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  本文關(guān)鍵詞：抗HBV多聚酶TP區(qū)細(xì)胞內(nèi)VH抗體體外抑制HBV復(fù)制的實(shí)驗(yàn)研究　出處：《第三軍醫(yī)大學(xué)》2006年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 肝炎病毒 乙型 細(xì)胞內(nèi)抗體 二氫葉酸還原酶 蛋白質(zhì)片段互補(bǔ)方法 單鏈可變區(qū)抗體 甲氧芐胺嘧啶 末端蛋白 重鏈可變區(qū)抗體

【摘要】： 實(shí)驗(yàn)背景 在我國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。到目前為止仍然缺乏高效的抗病毒治療藥物。在目前可用的抗病毒藥物中,α-干擾素是最為常用的,然而HBeAg陰轉(zhuǎn)率僅有20%～30%。核苷類似物如拉米夫定經(jīng)臨床使用,發(fā)現(xiàn)雖然有明顯的抗病毒作用,但是停藥后易復(fù)發(fā)并易誘導(dǎo)耐藥毒株的產(chǎn)生。近年來,細(xì)胞內(nèi)抗體(intrabody)的研究給乙型病毒性肝炎的抗病毒治療帶來了希望。細(xì)胞內(nèi)抗體是利用抗體工程技術(shù)將抗體(片段)表達(dá)在細(xì)胞內(nèi),成為細(xì)胞內(nèi)蛋白,然后特異性的與靶蛋白(抗原)結(jié)合,選擇性地抑制其功能。 細(xì)胞內(nèi)抗體指細(xì)胞內(nèi)合成且作用于細(xì)胞內(nèi)組分的抗體,通常指的是scFv(single chain variable fragments,scFv,單鏈可變區(qū)抗體),但scFv幾乎不能在細(xì)胞內(nèi)發(fā)揮作用。因?yàn)閟cFv在細(xì)胞內(nèi)質(zhì)網(wǎng)成熟,細(xì)胞內(nèi)的還原環(huán)境使scFv不能形成鏈內(nèi)二硫鍵,而這對抗體的正確折疊至關(guān)重要,從而影響scFv的可溶性與穩(wěn)定性。而VH(variable fragments of heavy chain,VH,重鏈可變區(qū))抗體不存在這樣的缺陷,VH抗體不需要鏈內(nèi)二硫鍵也同樣具有活性。VH抗體可在原核細(xì)胞或真核細(xì)胞內(nèi)表達(dá),已證明它們有很強(qiáng)的中和抗原的作用。還有一點(diǎn),在選擇特異性抗體時,VH抗體庫的多樣性要比scFv抗體庫的多樣性小的多,這就減少了抗體選擇的復(fù)雜性。 酵母展示scFv抗體庫(yeast display scFv antibody library)包含有超過1010個人類scFv抗體。其表達(dá)載體為釀酒酵母(Saccharomyces cerevisiae)中的質(zhì)粒pPNL-6。因此可以把酵母展示scFv抗體庫轉(zhuǎn)變?yōu)槿祟怴H抗體庫,從中進(jìn)行特異性VH抗體的選擇。 從抗體庫中選擇有效抗體目前有很多方法,如噬菌體展示技術(shù)(phage display)、核糖體展示技術(shù)(ribosome display)、酵母雙雜交技術(shù)(yeast two-hybrid system)等。這些方法各有利弊,但PCA(protein fragment complementation assay,PCA,蛋白質(zhì)片段互補(bǔ)方法)在抗體選擇中顯示出很多優(yōu)點(diǎn)。比如作為與抗體結(jié)合的抗原不需要表達(dá)與純化,只需要抗原的基因,這大大減少了實(shí)驗(yàn)難度;由于抗原抗體的結(jié)合是在細(xì)胞內(nèi)自然環(huán)境下發(fā)生,與選擇出的特異性抗體發(fā)揮作用的環(huán)境一致;不需要相應(yīng)的報告基因表達(dá),
[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 偽 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,
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2006
【分類號】：R392

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本文編號：1414953