【作者】 郭龙华；

【导师】 董长垣；

【作者基本信息】 武汉大学 ， 病原生物学， 2005， 硕士

【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。更多还原

【Abstract】 Hepatitis B virus (HBV) is the prototype member of the hepadnavirus family. It has a circular and partially double-stranded DNA genome of about 3.2 kb containing four overlapping open reading frames: the C region, S region, P region and X region. The whole HBV particle is Dane particle, and it is a 42 nm global particle. HBV is known as the minimal DNA virus that is harmful to human at present. The high efficiency of the genetic information of HBV DNA is rare in the nature world. The replication of HBV DNA passes through reverse transcription. As the reverse transcriptase is lack of effective function of proofreading the bases, the HBV genome is easier to mutate than other DNA viruses. It will bring new problems to the preventability, diagnosis, remedy and prognosis of the diseases.In this experiment, we meliorated the way of seething with disruption liquid and took the way to extract HBV DNA templates from the serum samples. Compared with another two ways, this one was quicker, simpler and more efficient. Consulting the HBV genome in GenBank , We designed three specific primers . The products of PCR include BCP(basal core promoter) and PreC gene. In the 16 samples of HBV DNA positive sera, 13 samples were negative and 3 samples were positive for HBeAg. The samples were all amplified by heminested-PCR. After purified and recovered, the products were cloned to pUCm-T vectors. The pUCm-HBV-403 recombinations were filtrated by α -complement and identified by PCR and restriction endonuclease. According to sequence and aligning HBV BCP and PreC gene, we found the mutations of BCP were grouped in TA1 TA2 and TA3 of TATA-like boxes. There were no insertion or deletion mutations, which were different from the report of Dong jing in Beijing. TA4 was very conservative, and there was no mutation. One of the hot mutations in BCP was at nucleotide 1799 position (C→G), that was synonymous mutation to X protein. The mutations in PreC gene were grouped at nucleotide 1896position (G—A), which rendered the progression. Furthermore, in this research a novel insertion mutation was found in DR1. DR1 and DR2 play an important role in the replication and annulus.Mutations in BCP and PreC gene can cause e antigen seroconversion and escape from the recognition by the host’s immune system. Some of the mutations will lead to serious liver damages, and some will affect the curative effect of interferon. In order to improve the level of diagnosis and remedy of HBV, we should attach more importance to the research of HBV BCP and PreC gene mutations.更多还原