本文選題：P-selectin + GPI��； 參考：《中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院》2008年碩士論文

【摘要】： 近年來(lái), P-selectin在炎癥反應(yīng)及腫瘤轉(zhuǎn)移過(guò)程中的作用越來(lái)越受到重視,以P-selectin為靶點(diǎn),阻斷P-selectin與其配體的結(jié)合,可能對(duì)相關(guān)疾病的治療具有較大潛力。目前國(guó)際還沒(méi)有關(guān)于P-selectin人源性抗體的報(bào)道。本研究以真核表達(dá)和原核表達(dá)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(kù)中篩選出針對(duì)P-selectin的人源性特異性抗體,力爭(zhēng)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(shí)現(xiàn)了可溶性表達(dá)。同時(shí)利用GPI錨定技術(shù)在真核細(xì)胞細(xì)胞膜上錨定表達(dá)P-selectin功能性片段。通過(guò)全合成的方法合成了GPI信號(hào)肽基因,融合于P-selectin基因下游構(gòu)建真核表達(dá)載體pMCEw2-P-selectin-GPI,轉(zhuǎn)染CHO細(xì)胞,利用GPI錨定將P-selectin的功能性基因片段表達(dá)在CHO細(xì)胞表面,獲得了在細(xì)胞表面長(zhǎng)期穩(wěn)定表達(dá)P-selectin功能性片段的CHO細(xì)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(kù),以原核表達(dá)產(chǎn)物的P-selectin和真核GPI錨定表達(dá)的P-selectin為抗原,分別進(jìn)行了特異性抗體的篩選。利用原核表達(dá)產(chǎn)物篩選到15株特異性較好的抗體;利用表達(dá)P-selectin功能性片段的P6細(xì)胞株,通過(guò)細(xì)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(xì)胞篩選方法得到的兩個(gè)抗體1H11、2F8改造為全抗體形式,并進(jìn)行體外抗黏附試驗(yàn)。實(shí)驗(yàn)結(jié)果初步表明,我們所篩選到的2個(gè)抗人P-selectin人源性抗體,特異性較好,親和力較高,分別達(dá)到150pM和427pM,在體外能夠抑制血小板與中性粒細(xì)胞結(jié)合,表明這兩個(gè)抗體在細(xì)胞學(xué)水平上具有一定的抗黏附功能,為進(jìn)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(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392

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