脂联素单链抗体基因的克隆及表达

Clone and expression of adiponectin single chain antibody gene

目的克隆人脂联素单链抗体基因并进行表达。方法提取分泌抗脂联素单克隆抗体杂交瘤细胞株的总RNA,通过RT-PCR技术,扩增VH和VL,与质粒pUC19载体连接,形成克隆,并对其进行鉴定分析。结果抗体重、轻链可变区扩增拼接后的重、轻链各约为340bp、350bp,基因全长约710bp,将拼接产物插入pUC19质粒中转化大肠杆菌JM109,得到数个克隆,经酶切分析约含710bp片段,与拼接结果基本相同,经测定特异性较好,与其他杂蛋白及载脂蛋白没有交叉反应。结论抗人脂联素单链抗体基因的克隆和表达较为成功。

Objective To construct and express the single chain antibody （scFv） in E. coli JM107 by cloning the variable region genes from habridoma against adiponectin. Methods Total RNA was extracted from hybridoma cell line secreting monoclonal antibodies （McAbs） against adiponectin, and the cDNAs of VL and VH were amplified by RT PCR. The products were fused by a short peptide linker and inserted into pUC19 vector and expressed in E. coli JM109, then their sequences were analyzed. Results The VH gene contained 350bp encoding 116 amino acid residues. The VL gene contained 340bp encoding 113 amino acid residues. Restriction endonuclease digestion and DNA sequencing proved that the scFv contained 710bp that was constructed correctly. ELISA results showed that scFv was successfully expressed in E. coli JM109 and the expression protein had specific antigen binding activity. Conclusion Anti-human adiponectin ScFv is successfully constructed for the use in clinical studies.