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

Expression and Purification of a Humanized Anti-IL-21R scFv and Determination of its Affinity Constant

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。

The anti-IL-21 R single-chain variable fragment(scFv) C2 screened from a human phage display library was expressed in Escherichia coli HB2151,the concentrations of isopropyl-|3-D-thiogalactopyranoside(IPTG) induced in expression was tested,then the scFv C2 was purified with the Ni-affinity column,and different concentrations of imidazole were tested to purify the target protein.Then the purified scFv C2 was detected by SDS-PAGE and HPLC.The affinity constant of scFv C2 was determined by noncompetitive Enzyme-Linked Immunosorbent Assay(ELISA).After a series of trials,the best concentration,the best time of IL-21 R to cover the plate and the proper binding time of IL-21 R to scFv C2 to reach an equilibrium were determined.The experimental results indicated that the scFv C2 was expressed in soluble form in cell periplasm with 0.1mmol/L IPTG hypothermia induction.The expressed scFv C2 could be purified by Ni-affinity chromatography,hybridproteins were eluted with low concentration of imidazole,the target protein was eluted with 200 mmol/L imidazole,and the purity of scFv C2 was 92%detected by HPLC.The quantitation of scFv C2 was detected by the coomassie brilliant blue kit,and the best yield of scFv C2 was 1.23mg/mL fermentation broth.The noncompetitive ELISA experimental results indicated that the best IL-21 R coating concentration were 20,10,5 and 2.5μg/mL,the best IL-21 R coating time was 16 hours,and the best antigen and antibody binding time was 8 hours.Then the standard binding curve of the binding reaction of IL-21 R and C2 was poltced,the antibody concentrations corresponding to OD50%according to different IL-21 R concentration(20,10,5 and 2.5μg/mL) binding curves were obtained,and the affinity constant of scFv C2 as(7.36 ±2.62) ×10~8 L/mol was calculated.This results indicated that the anti-IL-21 R scFv C2 could strongly bind to the target antigen,and provided a theoretical basis for its tumor-target clinical use.