抗人P-selectin人源性抗体的制备及功能研究

发布时间：2018-06-18 06:53

本文选题：P-selectin + GPI　；参考：《中国人民解放军军事医学科学院》2008年硕士论文

【摘要】： 近年来, P-selectin在炎症反应及肿瘤转移过程中的作用越来越受到重视,以P-selectin为靶点,阻断P-selectin与其配体的结合,可能对相关疾病的治疗具有较大潜力。目前国际还没有关于P-selectin人源性抗体的报道。本研究以真核表达和原核表达系统获得P-selectin功能性片段为抗原,利用本室构建的大容量全合成人抗体库中筛选出针对P-selectin的人源性特异性抗体,力争获得能够阻断P-selectin与配体结合的中和抗体,从而为相关疾病的治疗奠定良好的基础。首先,利用RT-PCR的方法从血小板中钓取P-selectin的功能性基因片段,在大肠杆菌中实现了可溶性表达。同时利用GPI锚定技术在真核细胞细胞膜上锚定表达P-selectin功能性片段。通过全合成的方法合成了GPI信号肽基因,融合于P-selectin基因下游构建真核表达载体pMCEw2-P-selectin-GPI,转染CHO细胞,利用GPI锚定将P-selectin的功能性基因片段表达在CHO细胞表面,获得了在细胞表面长期稳定表达P-selectin功能性片段的CHO细胞株P6。其次,利用本室构建的1.35×1010的全合成人源性抗体库,以原核表达产物的P-selectin和真核GPI锚定表达的P-selectin为抗原,分别进行了特异性抗体的筛选。利用原核表达产物筛选到15株特异性较好的抗体;利用表达P-selectin功能性片段的P6细胞株,通过细胞扣除筛选的方法得到2株在噬菌体水平上特异性较好抗体1H11、2F8。最后,将利用细胞筛选方法得到的两个抗体1H11、2F8改造为全抗体形式,并进行体外抗黏附试验。实验结果初步表明,我们所筛选到的2个抗人P-selectin人源性抗体,特异性较好,亲和力较高,分别达到150pM和427pM,在体外能够抑制血小板与中性粒细胞结合,表明这两个抗体在细胞学水平上具有一定的抗黏附功能,为进一步抗体介导的相关疾病治疗研究奠定了良好基础。
[Abstract]:In recent years, the role of P-selectin in the process of inflammation and tumor metastasis has been paid more and more attention. Taking P-selectin as a target to block the binding of P-selectin with its ligand may have great potential for the treatment of related diseases. At present, there are no reports on human-derived antibodies against P-selectin. In this study, the functional fragment of P-selectin was obtained from eukaryotic expression and prokaryotic expression system as antigen, and the human specific antibody against P-selectin was screened from the large volume synthetic human antibody library constructed in our laboratory. To obtain the neutralizing antibody which can block the ligand binding of P-selectin, so as to lay a good foundation for the treatment of related diseases. Firstly, the functional gene fragment of P-selectin was isolated from platelets by RT-PCR, and the soluble expression was achieved in E. coli. At the same time, P-selectin functional fragment was expressed on eukaryotic cell membrane by GPI anchoring technique. The GPI signaling peptide gene was synthesized by full synthesis. The eukaryotic expression vector pMCEw2-P-selectin-GPIwas constructed by fusion with P-selectin gene downstream, and then transfected into Cho cells. The functional gene fragment of P-selectin was expressed on the surface of Cho cells by GPI anchoring. Cho cell line P6 was obtained which expressed P-selectin functional fragment stably on cell surface for a long time. Secondly, a 1. 35 脳 1010 full-synthetic human antibody library was constructed in our laboratory. The specific antibodies were screened using P-selectin of prokaryotic expression product and P-selectin anchored by eukaryotic GPI as antigens, respectively. Fifteen P6 cell lines expressing P-selectin functional fragments were screened by prokaryotic expression products, and two P6 cell lines with good specificity at phage level were obtained by cell subtractive screening. Finally, the two antibodies 1H112F8 obtained by cell screening method were transformed into the whole antibody form, and the anti-adhesion test was carried out in vitro. The results showed that the two anti-human P-selectin antibodies were specific and highly affinity, up to 150pM and 427pM, respectively, which could inhibit the binding of platelets to neutrophils in vitro. The results indicated that the two antibodies had anti-adhesion function at the cytological level, which laid a good foundation for the further research on the antibody-mediated treatment of related diseases.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

【参考文献】