B基因型和C基因型HBV转录活性差异的机制研究

发布时间：2018-05-16 12:51

本文选题：乙型肝炎病毒 + 基因型　；参考：《苏州大学》2014年硕士论文

【摘要】：目前全世界大约有3.5亿人群感染了乙型肝炎病毒（HBV），HBV属于嗜肝病毒，在复制周期中需要以前基因组RNA（pgRNA）为模板逆转录成共价闭合环状DNA（cccDNA），而逆转录酶缺少校对功能，因此在复制过程中容易发生碱基突变，从而形成不同的准种。当病毒基因组的核苷酸差异大于8%时，即形成了不同的基因型。根据HBV全基因组序列的差异，HBV可以分为10种主要的基因型（A-J），这10种基因型在全世界呈地域性分布，我国最常见的是基因型B和C。在临床上，HBV前C区（PC）G1896A突变和核心启动子区（BCP）A1762T/G1764A突变可分别会减弱和阻断HBeAg的表达，常见于HBeAg阴性慢性乙型肝炎（CHB）患者；与B基因型慢性HBV感染者相比，C基因型感染者的BCP区突变率较高，HBV活动性复制的持续时间较长，HBeAg血清学转换时间较晚，且更容易发展至终末期肝病和肝细胞肝癌（HCC）。在HBeAg阳性的慢性乙型肝炎患者中，基因型C患者的HBV DNA滴度和HBeAg滴度较高，且更容易发生BCP区A1762T/G1764A突变，而基因型B更容易发生前C区G1896A突变。近年来，国内外对来源于B和C基因型CHB患者的临床分离毒株的生物学特性报道较少。Qin等通过体外分析的方法发现BCP区未发生A1762T/G1764A突变的基因型C pcRNA和前基因组RNA（pg RNA）的转录水平比基因型B低，BCP区发生A1762T/G1764A突变的基因型C转录水平均明显增强，在野毒株BCP区人为的引入A1762T/G1764A双突变也可以明显增强HBV的转录水平。因为核心启动子直接启动pgRNA和pcRNA的转录，进而影响基因组复制，根据以上研究结果我们假设核心启动子的活性是影响B基因型和C基因型HBV转录活性差异的主要因素，本实验中我们对以上假设开展了三部分的研究工作。第一部分研究是B基因型和C基因型HBV核心启动子区序列分析。我们从Genbank下载了453条B基因型HBV全基因序列、525条C基因型序列；另外从慢乙肝患者血清中抽提HBV基因组，构建病毒群全基因质粒，测定基因型，然后挑取100条B基因型和100条C基因型单克隆质粒。用Vector NTI软件比对两种基因型样本的核心启动子序列，结果发现：（1）B基因型HBV有32%存在A1762T/G1764A突变，C基因型HBV有68%存在A1762T/G1764A突变。（2）B基因型和C基因型核心启动子区在nt1633、nt1635、nt1636、nt1652、nt1673、nt1730的位置存在差异，并且碱基类型与基因型有关。第二部分的工作主要是对B基因型和C基因型HBV标本的核心启动子活性进行分析。我们通过构建pGL2表达质粒，分析外源性启动子对pGL2表达萤火虫（Fluc）和海肾荧光素酶（Rluc）的影响。我们在nt1611-nt1632之间设计一条包含SacI酶切位点的上游引物，，在nt1862-nt1886之间设计一条包含HindIII酶切位点的下游引物，PCR扩增启动子片段，构建含有HBV核心启动子序列的pGL2重组质粒，将质粒转染至Huh7和HepG2细胞系，通过双荧光素酶报告系统分析Fluc/Rluc的比值来反映外源性启动子活性。结果发现：（1）在Huh7和HepG2细胞系中HBV B基因型核心启动子活性显著强于C基因型（P0.05）。（2）相同基因型的标本之间的核心启动子活性存在差异，对应的核心启动子序列也不相同。（3）核心启动子活性和HBV体外转录活性相关。该结果初步验证了HBV基因型B和基因型C启动子活性的差异是影响这两种基因型体外转录活性差异的主要因素的假设。在第三部分中，根据本研究第一部分HBV基因型B和C启动子区序列比对结果，我们采用定点突变的方法对上述构建的pGL2质粒中HBV基因型B和基因型C启动子区进行定点突变，把nt1633、nt1635、nt1636、nt1652、nt1673、nt1730位置的碱基突变成另一种基因型相同位点的碱基类型，然后分析碱基突变前后双荧光素酶表达的变化。结果发现：（1）nt1633、nt1635、nt1636、nt1652联合突变时， B基因型核心启动子活性明显减弱，C基因型核心启动子活性明显增强。（2）nt1673和nt1730分别突变后对B基因型核心启动子活性影响差异不显著，对C基因型核心启动子活性影响显著。结果进一步说明了上述碱基位点对核心启动子的活性影响较大，核心启动子活性是影响B基因型和C基因型HBV体外转录活性差异的主要因素。
[Abstract]:At present, about 350 million people all over the world are infected with hepatitis B virus (HBV), and HBV belongs to the liver virus. In the replication cycle, the previous genome RNA (pgRNA) is needed as a template to reverse the covalently closed circular DNA (cccDNA), while the reverse transcriptase lacks the proofreading function. When the nucleotide difference of the virus genome is greater than 8%, different genotypes are formed. According to the difference of the whole genome sequence of HBV, HBV can be divided into 10 major genotypes (A-J), the 10 genotypes are distributed all over the world, and the most common in our country is the genotype B and C. in the clinical, HBV before C region (PC) G1896A mutation and core initiation The BCP A1762T/G1764A mutation can weaken and block the expression of HBeAg, which is common in patients with HBeAg negative chronic hepatitis B (CHB). Compared with the B genotype chronic HBV infection, the BCP region mutation rate of the C genotype infected persons is higher, the HBV active replication duration is longer, the HBeAg serological conversion time is late, and it is easier to hair. To end-stage liver disease and hepatocellular carcinoma (HCC). In patients with HBeAg positive chronic hepatitis B, the HBV DNA titer and HBeAg titer of the genotype C patients are higher, and the BCP region A1762T/G1764A mutation is more likely to occur, and the genotype B is more likely to occur in the G1896A mutation in the anterior C region. The biological characteristics of the isolates were reported less.Qin and so on. The transcriptional level of the genotype C pcRNA and the pre genomic RNA (PG RNA) without A1762T/G1764A mutation in the BCP region was lower than that of the genotype B, and the C transcriptional level of the A1762T/G1764A mutation in the BCP region was obviously enhanced. 1762T/G1764A double mutation can also significantly enhance the transcriptional level of HBV, because the core promoter directly starts the transcription of pgRNA and pcRNA and then affects genome replication. According to the results, we hypothesized that the activity of the core promoter is the main factor affecting the difference in the transcriptional activity of the B genotypes and the C genotypes of HBV. It is assumed that three parts of the research work have been carried out.
The first part of the study is the sequence analysis of the B genotype and the C genotype HBV core promoter region. We download 453 B genotypic HBV full gene sequences and 525 C genotypes from Genbank, and also extract the HBV genome from the sera of the patients with chronic hepatitis B, construct the whole gene plasmid of the virus group, determine the genotypes, and then pick up 100 B genotypes and 100 strips. C genotype monoclonal plasmids were used to compare the core promoter sequences of two genotypes with Vector NTI software. The results were as follows: (1) 32% of B genotype HBV had A1762T/G1764A mutations, and 68% of C genotype HBV had A1762T/G1764A mutation. (2) the B gene type and C genotype promoter region were in nt1633, There are differences in the location, and the base type is related to genotype.
The second part of the work is mainly to analyze the core promoter activity of the B genotype and the C genotype HBV. By constructing the pGL2 expression plasmid, we analyzed the effect of exogenous promoter on the expression of Fluc and the sea kidney luciferase (Rluc) by the exogenous promoter. We design an upstream of the SacI enzyme cut site between the nt1611-nt1632. Primers were designed to design a downstream primer containing HindIII enzyme cut site between nt1862-nt1886, PCR amplification promoter fragment and pGL2 recombinant plasmid containing HBV core promoter sequence. The plasmid was transfected into Huh7 and HepG2 cell lines, and the ratio of Fluc/Rluc was analyzed by double luciferase reporter system to reflect the activity of exogenous promoter. The results were as follows: (1) the activity of HBV B genotype core promoter activity in Huh7 and HepG2 cell lines was significantly stronger than that of C genotype (P0.05). (2) the activity of core promoter between the specimens of the same genotypes was different, and the corresponding core promoter sequences were also different. (3) the activity of the core promoter was related to the transcriptional activity of HBV in vitro. The results were preliminarily verified. The difference in promoter activity between HBV genotype B and genotype C is the main factor affecting the difference in transcriptional activity between the two genotypes.
In the third part, according to the sequence alignment of the HBV genotype B and C promoter in the first part of this study, we use site directed mutagenesis to mutagenesis the HBV genotypic B and genotype C promoter in the pGL2 plasmids constructed above, and turn the bases of nt1633, nt1635, nt1636, nt1652, nt1673, and locations into another gene. Based on the base type of the same loci, the changes in the expression of the double luciferase before and after the base mutation were analyzed. The results were as follows: (1) when nt1633, nt1635, nt1636, nt1652 were combined, the activity of the core promoter of the B genotypes was obviously weakened and the activity of the core promoter of the C genotypes increased significantly. (2) the B genotype core was initiated after the mutation of nt1673 and nt1730 respectively. There is no significant difference in the effect of the activity of the promoter, which has a significant effect on the activity of the core promoter of the C genotype. The results further indicate that the above base site has a great influence on the activity of the core promoter, and the core promoter activity is the main factor affecting the difference in the transcriptional activity of the B genotype and the C genotype HBV in vitro.

【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R512.62

【共引文献】