抗AIF单链抗体的构建与高效表达

Construction and expression of a single-chain Fv antibody fragment against acidic isoferritin in Escherichia coli

目的获得具有生物活性的抗酸性同功铁蛋白(AIF)单链抗体。方法利用重组噬菌体抗体库技术从杂交瘤细胞4c9中分别克隆小鼠抗体重链可变区(VH)和轻链可变区(VL)基因,成功构建AIF4c9scFv基因,并将其亚克隆于原核表达载体pET28a,转化E.coliBL21(DE3),经IPTG诱导表达。表达产物经镍鏊合层析柱复性后,用ELISA和Westernblotting检测表达蛋白与AIF的结合特性。结果AIF4c9scFv表达蛋白在细胞质中主要以不溶性包涵体形式存在,优化后的柱内复性程序可从高浓度的包涵体变性蛋白中高效复性AIF4c9scFv表达蛋白,其回收率可达75%左右。AIF4c9scFv复性蛋白可特异识别AIF抗原,具有良好的AIF抗体活性,其亲和力常数(KDscFv)为7.29×10-8mol/L。表达菌体的功能性AIF4c9scFv抗体产量约为62mg/L。结论在大肠杆菌中高效表达的抗AIFScfv经柱内复性后具有较好的生物学活性。

Objective To obtain the functional single chain Fv antibody （scFv） against acidic isoferritin （AIF）. Methods The variable regions of heavy chain （ VH ） and light chain （ VL ） from the hybridoma 4c9 were connected with a flexible linker using an assembly polymerase chain reaction. The construction of Vn-linker-VL was inserted into a phagemid pCANTAB 5 E, and then selected with the recombinant phage antibody system （RPAS）. Anti-AIF scFv gene from the recombinant phagemid pCAN4c9 was subcloned into pET28a and expressed in E. coli BL21（DE3）. The recombinant anti-AIF scFv was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and enzyme-linked immunosorbent assay after refold with an on-column refolding procedure based on Ni-chelating chromatography. Results The active anti-AIF scFv was recovered efficiently from inclusion bodies with a refolding yield of approximate 75 % confirmed by spectrophotometer. The results showed that the anti-AIF scFv retained the specific binding activity to AIF with an affinity constant of 7.29×10^-8 mol/L. The overall yield of anti-AIF scFv with bioactivity in E. coli flask culture was more than 62 mg/L. Condusion A functional scFv antibody fragment specific against AIF is produced at high level in Escherichia coli.