發(fā)布時(shí)間：2018-11-27 18:00

【摘要】： 目的從噬菌體抗體庫(kù)中篩選獲得的肝癌細(xì)胞HepG2特異性結(jié)合單鏈抗體(single chain Fv, scFv),為進(jìn)一步提高其親合力(avidity),構(gòu)建和表達(dá)與肝癌細(xì)胞HepG2特異結(jié)合的雙價(jià)抗體(Bivalent single chain Fv, BsFv),鑒定其生物學(xué)活性,并和親本單鏈抗體進(jìn)行比較。 方法①以與肝癌細(xì)胞特異結(jié)合的單鏈抗體SLH10基因?yàn)槟０?設(shè)計(jì)引物引入中間連接肽(G4S)及酶切位點(diǎn)AscI,通過PCR方法擴(kuò)增出兩個(gè)scFv片段,將其連接,并克隆至原核表達(dá)載體pAB1;②將表達(dá)載體轉(zhuǎn)化大腸桿菌TG1,挑取單克隆細(xì)菌進(jìn)行擴(kuò)增,提取質(zhì)粒酶切、測(cè)序鑒定;③陽(yáng)性菌于23℃經(jīng)IPTG誘導(dǎo)表達(dá),在不同時(shí)間點(diǎn)收集菌液進(jìn)行SDS-PAGE分析,并進(jìn)行亞細(xì)胞定位分析,確定最適表達(dá)條件后進(jìn)行大量表達(dá),Ni2+-NTA層析柱純化抗體,對(duì)純化產(chǎn)物進(jìn)行SDS-PAGE分析和Western blot鑒定;④通過FCM測(cè)定純化產(chǎn)物與L02細(xì)胞和HepG2細(xì)胞結(jié)合活性,并進(jìn)一步測(cè)定與其他幾種腫瘤細(xì)胞株結(jié)合特性,比較BsFv、親本scFv親合力。 結(jié)果①提取重組菌質(zhì)粒,酶切經(jīng)凝膠電泳可見約1500bp大小片段條帶,與BsFv基因大小相符;測(cè)序結(jié)果提示兩個(gè)擴(kuò)增基因片段以及中間連接肽序列正確無(wú)誤,表明BsFv構(gòu)建成功;②scFv及BsFv表達(dá)菌于23℃經(jīng)IPTG誘導(dǎo)隨時(shí)間的延長(zhǎng)，蛋白產(chǎn)量上升;收集處理誘導(dǎo)12h菌體，亞細(xì)胞定位分析提示細(xì)菌外周質(zhì)中存在目的蛋白表達(dá)，分子量大小分別為30kD、60kD左右，與理論值相符;③經(jīng)IPTG誘導(dǎo)大量表達(dá)，收集上清超濾濃縮，收集菌體經(jīng)處理得到外周質(zhì)產(chǎn)物，所有產(chǎn)物經(jīng)NiZ+一NTA層析柱純化，并通過SDS一PAGE分析、W己stem blot鑒定表明表達(dá)蛋白的分子量與理論值一致;④FCM分析純化抗體分別與幾種細(xì)胞株的結(jié)合活性，，結(jié)果表明BsFv與親本scFv比較，與HePGZ細(xì)胞結(jié)合率增加，與L02細(xì)胞及其他腫瘤細(xì)胞株結(jié)合率下降，表明BsFv與HePGZ細(xì)胞株親合力有所增加。 結(jié)論成功構(gòu)建與表達(dá)雙價(jià)抗體BsFv;與親本抗體scFv比較，BsFv與細(xì)胞結(jié)合的親合力增加，本實(shí)驗(yàn)為進(jìn)一步將其作為腫瘤靶向分子研究奠定了基礎(chǔ)。
[Abstract]:Objective to construct and express bivalent antibody (Bivalent single chain Fv, a bivalent antibody to hepatoma cell line HepG2, which was selected from phage antibody library to construct and express bivalent antibody (Bivalent single chain Fv, which is specific for HepG2 binding to single chain antibody (single chain Fv, scFv),) in order to further enhance its affinity (avidity),. BsFv), identified its biological activity and compared it with parent scFv. Methods 1 using the SLH10 base of single chain antibody (scFV) specifically binding to hepatoma cells as template, two scFv fragments were amplified by PCR and cloned into prokaryotic expression vector pAB1; by introducing intermediate junction peptide (G4S) and restriction enzyme site (AscI,) into the primer. (2) the expression vector was transformed into Escherichia coli TG1, and the monoclonal bacteria were amplified, the plasmids were digested and sequenced. (3) the positive bacteria were induced by IPTG at 23 鈩

本文編號(hào)：2361612