本文選題：核苷類似物 + HBV突變株��； 參考：《河北醫(yī)科大學》2010年碩士論文

【摘要】： 乙型肝炎病毒(HBV)持續(xù)感染是一世界性的衛(wèi)生問題,慢性肝炎終至肝硬化甚至肝細胞癌的發(fā)生。隨著疫苗的普遍接種及抗病毒藥物的廣泛應用,有效的降低了發(fā)病率及死亡率,但是也給人類帶來一系列的問題:疫苗/診斷逃逸突變以及核苷類似物長期應用引發(fā)耐藥導致檢測和治療的失敗。這些突變株與野生株相比,在生物學特性方面必然有其特殊性。為了進一步了解突變株對病毒復制力、疫苗接種效能所帶來的影響,以及研究核苷類似物的耐藥機制、篩選新型藥物,有必要建立全基因組的細胞模型。國內(nèi)外研究表明,HepG2和Huh7細胞瞬時轉(zhuǎn)染乙肝病毒質(zhì)粒后,能夠支持乙肝病毒的復制。然而,由于缺乏復制的穩(wěn)定性,對藥物篩選等高要求的實驗是不適用的。HepG2.2.15細胞系是目前公認的應用最廣泛的乙型肝炎細胞模型,并且被廣泛應用于抗病毒研究。不僅病毒復制均衡、變異小,而且經(jīng)多代培養(yǎng)仍可以長時間地維持病毒復制。但是病毒復制水平較低,并且不能人為控制。為解決目前面臨的難題,有必要構(gòu)建一個穩(wěn)定的、可誘導表達的HBV突變株細胞系。四環(huán)素(Tet)誘導的基因表達系統(tǒng)是目前研究和應用最為廣泛的真核細胞基因表達系統(tǒng)。本研究就是利用PCR、酶切等手段構(gòu)建多個HBV全基因組突變株,轉(zhuǎn)染前期已構(gòu)建成功的tTA細胞系,用潮霉素篩選,構(gòu)建多個突變株可調(diào)控性表達細胞系,并作為細胞模型檢測已知的HBV抑制劑的作用。為進一步完善HBsAg診斷試劑盒及其變異監(jiān)測、研究耐藥機制、篩選新型抗病毒藥物奠定了實驗基礎。本研究共分2部分完成。 第一部分多種HBV突變株可調(diào)控性表達細胞系的構(gòu)建 目的:應用分子生物學技術(shù),將HBV突變株克隆到pTRE2Hyg載體上,構(gòu)建8個HBV全基因組突變株。通過轉(zhuǎn)染前期實驗構(gòu)建成功的tTA細胞系,構(gòu)建多種突變株可調(diào)控性表達細胞系,觀察病毒表達情況,證明穩(wěn)定復制能力及可調(diào)控性。 方法: 1應用PCR和酶切重組技術(shù),在PCH-3093質(zhì)粒P基因RT區(qū)B, C, D亞功能域173, 180, 181, 204, 236位點及S基因區(qū)100, 120, 145位點實現(xiàn)定點突變,并克隆到pTRE2Hyg載體上,構(gòu)建具有潮霉素抗性的pTRE-HBV-A181T, pTRE-HBV-N236T, pTRE-HBV-A181T-N236T, pTRE-HBV-M204I, pTRE-HBV-L180M-M204V, pTRE-HBV-V173L-L180M -M204V, pTRE-HBV-G145R, pTRE-HBV-Y100C-P120T 8個質(zhì)粒。 2通過酶切及測序鑒定新構(gòu)建質(zhì)粒的正確性后,大量提取質(zhì)粒。 3前期實驗已經(jīng)成功構(gòu)建了整合有ptTA2質(zhì)粒的HepG2細胞系(G2TA2-7)。上述8個新構(gòu)建質(zhì)粒和pTRE-WMHBV分別轉(zhuǎn)染此細胞,經(jīng)潮霉素篩選,獲得陽性細胞克隆。 4分別經(jīng)過PCR篩選出高分泌突變HBV DNA及WMHBV DNA顆粒的細胞系。 5在無抗生素篩選條件下,連續(xù)傳代20代,Southern blot觀察其穩(wěn)定復制能力,并挑選最佳細胞系。 6細胞系可調(diào)控性的鑒定應用southern blot驗證各細胞系在強力霉素去除或加入時HBV DNA的表達情況。 7檢測突變對病毒復制能力的影響應用質(zhì)粒轉(zhuǎn)染技術(shù)及Southern blot檢測病毒復制能力的變化情況。 結(jié)果: 1成功構(gòu)建了8個具有潮霉素抗性的突變株質(zhì)粒。 2轉(zhuǎn)染后,經(jīng)潮霉素篩選,分別獲得60-100個細胞克隆。 3分別挑選其中48個單細胞克隆進行擴大培養(yǎng),經(jīng)PCR檢測細胞上清液中HBV DNA及WMHBV DNA,選取4個表達量較高者。 4在無抗生素篩選條件下,繼續(xù)傳代培養(yǎng)20代(約4個月),通過southern blot檢測,在細胞裂解液中能夠形成HBV復制中間體,并且均強于G2.2.15細胞。最終選定A181T55, N236T13, A181T-N236T1, M204I43, L180M- M204V16, V173L-L180M-M204V44, G145R1, Y100C-P120T3, WMHBV42九個細胞株。 5通過southern blot檢測,證明了所構(gòu)建的幾個細胞系為可誘導性表達,即去除強力霉素時,細胞裂解液中能夠形成HBV復制中間體;加入時則停止表達。 6與野生株HBV相比,突變株M204I, M204V及N236T的復制能力明顯下降;而L180M-M204V及G145R, Y100C-P120T變化不大。 結(jié)論:成功構(gòu)建了八個突變株和一個WMHBV可調(diào)控性表達細胞系,有助于研究某一基因在各個不同發(fā)育時期的功能,也為篩選新型抗病毒藥物、完善檢測方法奠定了實驗基礎。 第二部分HBV突變株細胞系對常用的HBV抑制劑的敏感性研究目的: 檢測新構(gòu)建的突變株及WMHBV細胞系對常用核苷類似物的敏感性。為完善檢測方法及進一步篩選新型藥物奠定了基礎。 方法: 1通過Southern blot,檢測新構(gòu)建的核苷類似物耐藥突變株對拉米夫定和阿德福韋酯的敏感性。 2通過Native southern,檢測新構(gòu)建細胞系對拉米夫定和阿德福韋酯的敏感性。 3通過質(zhì)粒瞬時轉(zhuǎn)染及Southern blot,檢測阿德福韋酯理論耐藥突變點對阿德福韋酯敏感性。 4經(jīng)PCR及基因測序技術(shù),驗證新構(gòu)建細胞系的阿德福韋酯耐藥突變位點。 結(jié)果: 1經(jīng)Southern blot檢測發(fā)現(xiàn)在A181T55, N236T13, A181T-N236T1及作為對照的野生型HBV株加拉米夫定組,隨著藥物濃度的不斷加大,HBV復制中間體表達水平逐漸減弱;而L180M-M204V16, M204I43及V173L-L180M-M204V44三組細胞系,HBV復制中間體表達水平不因加拉米夫定而改變,并且不受藥物濃度的影響;所有細胞系加阿德福韋酯組,包括A181T55, N236T13及A181T-N236T1均對阿德福韋酯敏感。 2經(jīng)Native southern發(fā)現(xiàn),野生型HBV病毒株、G145R1, Y100C-P120T3及WMHBV42的復制水平均能被拉米夫定及阿德福韋酯有效的抑制;L180M-M204V16及M204I43拉米夫定組,病毒復制水平未見減弱;所有細胞系加阿德福韋酯組包括N236T13,復制水平均可見明顯減弱。 3經(jīng)質(zhì)粒瞬時轉(zhuǎn)染及Southern blot檢測發(fā)現(xiàn),阿德福韋酯理論耐藥突變點N236T,與野生型HBV相似,同樣對阿德福韋酯敏感。 4經(jīng)基因測序,證實新構(gòu)建細胞系A181T55的181位點、N236T13的236位點及A181T-N236T1的181和236位點上均有突變,并且突變與設計完全一致。 結(jié)論:作為細胞模型部分驗證了常見的HBV抑制劑的作用,為進一步篩選新型抗HBV藥物、進一步構(gòu)建動物模型、開展體內(nèi)實驗奠定基礎。
[Abstract]:The continuous infection of hepatitis B virus (HBV) is a worldwide health problem, chronic hepatitis and even liver cell carcinoma. With the widespread vaccination and the widespread use of antiviral drugs, the incidence and mortality of the hepatitis B virus are effectively reduced, but a series of problems are also brought to human beings: vaccine / diagnosis escape mutation and nuclear The long-term application of glucoside analogues causes resistance to the failure of detection and treatment. These mutant strains are bound to be specific in biological characteristics compared with wild plants. In order to further understand the effects of mutant strains on virus replication, vaccine efficacy, and the mechanism of resistance to nucleoside analogues, new drugs are screened. It is necessary to establish a whole genome cell model. Domestic and foreign studies have shown that HepG2 and Huh7 cells can be transiently transfected into HBV plasmids and can support the replication of HBV. However, due to the lack of replication stability, the.HepG2.2.15 cell line, which is not suitable for the high requirement of drug screening, is the most widely accepted application. Hepatitis B cell model, which is widely used in antiviral research, is not only balanced and small, but can still maintain virus replication for a long time. But the replication level of the virus is low and can not be controlled artificially. It is necessary to build a stable, inducible HBV process to solve the current problem. The gene expression system induced by tetracycline (Tet) is the most widely used gene expression system for eukaryotic cells. This study is to construct a number of all HBV mutant strains by means of PCR and enzyme cutting. The successful tTA cell line has been constructed in the early stage of transfection, and a variety of mutant strains can be constructed and regulated by hygromycin. The expression of cell lines is expressed as a cell model to detect the known HBV inhibitors. It provides an experimental basis for further improving the HBsAg diagnostic kit and its variation monitoring, studying the mechanism of drug resistance, and screening new antiviral drugs. This study is divided into 2 parts.
The first part is the construction of a variety of HBV mutant cell lines.
Objective: to construct 8 HBV whole genome mutant strains by cloning the HBV mutant strain on pTRE2Hyg vector by molecular biology technology, and construct a successful tTA cell line through the preliminary transfection experiment to construct a variety of mutant strains to express the cell lines, observe the virus expression, and verify the stable replication ability and regulation.
Method:
1 using PCR and enzyme cut recombination technology, the fixed point mutation was realized in the RT region of the RT region B, C, D subdomain 173, 180, 181, 204, 236, and S gene region 100, 120 and 145, and was cloned on the pTRE2Hyg vector to construct pTRE-HBV-A181T for the hygromycin resistance, pTRE-HBV-N236T, pTRE-HBV-A181T-N236T and dialectical. L180M-M204V, pTRE-HBV-V173L-L180M -M204V, pTRE-HBV-G145R, pTRE-HBV-Y100C-P120T 8 plasmids.
2 after digested and sequenced to identify the correctness of the new plasmid, a large number of plasmids were extracted.
3 the HepG2 cell line (G2TA2-7) integrated with ptTA2 plasmid has been successfully constructed in the previous experiments. The above 8 newly constructed plasmids and pTRE-WMHBV were transfected to the cells respectively, and the positive cell clones were obtained by hygromycin screening.
4 the cell lines of HBV DNA and WMHBV DNA granules were screened by PCR respectively.
5 under the condition of no antibiotic screening, 20 generations were passaged continuously. Southern blot was used to observe its stable replication ability and the best cell line was selected.
6 cell line identification. Southern blot was used to verify the expression of HBV DNA in each cell line when doxycycline was removed or added.
7 detect the effect of mutation on virus replication ability, and detect the change of virus replication ability by plasmid transfection and Southern blot.
Result:
1 8 plasmids with hygromycin resistance were successfully constructed.
2 after transfection, 60-100 cell clones were obtained by hygromycin screening.
3 48 single cell clones were selected and cultured respectively. The HBV DNA and WMHBV DNA in the supernatant were detected by PCR, and 4 higher expression levels were selected.
4 under the condition of no antibiotic screening, continuous subculture for 20 generations (about 4 months), through southern blot detection, can form HBV replication intermediates in cell lysate, and are stronger than G2.2.15 cells. Finally, A181T55, N236T13, A181T-N236T1, M204I43, L180M- M204V16, V173L-L180M-M204V44, G145R1, nine A cell line.
5 through southern blot detection, it is proved that several cell lines are inducible expression, that is, when doxycycline is removed, HBV replication intermediates can be formed in the cell lysate, and the expression stops when added.
6 compared with wild strain HBV, the replication ability of mutant M204I, M204V and N236T decreased significantly, while L180M-M204V and G145R did not change significantly.
Conclusion: eight mutant strains and a WMHBV controllable expression cell line have been successfully constructed. It is helpful to study the function of one gene in different developmental stages, and also provides an experimental basis for screening new antiviral drugs and improving detection methods.
The second part is the sensitivity of HBV mutant cell lines to commonly used HBV inhibitors.
The sensitivity of the newly constructed mutant and WMHBV cell lines to the nucleoside analogues was tested, which laid the foundation for improving the detection methods and further screening new drugs.
Method:
1 the sensitivity of the newly constructed nucleoside analogs resistant mutants to lamivudine and adefovir dipivoxil was detected by Southern blot.
2 the sensitivity of newly constructed cell lines to lamivudine and adefovir dipivoxil was detected by Native Southern.
3 through plasmid transiently transfection and Southern blot, the sensitivity of adefovir dipivoxil resistant mutation to adefovir dipivoxil was detected.
4 PCR and gene sequencing technology were used to verify the resistance mutations of the newly constructed cell line.
Result:
1 by Southern blot detection, A181T55, N236T13, A181T-N236T1 and the control group of wild type HBV strain plus lamivudine group, with the increase of drug concentration, the expression level of HBV replication intermediates gradually weakened; while L180M-M204V16, M204I43 and V173L-L180M-M204V44 three groups, HBV replication intermediates were not expressed by Lamy All cell lines and adefovir groups, including A181T55, N236T13 and A181T-N236T1, were sensitive to adefovir esters.
2 Native Southern found that the replication level of wild type HBV virus strains, G145R1, Y100C-P120T3 and WMHBV42 could be effectively suppressed by lamivudine and adefovir ester. The replication level of the virus was not weakened in L180M-M204V16 and M204I43 lamivudine group; all cell lines and A De fovir group including N236T13, the level of replication was obviously reduced. Weak.
3 plasmid transiently transfection and Southern blot detection showed that the drug-resistant mutation point N236T of adefovir dipivoxil was similar to that of wild-type HBV and was also sensitive to adefovir dipivoxil.
4 the 181 site of the newly constructed cell line A181T55 was confirmed by gene sequencing, and there was a mutation at the 181 and 236 loci of the 236 site of N236T13 and A181T-N236T1, and the mutation was exactly the same as that of the design.
Conclusion: as part of the cell model, the role of the common HBV inhibitors is verified, which lays the foundation for further screening new anti HBV drugs, further building animal models and carrying out in vivo experiments.
【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R373

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