本文選題：DR5 + 多價(jià)抗體�。� 參考：《河南大學(xué)》2011年碩士論文

【摘要】：背景 腫瘤壞死因子(Tumor necrosis factor,TNF)相關(guān)的凋亡誘導(dǎo)配體(TNF-related apoptosis inducing ligand,TRAIL)能夠誘導(dǎo)多種腫瘤細(xì)胞和轉(zhuǎn)化細(xì)胞發(fā)生凋亡。TRAIL誘導(dǎo)腫瘤細(xì)胞凋亡是通過(guò)其受體TRAIL-R1(Death receptor 4,DR4)及TRAIL-R2(Death receptor 5,DR5)介導(dǎo)的,但一些研究發(fā)現(xiàn)TRAIL對(duì)正常的肝細(xì)胞有毒性作用,限制了其在臨床上的應(yīng)用。一些抗TRAIL死亡受體的單克隆抗體具有死亡受體配體的功能活性,能模擬TRAIL的作用,誘導(dǎo)腫瘤細(xì)胞凋亡,而且對(duì)正常人的肝細(xì)胞等沒(méi)有毒副作用。本實(shí)驗(yàn)室利用人DR5蛋白制備出分泌抗DR5抗體的雜交瘤細(xì)胞-YM366EC,研究發(fā)現(xiàn)抗DR5抗體交聯(lián)后對(duì)腫瘤細(xì)胞的增殖有明顯的抑制作用,可以誘導(dǎo)對(duì)TRAIL敏感的腫瘤細(xì)胞的凋亡。但是,單克隆抗體存在一些缺陷,如完整的抗體分子量大,而且大部分抗體是鼠源性抗體,應(yīng)用于人體會(huì)產(chǎn)生人抗鼠抗體反應(yīng)(Human anti-mouse antibody,HAMA)等,引起免疫排斥反應(yīng),阻斷抗體效用的發(fā)揮,因而妨礙了其在臨床上的應(yīng)用。隨著DNA重組技術(shù)的發(fā)展,可以應(yīng)用基因工程技術(shù)制備基因工程抗體,既能保持單克隆抗體均一性,特異性強(qiáng)的優(yōu)點(diǎn),又能克服其鼠源性的弊端。多價(jià)抗體是基因工程抗體之一,近年來(lái)人們對(duì)用基因工程技術(shù)生產(chǎn)多價(jià)抗體已進(jìn)行了大量的實(shí)驗(yàn),取得了迅速地發(fā)展。 目的 通過(guò)構(gòu)建YM366EC的多價(jià)抗體,使得DR5發(fā)生交聯(lián),誘導(dǎo)腫瘤細(xì)胞凋亡。 方法 應(yīng)用兼并PCR、5′-RACE技術(shù)從抗DR5雜交瘤細(xì)胞YM366EC中釣取抗DR5抗體可變區(qū)基因VL、VH,用連接肽基因(Gly4Ser)3將VL和VH連接成單鏈抗體scFv,命名為3D。隨后利用重疊延伸PCR技術(shù)分別將單鏈抗體scFv與人IgG3上游鉸鏈區(qū)/p53四價(jià)功能域融合基因、C4結(jié)合蛋白融合基因連接,得到四價(jià)和八價(jià)抗體融合基因,分別命名為3S及3B。測(cè)序正確后,利用HindⅢ和XhoⅠ酶切位點(diǎn),將三段融合基因分別克隆入真核表達(dá)載體pSecTag2-A中,得到p-3D,p-3S及p-3B,將上述質(zhì)粒分別轉(zhuǎn)染CHO細(xì)胞,進(jìn)行真核表達(dá)。利用SDS-PAGE和Western blot實(shí)驗(yàn)鑒定重組蛋白的表達(dá),表達(dá)產(chǎn)物經(jīng)Ni2+-NTAsuperflow親和柱純化后,通過(guò)MTT實(shí)驗(yàn)檢測(cè)重組抗體對(duì)腫瘤細(xì)胞增殖的影響,利用流式細(xì)胞儀測(cè)定其抗腫瘤活性。 結(jié)果 成功克隆了抗DR5抗體的可變區(qū)VL、VH基因,構(gòu)建了單鏈、四價(jià)、八價(jià)真核表達(dá)載體;SDS-PAGE和Western blot實(shí)驗(yàn)證明:p-3D、p-3S及p-3B質(zhì)粒表達(dá)產(chǎn)物中分別含有約35 KD、40 KD及40 KD大小的特異性蛋白條帶;MTT實(shí)驗(yàn)結(jié)果顯示四價(jià)和八價(jià)重組抗體對(duì)腫瘤細(xì)胞的增殖有明顯地抑制作用,流式細(xì)胞儀結(jié)果顯示:單鏈抗體沒(méi)有抗腫瘤活性,四價(jià)和八價(jià)重組抗體都具有很好的抗腫瘤活性。 結(jié)論 成功克隆抗DR5抗體的可變區(qū)基因;成功構(gòu)建了單鏈、四價(jià)、八價(jià)真核表達(dá)載體;制備了具有抗腫瘤活性的抗DR5多價(jià)抗體,為進(jìn)一步的體內(nèi)實(shí)驗(yàn)奠定基礎(chǔ)。
[Abstract]:Background TNF-related apoptosis inducing ligand trail can induce apoptosis in various tumor cells and transformed cells. Trail induces apoptosis of tumor cells through its receptors TRAIL-R1, death receptor 4DR4) and TRAIL-R2 death receptor 5DR5. However, some studies have found that trail is toxic to normal hepatocytes, limiting its clinical application. Some monoclonal antibodies against trail death receptors have the functional activity of death receptor ligands, which can mimic the effect of trail, induce tumor cell apoptosis, and have no toxic side effects on normal human hepatocytes. Hybridoma cells YM366ECs secreting anti-DR5 antibodies were prepared by using human DR5 protein in our laboratory. It was found that anti-DR5 antibodies cross-linked could inhibit the proliferation of tumor cells and induce apoptosis of tumor cells sensitive to trail. However, there are some defects in monoclonal antibodies, such as the large molecular weight of complete antibodies, and the fact that most of the antibodies are murine antibodies. When used in human bodies, human anti-mouse antibody reactions and human anti-mouse antibodyHAMAs will be produced, resulting in immune rejection. Blocking the effect of antibody hinders its clinical application. With the development of DNA recombination technology, genetic engineering technology can be used to prepare genetic engineering antibodies, which can not only maintain the uniformity and specificity of monoclonal antibodies, but also overcome its murine drawbacks. Polyvalent antibody is one of the genetic engineering antibodies. In recent years, a large number of experiments have been carried out to produce polyvalent antibodies using genetic engineering technology, and rapid development has been made. Objective to construct polyvalent antibodies of YM366EC and make DR5 cross-link. Methods the variable region of anti-DR5 antibody VHs was isolated from YM366EC cells by degenerate PCR5 5- race technique. The VL and VH were ligated into single chain antibody scFv3 by gly4Serin3, which was named as 3D. Then the single chain antibody (scFv) was linked to the fusion gene of p53-p54-binding protein by overlapping extension PCR, and the fusion genes of tetravalent and octavalent antibodies were named 3s and 3Brespectively. After the sequencing was correct, the three fusion genes were cloned into eukaryotic expression vector pSecTag2-A using Hind 鈪，

本文編號(hào)：2003082