电鳐乙酰胆碱酯酶单链抗体基因的克隆及表达(英文)

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

Gene cloning and expression of single-chain antibody against Torpedo acetylcholinesterase

AIM In study of the molecular basis of the inhibition of anti-acetylcholinesterase(AChE) monoclonal antibody 3F3(3F3Ab) by means of computer simulation and molecular docking, the size of 3F3 seems too big to be manipulated. Thus a single-chain antibody(ScFv) was designed and used as a tool in the study. This paper aimed to clone and express the anti-AChE 3F3Ab single chain(Sc3F3) gene, to deduce the amino acid sequence of the recombinant Sc3F3, and to exhibit the inhibition effect of Sc3F3 on AChE. METHODS The total cellular RNA was prepared from hybridoma cells that secreted anti-AChE 3F3Ab. The cDNAs of variable regions of the heavy chain (V H) and light chain (V L) were amplified by RT-PCR. The Sc3F3 gene was obtained by joining V H and V L genes via a flexible gene linker coding for (Gly 4Ser) 3. Sc3F3 gene was inserted into the vector pCANTAB5E for expression. The expressed Sc3F3 in E.coli was purified by Sephadex G 75 gel filtration and then by QAE Sephadex A-50 ion exchange chromatography. Immuno-reactivity between AChE and antibodies was measured by ELISA. AChE activity was assayed by a microcolorimetric method. RESULTS The sequence analysis demonstrated that the Sc3F3 gene is functionally rearranged in length of 723 bp. The V H and V L genes were 357 bp and 321 bp in length, respectively. The molecular weight of the purified recombinant Sc3F3 encoded by Sc3F3 gene (containing the linker peptide gene) was estimated at 31.6 ku, and it reacted well with AChE, however the inhibition of Sc3F3 on AChE activity was weaker than that of 3F3Ab, about one tenth of the efficiency of its parent antibody. CONCLUSION The utilization of the designed Sc3F3 should be dependable in the computer simulation and molecular docking research.