抗利巴韦林单链抗体基因构建及其结构分析

Construction and Structure of Anti-ribavirin Single-chain Antibody Gene

【目的】克隆构建利巴韦林(RBV)单链抗体(scFv)基因,对其理化特性进行分析并对蛋白质结构进行模拟,为后期检测方法的建立及分子改造提供参考依据。【方法】以分泌利巴韦林抗体的杂交瘤细胞株总RNA为模板,通过RT-PCR技术扩增抗体的重链可变区(VH)和轻链可变区(VL),然后以柔性连接短肽(Gly4Ser)3为接头拼接完整的scFv-RBV。利用生物信息学方法对scFv-RBV的理化性质及蛋白结构功能进行预测分析。【结果】构建的scFv基因编码240个氨基酸,相对分子质量为26162.27 Da,理论等电点(pI)为8.57。在二级结构中,β-折叠(39.17%)和无规卷曲(45.41%)占主导地位,α-螺旋(5.42%)和β-转角(10%)相对较少。在三级结构中,VH和VL区域被Linker相互牵拉靠近,形成典型的口袋样形状的空间构象,符合单链抗体的结构特征,理论上可以与RBV抗原特异性结合。【结论】成功构建scFv-RBV基因,并利用生物信息学方法预测分析scFv的二级、三级结构,该结果可为后期进行单链抗体的表达、纯化及定向进化提供理论支撑。

【Objective】The ribavirin(RBV)single-chain antibody(scFv)gene was constructed,cloned,and physiochemically analyzed to establish a model for the detection method development and molecular modification.【Method】Using the total RNA of the hybridoma cell line secreting RBV antibody as a template,both heavy-chain(VH)and light-chain variable(VL)regions of the antibody were amplified by RT-PCR.Then the short peptide(Gly4Ser)3 was employed as the splicing joint to construct the complete scFv-RBV.Bioinformatics methods were applied to predict and analyze the physiochemical properties,protein structure,and functions of the gene.【Results】The constructed scFv-RBV encoded 240 amino acids with a relative molecular mass of 26,162.27 Da and a theoretical isoelectric point of 8.57.The secondary structure of the protein consisted of 39.17%β-sheets,45.41%random coils,5.42%α-helices,and 10%β-turns.In the tertiary structure,the VH and VL regions were pulled close by Linker,forming a typical pocket-like spatial configuration that conforms to the structural characteristics of a single-chain antibody.Theoretically,it could bind specifically to RBV antigens.【Conclusion】The successfully constructed scFv-RBV in this study afforded the utilization of bioinformatics methods to predict and analyze the secondary and tertiary structures of scFv gene paving the way for further studies on the expression,purification,and directed evolution of the single-chain antibodies.