抗人CD25分子单链抗体基因的构建、表达及初步鉴定

Construction,expression and identification of a single chain antibody variable(scFv) against human CD25 molecule

目的:构建和表达抗人CD25分子单链抗体(scFv)蛋白,并测定其生物学活性。方法:用RT-PCR方法从能分泌特异性抗CD25单克隆抗体(mAb)的杂交瘤细胞中分离纯化抗体VH和VL基因。用重叠延伸PCR方法将VH和VL拼接在一起,构建抗CD25分子scFv的基因。将scFv基因克隆至pMD18T,用限制性内切酶切以及测序鉴定。将scFv基因连接到pBAD/gⅢA表达载体,转化Top10表达菌。阳性克隆用左旋阿拉伯糖诱导4 h,SDS-PAGE电泳检测蛋白纯度,竞争抑制ELISA实验检测其活性。结果:scFv基因长度约为700 bp。通过DNA序列测定和分析,构建出VL-(GGGGS)3-VH(但其中349位G突变为A,使Linker其中一位Gly→Ser)。其VH隶属于小鼠Ig重链可变区Ⅲ(C)亚类,全长351 bp,可编码117个氨基酸;其VL隶属于小鼠Igκ轻链可变区Ⅳ亚类,全长318 bp,可编码106个氨基酸。TOP10中表达的scFv抗体加上同时融合表达的两个标签6×His和C-mycMr约为31 000,结果符合scFv的Mr。竞争抑制细胞ELISA实验显示表达的scFv具有活性。结论:此scFv基因的表达产物具有一定的特异结合活性。为抗CD25 scFv的临床应用打下了基础。

AIM： To construct and express a single chain fragment variable （scFv） fragment against human CD25 molecule and identify its bioactivity. METHODS： VH and VL genes of anti-mudne CD25 monoclonal antibody were cloned by RT-PCR from hybridoma cell WuTac secreting anti-CD25 mAb. scFv gene was spliced by sequence overlap extending （SOE） PCR, and then it was ligated into pMD18T vector to be identified by endonuclease digestion, PCR and sequencing, scFv gene was cloned into pBAD/glllA expression vector and transformed into TOP10 E. coli. After positive clones were induced by L-arabinose for 4 hours, the purity of protein was detected by SDS-PAGE and its bioactivity was identified by competitive inhibition ELISA test. RESULTS： scFv genes of VL-（GGGGSGGGGSSGGGS）- VH was constructed successfully. The VH chain consisted of 351 bp and encoded 117 amino acids, which belonged to heavy chain subgroup III （C） of mouse immunoglobulin variable region. The VL chain consisted of 318 bp and encoded 106 amino acids, which belonged to light chain subgroup Ⅳ of mouse immunoglobulin variable region. The scFv antibody expressed by TOP10 fused with 6 x His and C-myc tag protein and the relative molecular mass of fusion protein was about 31 000. Competitive inhibition ELISA test indicated the scFv antibody had specific activity. CONCLUSION： The expressed product of the single-chain antibody shows some specific binding capacity, which provides a basis for the clinical application of anti-CD25 single-chain antibody.