α-Gal抗原模拟表位分析及模拟肽活性鉴定

发布时间：2018-04-14 09:04

  本文选题：噬菌体展示 + 模拟表位　； 参考：《浙江大学》2006年硕士论文

【摘要】：阻断人天然抗α-Gal抗体与猪细胞表面大量表达的α-Gal抗原是异种器官移植成功的一个重要前提。在异种器官移植中,这些α-Gal抗原可与人天然抗α-Gal抗体诱发超急排斥反应。为找到能够运用于异种器官移植中的特异性结合抗体的多肽,对两种肽库进行了亲和筛选:线性7肽库和C7C肽库。采用抗B型血单克隆抗体为靶分子,经过4轮筛选,随机挑取了160个噬菌体克隆,经ELISA实验检验22个克隆特异地结合抗体。DNA测序结果显示,这些筛选出来的多肽具有高度同源序列PT和STL。水苏糖竞争实验证明这些多肽都特异性地结合于α-Gal抗原表位结合位点。血凝实验表明8个多肽可以竞争性抑制由人天然抗α-Gal抗体诱发的凝血反应。这些结果说明筛选出来的多肽能够在空间构象上模拟α-Gal抗原表位。这些α-Gal抗原表位模拟肽所提供的分子结构信息为将来进一步开发抗异种器官移植超急排斥反应的药物提供了基础。 1 噬菌体展示肽库的筛选 以抗B型血单克隆抗体包被酶标板,5%牛血清白蛋白37℃封闭2小时,TBS洗板3次,加入噬菌体肽库室温反应1小时,含0.1%Tween-20的TBS洗板10次,每孔加入酸洗脱液洗脱10分钟,然后用中和液中和。将洗脱的噬菌体感染感受态E.coli ER2738扩增,纯化,进行下一轮的筛选。噬菌体线性7肽库和C7C肽库经过4轮筛选,各出现不同程度的富集现象。 2 酶联免疫吸附实验筛选阳性噬菌体 第4轮洗脱下来的噬菌体感染E. coli ER2738,从平板中随机挑取了160个噬
[Abstract]:It is an important prerequisite for the success of xenotransplantation to block human natural anti-伪 -Gal antibody and 伪 -Gal antigen expressed on porcine cell surface.In xenogeneic organ transplantation, these 伪 -Gal antigens can induce hyperacute rejection with human natural anti 伪 -Gal antibodies.In order to find the peptides that can be used in xenotransplantation, two peptide libraries were screened by affinity: linear 7 peptide library and C7C peptide library.After 4 rounds of screening, 160 phage clones were randomly selected by using monoclonal antibody against type B blood as target molecules. The results of ELISA assay showed that 22 clones specifically bound to antibody.These selected polypeptides have high homologous sequences PT and STL.These peptides were specifically bound to 伪 -Gal epitope binding sites.The results of hemagglutination test showed that 8 peptides could competitively inhibit the coagulation reaction induced by human natural anti-伪 -Gal antibody.These results suggest that the selected peptides can mimic 伪 -Gal epitopes in spatial conformation.The molecular structure information provided by these 伪 -Gal epitope mimic peptides provides a basis for the further development of anti-xenograft superacute rejection drugs in the future.Screening of phage display peptide libraryThe anti-B blood monoclonal antibody was coated with enzyme labeled plate 5% bovine serum albumin for 3 times at 37 鈩，

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