【摘要】：研究背景及目的：肝炎是一種嚴(yán)重危害人類健康的傳染病，目前已發(fā)現(xiàn)和確認(rèn)的肝炎病毒有甲、乙、丙、丁、戊、庚型。1997年底日本學(xué)者Nishizawa運(yùn)用代表性差異技術(shù)(Reprentational difference analysis)從一名非甲-庚型肝炎病人(TTV)的血清中發(fā)現(xiàn)了輸血傳播病毒(Transfusion transmitted virus，TTV)。TTV基因組為單股線狀DNA，目前已測定的核苷酸序列長約3.8kb，有兩個開放讀碼框架(ORF)，ORF1位于該基因組的589-2898位核苷酸，編碼770個氨基酸，ORF2位于107-712位核苷酸，，編碼202個氨基酸；ORF1可能編碼病毒的結(jié)構(gòu)蛋白，ORF2可能編碼病毒的非結(jié)構(gòu)蛋白；可以通過垂直傳播，生物學(xué)特征與某些動物單鏈DNA病毒(微小病毒B19)相似。TTV的基因分型從已報道的TTV DNA部分基因序列來看，TTV DNA具有高度變異性，采用聚合酶鏈反應(yīng)(PCR)檢測血清TTV DNA是診斷TTV感染的主要手段。目前還未證實(shí)TTV感染引起肝損傷和影響肝功能，但TTV可引起ALT升高，出現(xiàn)病毒血癥，并且在局部地區(qū)爆發(fā)性流行，同時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較高，可能是非甲至非庚型肝炎的主要致病因子，現(xiàn)國內(nèi)對TTV的研究資料還很缺乏，本實(shí)驗(yàn)的意義在于將TTV-ORF1 DNA構(gòu)建原核表達(dá)載體并獲得高效表達(dá)，為TTV的ELISA檢測和疫苗的抗原研究提供基礎(chǔ)。 方法：從TTV病毒感染者外周血中提取DNA，用PCR從中擴(kuò)增出目的基因，酶切PCR擴(kuò)增產(chǎn)物并進(jìn)行純化獲得TTV-ORF1 DNA，插入載體pGEM-T Easy中，轉(zhuǎn)化JM109感受態(tài)細(xì)菌中，隨受體菌的生長繁殖，重組DNA分子得以復(fù)制擴(kuò)增；運(yùn)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
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：R392

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