Expression of single-chain bifunctional antibody fusion protein and its forecast with softwares

作者：Zhang Fangli · 发表于：Xinxiang yixueyuan xuebao · 年份：2008 · 研究领域：Monoclonal and Polyclonal Antibodies Research、Viral Infectious Diseases and Gene Expression in Insects、Virus-based gene therapy research

Objective To analyze secondary structure and the physico-chemical property of the fusion protein with software,and to investigate the expression of single-chain bifunctional antibody gene of secreting type anti-osteogenic sarcoma.Methods By the technology of polymerase chain reaction(PCR),the sequenced genes of secretary signal peptide of artificial synthetical antibody were added to single-chain antibody(scFv)genes′5′ of anti-osteogenic sarcoma and the interleukin-2(IL-2) genes were added to scFv-IL-2 genes,the sequenced genes were subcloned into corresponding restriction sites of PLxSN in turn to generate coexpression vector.The pL(scFv-IL-2)SN was packaged with PA317 and selected in G418 to obtain the positive clones,which were able to produce stable retrovirus,and then OSC9901 cells were infected by the recombinant retrovirus.The positive clones were obtained after G418 selection and were termed OSC/ scFv-IL-2,and fusion protein were identified by Western bloting in the transfected OSC9901 cells.In the works,DNAssist and ANTHEPROT V5 were used to analyze the sequence,the secondary structure,and the physical and chymic character.Results The inserted fragments in all constructed plasmids were structurally confirmed to be consistent with that of the published data.Similarly,the G418 selected vectors PL(scFv-IL-2)SN was confirmed to be successful in the stable expression of the objective proteins in the target cells,and expression of recombinant protein was confirmed by Weste...