Expression of singleª²chain bifunctional antibody fusion protein ScFvª²TNFª²¦Á and its forecast with softwares

    ZHANG Jianª²Hua

    Faculty of Biomedical Engineering, School of Electrical Engineering, Zhengzhou University, Zhengzhou 45000, China

    ¡¾Abstract¡¿ AIM: To analyze the secondary structure of the fusion protein with bioinformatical approach, and to establish and express a recombinant secretory antiª²osteogenic sarcoma singleª²chain biª²functional antibody gene. METHODS: By the technology of directional cloning, the sequenced genes were subcloned into corresponding restriction sites of PLxSN in turn to generate coexpression vector. The pL(ScFvª²TNFª²¦Á)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/ScFvª²TNFª²¦Á, and fusion protein was identified by Western blot in the transfected OSC9901 cells. In our works, DNAssist and ANTHEPROT V5 were used to analyze the fusion genes sequence, the secondary structure, and the physical and chemical characteristics. RESULTS: The inserted fragments in all constructed plasmids were structurally confirmed to be consistent with that of the published data. Similarly, the vectors pL(ScFvª²TNFª²¦Á)SN was confirmed to be successful in the stable expression of the objective proteins in the target cells, and the expression of the recombinant protein was confirmed by Western blot. On the fusion genes, the secondary structure of the protein was analyzed with DNAssist, and the physical and chemical characteristics of the protein was forecasted with ANTHEPROT V5. CONCLUSION: The softwares which were downloaded from web should be fully used to analyze biologic information, and the approach may have application in a gene therapy context. ......