover time; 3. After the VP1transfected into Hela cells12h, a typical cytopathetic effectwas observed in transfected HeLa cells; The reusult of MTT assay show thattransfection of recombinant plasmid pBudCE4.1-VP1can significantly inhibit theproliferation of HeLa cells;4. The reusult of double immunolabelled GM130and VP1showed that: withthe increased of VP1protein, the GM130was ribbon-like in normal cells and becomedisperse in the transfected cells;5. Flow cytometry analysis revealed: Compared with the mock transfectedgroup which transfected with vector pBudCE4.1, the proportion of G1phase wasincreased to58.81%(P＜0.05),57.15%(P＜0.05),54.87%(P＜0.01),55.46%(P＜0.05) and56.96%(P＜0.05) at16h,20h,24h,28h and32h post transfection.Conclusions:1. The eukaryotic expression plasmid VP1gene of CVB3was successfullyconstructed and express in HeLa cells;2. The VP1can cause significant cytopathic effect and inhibit the proliferationof HeLa cells;3. The golgi ribbon was brokendown and the structure of golgi apparatus wasdestroyed after transfection. VP1would be the key structure protein of causing Golgiapparatus destruction in the CVB3infected cells;4. After tansfecion with pBudCE4.1-VP1, the HeLa cell cycle should be mainlyarrest in G1phage.更多还原