本文選題：乙型肝炎病毒 + 基因型　； 參考：《蘇州大學(xué)》2014年碩士論文

【摘要】：目前全世界大約有3.5億人群感染了乙型肝炎病毒（HBV），HBV屬于嗜肝病毒，在復(fù)制周期中需要以前基因組RNA（pgRNA）為模板逆轉(zhuǎn)錄成共價(jià)閉合環(huán)狀DNA（cccDNA），而逆轉(zhuǎn)錄酶缺少校對(duì)功能，因此在復(fù)制過(guò)程中容易發(fā)生堿基突變，從而形成不同的準(zhǔn)種。當(dāng)病毒基因組的核苷酸差異大于8%時(shí)，即形成了不同的基因型。根據(jù)HBV全基因組序列的差異，HBV可以分為10種主要的基因型（A-J），這10種基因型在全世界呈地域性分布，我國(guó)最常見(jiàn)的是基因型B和C。在臨床上，HBV前C區(qū)（PC）G1896A突變和核心啟動(dòng)子區(qū)（BCP）A1762T/G1764A突變可分別會(huì)減弱和阻斷HBeAg的表達(dá)，常見(jiàn)于HBeAg陰性慢性乙型肝炎（CHB）患者；與B基因型慢性HBV感染者相比，C基因型感染者的BCP區(qū)突變率較高，HBV活動(dòng)性復(fù)制的持續(xù)時(shí)間較長(zhǎng)，HBeAg血清學(xué)轉(zhuǎn)換時(shí)間較晚，且更容易發(fā)展至終末期肝病和肝細(xì)胞肝癌（HCC）。在HBeAg陽(yáng)性的慢性乙型肝炎患者中，基因型C患者的HBV DNA滴度和HBeAg滴度較高，且更容易發(fā)生BCP區(qū)A1762T/G1764A突變，而基因型B更容易發(fā)生前C區(qū)G1896A突變。近年來(lái)，國(guó)內(nèi)外對(duì)來(lái)源于B和C基因型CHB患者的臨床分離毒株的生物學(xué)特性報(bào)道較少。Qin等通過(guò)體外分析的方法發(fā)現(xiàn)BCP區(qū)未發(fā)生A1762T/G1764A突變的基因型C pcRNA和前基因組RNA（pg RNA）的轉(zhuǎn)錄水平比基因型B低，BCP區(qū)發(fā)生A1762T/G1764A突變的基因型C轉(zhuǎn)錄水平均明顯增強(qiáng)，在野毒株BCP區(qū)人為的引入A1762T/G1764A雙突變也可以明顯增強(qiáng)HBV的轉(zhuǎn)錄水平。因?yàn)楹诵膯?dòng)子直接啟動(dòng)pgRNA和pcRNA的轉(zhuǎn)錄，進(jìn)而影響基因組復(fù)制，根據(jù)以上研究結(jié)果我們假設(shè)核心啟動(dòng)子的活性是影響B(tài)基因型和C基因型HBV轉(zhuǎn)錄活性差異的主要因素，本實(shí)驗(yàn)中我們對(duì)以上假設(shè)開(kāi)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核心啟動(dòng)子區(qū)序列分析。我們從Genbank下載了453條B基因型HBV全基因序列、525條C基因型序列；另外從慢乙肝患者血清中抽提HBV基因組，構(gòu)建病毒群全基因質(zhì)粒，測(cè)定基因型，然后挑取100條B基因型和100條C基因型單克隆質(zhì)粒。用Vector NTI軟件比對(duì)兩種基因型樣本的核心啟動(dòng)子序列，結(jié)果發(fā)現(xiàn)：（1）B基因型HBV有32%存在A1762T/G1764A突變，C基因型HBV有68%存在A1762T/G1764A突變。（2）B基因型和C基因型核心啟動(dòng)子區(qū)在nt1633、nt1635、nt1636、nt1652、nt1673、nt1730的位置存在差異，并且堿基類(lèi)型與基因型有關(guān)。 第二部分的工作主要是對(duì)B基因型和C基因型HBV標(biāo)本的核心啟動(dòng)子活性進(jìn)行分析。我們通過(guò)構(gòu)建pGL2表達(dá)質(zhì)粒，分析外源性啟動(dòng)子對(duì)pGL2表達(dá)螢火蟲(chóng)（Fluc）和海腎熒光素酶（Rluc）的影響。我們?cè)趎t1611-nt1632之間設(shè)計(jì)一條包含SacI酶切位點(diǎn)的上游引物，，在nt1862-nt1886之間設(shè)計(jì)一條包含HindIII酶切位點(diǎn)的下游引物，PCR擴(kuò)增啟動(dòng)子片段，構(gòu)建含有HBV核心啟動(dòng)子序列的pGL2重組質(zhì)粒，將質(zhì)粒轉(zhuǎn)染至Huh7和HepG2細(xì)胞系，通過(guò)雙熒光素酶報(bào)告系統(tǒng)分析Fluc/Rluc的比值來(lái)反映外源性啟動(dòng)子活性。結(jié)果發(fā)現(xiàn)：（1）在Huh7和HepG2細(xì)胞系中HBV B基因型核心啟動(dòng)子活性顯著強(qiáng)于C基因型（P0.05）。（2）相同基因型的標(biāo)本之間的核心啟動(dòng)子活性存在差異，對(duì)應(yīng)的核心啟動(dòng)子序列也不相同。（3）核心啟動(dòng)子活性和HBV體外轉(zhuǎn)錄活性相關(guān)。該結(jié)果初步驗(yàn)證了HBV基因型B和基因型C啟動(dòng)子活性的差異是影響這兩種基因型體外轉(zhuǎn)錄活性差異的主要因素的假設(shè)。 在第三部分中，根據(jù)本研究第一部分HBV基因型B和C啟動(dòng)子區(qū)序列比對(duì)結(jié)果，我們采用定點(diǎn)突變的方法對(duì)上述構(gòu)建的pGL2質(zhì)粒中HBV基因型B和基因型C啟動(dòng)子區(qū)進(jìn)行定點(diǎn)突變，把nt1633、nt1635、nt1636、nt1652、nt1673、nt1730位置的堿基突變成另一種基因型相同位點(diǎn)的堿基類(lèi)型，然后分析堿基突變前后雙熒光素酶表達(dá)的變化。結(jié)果發(fā)現(xiàn)：（1）nt1633、nt1635、nt1636、nt1652聯(lián)合突變時(shí)， B基因型核心啟動(dòng)子活性明顯減弱，C基因型核心啟動(dòng)子活性明顯增強(qiáng)。（2）nt1673和nt1730分別突變后對(duì)B基因型核心啟動(dòng)子活性影響差異不顯著，對(duì)C基因型核心啟動(dòng)子活性影響顯著。結(jié)果進(jìn)一步說(shuō)明了上述堿基位點(diǎn)對(duì)核心啟動(dòng)子的活性影響較大，核心啟動(dòng)子活性是影響B(tài)基因型和C基因型HBV體外轉(zhuǎn)錄活性差異的主要因素。
[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.

【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R512.62

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

1 Jun Cheng;Min Quan;Min Li;Shun-ai Liu;Qi Wang;;Quasispecies of Hepatitis B Virus[J];Infection International(Electronic Edition);2012年04期

2 劉魚(yú);王憬惺;何苗;楊通漢;

本文編號(hào)：1896879