抗克伦特罗单链抗体基因构建及蛋白结构模拟

目的：构建抗克伦特罗单链抗体(scFv)基因cbl,进行蛋白质结构模拟并对其理化特性进行分析。方法：采用重叠延伸PCR 方法,以能特异性分泌抗克伦特罗mAb 的杂交瘤细胞株5D1 为原料,构建抗克伦特罗scFv 基因cbl,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：抗克伦特罗scFv 基因cbl 全长720bp,对应225 个氨基酸,单链抗体分子量约为25826.6,等电点预测值8.78,为碱性蛋白质;二级结构显示抗克伦特罗单链抗体含α螺旋20 处,β折叠99 处,β转角28 处,随机卷曲93 处。cbl 三级结构建模显示重链(VH)和轻链(VL)形成一个疏水的" 口袋",Linker 游离于此结构之外,符合scFv 的结构特点,明显具有抗原结合位点的空间构象,理论上应该具有良好的抗原结合活性。结论：构建一个抗克伦特罗scFv 基因cbl,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信息。

Gene Construction and Protein Structure Prediction of Single Chain Fv against Cenbuterol

Objective: To construct the gene of single chain Fv (scFv) fragment against clenbuterol, to predict secondary and tertiary structures of its encoding protein by computer assisted modeling and to analyze its physical and chemical properties. Methods: The gene of clenbuterol scFv was constructed with 5D1 hybridoma cells that is able to secret monoclonal antibodies (mAb) with high activity and specificity against clenbuterol as the raw material, and sequenced by splicing overlap extension (SOE) PCR assay. Furthermore, its amino acid sequence was deduced and the secondary and tertiary structures were predicted using biological information software. Meanwhile, its physical and chemical properties were analyzed. Results: The full-length scFv gene had 720 bp and encoded 225 amino acids. The relative molecular weight of the scFv was 25826.6, its predictive isoelectric point (pI) was 8.78 and the antibody belonged to alkaline protein. The predicted secondary structure showed that the protein contained 20 α-helix, 99 β-sheet, 28 β-turn and 93 random coli. The mimic tertiary structure model of the scFv indicted that the VL and VH was involved in composing the hydrophobic“ pocket” which was beneficial to the antigen binding, but the linker was free from this structure. Conclusion: The successful construction of a scFv gene cbl against clenbuterol by us can laid the foundation for the further research into the expression, purification and bioactivity of genetic engineering antibody.