慢病毒载体介导HBV X基因稳定表达对HL7702细胞线粒体结构和功能的影响

发布时间：2018-03-11 17:31

本文选题：慢病毒　切入点：X蛋白　出处：《福建医科大学》2014年博士论文　论文类型：学位论文

【摘要】：目的:以慢病毒为载体构建稳定表达乙型肝炎病毒X基因(HBV X)的HL7702细胞株,探讨HBV X稳定表达对HL7702细胞线粒体结构和功能的影响。方法:首先根据In-Fusion技术原理设计克隆引物,采用PCR技术从真核质粒pc DNA3.1-X中扩增出含HBV X基因DNA全长片段,用In-Fusion交换酶将PCR产物与线性化慢病毒载体质粒连接,经PCR、测序、质粒表达检测鉴定。将构建成功的重组慢病毒质粒PLOV.CMV.e GFP.2A.3×FlagHBx.EF1a.Pura与包装质粒ps PAX2、包膜质粒p MD2.G三质粒瞬时共转染293T细胞以包装慢病毒颗粒,慢病毒颗粒的滴度采用荧光定量PCR的方法检测。包装好的重组慢病毒颗粒感染HL7702细胞,并经过嘌呤霉素筛选获得稳定表达HBV X的细胞株HL7702/HBx,并经过RT-PCR和Western-blot实验证实HL7702/HBx细胞内有HBV X稳定持续的表达。以半定量RT-PCR方法和Western-blot方法检测细胞色素C氧化酶III(COXIII)表达水平变化,酶动力学方法检测线粒体细胞色素C氧化酶活性,流式细胞术检测细胞内ROS水平和线粒体跨膜电位,利用激光扫描共聚焦显微镜采集荧光信号观察COXIII与HBV X蛋白(HBx)在肝细胞HL7702内的定位,扫描电镜下观察HL7702/HBx细胞线粒体超微结构的改变。结果:成功构建表达3×FlagHBx融合基因的重组慢病毒表达载体,经293T细胞包装和浓缩后,重组慢病毒滴度为8.61×107IU/ml。慢病毒感染HL7702细胞后经嘌呤霉素药物筛选,得到稳定表达HBx的新细胞株HL7702/HBx,经RT-PCR和Western-blot分析结果显示HL7702/HBx细胞能够持续稳定表达HBx。HBx在细胞浆和细胞核中表达,而细胞浆中HBx可分布于线粒体,并与COXIII共定位表达。扫描电镜下发现稳定表达HBx的细胞线粒体较空白对照组和空载对照组线粒体脊稍肿胀,细胞形态及结构无明显改变。半定量RT-PCR实验结果发现COXIII m RNA表达水平无明显改变,Western-blot实验结果表明HL7702/HBx细胞内COXIII蛋白表达上调,线粒体细胞色素C氧化酶活性水平升高,同时也发现细胞内ROS水平上升,而线粒体跨膜电位没有发生明显改变。结论:成功构建稳定表达HBx的细胞株HL7702/HBx。细胞浆中HBx可定位于线粒体并与COXIII相互作用,通过转录后水平调节COXIII表达,从而提高线粒体细胞色素C氧化酶活性和细胞内ROS水平,导致线粒体脊稍肿胀,但不改变线粒体跨膜电位和细胞形态。
[Abstract]:Objective: to construct a HL7702 cell line expressing HBV X gene stably by lentivirus and to investigate the effect of stable expression of HBV X on the mitochondrial structure and function of HL7702 cells. Methods: firstly, primers were designed according to the principle of In-Fusion technique. The full-length DNA fragment containing HBV X gene was amplified by PCR from eukaryotic plasmid PC DNA3.1-X. The PCR product was ligated with the linearized lentivirus vector plasmid by In-Fusion exchange enzyme. The recombinant lentivirus plasmid PLOV.CMV.e GFP.2A.3 脳 FlagHBx.EF1a.Pura and the packaging plasmid PS PAX2 were constructed and cotransfected into 293T cells to package the lentivirus particles. The titer of lentivirus particles was detected by fluorescent quantitative PCR. Packaged recombinant lentivirus particles infected HL7702 cells. The cell line HL7702 / HBxexpressing HBV X stably was obtained by purine mycin screening, and the expression of HBV X was confirmed by RT-PCR and Western-blot experiments. The expression of HBV X was detected by semi-quantitative RT-PCR method and Western-blot method. The activity of cytochrome C oxidase in mitochondria was detected by enzyme kinetic method, the intracellular ROS level and mitochondrial transmembrane potential were detected by flow cytometry. Fluorescence signals were collected by laser scanning confocal microscope to observe the localization of COXIII and HBV X protein in HL7702 of hepatocytes. Results: the recombinant lentivirus expression vector expressing 3 脳 FlagHBx fusion gene was successfully constructed and packaged and concentrated by 293T cells. The recombinant lentivirus titer was 8.61 脳 10 ~ (7) IU / ml. After infection of lentivirus with HL7702 cells, a novel cell line HL7702 / HBxexpressing HBx was obtained by purine mycin drug screening. The results of RT-PCR and Western-blot analysis showed that HL7702/HBx cells could express HBx.HBx in cytoplasm and nucleus steadily. However, HBx in cytoplasm could be distributed in mitochondria and co-expressed with COXIII. Under scanning electron microscope, mitochondria with stable expression of HBx were slightly swollen than those of blank control group and no-load control group. The results of semi-quantitative RT-PCR assay showed that the expression of COXIII m RNA did not change significantly. The results of Western-blot analysis showed that the expression of COXIII protein was up-regulated and the activity of cytochrome C oxidase in mitochondria was increased in HL7702/HBx cells. At the same time, it was also found that the level of ROS increased, but the mitochondrial transmembrane potential did not change significantly. Conclusion: the stable expression of HBx in HL7702 / HBx. the HBx in cytoplasm can be located in mitochondria and interact with COXIII. The expression of COXIII was regulated at posttranscriptional level, which increased the activity of cytochrome C oxidase and the level of intracellular ROS, which resulted in a slight swelling of mitochondria ridges, but did not change the mitochondrial transmembrane potential and cell morphology.
【学位授予单位】：福建医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R512.6;R735.7

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