發(fā)布時(shí)間：2018-01-11 23:02

  本文關(guān)鍵詞：全人源抗IL-21受體單鏈抗體的表達(dá)純化及其親和常數(shù)的測(cè)定　出處：《藥物生物技術(shù)》2014年06期 　論文類(lèi)型：期刊論文

  更多相關(guān)文章： 單鏈抗體 白介素受體 表達(dá) 純化 抗體親和力 酶聯(lián)免疫吸附法

【摘要】：將從全人源噬菌體單鏈抗體庫(kù)中經(jīng)親和篩選獲得的抗白介素21受體單鏈抗體C2進(jìn)行表達(dá),摸索IPTG誘導(dǎo)表達(dá)的最佳濃度,經(jīng)鎳柱親和層析分離純化,HPLC鑒定單鏈抗體C2的純度,并通過(guò)非競(jìng)爭(zhēng)性ELISA固相法,經(jīng)確定最佳抗原包板濃度、最佳抗原包板時(shí)間及最佳抗原與抗體結(jié)合反應(yīng)時(shí)間,測(cè)定單鏈抗體C2的親和常數(shù)。實(shí)驗(yàn)結(jié)果表明在0.1 mmol/L IPTG誘導(dǎo)下單鏈抗體C2可溶性表達(dá)在細(xì)胞周質(zhì),鎳柱親和層析純化C2,純度為92%,其最佳表達(dá)量為1.23 mg/L發(fā)酵液。采用非競(jìng)爭(zhēng)性ELISA法,通過(guò)繪制不同濃度白介素21受體與單鏈抗體C2的結(jié)合反應(yīng)曲線(xiàn),計(jì)算出C2的親和常數(shù)為(7.36±2.62)×10~8L/mol,與IL-21R有較強(qiáng)的親和力,為其進(jìn)一步開(kāi)發(fā)進(jìn)行腫瘤靶向治療提供理論基礎(chǔ)。
[Abstract]:The anti-interleukin-21 receptor scFv C2 obtained from the full-human phage phage single-chain antibody library was expressed by affinity screening. The optimal concentration of IPTG induced expression was explored and purified by nickel affinity chromatography. The purity of scFv C2 was identified by HPLC, and the optimal concentration of antigen plate, the best time of antigen encapsulation and the reaction time of antigen binding to antibody were determined by non-competitive ELISA solid phase method. The affinity constant of scFv C2 was determined. The results showed that SFV C2 was soluble expressed in the peripheral cytoplasm of cells induced by 0.1 mmol/L IPTG, and C2 was purified by nickel column affinity chromatography. The purity was 92 and the best expression was 1.23 mg/L fermentation broth. By using non-competitive ELISA method, the binding reaction curve of interleukin-21 receptor with single chain antibody C2 was drawn by using different concentrations of interleukin-21 receptor. The affinity constant of C2 is 7.36 鹵2.62 脳 10 ~ (8) L / mol, which has strong affinity with IL-21R, which provides a theoretical basis for further development of tumor targeting therapy.
【作者單位】： 南京中醫(yī)藥大學(xué)藥學(xué)院;中國(guó)藥科大學(xué)生命科學(xué)與技術(shù)學(xué)院;
【基金】：南京中醫(yī)藥大學(xué)自然科學(xué)青年基金項(xiàng)目(No.12XZR26) 江蘇高校優(yōu)勢(shì)學(xué)科建設(shè)工程資助項(xiàng)目
【分類(lèi)號(hào)】：R392
【正文快照】： 白介素21受體(Interleukin 21 Receptor,IL-21R)具有 隆抗體(mAb 9E10)購(gòu)自美國(guó)Santa Cruz公司;辣根過(guò)氧化廣泛的淋巴細(xì)胞分布,IL-21R的異常表達(dá)可破壞機(jī)體的免 物酶(HRP)標(biāo)記的羊抗鼠IgG、96孔微孔板購(gòu)自上�；蛞咂胶�,導(dǎo)致腫瘤、炎癥及多種自身免疫性疾病,例如急性 生物工

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