乙型肝炎病毒負(fù)性調(diào)節(jié)元件亞元件的鑒定及功能研究
發(fā)布時(shí)間：2019-06-24 21:55
【摘要】： 乙型肝炎病毒(hepatitis B virus,HBV)屬嗜肝DNA病毒,是一種嚴(yán)重危害人類健康的病原體。乙型肝炎病毒感染人體后將導(dǎo)致急、慢性乙型肝炎,部分患者演變?yōu)楦斡不?甚至肝細(xì)胞癌,乙型肝炎在我國(guó)危害尤其嚴(yán)重。 HBV可以從復(fù)制、表達(dá)、轉(zhuǎn)錄、轉(zhuǎn)錄后等多個(gè)環(huán)節(jié)調(diào)節(jié)自身復(fù)制。HBV在宿主細(xì)胞核內(nèi)形成穩(wěn)定的病毒遺傳物質(zhì),使病毒持續(xù)復(fù)制增殖,在體內(nèi)建立持久的感染,難以被抗病毒藥物徹底清除。目前,人們對(duì)HBV負(fù)鏈轉(zhuǎn)錄調(diào)控的研究已較透徹,而對(duì)HBV正鏈的轉(zhuǎn)錄及調(diào)節(jié)還有許多未知之處。本實(shí)驗(yàn)室前期研究HBV正鏈轉(zhuǎn)錄體的工作中發(fā)現(xiàn)HBV nt250-453之間的序列是一新的負(fù)性調(diào)節(jié)元件。 本課題在前期研究基礎(chǔ)上,進(jìn)一步研究HBV nt250-453負(fù)性調(diào)節(jié)元件所包含的亞元件,分析其對(duì)HBV復(fù)制的影響,為深入理解HBV復(fù)制表達(dá)的自身調(diào)控提供新的知識(shí),為抗HBV感染的治療提供新的思路。 第一部分HBV負(fù)性調(diào)節(jié)元件亞元件的鑒定 目的:鑒定HBV nt250-453負(fù)性調(diào)節(jié)元件中存在的重要功能亞元件。 方法:構(gòu)建包含HBV nt453-434、nt403-384和nt283-264序列的蟲(chóng)熒光素酶基因表達(dá)質(zhì)粒,分別與內(nèi)參海參熒光素酶表達(dá)質(zhì)粒共轉(zhuǎn)染HepG2細(xì)胞,利用雙熒光素酶報(bào)告基因檢測(cè)系統(tǒng),檢測(cè)蟲(chóng)熒光素酶的表達(dá)情況。逆轉(zhuǎn)錄PCR檢測(cè)蟲(chóng)熒光素酶基因的mRNA水平。 結(jié)果:與pGL3-control組比較,含有單個(gè)亞元件的重組質(zhì)粒組相對(duì)熒光素酶活性降低,分別為79.66%、80.79%、78.36%;含有2個(gè)亞元件的重組質(zhì)粒組進(jìn)一步降低至55.83%、56.27%、61.44%;含有3個(gè)亞元件的重組質(zhì)粒組的相對(duì)熒光素酶活性明顯降低,為pGL3-control組的38.83%。逆轉(zhuǎn)錄PCR顯示熒光素酶的mRNA水平降低,與熒光素酶活性檢測(cè)結(jié)果相符合。 結(jié)論:HBV nt453-434、nt403-384和nt283-264序列為HBV nt250-453負(fù)性調(diào)節(jié)元件中發(fā)揮關(guān)鍵作用的三個(gè)亞元件;這三個(gè)亞元件可以獨(dú)立發(fā)揮作用;當(dāng)以協(xié)同的方式發(fā)揮作用時(shí),則表現(xiàn)出最強(qiáng)的負(fù)性調(diào)節(jié)作用。 第二部分負(fù)性調(diào)節(jié)元件對(duì)HBV復(fù)制的影響 目的:研究HBV nt250-453負(fù)性調(diào)節(jié)元件對(duì)HBV復(fù)制的影響。 方法:將缺失HBV nt250-453序列的突變質(zhì)粒MLJ196與輔助質(zhì)粒LJ96共轉(zhuǎn)染HepG2細(xì)胞,同時(shí)設(shè)立野生型質(zhì)粒LJ196與LJ96共轉(zhuǎn)染組為對(duì)照,在輔助質(zhì)粒LJ96提供的HBV病毒C蛋白、P蛋白作用下形成HBV復(fù)制中間體。轉(zhuǎn)染后72h提取細(xì)胞內(nèi)總DNA,利用Southern雜交檢測(cè)缺失HBV nt250-453序列后對(duì)HBV復(fù)制中間體的影響。 結(jié)果:Southern雜交檢測(cè)到HBV DNA復(fù)制中間體,與轉(zhuǎn)染野生型質(zhì)粒LJ196+LJ96組相比較,轉(zhuǎn)染突變質(zhì)粒MLJ196+LJ96組的HBV rcDNA條帶無(wú)明顯差別。陰性對(duì)照組無(wú)雜交信號(hào)。 結(jié)論:HBV nt250-453負(fù)性調(diào)節(jié)元件對(duì)形成HBV復(fù)制中間體無(wú)影響,推測(cè)該負(fù)性調(diào)節(jié)元件對(duì)HBV轉(zhuǎn)錄水平的抑制作用并未影響到病毒的復(fù)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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R373
本文編號(hào)：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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R373
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