【摘要】： 目的:將我室構(gòu)建的抗肝癌噬菌體單鏈抗體二聚體(scFv-BDM2)在大腸桿菌可溶性表達(dá)、純化,并對(duì)其親和力與特異性行初步鑒定,為下一步構(gòu)建~(99m)Tc標(biāo)記的scFv-BDM2及scFv-BDM2-發(fā)熱磁性納米顆粒的臨床應(yīng)用奠定基礎(chǔ)。 方法:1.將抗肝癌scFv-BDM2由E.coli TGl轉(zhuǎn)入E.coli HB2151中,IPTG誘導(dǎo)可溶性表達(dá),制備大量scFv-BDM2;利用HiTrap Anti-E Tag親和層析柱對(duì)表達(dá)的二聚體進(jìn)行純化。 2.將純化后的scFv-BDM2進(jìn)行聚丙烯酰胺凝膠電泳,以Bradford法測(cè)定其濃度,酶聯(lián)免疫吸附實(shí)驗(yàn)法(ELISA)檢測(cè)其生物活性和特異性;采用非競(jìng)爭(zhēng)酶免疫技術(shù)測(cè)定上述純化的scFv-BDM2親和力常數(shù),并比較其大小變化。 結(jié)果:1.將抗肝癌scFv DM二聚體在E.coli HB2151大量表達(dá)并純化后經(jīng)聚丙烯酰胺凝膠電泳分析,可見(jiàn)一清晰蛋白條帶;經(jīng)分子相對(duì)遷移率初步計(jì)算估計(jì),目的條帶的分子量約為60KD,純化后的二聚體濃度約為16μg/ml。 2.采用非競(jìng)爭(zhēng)酶免疫法測(cè)定上述純化的單鏈抗體二聚體的活性效價(jià)和親和力常數(shù)分別為1:64、3.735×10~7mol/L~(-1),而scFv DM的親和力常數(shù)為2.44×10~7mol/L(-1),二聚體的親和力為scFv DM的1.53倍。以ELISA法對(duì)所獲陽(yáng)性克隆的特異性分析發(fā)現(xiàn),抗肝癌scFv-BDM2均與三種肝癌細(xì)胞結(jié)合,而與正常肝細(xì)胞、胃癌細(xì)胞和膀胱癌細(xì)胞不結(jié)合。 結(jié)論:經(jīng)大腸桿菌表達(dá)及親和層析純化后獲得一定濃度的scFv DM二聚體,其親和力較前提高,和抗肝癌噬菌體單鏈抗體同樣具有良好的抗原結(jié)合特異性。
[Abstract]:Objective: to express and purify anti-hepatoma phage single-chain antibody dimer (scFv-BDM2) in Escherichia coli, and to identify its affinity and specificity. It lays a foundation for the clinical application of ~ (99m) Tc labeled scFv-BDM2 and scFv-BDM2- febrile magnetic nanoparticles. Methods: 1. The anti-hepatoma scFv-BDM2 was transferred from E.coli TGl to E.coli HB2151, and the soluble expression was induced by IPTG. A large number of scFv-BDM2; was purified by HiTrap Anti-E Tag affinity chromatography. 2. The purified scFv-BDM2 was detected by polyacrylamide gel electrophoresis and its concentration was determined by Bradford method. The bioactivity and specificity of the purified scFv-BDM2 were detected by Elisa (ELISA). The affinity constants of the purified scFv-BDM2 were determined by non-competitive enzyme immunoassay and the changes of its size were compared. Results: 1. The anti-hepatoma scFv DM dimer was expressed in E.coli HB2151 and purified by polyacrylamide gel electrophoresis, and a clear protein band was found. The molecular weight of the target band is about 60 KD and the concentration of the purified dimer is about 16 渭 g / ml. 2. The activity titers and affinity constants of the purified scFv dimer were 1: 64-3.735 脳 10 ~ (7) mol / L ~ (-1) and 2.44 脳 10~7mol/L (-1) by non-competitive enzyme immunoassay, respectively. The affinity of dimer was 1.53 times of that of scFv DM. The specific analysis of the positive clones by ELISA method showed that anti-HCC scFv-BDM2 was bound to three kinds of hepatoma cells, but not to normal hepatocytes, gastric cancer cells and bladder cancer cells. Conclusion: a certain concentration of scFv DM dimer was obtained after expression of Escherichia coli and purification by affinity chromatography. The affinity of scFv DM dimer was improved compared with that before, and it had good antigen-binding specificity as well as anti-hepatoma phage scFv.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類(lèi)號(hào)】：R392;R735.7

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相關(guān)期刊論文 前4條

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