Prediction of three dimensional structure of bispecific antibodies linked by flexible peptide

SHAO Changli; GAO Yanhong; GAO Jing; WANG Guohua; DONG Zhennan

Abstract：Objective To construct the gene of a bispecific antibodies against red blood cells(RBCs)and HIV-1p24 antigen with different length peptides and simulate the tertiary structure of the bispecific antibodies for confirming the proper formulation of the protein expression.Methods The variable region genes of monoclonal antibodies were respectively amplified using RT-PCR from 3 D7 hybridoma cells which secrets monoclonal antibodies against HIV-1p24 antigen.The variable region genes of heavy and light chains were spliced by overlap extension(SOE)PCR to assemble SeFv(ScFv_(3D7))gene encoding the anti-HIV-1p24 antigen.Based on the sequence,the physical and chemical characteristics and the secondary structure of ScFv_(3D7) were analyzed by the software of ANTHEPROT5.0.Meanwhile,the tertiary structure prediction of ScFv3D7 was performed by SWISS-MODLE software.On the basis of sequence from ScFv_(3D7) and ScFv_(2E8) gene encoding the anti-RBC(red blood cell),which was prepared before,the gene of bispecific antibodies against RBC and HIV-1 p24 antigen was constructed by different length peptides to simulate the tertiary structure with SWISS-MODLE software.Results The assembled ScFv_(3D7) gene consisted of 729 bp,including the sequence of linker peptide(G_4S)_3.The isoelectric point(pI)and the relative molecular masses of ScFv_(3D7) encoded 243 amino acids were 5.665 and 26 147.937 Da respectively.The secondary structure analysis of ScFv_(3D7) showed the different ratio of helix,sheet and turn structures by ANTHEPROT5.0 software.The predicting tertiary structure of ScFv_(3D7) indicated that it met the antibody structure of immunoglobulin family by SWISS-MODLE.Finally,the bispecific antibodies against RBC and HIV-1 p24 antigen was simulated to construct tertiary structure as a integrity molecule by SWISS-MODLE with 75 peptide(G4S)15.Conclusion The bispecific antibodies against RBC and HIV-1 p24 antigen linked by flexible peptides with 75 amino acids are confirmed as the proper formulation for the protein expression.