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目的构建抗人红细胞与抗HIV-1p24抗原双特异性抗体分子基因并进行三维立体结构模拟,为双特异性抗体蛋白的表达奠定理论基础。方法采用RT-PCR法从特异性分泌抗HIV-1p24抗原单克隆抗体的杂交瘤细胞株3D7中克隆单抗的可变区基因,构建抗HIV-1p24抗原单链抗体基因ScFv3D7,并通过ANTHEPROT5.0软件进行物理和化学以及二级结构预测,利用SWISS-MODLE软件进行立体结构模拟。通过15、30、45、60、75肽模拟连接单链抗体基因ScFv3D7与ScFv2E8(抗人红细胞H抗原,已制备),构建双特异性抗体分子基因,并进行立体结构预测。结果由柔性肽(G4S)3连接的单链抗体基因ScFv3D7,大小为729bp,编码243个氨基酸,等电点为5.665,相对分子质量为26147.937。其二级结构分析显示了不同比例的螺旋、片层和转角结构,三维立体结构预测模型符合免疫球蛋白家族特征。采用不同长度的柔性肽模拟连接两个单链抗体基因,其中以75肽连接的双特异性抗体分子的三维立体模型形成了完整的蛋白分子。结论基于75肽连接的双特异性抗体分子基因所模拟的立体结构可以认为是适合蛋白表达的分子模型。
Objective To construct the biospecimens of bispecific antibody against human erythrocyte and anti-HIV-1p24 antigen and to construct the theoretical basis for the expression of bispecific antibody. Methods The variable region gene of monoclonal antibody (mAb) was cloned from hybridoma cell line 3D7 specifically secreting anti-HIV-1p24 monoclonal antibody by RT-PCR and the anti-HIV-1p24 single chain antibody gene ScFv3D7 was constructed and passed ANTHEPROT5. 0 Software for physical and chemical as well as secondary structure prediction, using SWISS-MODLE software for structural simulation. ScFv3D7 and ScFv2E8 (anti-human erythrocyte H antigen) were mimicked by 15, 30, 45, 60, 75 peptide to construct bispecific antibody gene and to predict the three-dimensional structure. Results The single chain antibody gene ScFv3D7 linked by flexible peptide (G4S) 3 was 729 bp in size and encoded 243 amino acids with an isoelectric point of 5.665 and a relative molecular mass of 26147.937. The secondary structure analysis showed different proportions of spiral, lamellar and corner structures, and the three-dimensional structure prediction model conformed to the immunoglobulin family characteristics. Two single-chain antibody genes were mimicked using flexible peptides of different lengths, in which the complete three-dimensional model of the bispecific antibody molecule linked by the 75 peptide formed an intact protein molecule. Conclusion The stereostructure simulated by 75-peptide-linked bispecific antibody molecule can be considered as a molecular model suitable for protein expression.