Wang Hong, Chen Dan, Deng Ning, Xiang Jun-Jian, Jin Ying-Jie, Huang Hong-Liang, Tang Yong, Yang Hong-Yu
Center of Antibody Engineering, Jinan University, Guangzhou 510632, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2007 Dec;23(12):1150-3.
To construct and express the single chain antibody (scFv) in E.coli HB2151 by cloning the variable region genes from hybridoma against bFGF.
Total RNA was extracted from hybridoma cell line B2F3 secreting mAbs against bFGF and the cDNA was amplified by retropolymerase chain reaction (RT-PCR). V(L) and V(H) were fused by a short peptide linker containing 15 amino acids (Gly(4)Ser)(3) using splice-overlap extension PCR to construct the scFv gene. The sequences of the scFv were analyzed by Shanghai Sangon Biological Engineering Technology and Services Co. Ltd and Ig Blast data base in GenBank. The scFv gene was inserted into pCANTAB-5E vector and expressed in E.coli HB2151.
The V(H) gene contained 375 base pairs and encoded 125 amine acid residues. The V(L) gene contained 399 base pairs and encoded 133 amine acid residues. There were four FRs, three CDRs and two characteristic cysteine residues in the V(H) gene and the V(L) gene, respectively. The scFv gene contained 789 base pairs and encoded 263 amine acid residues with the structure of V(H)-linker-V(L). Restriction endonuclease digestion and DNA sequencing proved that the expression vector of pCANTAB-5E-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.
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.
通过克隆抗碱性成纤维细胞生长因子（bFGF）杂交瘤的可变区基因，在大肠杆菌HB2151中构建并表达单链抗体（scFv）。
从分泌抗bFGF单克隆抗体的杂交瘤细胞系B2F3中提取总RNA，通过逆转录聚合酶链反应（RT-PCR）扩增cDNA。使用重叠延伸PCR法，通过含15个氨基酸（Gly(4)Ser）(3)的短肽接头将轻链可变区（V(L)）和重链可变区（V(H)）融合，构建scFv基因。scFv序列由上海生工生物工程技术服务有限公司和GenBank中的Ig Blast数据库进行分析。将scFv基因插入pCANTAB-5E载体并在大肠杆菌HB2151中表达。
V(H)基因包含375个碱基对，编码125个氨基酸残基。V(L)基因包含399个碱基对，编码133个氨基酸残基。V(H)基因和V(L)基因分别有四个框架区（FRs）、三个互补决定区（CDRs）和两个特征性半胱氨酸残基。scFv基因包含789个碱基对，编码263个氨基酸残基，具有V(H)-接头-V(L)结构。限制性内切酶消化和DNA测序证明pCANTAB-5E-scFv表达载体构建正确。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和酶联免疫吸附测定（ELISA）分析表明，scFv在大肠杆菌HB2151中成功表达，表达蛋白具有特异性抗原结合活性。
成功克隆了抗bFGF单克隆抗体的可变区基因，构建并表达了单链抗体片段，这将对研究抗bFGF人源化抗体有很大帮助。
