本文選題：乙型肝炎病毒 + 變異�。� 參考：《武漢大學(xué)》2005年碩士論文

【摘要】：乙型肝炎病毒(hepatitis B virus, HBV)屬嗜肝DNA病毒科,為不完全環(huán)狀雙鏈DNA病毒。完整的HBV顆粒為42nm大小的球形顆粒,即Dane顆粒。HBV是目前已知的對(duì)人類(lèi)致病的最小DNA病毒,基因組全長(zhǎng)約3.2kb。HBV有四個(gè)主要的開(kāi)放讀碼框,分別稱(chēng)為S區(qū)、C區(qū)、P區(qū)、X區(qū),有些區(qū)是重疊的,HBV這種利用基因組DNA的遺傳信息的高效性在自然界中是罕見(jiàn)的。HBV復(fù)制過(guò)程中要經(jīng)過(guò)逆轉(zhuǎn)錄,由于逆轉(zhuǎn)錄酶缺乏有效的堿基校對(duì)功能,故HBV基因組比一般DNA病毒易發(fā)生變異。這給疾病的預(yù)防、診斷、治療和預(yù)后帶來(lái)新問(wèn)題。 本實(shí)驗(yàn)采用裂解液煮沸法提取血清中HBV DNA。該法經(jīng)過(guò)改良后,與堿裂解法和經(jīng)典的苯酚法進(jìn)行了效果比較,證實(shí)了本改良法具有快速、簡(jiǎn)便、高效等特點(diǎn)。本研究參照GenBank中的HBV基因組序列,用PrimerPremier5.0軟件設(shè)計(jì)3條定義HBV基本核心啟動(dòng)子(basal core promoter, BCP)和前C區(qū)(precore, PreC)的特異性引物P1、P2、P3,進(jìn)行半巢式PCR,擴(kuò)增16例HBV DNA陽(yáng)性血清,其中HBeAg陰性血清13例,HBeAg陽(yáng)性血清3例(作對(duì)照)。將PCR產(chǎn)物純化回收,克隆入pUCm-T載體,利用α互補(bǔ)篩選pUCm-HBV-403重組子,采用PCR和酶切重組子鑒定。通過(guò)測(cè)序和比對(duì)分析HBV BCP和PreC基因序列,發(fā)現(xiàn)BCP變異主要集中在TATA樣盒的TA1、TA2、TA3,未見(jiàn)插入和缺失突變,與北京董菁的報(bào)道顯著不同。TA4極為保守,TA4兩處均未見(jiàn)變異。BCP中一個(gè)變異熱點(diǎn)在nt1799位C→G,對(duì)于X蛋白來(lái)說(shuō)是同義突變。PreC基因變異主要集中在nt1896位G→A,使第28位密碼子TGG→TAG(終止子),它導(dǎo)致蛋白質(zhì)翻譯提前終止,使HBV不產(chǎn)生HBeAg。本次研究發(fā)現(xiàn)一例新奇的插入突變,發(fā)生在DR1(direct repeat sequence, DR)內(nèi),DR1和DR2在病毒復(fù)制和成環(huán)過(guò)程中起重要作用。 BCP和PreC基因變異可使HBeAg陰轉(zhuǎn),易使人們對(duì)體內(nèi)HBV掉以輕心。某些BCP和PreC基因位點(diǎn)的變異,可引起嚴(yán)重的肝損害,有些位點(diǎn)的變異影響干擾素的療效。為提高對(duì)HBV的診治水平,人們應(yīng)重視對(duì)HBV BCP和PreC基因變異的研究。
[Abstract]:Hepatitis B virus (HBV) belongs to the Hepatitis B virus family, which is an incomplete circular double stranded DNA virus. The complete HBV particles are spherical particles of 42nm size, that is, Dane particles. HBV particles are the smallest known virus to cause human disease. There are four main open reading codes for the whole genome of 3.2kb.HBV, which are called S region C region P region X region, respectively. Some regions are overlapped. The high efficiency of genetic information using genomic DNA, which is rare in nature, requires reverse transcription in the replication process, due to the lack of effective base proofreading by reverse transcriptase. Therefore, the HBV genome is more likely to mutate than the normal DNA virus. This poses new problems in disease prevention, diagnosis, treatment and prognosis. In this experiment, HBV DNA in serum was extracted by boiling method of pyrolysis solution. The improved method was compared with the alkali cracking method and the classical phenol method. It was proved that the improved method had the advantages of rapidity, simplicity and high efficiency. In this study, referring to the HBV genome sequence in GenBank, we designed three specific primers, P1P2P3, which defined the basic core promoter of HBV core promoter, BCP) and precore core promoter, BCP) (PreC3) by PrimerPremier5.0 software, and amplified 16 HBV DNA positive sera by semi-nested PCR. There were 13 cases of HBeAg negative serum and 3 cases of HBeAg positive serum. The PCR product was purified and recovered and cloned into the pUCm-T vector. The pUCm-HBV-403 recombinant was screened by 偽 -complementary method and identified by PCR and restriction enzyme digestion. By sequencing and comparing the sequence of HBV BCP and PreC gene, it was found that the BCP mutation was mainly in TA1, TA2, TA3, and no insertion or deletion mutation was found. There is a significant difference from the report of Dong Jing in Beijing. TA4 is extremely conserved. There is no mutation. One of the hotspots of variation in BCP is nt1799 C G. For X protein, the synonymous mutation. PreC gene mutation is mainly concentrated in nt1896 G, making the 28th position. Codon TGG / tag, which leads to early termination of protein translation, HBV does not produce HBeAg. In this study, we found a novel insertion mutation, which occurs in DR1(direct repeat sequence (DR1) and DR2 plays an important role in viral replication and ring formation. Mutations in BCP and PreC genes can turn HBeAg negative and make people take HBV lightly. Mutations in some BCP and PreC loci can cause severe liver damage, and some loci affect the efficacy of interferon. In order to improve the diagnosis and treatment of HBV, people should pay attention to the study of HBV BCP and PreC gene mutation.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類(lèi)號(hào)】：R373

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 劉冰;等溫?cái)U(kuò)增檢測(cè)HBV S基因方法的建立及臨床應(yīng)用的初步研究[D];西北大學(xué);2007年

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本文編號(hào)：1849357