【作者】 张晖；

【导师】 詹金彪；

【作者基本信息】 浙江大学 ， 生物化学与分子生物学， 2003， 硕士

【摘要】 猪器官直接移植至人体时，可触发超急性排斥反应（hyperacute rejection，HAR）。人体内预存的天然抗体与猪细胞表面的糖链结构Gal-α 1，3-Gal结合是引发HAR的主要原因。有证据表明，可通过阻断人体内预存的天然抗体与猪细胞表面的Gal-α 1，3-Gal抗原表位结合而阻止HAR的发生。因此，通过筛选到能与人体内预有的天然抗体结合或使其失效的小肽，就可能会找到简易的解决此HAR的治疗试剂。 噬菌体随机肽库展示了各种不同排列的小肽，能于抗体、受体或其他蛋白结合。我们利用噬菌体展示技术在XCX₁₅随机肽库中进行了筛选，最终筛选到能与单克隆抗Gal-α 1，3-Gal抗体（抗B型血单克隆抗体，mAB anti-B，能与Gal-α 1，3-Gal结构特异性结合）特异性结合的噬菌体克隆。我们进一步实验证实了这个阳性噬菌体克隆能阻断人体内预存的天然抗体或西非单叶豆凝集素（BS-I-B4，能与Gal-α 1，3-Gal结构特异性结合）介导所引起的猪红细胞的凝集。说明这一小肽可以有效地阻断人天然抗体。 （一） 用噬菌体展示亲和筛选法筛选与mAB anti-B结合的噬菌体 以mAB anti-B（滴度1：128）包被96孔酶标板，4℃过夜。5％BSA封闭后，噬菌体肽库扩增液与mAB anti-B室温轻摇结合1小时后，用TBST洗板洗去没有于mAB anti-B结合的噬菌体，再用100mg/ml的蜜二糖(Melibiose，分子结构为 2003浙江大学硕土学位论文(ialal，6｛ 门 能与 mAB anti化结合的噬菌体亲和洗脱下来。这些噬菌体用kankgl大肠杆菌感染扩增。这样的亲和筛选过程重复 4次，其中第 1、2两轮用 0．1W（吐温与1”BS溶液的体积比）TBST洗板，第3、4两轮分别用0．2％、0．5q0’rBST洗板。虽然洗板的TfBST浓度在逐渐加大，仍看到了阳性噬菌体克隆的富集，第4轮的产出比第 1轮要大5倍。挑取筛选出来的噬菌体做ELISA鉴定，并测定与InAB anti－11的结合力。（一 ［｛I。ISA鉴定噬菌体阳性克隆与InAB anti上的结合力 j 1＊0卜＊子的 mAB an t i－B（滴度 1：128）包被 96了酶标板，4”C过夜。5 W；BSA室温f们A］2小时，用 5 mM的 TBS缓冲液洗板 3次后，加入噬菌体原液与 InAB anti个结合I小时。川m引’洗板3次后，加入m卜兔抗m 抗体，室温下结合1小时。用’I’BSI’洗板10次后，加入四甲基联苯胺（’fMB）显色剂显色，此显色反应控制在J）分钟，用50 HL ZM H。SO。终止反应，在姑0 urn处用酶标仅读取吸光度4值。 挑选出了17个噬菌体克隆来进行检测，从中又挑出5个结合率较高的克隆重做了＊。ISA来比较它们的结合力。结果表明所有挑出的克隆均能与 InAB ant卜B结合，其中：记号噬菌体克隆的结合力最强，再用蜜二糖竞争抑制试验，证明其结合的特异性。(个紧二：糖竞争抑制ELISA试验 为了检测资二糖（结构为 Gal－al，：3－Glc）是否能抑制阳性克隆 35与 dB anti心的结合，各种不同浓度的蜜二糖与噬菌体原液混合加入到己包被汹B。川卜B的孔中，其余所有步骤同前述ELISA方法一样。结果表明，从终浓度为300 InM－0．00：3 InM的蜜：二额都能扣片创阳性克隆与 InAB an t i化的结合，说明阳性克隆与 mAB an t i十的结合位点与安：二糖结合的位点相同。恻小肽序列的测定 阳性噬菌体单链的DNA 按分子克隆实验手册方法抽提与纯化。川5，CTGAAGAGAGTCAAAAGC－3’引物测序。由DNA序列推导出插入的随机小肽序列，最后得到的小肽氨基酸序列为：CCWLLRQPWll7VRSIRS、 3 2003浙江大学硕土学位论文SCVmSSTNZ RLRLCNQu、DCP WsWs AWAWS RARASC“小m工、KCRSL GPGP KRQKRQ LTMLTM pp用噬菌体展示肽抑制人血清与猪红细胞的凝集反应 此反应在血凝板上操作。先把制备好的新鲜 2 ％（压积比）猪红细胞悬浮在！。BS缓冲液中备用。凝集反应体系包括逐孔用PBS倍比稀释的40 pl阳性克隆提取液、4（）pl人血清(包含有人天然抗体X40 VIZ％的猪红细胞悬液。混合后先用震荡器震荡 1分钟，静置 l小时后观察凝集结果。阴性克隆（与 mAB an t i－B无结合的噬菌体）作为对照实验。如果出现凝集，可见红细胞分布在孔中呈网状。相对的，如果红细胞沉淀到孔的底部，呈一边缘光滑的圆点，则此为没有出现凝集。实验结果拍照保存以便分析。最后结果表明该噬菌体展示肽能够阻断人预存大然抗体所引起的猪红细胞的凝集。闪噬菌体展示肽抑制BS－I干4与猪红细胞的凝集反应 JZing／1。的西非单叶豆凝集素（8＊1－＊，它与具有h－al，＊Gal的结构结合）替代人血伤加入到反应体系中。别的实验步骤都同上面的凝集试验。结果表明该小肽同时也能阻断凝集素所引起的猪红细胞凝集。(0不同血型的人血清对猪红细胞的凝集作用更多还原

【Abstract】 The obstacle for pig-to-human transplantation is hyperacute rejection (HAR) triggered by the interaction between human natural antibodies and the antigenic epitope Gal-α1,3-Gal. The a-Gal structure is considered to be the major enoantigenic epitopes present on porcine tissues. Evidences indicate that HAR may be prevented by blocking the interaction of human natural anti-α Gal antibodies with antigenic epitope Gal-α1,3-Gal on pig tissues. The prospect of finding peptides that are able to block or remove human natural antibodies may simplify the development of potent therapeutic agents.Random peptides displayed on the phage surface offer a rich source of molecular diversity in search for ligand peptides that bind to an antibody, receptor or other binding proteins We attempted to identify peptides using a phage-displayed library XCX15. A peptide was identified that appears to bind the monoclonal anti-B antibody (mAB anti-B, which can bind Gal-α-l,3-Gal structure) at the binding site of Gal-αl,3-Gal. Our experiments have demonstrated that the positive phage bearing the peptide can block the human natural antibody-mediated agglutination of pig RBCs. The peptide can also block the agglution of pig RBCs caused by lectin BS-I-B4 (which binds the structure ofGal-α-l,3-Gal).(1) Selection of antibody-binding phage(Biopanning)mAB anti-B (titer 1:128) was coated on microtiter wells at 4癈 overnight. Wells were saturatedwith 5 % BSA. In the first panning, the phage library was incubated at room temperature for 1 hour with slowly shaking. Nonbinding phages were removed by extensive washing with TBST [Tris-buffered saline (TBS) supplemented with Tween-20 (TBST)]. The bound phages were eluted with 100 nig/ml of melibiose(which has Gal-α,6-Glc structure). Phages were amplified in Escherichici coli (kank91). Affinity selection was repeated for four times. The plate was washed with 0.1 % TBST in first two rounds, and with 0.2 and 0.5 % TBST in round 3 and 4, respectively. Although the concentration of TBST increased very much, the enrichment of positive phage clones was observed. The yield of the fourth round was approximately 5-time more than that of the first round.(2) Detection of binding of phage to antibody by ELISAA flat-bottomed microtiter plate was coated overnight at 4℃ with 100 ul of theanti-B antibody. Wells were then saturated with 5% BSA and then washed with 5 mM TBS for three times. Purified phages (50 ul) was added into each well. The wells were washed with 0.2 % TBST to remove unbound phages, and then rabbit anti-M13-HRP was added into each well and incubated at room temperature for 1 hour. Unbound rabbit anti-Mi3-HRP was removed by washing with 0.1% TBST for 10 times. The substrate 3,3’,5,5’-tetramethylbenzidine (TMB) was used for development, and the reaction was carried on for 30 minutes, and then terminated by adding 50 ul of 2 M H2SO4. Solute optical density in each well was measured at 450 nm by a microplate reader. After biopanning, 17 clones were randomly picked and screened. 5 out of them were performed the ELISA again to compare the binding activity. All clones selected were able to bind the antibody. Among them, clone 35 had strongest binding ability, and therefore was chosen for the competitive binding assay with melibiose.(3) Inhibition of binding of antibody to peptide by melibioseIn order to test the melibiose(Gal-α 1,3-Glc) inhibition of peptide binding to anti-B antibody, different concentrations of melibiose were mixed with the phage solution before the phage was added into the antibody-coated wells. All other procedures were same as the above ELISA method.The binding to monoclonal anti-B antibody of phage 35 was competitively inhibited by melibiose at the final concentration range of 0.003-300 mM. This result suggested that the phage was bound to monoclonal anti-B antibody at the melibiose binding site. (4) sequence to identify consensus motifsThe positive phage single-stranded DNA was extracted and purified according to Molecular Clonin更多还原