抗人ICAM-1单链抗体表达载体的构建及在大肠杆菌中的表达

Construction of Expression Vector for Anti-Human Intercellular Adhesion Molecule-1 Single Chain Fragment Variable and Its Expression in E.coli

目的构建抗人细胞间黏附分子-1(ICAM-1)单链抗体(ScFv)的表达载体,并在大肠肝菌中表达。方法从分泌ICAM-1单抗的杂交瘤细胞中提取RNA,用RT-PCR扩增抗体VH和VL基因,重叠延伸PCR扩增人ICAM-1-ScFv基因,将其连接到pET-22b(+)载体上,转化大肠杆菌BL2(DE3),IPTG诱导表达,表达产物纯化及复性后,检测特异性及活性。结果序列分析表明抗ICAM-1 ScFv基因全长为744bp,编码247个氨基酸,其中含357bp的VH基因片段和342bp的VL基因片段。表达蛋白以包涵体形式存在,表达量占菌体总蛋白的32%。经变性和复性后,纯度达80%以上,复性率达25%。Western blot和ELISA检测,ScFv均可与ICAM-1抗原特异性结合。细胞黏附试验表明ScFv能抑制ICAM-1与HSB2细胞间的黏附,其作用稍弱于亲本mAb。结论已成功构建了抗人ICAM-1的ScFv表达载体,其表达产物具有抗体特异性和活性。

Objective To construct the expression vector for anti-human intercellular adhesion molecule-1 （ICAM-1） single chain fragment variable （ScFv） and express in E. coli .Methods Extract RNA from the hybridoma cells secreting McAb against ICAM-1 for amplifi- cation of VH and VL genes by RT-PCR. Amplify human ICAM-1 ScFv gene by SOE-PCR and insert into vector pET-22b（ ＋ ）. Transform the constructed recombinant plasmid to E. coil for expression under induction of IFIG. The expressed product were purified,re-naturalized and de- termined for specificity and activity. Results Sequencing result showed that the amplified ICAM-1 ScFv gene fragment, at a full-length of 744 bp, encoded 247 amino acids and consisted of Vii and VL genes,at lengths of 357 bp and 342 bp respectively,and a 45 bp tlexible linker. The expressed product,in a form of inclusion body,contained 32% of total somatic protein and reached a purity of more than 80% after denatu- ration and renaturation. The refolding rate was 25%. Both Western blot and ELISA showed specific binding of ScFv to ICAM-1 antigen. Cell ad- hesion test proved that the ScFv inhibited the adhesion of ICAM-1 to HSB2 cells. However, compared with that of its parental McAb, the in- hibitory effect of ScFv was slightly weak. Conclusion The expression vector for anti-human ICAM-1 ScFv was successfully constructed, and the expressed product showed antibody specificity and activity.