乙型肝炎病毒负性调节元件亚元件的鉴定及功能研究
发布时间：2019-06-24 21:55
【摘要】： 乙型肝炎病毒(hepatitis B virus,HBV)属嗜肝DNA病毒,是一种严重危害人类健康的病原体。乙型肝炎病毒感染人体后将导致急、慢性乙型肝炎,部分患者演变为肝硬化,甚至肝细胞癌,乙型肝炎在我国危害尤其严重。 HBV可以从复制、表达、转录、转录后等多个环节调节自身复制。HBV在宿主细胞核内形成稳定的病毒遗传物质,使病毒持续复制增殖,在体内建立持久的感染,难以被抗病毒药物彻底清除。目前,人们对HBV负链转录调控的研究已较透彻,而对HBV正链的转录及调节还有许多未知之处。本实验室前期研究HBV正链转录体的工作中发现HBV nt250-453之间的序列是一新的负性调节元件。 本课题在前期研究基础上,进一步研究HBV nt250-453负性调节元件所包含的亚元件,分析其对HBV复制的影响,为深入理解HBV复制表达的自身调控提供新的知识,为抗HBV感染的治疗提供新的思路。 第一部分HBV负性调节元件亚元件的鉴定 目的:鉴定HBV nt250-453负性调节元件中存在的重要功能亚元件。 方法:构建包含HBV nt453-434、nt403-384和nt283-264序列的虫荧光素酶基因表达质粒,分别与内参海参荧光素酶表达质粒共转染HepG2细胞,利用双荧光素酶报告基因检测系统,检测虫荧光素酶的表达情况。逆转录PCR检测虫荧光素酶基因的mRNA水平。 结果:与pGL3-control组比较,含有单个亚元件的重组质粒组相对荧光素酶活性降低,分别为79.66%、80.79%、78.36%;含有2个亚元件的重组质粒组进一步降低至55.83%、56.27%、61.44%;含有3个亚元件的重组质粒组的相对荧光素酶活性明显降低,为pGL3-control组的38.83%。逆转录PCR显示荧光素酶的mRNA水平降低,与荧光素酶活性检测结果相符合。 结论:HBV nt453-434、nt403-384和nt283-264序列为HBV nt250-453负性调节元件中发挥关键作用的三个亚元件;这三个亚元件可以独立发挥作用;当以协同的方式发挥作用时,则表现出最强的负性调节作用。 第二部分负性调节元件对HBV复制的影响 目的:研究HBV nt250-453负性调节元件对HBV复制的影响。 方法:将缺失HBV nt250-453序列的突变质粒MLJ196与辅助质粒LJ96共转染HepG2细胞,同时设立野生型质粒LJ196与LJ96共转染组为对照,在辅助质粒LJ96提供的HBV病毒C蛋白、P蛋白作用下形成HBV复制中间体。转染后72h提取细胞内总DNA,利用Southern杂交检测缺失HBV nt250-453序列后对HBV复制中间体的影响。 结果:Southern杂交检测到HBV DNA复制中间体,与转染野生型质粒LJ196+LJ96组相比较,转染突变质粒MLJ196+LJ96组的HBV rcDNA条带无明显差别。阴性对照组无杂交信号。 结论:HBV nt250-453负性调节元件对形成HBV复制中间体无影响,推测该负性调节元件对HBV转录水平的抑制作用并未影响到病毒的复制。
[Abstract]:Hepatitis B virus (hepatitis B virus,HBV), a hepatophilic DNA virus, is a serious pathogen that endangers human health. Hepatitis B virus infection will lead to acute, chronic hepatitis B, some patients evolved into liver cirrhosis, and even hepatocellular carcinoma. Hepatitis B is especially harmful in our country. HBV can regulate self-replication from replication, expression, post-transcriptional and other links. HBV forms stable viral genetic material in the host nucleus, which makes the virus continue to replicate and proliferate, establish lasting infection in vivo, and is difficult to be completely eliminated by antiviral drugs. At present, the transcriptional regulation of HBV negative chain has been thoroughly studied, but there are still many unknown aspects of the transcription and regulation of HBV positive chain. In the previous study of HBV positive chain transcripts, it was found that the sequences between HBV nt250-453 were a new negative regulator. On the basis of previous research, this paper further studies the subcomponents contained in HBV nt250-453 negative regulatory elements, analyzes its influence on HBV replication, and provides a new knowledge for further understanding the self-regulation of HBV replication expression and a new way of thinking for the treatment of HBV infection. Part 1 Identification of HBV negative regulating element objective: to identify the important functional subelements in HBV nt250-453 negative regulating element. Methods: the luciferase gene expression plasmid containing HBV nt453-434,nt403-384 and nt283-264 sequences was constructed and co-transfected with the luciferase expression plasmid of Panax quinquefolium respectively. The expression of luciferase was detected by double luciferase reporter gene detection system. The mRNA level of luciferase gene was detected by reverse transcription PCR. Results: compared with the pGL3-control group, the relative luciferase activity of the recombinant plasmid group containing a single subelement was 79.66%, 80.79%, 78.36%, the recombinant plasmid group containing two subelements was further reduced to 55.83%, 56.27%, 61.44%, and the relative luciferase activity of the recombinant plasmid group containing three subelements was 38.83% of that of the pGL3-control group. Reverse transcription PCR showed that the mRNA level of luciferase decreased, which was consistent with the results of luciferase activity. Conclusion: HBV nt453-434,nt403-384 and nt283-264 sequences are three subelements that play a key role in HBV nt250-453 negative regulatory elements, and these three subelements can play an independent role, and show the strongest negative regulatory effect when they work in a cooperative way. The second part is the effect of negative regulating elements on HBV replication objective: to study the effect of HBV nt250-453 negative regulatory elements on HBV replication. Methods: the mutant plasmid MLJ196 without HBV nt250-453 sequence and the auxiliary plasmid LJ96 were co-transfected into HepG2 cells. At the same time, the wild plasmid LJ196 and LJ96 co-infected group were used as control to form HBV replication intermediate under the action of HBV virus C protein and P protein provided by auxiliary plasmid LJ96. The total DNA, was extracted 72 hours after transfection. The effect of deletion of HBV nt250-453 sequence on HBV replication intermediates was detected by Southern hybridization. Results: HBV DNA replication intermediates were detected by Southern hybridom. compared with the wild plasmid LJ196 LJ96 group, there was no significant difference in the HBV rcDNA band in the mutant plasmid MLJ196 LJ96 group. There was no hybrid signal in the negative control group. Conclusion: the negative regulatory element of HBV nt250-453 has no effect on the formation of HBV replication intermediate. It is speculated that the inhibitory effect of the negative regulatory element on the transcriptional level of HBV does not affect the replication of the virus.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R373
本文编号：2505393
[Abstract]:Hepatitis B virus (hepatitis B virus,HBV), a hepatophilic DNA virus, is a serious pathogen that endangers human health. Hepatitis B virus infection will lead to acute, chronic hepatitis B, some patients evolved into liver cirrhosis, and even hepatocellular carcinoma. Hepatitis B is especially harmful in our country. HBV can regulate self-replication from replication, expression, post-transcriptional and other links. HBV forms stable viral genetic material in the host nucleus, which makes the virus continue to replicate and proliferate, establish lasting infection in vivo, and is difficult to be completely eliminated by antiviral drugs. At present, the transcriptional regulation of HBV negative chain has been thoroughly studied, but there are still many unknown aspects of the transcription and regulation of HBV positive chain. In the previous study of HBV positive chain transcripts, it was found that the sequences between HBV nt250-453 were a new negative regulator. On the basis of previous research, this paper further studies the subcomponents contained in HBV nt250-453 negative regulatory elements, analyzes its influence on HBV replication, and provides a new knowledge for further understanding the self-regulation of HBV replication expression and a new way of thinking for the treatment of HBV infection. Part 1 Identification of HBV negative regulating element objective: to identify the important functional subelements in HBV nt250-453 negative regulating element. Methods: the luciferase gene expression plasmid containing HBV nt453-434,nt403-384 and nt283-264 sequences was constructed and co-transfected with the luciferase expression plasmid of Panax quinquefolium respectively. The expression of luciferase was detected by double luciferase reporter gene detection system. The mRNA level of luciferase gene was detected by reverse transcription PCR. Results: compared with the pGL3-control group, the relative luciferase activity of the recombinant plasmid group containing a single subelement was 79.66%, 80.79%, 78.36%, the recombinant plasmid group containing two subelements was further reduced to 55.83%, 56.27%, 61.44%, and the relative luciferase activity of the recombinant plasmid group containing three subelements was 38.83% of that of the pGL3-control group. Reverse transcription PCR showed that the mRNA level of luciferase decreased, which was consistent with the results of luciferase activity. Conclusion: HBV nt453-434,nt403-384 and nt283-264 sequences are three subelements that play a key role in HBV nt250-453 negative regulatory elements, and these three subelements can play an independent role, and show the strongest negative regulatory effect when they work in a cooperative way. The second part is the effect of negative regulating elements on HBV replication objective: to study the effect of HBV nt250-453 negative regulatory elements on HBV replication. Methods: the mutant plasmid MLJ196 without HBV nt250-453 sequence and the auxiliary plasmid LJ96 were co-transfected into HepG2 cells. At the same time, the wild plasmid LJ196 and LJ96 co-infected group were used as control to form HBV replication intermediate under the action of HBV virus C protein and P protein provided by auxiliary plasmid LJ96. The total DNA, was extracted 72 hours after transfection. The effect of deletion of HBV nt250-453 sequence on HBV replication intermediates was detected by Southern hybridization. Results: HBV DNA replication intermediates were detected by Southern hybridom. compared with the wild plasmid LJ196 LJ96 group, there was no significant difference in the HBV rcDNA band in the mutant plasmid MLJ196 LJ96 group. There was no hybrid signal in the negative control group. Conclusion: the negative regulatory element of HBV nt250-453 has no effect on the formation of HBV replication intermediate. It is speculated that the inhibitory effect of the negative regulatory element on the transcriptional level of HBV does not affect the replication of the virus.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R373
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