抗四环素单链抗体基因的构建与序列测定

摘要

目的：构建抗盐酸四环素的单链抗体（scFv）基因。方法：以抗盐酸四环素单克隆抗体杂交瘤细胞株的总RNA为模板，用RT-PCR法扩增全套抗体轻、重链基因；经重叠延伸反应，以编码柔性多肽（Gly4Ser）3的基因为接头，将轻、重链基因组装为完整的scFv基因，并克隆到pGEMT-Easy载体中进行测序分析。结果：所克隆的四环素scFV基因全长为735 bp，为VH-Linker-VL结构，VH基因为354 bp，Linker为（Gly4Ser）3多肽的核酸序列，VL基因为336 bp。结论：构建了抗四环素的单链抗体基因，为进一步用于四环素的残留检测奠定基础。%Objective: To construct the single chain variable fragment(scFv) gene specific for anti-tetracycline. Methods: The total RNA was isolated from anti-tetracycline monoclonal hybridoma cell mRNA. Variable light and heavy domains of the immunoglobulin genes were amplified by PCR and assembled to produce full length of scFv by overlap extension PCR using a linker primer containing flexible polypeptide (Gly4Ser)3. The scFv fragment was cloned into the vector pGEMT-Easy and sequenced with auto-DNA sequencer. Results: Cloned scFv gene was the structure of VH-Linker-VL, consisting 735 bp. The VH gene was 354 bp, while the VL gene was 336 bp. The sequence of the linker was (Gly4Ser)3. Conclusion: We built the single-chain antibody genes for tetracycline, and lay the foundation for further used for the detection of tetracycline residues.