全人源抗IL-21受体单链抗体的表达纯化及其亲和常数的测定

发布时间：2018-01-11 23:02

本文关键词：全人源抗IL-21受体单链抗体的表达纯化及其亲和常数的测定　出处：《药物生物技术》2014年06期 　论文类型：期刊论文

【摘要】：将从全人源噬菌体单链抗体库中经亲和筛选获得的抗白介素21受体单链抗体C2进行表达,摸索IPTG诱导表达的最佳浓度,经镍柱亲和层析分离纯化,HPLC鉴定单链抗体C2的纯度,并通过非竞争性ELISA固相法,经确定最佳抗原包板浓度、最佳抗原包板时间及最佳抗原与抗体结合反应时间,测定单链抗体C2的亲和常数。实验结果表明在0.1 mmol/L IPTG诱导下单链抗体C2可溶性表达在细胞周质,镍柱亲和层析纯化C2,纯度为92%,其最佳表达量为1.23 mg/L发酵液。采用非竞争性ELISA法,通过绘制不同浓度白介素21受体与单链抗体C2的结合反应曲线,计算出C2的亲和常数为(7.36±2.62)×10~8L/mol,与IL-21R有较强的亲和力,为其进一步开发进行肿瘤靶向治疗提供理论基础。
[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 卤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.
【作者单位】：南京中医药大学药学院;中国药科大学生命科学与技术学院;
【基金】：南京中医药大学自然科学青年基金项目(No.12XZR26) 江苏高校优势学科建设工程资助项目
【分类号】：R392
【正文快照】： 白介素21受体(Interleukin 21 Receptor,IL-21R)具有隆抗体(mAb 9E10)购自美国Santa Cruz公司;辣根过氧化广泛的淋巴细胞分布,IL-21R的异常表达可破坏机体的免物酶(HRP)标记的羊抗鼠IgG、96孔微孔板购自上海基因疫平衡,导致肿瘤、炎症及多种自身免疫性疾病,例如急性生物工

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