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目的 欲用计算机模拟及分子对接技术揭示抗乙酰胆碱酯酶单克隆抗体 3F3抑酶的分子基础 ,但 3F3分子远大于乙酰胆碱酯酶 (AChE) ,难以操作 ,故拟用其单链抗体为工具进行研究。本研究旨在设计、克隆并表达抗AChE单链抗体Sc3F3基因 ,推演重组单链抗体的氨基酸顺序 ,并证明纯化的重组单链抗体有抑制AChE的作用。方法 使用分泌抗电鳐AChE单克隆抗体 3F3(3F3Ab)的杂交瘤细胞提取总RNA。用RT PCR技术扩增重链可变区 (VH)cDNA及轻链可变区 (VL)cDNA基因 ,通过连接肽(Gly4 Ser) 3 基因将VH 基因和VL 基因连接 ,制成单链抗体基因 (ScFv)。将ScFv基因插入载体pCANTAB 5E用于表达。在大肠杆菌中表达的 3F3单链抗体(Sc3F3)用SephadexG75凝胶过滤和QAE SephadexA 5 0离子交换层析纯化。AChE与抗体的免疫反应性用ELISA法测定。AChE活性用微量羟胺比色法测定。结果 测序结果证明所克隆的ScFv基因是功能重排基因 ,长度为 72 3bp。重、轻链基因长度分别为 35 7bp和 32 1bp。ScFv基因 (含连接肽基因 )所编码的纯化的重组Sc3F3的分子量为 31.6ku。Sc3F3与AChE反应良好 ,但对AChE活性的抑制明显弱于 3F3Ab。Sc3F3的抑制效力约为 3F3Ab的 1/10。结论 在计算机模拟及分子对接研究中使用本研究设计的单链抗体Sc3F3应是可靠的
Objective To reveal the molecular basis of anti-acetylcholinesterase monoclonal antibody 3F3 aprotein by computer simulation and molecular docking, but the 3F3 molecule is much larger than acetylcholinesterase (AChE), so it is difficult to operate. Therefore, it is proposed to use its single chain antibody as a tool . The purpose of this study was to design, clone and express Sc3F3 gene against AChE scFv and to deduce the amino acid sequence of the recombinant scFv antibody. The purified recombinant scFv antibody could inhibit the AChE activity. Methods Total RNA was extracted from hybridoma cells secreting monoclonal antibody against AChE monoclonal antibody 3F3 (3F3Ab). The heavy chain variable region (VH) cDNA and the light chain variable region (VL) cDNA were amplified by RT PCR. The VH gene and the VL gene were linked by the peptide (Gly4 Ser) 3 gene to form the single chain antibody gene (ScFv). The ScFv gene was inserted into vector pCANTAB 5E for expression. The 3F3 single chain antibody (Sc3F3) expressed in E. coli was purified by Sephadex G75 gel filtration and QAE Sephadex A 50 ion exchange chromatography. The immunoreactivity of AChE with antibodies was determined by ELISA. AChE activity was measured by micro hydroxylamine colorimetry. Results The sequencing results showed that the cloned ScFv gene was a functionally rearranged gene with a length of 72 3bp. The heavy and light chain genes were 35 7 bp and 32 bp, respectively. The molecular weight of the purified recombinant Sc3F3 encoded by the ScFv gene (including the linker peptide gene) was 31.6ku. Sc3F3 reacted well with AChE but inhibited AChE activity significantly less than 3F3Ab. The inhibitory potency of Sc3F3 is about 1/10 of that of 3F3Ab. Conclusion The single chain antibody Sc3F3 designed in this study should be reliable in computer simulation and molecular docking studies