抗DR5多价抗体的抗肿瘤作用

发布时间：2018-06-10 11:36

  本文选题：DR5 + 多价抗体　； 参考：《河南大学》2011年硕士论文

【摘要】：背景 肿瘤坏死因子(Tumor necrosis factor,TNF)相关的凋亡诱导配体(TNF-related apoptosis inducing ligand,TRAIL)能够诱导多种肿瘤细胞和转化细胞发生凋亡。TRAIL诱导肿瘤细胞凋亡是通过其受体TRAIL-R1(Death receptor 4,DR4)及TRAIL-R2(Death receptor 5,DR5)介导的,但一些研究发现TRAIL对正常的肝细胞有毒性作用,限制了其在临床上的应用。一些抗TRAIL死亡受体的单克隆抗体具有死亡受体配体的功能活性,能模拟TRAIL的作用,诱导肿瘤细胞凋亡,而且对正常人的肝细胞等没有毒副作用。本实验室利用人DR5蛋白制备出分泌抗DR5抗体的杂交瘤细胞-YM366EC,研究发现抗DR5抗体交联后对肿瘤细胞的增殖有明显的抑制作用,可以诱导对TRAIL敏感的肿瘤细胞的凋亡。但是,单克隆抗体存在一些缺陷,如完整的抗体分子量大,而且大部分抗体是鼠源性抗体,应用于人体会产生人抗鼠抗体反应(Human anti-mouse antibody,HAMA)等,引起免疫排斥反应,阻断抗体效用的发挥,因而妨碍了其在临床上的应用。随着DNA重组技术的发展,可以应用基因工程技术制备基因工程抗体,既能保持单克隆抗体均一性,特异性强的优点,又能克服其鼠源性的弊端。多价抗体是基因工程抗体之一,近年来人们对用基因工程技术生产多价抗体已进行了大量的实验,取得了迅速地发展。 目的 通过构建YM366EC的多价抗体,使得DR5发生交联,诱导肿瘤细胞凋亡。 方法 应用兼并PCR、5′-RACE技术从抗DR5杂交瘤细胞YM366EC中钓取抗DR5抗体可变区基因VL、VH,用连接肽基因(Gly4Ser)3将VL和VH连接成单链抗体scFv,命名为3D。随后利用重叠延伸PCR技术分别将单链抗体scFv与人IgG3上游铰链区/p53四价功能域融合基因、C4结合蛋白融合基因连接,得到四价和八价抗体融合基因,分别命名为3S及3B。测序正确后,利用HindⅢ和XhoⅠ酶切位点,将三段融合基因分别克隆入真核表达载体pSecTag2-A中,得到p-3D,p-3S及p-3B,将上述质粒分别转染CHO细胞,进行真核表达。利用SDS-PAGE和Western blot实验鉴定重组蛋白的表达,表达产物经Ni2+-NTAsuperflow亲和柱纯化后,通过MTT实验检测重组抗体对肿瘤细胞增殖的影响,利用流式细胞仪测定其抗肿瘤活性。 结果 成功克隆了抗DR5抗体的可变区VL、VH基因,构建了单链、四价、八价真核表达载体;SDS-PAGE和Western blot实验证明:p-3D、p-3S及p-3B质粒表达产物中分别含有约35 KD、40 KD及40 KD大小的特异性蛋白条带;MTT实验结果显示四价和八价重组抗体对肿瘤细胞的增殖有明显地抑制作用,流式细胞仪结果显示:单链抗体没有抗肿瘤活性,四价和八价重组抗体都具有很好的抗肿瘤活性。 结论 成功克隆抗DR5抗体的可变区基因;成功构建了单链、四价、八价真核表达载体;制备了具有抗肿瘤活性的抗DR5多价抗体,为进一步的体内实验奠定基础。
[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，

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