本文選題：P-selectin + GPI�。� 參考：《中國人民解放軍軍事醫(yī)學科學院》2008年碩士論文

【摘要】： 近年來, P-selectin在炎癥反應(yīng)及腫瘤轉(zhuǎn)移過程中的作用越來越受到重視,以P-selectin為靶點,阻斷P-selectin與其配體的結(jié)合,可能對相關(guān)疾病的治療具有較大潛力。目前國際還沒有關(guān)于P-selectin人源性抗體的報道。本研究以真核表達和原核表達系統(tǒng)獲得P-selectin功能性片段為抗原,利用本室構(gòu)建的大容量全合成人抗體庫中篩選出針對P-selectin的人源性特異性抗體,力爭獲得能夠阻斷P-selectin與配體結(jié)合的中和抗體,從而為相關(guān)疾病的治療奠定良好的基礎(chǔ)。 首先,利用RT-PCR的方法從血小板中釣取P-selectin的功能性基因片段,在大腸桿菌中實現(xiàn)了可溶性表達。同時利用GPI錨定技術(shù)在真核細胞細胞膜上錨定表達P-selectin功能性片段。通過全合成的方法合成了GPI信號肽基因,融合于P-selectin基因下游構(gòu)建真核表達載體pMCEw2-P-selectin-GPI,轉(zhuǎn)染CHO細胞,利用GPI錨定將P-selectin的功能性基因片段表達在CHO細胞表面,獲得了在細胞表面長期穩(wěn)定表達P-selectin功能性片段的CHO細胞株P(guān)6。 其次,利用本室構(gòu)建的1.35×1010的全合成人源性抗體庫,以原核表達產(chǎn)物的P-selectin和真核GPI錨定表達的P-selectin為抗原,分別進行了特異性抗體的篩選。利用原核表達產(chǎn)物篩選到15株特異性較好的抗體;利用表達P-selectin功能性片段的P6細胞株,通過細胞扣除篩選的方法得到2株在噬菌體水平上特異性較好抗體1H11、2F8。 最后,將利用細胞篩選方法得到的兩個抗體1H11、2F8改造為全抗體形式,并進行體外抗黏附試驗。實驗結(jié)果初步表明,我們所篩選到的2個抗人P-selectin人源性抗體,特異性較好,親和力較高,分別達到150pM和427pM,在體外能夠抑制血小板與中性粒細胞結(jié)合,表明這兩個抗體在細胞學水平上具有一定的抗黏附功能,為進一步抗體介導(dǎo)的相關(guān)疾病治療研究奠定了良好基礎(chǔ)。
[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.
【學位授予單位】：中國人民解放軍軍事醫(yī)學科學院
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R392

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