本文選題：噬菌體展示 + 模擬表位　； 參考：《浙江大學(xué)》2006年碩士論文

【摘要】：阻斷人天然抗α-Gal抗體與豬細(xì)胞表面大量表達(dá)的α-Gal抗原是異種器官移植成功的一個(gè)重要前提。在異種器官移植中,這些α-Gal抗原可與人天然抗α-Gal抗體誘發(fā)超急排斥反應(yīng)。為找到能夠運(yùn)用于異種器官移植中的特異性結(jié)合抗體的多肽,對兩種肽庫進(jìn)行了親和篩選:線性7肽庫和C7C肽庫。采用抗B型血單克隆抗體為靶分子,經(jīng)過4輪篩選,隨機(jī)挑取了160個(gè)噬菌體克隆,經(jīng)ELISA實(shí)驗(yàn)檢驗(yàn)22個(gè)克隆特異地結(jié)合抗體。DNA測序結(jié)果顯示,這些篩選出來的多肽具有高度同源序列PT和STL。水蘇糖競爭實(shí)驗(yàn)證明這些多肽都特異性地結(jié)合于α-Gal抗原表位結(jié)合位點(diǎn)。血凝實(shí)驗(yàn)表明8個(gè)多肽可以競爭性抑制由人天然抗α-Gal抗體誘發(fā)的凝血反應(yīng)。這些結(jié)果說明篩選出來的多肽能夠在空間構(gòu)象上模擬α-Gal抗原表位。這些α-Gal抗原表位模擬肽所提供的分子結(jié)構(gòu)信息為將來進(jìn)一步開發(fā)抗異種器官移植超急排斥反應(yīng)的藥物提供了基礎(chǔ)。 1 噬菌體展示肽庫的篩選 以抗B型血單克隆抗體包被酶標(biāo)板,5%牛血清白蛋白37℃封閉2小時(shí),TBS洗板3次,加入噬菌體肽庫室溫反應(yīng)1小時(shí),含0.1%Tween-20的TBS洗板10次,每孔加入酸洗脫液洗脫10分鐘,然后用中和液中和。將洗脫的噬菌體感染感受態(tài)E.coli ER2738擴(kuò)增,純化,進(jìn)行下一輪的篩選。噬菌體線性7肽庫和C7C肽庫經(jīng)過4輪篩選,各出現(xiàn)不同程度的富集現(xiàn)象。 2 酶聯(lián)免疫吸附實(shí)驗(yàn)篩選陽性噬菌體 第4輪洗脫下來的噬菌體感染E. coli ER2738,從平板中隨機(jī)挑取了160個(gè)噬
[Abstract]:It is an important prerequisite for the success of xenotransplantation to block human natural anti-偽 -Gal antibody and 偽 -Gal antigen expressed on porcine cell surface.In xenogeneic organ transplantation, these 偽 -Gal antigens can induce hyperacute rejection with human natural anti 偽 -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 偽 -Gal epitope binding sites.The results of hemagglutination test showed that 8 peptides could competitively inhibit the coagulation reaction induced by human natural anti-偽 -Gal antibody.These results suggest that the selected peptides can mimic 偽 -Gal epitopes in spatial conformation.The molecular structure information provided by these 偽 -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，

本文編號：1748604