多种乙型肝炎病毒突变株可调控性表达细胞系的构建

发布时间：2018-07-11 15:29

本文选题：核苷类似物 + HBV突变株　；参考：《河北医科大学》2010年硕士论文

【摘要】： 乙型肝炎病毒(HBV)持续感染是一世界性的卫生问题,慢性肝炎终至肝硬化甚至肝细胞癌的发生。随着疫苗的普遍接种及抗病毒药物的广泛应用,有效的降低了发病率及死亡率,但是也给人类带来一系列的问题:疫苗/诊断逃逸突变以及核苷类似物长期应用引发耐药导致检测和治疗的失败。这些突变株与野生株相比,在生物学特性方面必然有其特殊性。为了进一步了解突变株对病毒复制力、疫苗接种效能所带来的影响,以及研究核苷类似物的耐药机制、筛选新型药物,有必要建立全基因组的细胞模型。国内外研究表明,HepG2和Huh7细胞瞬时转染乙肝病毒质粒后,能够支持乙肝病毒的复制。然而,由于缺乏复制的稳定性,对药物筛选等高要求的实验是不适用的。HepG2.2.15细胞系是目前公认的应用最广泛的乙型肝炎细胞模型,并且被广泛应用于抗病毒研究。不仅病毒复制均衡、变异小,而且经多代培养仍可以长时间地维持病毒复制。但是病毒复制水平较低,并且不能人为控制。为解决目前面临的难题,有必要构建一个稳定的、可诱导表达的HBV突变株细胞系。四环素(Tet)诱导的基因表达系统是目前研究和应用最为广泛的真核细胞基因表达系统。本研究就是利用PCR、酶切等手段构建多个HBV全基因组突变株,转染前期已构建成功的tTA细胞系,用潮霉素筛选,构建多个突变株可调控性表达细胞系,并作为细胞模型检测已知的HBV抑制剂的作用。为进一步完善HBsAg诊断试剂盒及其变异监测、研究耐药机制、筛选新型抗病毒药物奠定了实验基础。本研究共分2部分完成。第一部分多种HBV突变株可调控性表达细胞系的构建目的:应用分子生物学技术,将HBV突变株克隆到pTRE2Hyg载体上,构建8个HBV全基因组突变株。通过转染前期实验构建成功的tTA细胞系,构建多种突变株可调控性表达细胞系,观察病毒表达情况,证明稳定复制能力及可调控性。方法: 1应用PCR和酶切重组技术,在PCH-3093质粒P基因RT区B, C, D亚功能域173, 180, 181, 204, 236位点及S基因区100, 120, 145位点实现定点突变,并克隆到pTRE2Hyg载体上,构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个质粒。 2通过酶切及测序鉴定新构建质粒的正确性后,大量提取质粒。 3前期实验已经成功构建了整合有ptTA2质粒的HepG2细胞系(G2TA2-7)。上述8个新构建质粒和pTRE-WMHBV分别转染此细胞,经潮霉素筛选,获得阳性细胞克隆。 4分别经过PCR筛选出高分泌突变HBV DNA及WMHBV DNA颗粒的细胞系。 5在无抗生素筛选条件下,连续传代20代,Southern blot观察其稳定复制能力,并挑选最佳细胞系。 6细胞系可调控性的鉴定应用southern blot验证各细胞系在强力霉素去除或加入时HBV DNA的表达情况。 7检测突变对病毒复制能力的影响应用质粒转染技术及Southern blot检测病毒复制能力的变化情况。结果: 1成功构建了8个具有潮霉素抗性的突变株质粒。 2转染后,经潮霉素筛选,分别获得60-100个细胞克隆。 3分别挑选其中48个单细胞克隆进行扩大培养,经PCR检测细胞上清液中HBV DNA及WMHBV DNA,选取4个表达量较高者。 4在无抗生素筛选条件下,继续传代培养20代(约4个月),通过southern blot检测,在细胞裂解液中能够形成HBV复制中间体,并且均强于G2.2.15细胞。最终选定A181T55, N236T13, A181T-N236T1, M204I43, L180M- M204V16, V173L-L180M-M204V44, G145R1, Y100C-P120T3, WMHBV42九个细胞株。 5通过southern blot检测,证明了所构建的几个细胞系为可诱导性表达,即去除强力霉素时,细胞裂解液中能够形成HBV复制中间体;加入时则停止表达。 6与野生株HBV相比,突变株M204I, M204V及N236T的复制能力明显下降;而L180M-M204V及G145R, Y100C-P120T变化不大。结论:成功构建了八个突变株和一个WMHBV可调控性表达细胞系,有助于研究某一基因在各个不同发育时期的功能,也为筛选新型抗病毒药物、完善检测方法奠定了实验基础。第二部分HBV突变株细胞系对常用的HBV抑制剂的敏感性研究目的: 检测新构建的突变株及WMHBV细胞系对常用核苷类似物的敏感性。为完善检测方法及进一步筛选新型药物奠定了基础。方法: 1通过Southern blot,检测新构建的核苷类似物耐药突变株对拉米夫定和阿德福韦酯的敏感性。 2通过Native southern,检测新构建细胞系对拉米夫定和阿德福韦酯的敏感性。 3通过质粒瞬时转染及Southern blot,检测阿德福韦酯理论耐药突变点对阿德福韦酯敏感性。 4经PCR及基因测序技术,验证新构建细胞系的阿德福韦酯耐药突变位点。结果: 1经Southern blot检测发现在A181T55, N236T13, A181T-N236T1及作为对照的野生型HBV株加拉米夫定组,随着药物浓度的不断加大,HBV复制中间体表达水平逐渐减弱;而L180M-M204V16, M204I43及V173L-L180M-M204V44三组细胞系,HBV复制中间体表达水平不因加拉米夫定而改变,并且不受药物浓度的影响;所有细胞系加阿德福韦酯组,包括A181T55, N236T13及A181T-N236T1均对阿德福韦酯敏感。 2经Native southern发现,野生型HBV病毒株、G145R1, Y100C-P120T3及WMHBV42的复制水平均能被拉米夫定及阿德福韦酯有效的抑制;L180M-M204V16及M204I43拉米夫定组,病毒复制水平未见减弱;所有细胞系加阿德福韦酯组包括N236T13,复制水平均可见明显减弱。 3经质粒瞬时转染及Southern blot检测发现,阿德福韦酯理论耐药突变点N236T,与野生型HBV相似,同样对阿德福韦酯敏感。 4经基因测序,证实新构建细胞系A181T55的181位点、N236T13的236位点及A181T-N236T1的181和236位点上均有突变,并且突变与设计完全一致。结论:作为细胞模型部分验证了常见的HBV抑制剂的作用,为进一步筛选新型抗HBV药物、进一步构建动物模型、开展体内实验奠定基础。
[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.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R373

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