农药克百威单链抗体基因构建及蛋白结构模拟

Construction and Structure Prediction of Murine Single Chain Fv Gene against Carbofuran

构建小分子农药克百威(CBF)单链抗体(scFv)基因cbf,进行蛋白质二级、三级预测并对其理化特性进行分析。采用重叠延伸PCR方法,以能特异性分泌抗CBF单克隆抗体的杂交瘤细胞株5D3为原料,构建抗CBFscFv基因cbf,进行测序,采用生物信息软件对氨基酸序列推导并进行结构预测,同时对理化性质进行分析。结果得出,抗CBFscFv基因cbl全长750bp,对应228个氨基酸,单链抗体分子量约为26315.1,等电点预测值5.66,为酸性蛋白质;二级结构显示抗克伦特罗单链抗体含α螺旋18处,β折叠108处,β转角24处,随机卷曲93处。cbf三级结构建模显示VL和VH符合scFv的结构特点,可观察到6个环区形成疏水的"口袋",具有抗原结合位点的空间构象。从而构建了一个抗CBFscFv基因cbf,应用信息学技术所获得的预测和分析结果为抗克伦特罗单链抗体的进一步表达、纯化和活性研究提供了大量的信息。

The gene of single chain Fv fragment （scFv） was constructed against carbofuran and the physical and chemical characteristics, secondary structure and tertiary structure of the scFv was predicted using computer assisted modeling. The variable region gene of the heavy and light chains were amplified respectively from 5D1 hybridoma cells that can secret monoclonal antibodies with high activity and specificity against carbofuran. These two fragments were then spliced together through a flexible linker to scFv by using splicing overlap extension （SOE） PCR. The scFv gene was named cbf and the sequence of scFv gene was measured. The physical and chemical characteristics, secondary and tertiary structure were predicted using Internet and corresponding software. The whole scFv gene contained 750 bp was cloned successfully and encoded 228 amino acids. Theoretically, the scFv was relative molecular masses 26315.1, the isoelectric point （pI） was 5.66 and the antibody belongs to acid protein. The predictied secondary structure showed the protein contained 18 α-hefix, 108 β-sheet, 24 β-turu and 93 random coli. The mimic tertiary structure model of scFv indicted the linker was isolated from VH and VL. The VL, as well as the VH, was involved in composing the hydrophobic＂pocket＂ which was beneficial to the antigen binding. The construction and analysis of scFv against carbofuran laid the foundation for the further research into expression, purification and bioactivity of the genetic engineering antibody.