HBeAg阴性血清中乙肝病毒基本核心启动子和前C区变异研究

发布时间：2018-05-05 21:25

本文选题：乙型肝炎病毒 + 变异　；参考：《武汉大学》2005年硕士论文

【摘要】：乙型肝炎病毒(hepatitis B virus, HBV)属嗜肝DNA病毒科,为不完全环状双链DNA病毒。完整的HBV颗粒为42nm大小的球形颗粒,即Dane颗粒。HBV是目前已知的对人类致病的最小DNA病毒,基因组全长约3.2kb。HBV有四个主要的开放读码框,分别称为S区、C区、P区、X区,有些区是重叠的,HBV这种利用基因组DNA的遗传信息的高效性在自然界中是罕见的。HBV复制过程中要经过逆转录,由于逆转录酶缺乏有效的碱基校对功能,故HBV基因组比一般DNA病毒易发生变异。这给疾病的预防、诊断、治疗和预后带来新问题。本实验采用裂解液煮沸法提取血清中HBV DNA。该法经过改良后,与碱裂解法和经典的苯酚法进行了效果比较,证实了本改良法具有快速、简便、高效等特点。本研究参照GenBank中的HBV基因组序列,用PrimerPremier5.0软件设计3条定义HBV基本核心启动子(basal core promoter, BCP)和前C区(precore, PreC)的特异性引物P1、P2、P3,进行半巢式PCR,扩增16例HBV DNA阳性血清,其中HBeAg阴性血清13例,HBeAg阳性血清3例(作对照)。将PCR产物纯化回收,克隆入pUCm-T载体,利用α互补筛选pUCm-HBV-403重组子,采用PCR和酶切重组子鉴定。通过测序和比对分析HBV BCP和PreC基因序列,发现BCP变异主要集中在TATA样盒的TA1、TA2、TA3,未见插入和缺失突变,与北京董菁的报道显著不同。TA4极为保守,TA4两处均未见变异。BCP中一个变异热点在nt1799位C→G,对于X蛋白来说是同义突变。PreC基因变异主要集中在nt1896位G→A,使第28位密码子TGG→TAG(终止子),它导致蛋白质翻译提前终止,使HBV不产生HBeAg。本次研究发现一例新奇的插入突变,发生在DR1(direct repeat sequence, DR)内,DR1和DR2在病毒复制和成环过程中起重要作用。 BCP和PreC基因变异可使HBeAg阴转,易使人们对体内HBV掉以轻心。某些BCP和PreC基因位点的变异,可引起严重的肝损害,有些位点的变异影响干扰素的疗效。为提高对HBV的诊治水平,人们应重视对HBV BCP和PreC基因变异的研究。
[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 伪 -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.
【学位授予单位】：武汉大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R373

【引证文献】