TTV抗原ORF1的基因克隆及原核表达

发布时间：2018-10-30 15:09

【摘要】：研究背景及目的：肝炎是一种严重危害人类健康的传染病，目前已发现和确认的肝炎病毒有甲、乙、丙、丁、戊、庚型。1997年底日本学者Nishizawa运用代表性差异技术(Reprentational difference analysis)从一名非甲-庚型肝炎病人(TTV)的血清中发现了输血传播病毒(Transfusion transmitted virus，TTV)。TTV基因组为单股线状DNA，目前已测定的核苷酸序列长约3.8kb，有两个开放读码框架(ORF)，ORF1位于该基因组的589-2898位核苷酸，编码770个氨基酸，ORF2位于107-712位核苷酸，，编码202个氨基酸；ORF1可能编码病毒的结构蛋白，ORF2可能编码病毒的非结构蛋白；可以通过垂直传播，生物学特征与某些动物单链DNA病毒(微小病毒B19)相似。TTV的基因分型从已报道的TTV DNA部分基因序列来看，TTV DNA具有高度变异性，采用聚合酶链反应(PCR)检测血清TTV DNA是诊断TTV感染的主要手段。目前还未证实TTV感染引起肝损伤和影响肝功能，但TTV可引起ALT升高，出现病毒血症，并且在局部地区爆发性流行，同时TTV常和HBV及HCV共同感染。TTV在非甲至非庚型肝炎患者中的感染率较高，可能是非甲至非庚型肝炎的主要致病因子，现国内对TTV的研究资料还很缺乏，本实验的意义在于将TTV-ORF1 DNA构建原核表达载体并获得高效表达，为TTV的ELISA检测和疫苗的抗原研究提供基础。方法：从TTV病毒感染者外周血中提取DNA，用PCR从中扩增出目的基因，酶切PCR扩增产物并进行纯化获得TTV-ORF1 DNA，插入载体pGEM-T Easy中，转化JM109感受态细菌中，随受体菌的生长繁殖，重组DNA分子得以复制扩增；运用
[Abstract]:Background and objective: hepatitis is a serious infectious disease that endangers human health. At present, hepatitis virus A, B, C, D, E has been found and confirmed. By the end of 1997, Nishizawa, a Japanese scholar, using representational differential technique (Reprentational difference analysis), found that the (Transfusion transmitted virus,TTV). TTV genome of the transfusion transmitted virus (Transfusion transmitted virus,TTV). TTV) was a single strand linear DNA, from the serum of a non-A-G hepatitis patient. The nucleotide sequence has been determined to be about 3.8kb. there are two open reading frame (ORF), ORF1 at 589-2898 nucleotides in the genome, encoding 770 amino acids and ORF2 at 107-712 nucleotides, encoding 202 amino acids. ORF1 may encode the structural protein of the virus and ORF2 may encode the non-structural protein of the virus. By vertical transmission, the biological characteristics are similar to those of some animal single-stranded DNA viruses (parvovirus B19). The genotyping of TTV shows that, TTV DNA is highly variable from some reported TTV DNA gene sequences. Detection of serum TTV DNA by polymerase chain reaction (PCR) is the main method for diagnosis of TTV infection. At present, it has not been confirmed that TTV infection can cause liver injury and affect liver function, but TTV can cause the increase of ALT, viremia and outbreak epidemic in local area. At the same time, TTV is often co-infected with HBV and HCV. The infection rate of TTV in patients with non-A to non-G hepatitis is relatively high, which may be the main pathogenic factor of non-A to non-G hepatitis. The significance of this experiment is to construct prokaryotic expression vector of TTV-ORF1 DNA and obtain high expression, which provides the basis for ELISA detection of TTV and antigen study of vaccine. Methods: DNA, was extracted from the peripheral blood of patients with TTV virus and PCR was used to amplify the target gene. The PCR amplification products were digested and purified. The TTV-ORF1 DNA, was inserted into the pGEM-T Easy vector and transformed into JM109 susceptible bacteria. With the growth and reproduction of the receptor bacteria, the recombinant DNA molecule was duplicated and amplified. Application
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392

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