基于特定位点广泛突变的聚合酶体外表型分析

发布时间：2018-05-29 14:12

本文选题：HBV + 聚合酶　；参考：《重庆医科大学》2017年硕士论文

【摘要】：核苷(酸)类似物应用于慢性乙肝治疗时常发生耐药。聚合酶特定位点突变与耐药有直接关系。在核苷类似物耐药相关研究中,对突变热点进行广泛突变表型分析有助于理解基因型与表型的相互关系。现有基于质粒转染的广泛突变分析方法存在一些不足。本文中,我们建立了一种聚合酶特定位点广泛突变表型分析的新策略,该方法结合了简并引物随机突变方法以及聚合酶反式互补策略。以拉米夫定(lamivudine,LAM)耐药位点rt204为例,我们检验了该策略的可行性与实用性。目的:通过结合简并引物随机突变方法以及聚合酶反式互补策略,构建一种聚合酶特定位点广泛突变表型分析的新策略。方法:1.通过简并引物随机突变和片断替换反应(Fragment Substitution Reaction,FSR),构建RT区位点特异性的多种突变;2.通过慢病毒包装混合质粒,将含特定位点多种突变引入293HBV-pol-稳定细胞系;通过反式互补,拯救293HBV-pol-中HBV复制,筛选获得稳定复制的单克隆细胞系进行体外耐药表型分析;3.以rt204(LAM耐药位点)为例,构建包含rt204位点的随机突变,经慢病毒包装后感染293HBV-pol-稳定细胞系,经抗性筛选及基因组DNA测序获得包含特定突变的单克隆细胞系,进行体外药物敏感性分析。结果简并引物随机突变可有效地获得含RT区特定位点的多种突变形式,我们通过随机引物Rpol204引入了rt204位点两个碱基的随机突变(NNT),FSR替换质粒p Lentipol-D相应区段,构建了慢病毒载体HBV重组表达质粒(野生型和突变型),经过一次聚合酶链式反应获得了rt204位点的野生型及14种突变型;经抗性筛选及基因组测序获得了6种单克隆细胞系用于耐药检测;与野生型相比,突变型有明显复制缺陷,rt M204I相对于野生型复制水平为3.039%,rt204N,RT204K与rt204I复制水平接近;部分突变类型复制能力显著降低,如rt204T,rt204R,分别为0.102%,0.005%;经LAM处理后,野生型复制水平下降了7倍左右(6.842±0.983),rt204I/T复制水平降低1倍左右(1.201±0.031,1.174±0.146),rt204N对LAM敏感性较高,经药物处理后,复制能力降低了近10倍,rt204K/R对LAM中度敏感。结论我们成功建立了一种新的突变表型分析方法,其特点有:1.通过一轮PCR及FSR,获得特定位点广泛突变;2.通过一次慢病毒包装,获得含多种突变形式的慢病毒池;3.通过反式互补的方式获得含聚合酶突变的稳定细胞系用于药物敏感性分析。该策略较传统方法而言,具有简便,高效,变异率低等特点。利用该策略,我们成功构建了rt204位点的多种突变形式,证实了各突变型较野生型有明显功能缺陷。在使用LAM处理的情况下,rt204I/T占竞争优势。这一结果符合既往文献报道,且能合理解释临床相关耐药现象。以上结果提示该方法具有可行性。我们建立的这一实验策略可用于HBV突变型及HBV准种复制水平及药物敏感性评估,为临床慢性乙型肝炎病人的诊断和核苷(酸)类药物治疗提供新思路,为HBV耐药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.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R512.62

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