抗重组人bFGF单克隆抗体可变区基因克隆及单链抗体的构建和表达

Cloning of the variable region genes from hybridoma against bFGF and expression of single chain antibody fragments in E.coli HB2151

目的:从分泌抗重组人碱性成纤维细胞生长因子(bFGF)单克隆抗体(mAb)杂交瘤细胞株B2F3中克隆抗体可变区(V)基因,构建bFGF单链抗体(scFv),并进行可溶性表达。方法:从分泌bFGF mAb杂交瘤细胞株B2F3提取总RNA,用RT-PCR方法扩增抗体重链可变区基因(VH)和轻链的可变区基因(VL);再通过重叠延伸拼接(SOE)PCR方法,在VH和VL基因之间引入linker(Gly4Ser)3,构建bFGFscFv。将测序正确的scFv基因克隆到表达载体pCANTAB5E中,选择非抑制型菌株E.coli HB2151进行可溶性表达;经SDS-PAGE检测抗体表达水平,ELISA鉴定其抗原结合活性。结果:测序分析结果显示,VH基因序列全长375碱基对,编码125个氨基酸,VL基因序列全长399碱基对,编码133个氨基酸,二者均符合小鼠免疫球蛋白可变区基因特征,含有4个框架区(FR)、3个抗原互补决定区(CDR)及抗体特征性的2个半胱氨酸残基;构建的scFv全长789碱基对,编码263个氨基酸,连接结构为VH-linker-VL。SDS-PAGE分析表明scFv基因在大肠杆菌为可溶性表达,表达产物主要位于周质腔中,表达产物的Mr为27000,与理论预期值相符;间接ELISA检测结果显示原核表达的scFv具有与bFGF特异性结合的活性。结论:成功地克隆bFGF mAb可变区基因,并构建表达bFGF scFv,为下一步研究bFGF抗体人源化改造奠定实验基础。

AIM： To construct and express the single chain antibody （scFv） in E. coli HB2151 by cloning the vriable region genes from habfidoma against bFGF. METHODS： Total RNA was extracted from hybridoma cell line B2F3 secreting mAbs against bFGF and the cDNA was amplified by retropolymerase chain reaction （RT-PCR）. VL and VH were fused by a short pepUde linker containing 15 amino acids （Gly4Ser）3 using splice-overlap extension PCR to construct the scFv gene. The sequences of the scFv were analyzed by Shanghai Sangon Biological Engineering Technology ＆ Services Co. Ltd and Ig Blast data base in GenBank. The scFv gene was inserted into pCANTAB-SE vector and expressed in E. coli HB2151, RESULTS： The VH gene contained 375 base pairs and encoded 125 amine acid residues. The VL gene contained 399 base pairs and encoded 133 amine acid residues. There were four FRs, three CDRs and two characteristic cysteine residues in the VH gene and the VL gene, respectively. The scFv gene contained 789 base pairs and encoded 263 amine acid residues with the structure of VH-linker-VL. Restriction endonuclease digestion and DNA sequencing proved that the expression vector of pCANTAB-SE-scFv was constructed correctly. SDS-PAGE and ELISA analysis showed that scFv was successfully expressed in E. coli HB2151 and the expression protein had specific antigen binding activity. CONCLUSION： The variable region genes of anti-bFGF mAbs have been cloned successfully and single chain antibody fragments have been constructed and expressed, which will be a great help to the study of humanized antibodies against bFGF.