本文選題：HBV + 聚合酶�。� 參考：《重慶醫(yī)科大學》2017年碩士論文

【摘要】：核苷(酸)類似物應用于慢性乙肝治療時常發(fā)生耐藥。聚合酶特定位點突變與耐藥有直接關系。在核苷類似物耐藥相關研究中,對突變熱點進行廣泛突變表型分析有助于理解基因型與表型的相互關系�，F(xiàn)有基于質粒轉染的廣泛突變分析方法存在一些不足。本文中,我們建立了一種聚合酶特定位點廣泛突變表型分析的新策略,該方法結合了簡并引物隨機突變方法以及聚合酶反式互補策略。以拉米夫定(lamivudine,LAM)耐藥位點rt204為例,我們檢驗了該策略的可行性與實用性。目的:通過結合簡并引物隨機突變方法以及聚合酶反式互補策略,構建一種聚合酶特定位點廣泛突變表型分析的新策略。方法:1.通過簡并引物隨機突變和片斷替換反應(Fragment Substitution Reaction,FSR),構建RT區(qū)位點特異性的多種突變;2.通過慢病毒包裝混合質粒,將含特定位點多種突變引入293HBV-pol-穩(wěn)定細胞系;通過反式互補,拯救293HBV-pol-中HBV復制,篩選獲得穩(wěn)定復制的單克隆細胞系進行體外耐藥表型分析;3.以rt204(LAM耐藥位點)為例,構建包含rt204位點的隨機突變,經(jīng)慢病毒包裝后感染293HBV-pol-穩(wěn)定細胞系,經(jīng)抗性篩選及基因組DNA測序獲得包含特定突變的單克隆細胞系,進行體外藥物敏感性分析。結果簡并引物隨機突變可有效地獲得含RT區(qū)特定位點的多種突變形式,我們通過隨機引物Rpol204引入了rt204位點兩個堿基的隨機突變(NNT),FSR替換質粒p Lentipol-D相應區(qū)段,構建了慢病毒載體HBV重組表達質粒(野生型和突變型),經(jīng)過一次聚合酶鏈式反應獲得了rt204位點的野生型及14種突變型;經(jīng)抗性篩選及基因組測序獲得了6種單克隆細胞系用于耐藥檢測;與野生型相比,突變型有明顯復制缺陷,rt M204I相對于野生型復制水平為3.039%,rt204N,RT204K與rt204I復制水平接近;部分突變類型復制能力顯著降低,如rt204T,rt204R,分別為0.102%,0.005%;經(jīng)LAM處理后,野生型復制水平下降了7倍左右(6.842±0.983),rt204I/T復制水平降低1倍左右(1.201±0.031,1.174±0.146),rt204N對LAM敏感性較高,經(jīng)藥物處理后,復制能力降低了近10倍,rt204K/R對LAM中度敏感。結論我們成功建立了一種新的突變表型分析方法,其特點有:1.通過一輪PCR及FSR,獲得特定位點廣泛突變;2.通過一次慢病毒包裝,獲得含多種突變形式的慢病毒池;3.通過反式互補的方式獲得含聚合酶突變的穩(wěn)定細胞系用于藥物敏感性分析。該策略較傳統(tǒng)方法而言,具有簡便,高效,變異率低等特點。利用該策略,我們成功構建了rt204位點的多種突變形式,證實了各突變型較野生型有明顯功能缺陷。在使用LAM處理的情況下,rt204I/T占競爭優(yōu)勢。這一結果符合既往文獻報道,且能合理解釋臨床相關耐藥現(xiàn)象。以上結果提示該方法具有可行性。我們建立的這一實驗策略可用于HBV突變型及HBV準種復制水平及藥物敏感性評估,為臨床慢性乙型肝炎病人的診斷和核苷(酸)類藥物治療提供新思路,為HBV耐藥表型分析提供了新方法。
[Abstract]:Nucleoside (acid) analogues are often resistant to chronic hepatitis B therapy. PCR mutation is directly related to resistance. In the study of nucleoside analogues resistance, extensive mutation phenotype analysis of mutation hot spots helps to understand the interrelationship between genotypes and phenotypes. Extensive mutation analysis based on plasmid transfection There are some shortcomings in this method. In this paper, we have established a new strategy for the phenotypic analysis of PCR wide mutation. This method combines the random mutation method of degenerate primers and the polymerase chain reaction strategy. The lamivudine (LAM) resistance locus rt204 is used as an example to test the feasibility and practicability of the strategy. Objective: to construct a new strategy for the analysis of the phenotypic mutation of PCR by combining the random mutation method of degenerate primers and the polymerase chain reaction strategy. Methods: 1. through the random mutation and fragment replacement reaction (Fragment Substitution Reaction, FSR) of the degenerate primers, a variety of mutations in the RT location point specificity are constructed; 2. links are constructed. The hybrid plasmids were packaged by the slow virus, and a variety of mutational mutations were introduced into 293HBV-pol- stable cell lines. By trans complementation, HBV replication in 293HBV-pol- was saved and the monoclonal cell lines that obtained stable replication were screened for drug resistance phenotype analysis in vitro. 3. a random mutation containing rt204 site was constructed with rt204 (LAM resistance site) as an example. After virus packaging, 293HBV-pol- stable cell lines were infected, and the monoclonal cell lines containing specific mutations were obtained by resistance screening and genomic DNA sequencing. The results of drug sensitivity analysis in vitro were carried out. Results the random mutation of degenerate primers could effectively obtain a variety of mutant forms containing the specific location of the RT region. We introduced rt204 by random primer Rpol204. The random mutation of two bases (NNT) and the corresponding region of FSR replacement plasmid P Lentipol-D were used to construct the recombinant expression plasmid (wild type and mutant type) of the lentivirus vector HBV. After a polymerase chain reaction, the wild type and 14 mutagenesis of the rt204 locus were obtained, and 6 monoclonal cell lines were obtained by resistance screening and genome sequencing. Compared with wild type, the mutant had obvious replicative defects. The replication level of RT M204I was 3.039% relative to the wild type, rt204N, RT204K and rt204I replication level, and the replication ability of partial mutation type decreased significantly, such as rt204T, rt204R, 0.102%, 0.005% respectively. After LAM treatment, the level of wild type replication decreased by about 7 times (6.84 2 + 0.983), rt204I/T replication level was reduced about 1 times (1.201 + 0.031,1.174 + 0.146), rt204N was more sensitive to LAM. After drug treatment, the replication ability was reduced by nearly 10 times, and rt204K/R was moderately sensitive to LAM. Conclusion we successfully established a new mutant phenotype analysis method, which features: 1. through a round of PCR and FSR, to obtain special location points. Extensive mutation; 2. by a lentivirus package, the lentivirus pool with multiple mutations is obtained; 3. a stable cell line containing polymerase mutation is obtained by the trans complementary method for drug sensitivity analysis. This strategy is simple, efficient, and low mutation rate compared with the traditional method. Using this strategy, we successfully constructed the rt20 A variety of mutations at the 4 locus have confirmed that each mutant has obvious functional defects than the wild type. In the case of LAM treatment, rt204I/T has a competitive advantage. This result is in line with previous literature and can reasonably explain the clinical resistance. The results suggest that the method is feasible. The experimental strategy established by us can be established. The use of HBV mutant and HBV quasi species replication level and drug sensitivity assessment provides new ideas for the diagnosis of chronic hepatitis B patients and the treatment of nucleoside (acid) drugs, and provides a new method for the analysis of HBV resistant phenotype.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R512.62

【參考文獻】

相關期刊論文 前2條

1 Akinobu Tawada;Tatsuo Kanda;Osamu Yokosuka;;Current and future directions for treating hepatitis B virus infection[J];World Journal of Hepatology;2015年11期

2 Rebecca Pastor;Franois Habersetzer;Samira Fafi-Kremer;Michel Doffoёl;Thomas F Baumert;Jean-Pierre Gut;Franoise Stoll-Keller;Evelyne Schvoerer;;Hepatitis B virus mutations potentially conferring adefovir/ tenofovir resistance in treatment-naive patients[J];World Journal of Gastroenterology;2009年06期

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本文編號：1951168