抗HBV多聚酶TP区细胞内VH抗体体外抑制HBV复制的实验研究

发布时间：2018-01-12 16:05

本文关键词：抗HBV多聚酶TP区细胞内VH抗体体外抑制HBV复制的实验研究　出处：《第三军医大学》2006年博士论文　论文类型：学位论文

【摘要】： 实验背景在我国乙型肝炎病毒是引起重型病毒性肝炎和慢性病毒性肝炎的最主要病因。到目前为止仍然缺乏高效的抗病毒治疗药物。在目前可用的抗病毒药物中,α-干扰素是最为常用的,然而HBeAg阴转率仅有20%～30%。核苷类似物如拉米夫定经临床使用,发现虽然有明显的抗病毒作用,但是停药后易复发并易诱导耐药毒株的产生。近年来,细胞内抗体(intrabody)的研究给乙型病毒性肝炎的抗病毒治疗带来了希望。细胞内抗体是利用抗体工程技术将抗体(片段)表达在细胞内,成为细胞内蛋白,然后特异性的与靶蛋白(抗原)结合,选择性地抑制其功能。细胞内抗体指细胞内合成且作用于细胞内组分的抗体,通常指的是scFv(single chain variable fragments,scFv,单链可变区抗体),但scFv几乎不能在细胞内发挥作用。因为scFv在细胞内质网成熟,细胞内的还原环境使scFv不能形成链内二硫键,而这对抗体的正确折叠至关重要,从而影响scFv的可溶性与稳定性。而VH(variable fragments of heavy chain,VH,重链可变区)抗体不存在这样的缺陷,VH抗体不需要链内二硫键也同样具有活性。VH抗体可在原核细胞或真核细胞内表达,已证明它们有很强的中和抗原的作用。还有一点,在选择特异性抗体时,VH抗体库的多样性要比scFv抗体库的多样性小的多,这就减少了抗体选择的复杂性。酵母展示scFv抗体库(yeast display scFv antibody library)包含有超过1010个人类scFv抗体。其表达载体为酿酒酵母(Saccharomyces cerevisiae)中的质粒pPNL-6。因此可以把酵母展示scFv抗体库转变为人类VH抗体库,从中进行特异性VH抗体的选择。从抗体库中选择有效抗体目前有很多方法,如噬菌体展示技术(phage display)、核糖体展示技术(ribosome display)、酵母双杂交技术(yeast two-hybrid system)等。这些方法各有利弊,但PCA(protein fragment complementation assay,PCA,蛋白质片段互补方法)在抗体选择中显示出很多优点。比如作为与抗体结合的抗原不需要表达与纯化,只需要抗原的基因,这大大减少了实验难度;由于抗原抗体的结合是在细胞内自然环境下发生,与选择出的特异性抗体发挥作用的环境一致;不需要相应的报告基因表达,
[Abstract]:Experimental background Hepatitis B virus is the main cause of severe viral hepatitis and chronic viral hepatitis in China. So far, there is still a lack of effective antiviral drugs. Interferon 伪 is the most commonly used, but the negative conversion rate of HBeAg is only 20%. The nucleoside analogues, such as lamivudine, have been found to have obvious antiviral effects. However, it is easy to recur after drug withdrawal and induce the production of drug-resistant strains in recent years. The study of intracellular antibody intrabody brings hope to the antiviral therapy of hepatitis B. intracellular antibody is expressed in cells by using antibody engineering technology. Become intracellular proteins, then specifically bind to target proteins (antigens) and selectively inhibit their function. Intracellular antibodies are antibodies that are synthesized in cells and act on intracellular components, usually referred to as scFv(single chain variable fragments. ScFv, a single strand variable region antibody, can hardly play a role in cells by scFv, because scFv matures in the endoplasmic reticulum and the reduction environment in the cells prevents scFv from forming disulfide bonds in the chain. This is crucial to the proper folding of antibodies, which affects the solubility and stability of scFv. And VH(variable fragments of heavy chain. VH (heavy chain variable region) antibody does not have such defects. The VH antibody does not require disulfide bonds in the chain and can also be expressed in prokaryotic cells or eukaryotic cells. It has been proved that they have a strong antigen-neutralizing effect. Also, the diversity of VH antibody library is much smaller than that of scFv antibody library in the selection of specific antibodies. This reduces the complexity of antibody selection. Yeast display of scFv antibody library, yeast display scFv antibody library1. Contains more than 1 010 human scFv antibodies. Its expression vector is Saccharomyces cerevisiaeae. Therefore, the yeast display scFv antibody library can be transformed into a human VH antibody library. The specific VH antibody was selected. There are many methods to select effective antibodies from antibody libraries, such as phage display technique and ribosome display technique. Yeast two-hybrid technique and so on. These methods have their own advantages and disadvantages. But PCA(protein fragment complementation assayPCA. The method of protein fragment complementation) shows many advantages in antibody selection. For example, as an antibody-bound antigen, it does not need to be expressed and purified, but only needs antigen gene, which greatly reduces the difficulty of experiment. Because the binding of antigen and antibody occurs in the natural environment of the cell, it is consistent with the environment in which the selected specific antibody plays its role. There is no need for corresponding reporter gene expression,
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R392

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