本文選題：乙型肝炎病毒 + 基因型��； 參考：《四川大學(xué)》2007年碩士論文

【摘要】： 目的：乙型肝炎是一種流行范圍廣、危害程度大的世界性傳染病。我國是此病的高發(fā)區(qū)，乙肝攜帶者占到國民總數(shù)1／10以上，正確認(rèn)識乙肝病毒(HBV)高突變、多亞型的遺傳異質(zhì)性，對發(fā)病機(jī)制研究和臨床病毒診療均有重大意義�；谄鋸V泛存在的準(zhǔn)種現(xiàn)象，本實(shí)驗(yàn)擬采用長片段高保真聚合酶鏈反應(yīng)(LA-PCR)方法，對成都地區(qū)HBV基因型分布試作調(diào)查。 方法：設(shè)計(jì)合成HBV基因組間同源性較高的表面抗原蛋白(HBsAg)編碼區(qū)特異引物，對90例已確診的HBV-DNA陽性感染者血清裂解物進(jìn)行高保真PCR擴(kuò)增。采用雙脫氧鏈末端終止法(Sanger法)測序技術(shù)對所獲得PCR產(chǎn)物進(jìn)行測序；測序結(jié)果與GeneBank標(biāo)準(zhǔn)序列比對分型，并分析其準(zhǔn)種特征。同時，采用文獻(xiàn)中常用的型特異引物分型法作為標(biāo)準(zhǔn)對照，通過使用B、C、D三種基因型的特異引物組進(jìn)行巢式PCR擴(kuò)增檢驗(yàn)，比較兩種方法的分型結(jié)果以評估其可靠性和準(zhǔn)確性�；蛐秃突旌闲�；基因型特異引物分型法檢測結(jié)果為：B型43例(47.8％)，C型21例(23.3％)，，D型3例(3.3％)，B／C混合型22例(24.4％)，B／D混合型1例(1.1％)。測序結(jié)果顯示了HBV基因組中存在高度變異性的遺傳特征，不僅同一基因型內(nèi)廣泛存在多態(tài)性和異質(zhì)性，有些患者體內(nèi)的HBV病毒株也以多克隆的準(zhǔn)種形式存在。 結(jié)論：基因分型結(jié)果表明成都地區(qū)HBV優(yōu)勢基因型以B型為主，C型次之，偶見D型。這與文獻(xiàn)報(bào)道我國南方主要流行基因型為B型一致。對乙型肝炎患者血清病毒的測序圖進(jìn)一步分析表明，LA-PCR擴(kuò)增產(chǎn)物并非序列完全一致的單一克隆，而是一個以優(yōu)勢株為主的相關(guān)突變株病毒群(準(zhǔn)種)。S基因直接測序法能夠準(zhǔn)確地鑒定出相關(guān)突變株病毒群中的優(yōu)勢株基因型。因而與型特異引物分型法相比，排除了非優(yōu)勢突變株產(chǎn)生的假陽性干擾，更好地反映出了HBV基因的多克隆準(zhǔn)種特征，此結(jié)果差異具有統(tǒng)計(jì)學(xué)顯著性意義(P＜0.001)。實(shí)驗(yàn)結(jié)果證實(shí)了HBV高突變易重組及存在準(zhǔn)種現(xiàn)象的遺傳學(xué)特征，其遺傳異質(zhì)性，特別是一些重要多態(tài)位點(diǎn)的進(jìn)化意義以及對于乙肝診療、預(yù)后的影響尚有待進(jìn)一步研究。
[Abstract]:Objective: hepatitis B is a worldwide infectious disease with wide epidemic scope and great harm. China is a high incidence area of this disease, hepatitis B carriers account for more than 1 / 10 of the total number of people. It is of great significance to correctly understand the high mutation of hepatitis B virus (HBV) and the genetic heterogeneity of multiple subtypes, which is of great significance to the study of pathogenesis and clinical virus diagnosis and treatment. Based on the widespread quasispecies phenomenon, the long fragment high fidelity polymerase chain reaction (LPCR) method was used to investigate the genotype distribution of HBV in Chengdu. Methods: specific primers for the coding region of surface antigen protein (HBsAg) with high homology among genomes of HBV were designed and synthesized, and high fidelity PCR amplification was performed on 90 serum lysates of HBV-DNA positive patients. The PCR products were sequenced by dideoxy chain terminating method (Sanger method), and the results were compared with the standard GeneBank sequence, and the quasi species characteristics were analyzed. At the same time, the type-specific primer typing method commonly used in literature was used as the standard control, and the nested PCR amplification test was carried out by using the specific primer groups of three genotypes of BCU D, and the results of the two methods were compared to evaluate the reliability and accuracy of the two methods. The results of genotypic and mixed genotyping were as follows: 43 cases of type B, 47.8% and 21 cases of type C, 23. 3 and D, 3 cases of B / C mixed type, 22 cases of B / C mixed type 24. 4% B / D mixed type, 1 case of B / D mixed type. Sequencing results showed that there was a high degree of genetic variability in the HBV genome. Not only was there polymorphism and heterogeneity in the same genotype, but also in some patients, the HBV virus strains existed in the form of polyclonal quasispecies. Conclusion: the results of genotyping showed that the dominant genotype of HBV in Chengdu was B type C, and occasionally D type. This is consistent with the literature reported that the main epidemic genotype in southern China is B type. Further analysis of serovirus sequencing in patients with hepatitis B showed that the amplification product of La-PCR was not a single clone with identical sequence. It is a dominant mutant virus group (quasispecies. S gene direct sequencing method can accurately identify the dominant strain genotype in the related mutant virus group. Compared with the type-specific primer typing method, the false positive interference produced by non-dominant mutant was excluded, and the polyclonal quasispecies characteristics of HBV gene were better reflected. The difference was statistically significant (P < 0.001). The results confirmed the genetic characteristics of high mutation and quasispecies of HBV, and its genetic heterogeneity, especially the evolutionary significance of some important polymorphic loci, and its influence on the diagnosis and treatment of hepatitis B need to be further studied.
【學(xué)位授予單位】：四川大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R373

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 樊和斌,郭亞兵,楊潔,侯金林,王戰(zhàn)會,朱幼芙,秦勁峰;慢性乙型肝炎患者HBV基因型及其臨床意義[J];第一軍醫(yī)大學(xué)學(xué)報(bào);2005年02期

2 劉映霞,胡國齡,譚德明;湖南省乙肝病毒基因型分布及臨床意義[J];湖南醫(yī)科大學(xué)學(xué)報(bào);2002年01期

3 閻麗,侯金林,王戰(zhàn)會,牛貞玉,郭亞兵,駱抗先;寧夏地區(qū)乙型肝炎病毒基因型分布及D基因型的測序鑒定[J];解放軍醫(yī)學(xué)雜志;2000年01期

4 成軍;董菁;劉妍;李莉;斯崇文;王勤環(huán);張玲霞;陳菊梅;;乙型肝炎病毒準(zhǔn)種研究的臨床意義[J];世界華人消化雜志;2002年02期

5 成軍;董菁;;乙型肝炎病毒基因組結(jié)構(gòu)與功能復(fù)雜性的新認(rèn)識[J];世界華人消化雜志;2003年08期

6 繆曉輝,孔憲濤;乙型肝炎病毒基因檢測必須重視的幾個問題[J];中華肝臟病雜志;2001年01期

7 許軍,王齊欣,蔣棟,陳紅松,魏來,王宇,楊柳明,趙延龍;乙型肝炎病毒基因型與病情輕重的關(guān)系[J];中華肝臟病雜志;2003年01期

8 梁曉峰,陳園生,王曉軍,賀雄,陳麗娟,王駿,林長纓,白呼群,嚴(yán)俊,崔鋼,于競進(jìn);中國3歲以上人群乙型肝炎血清流行病學(xué)研究[J];中華流行病學(xué)雜志;2005年09期

9 范金水,莊輝,李遠(yuǎn)貴,朱曉潔,徐德忠,馬為民,王躍民,陳雅潔,婁國強(qiáng),馬廷賢;我國8城市HBsAg陽性和陰性乙肝患者的病毒血清型和基因型分析[J];中華微生物學(xué)和免疫學(xué)雜志;1998年02期

10 李雁,湯釗猷,葉勝龍,劉銀坤,陳潔,薛瓊,黃曉武,陳軍,鮑衛(wèi)華,楊炯,高東梅;體內(nèi)連續(xù)篩選法建立自發(fā)性肺轉(zhuǎn)移人肝癌細(xì)胞系[J];中華醫(yī)學(xué)雜志;2002年09期

本文編號：1991245