中国药理学与毒理学杂志2003，Vol.17Issue(1)：1-5,5.
电鳐乙酰胆碱酯酶单链抗体基因的克隆及表达
Gene cloning and expression of single-chain antibody against Torpedo acetylcholinesterase
摘要
Abstract
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 (VH) and light chain (VL) were amplified by RT-PCR. The Sc3F3 gene was obtained by joining VH and VL genes via a flexible gene linker coding for (Gly4Ser)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 VH and VL 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.关键词
抗体,单链/乙酰胆碱酯酶/基因克隆/基因表达Key words
antibody, single-chain/acetylcholine- sterase/gene clone/gene expression分类
医药卫生引用本文复制引用
王玉霞,武军华,滕霞,郭崇志,孙曼霁..电鳐乙酰胆碱酯酶单链抗体基因的克隆及表达[J].中国药理学与毒理学杂志,2003,17(1):1-5,5.基金项目
"九五"全军医药基金资助项目(96-Z-016) （96-Z-016）
