Secondary structure and physico-chemical property of fusion proteins: Analysis with bioinformatics network resource / 中国组织工程研究

ABSTRACT

AIM: To analyze secondary structure and physico-chemical property of the fusion protein with bioinformatics network resource, and explore the expression of a secretory anti-osteoblastic carcinoma single-chain bi-functional antibody gene. METHODS: ①The single-chain variable fragment (ScFv) antibody gene and interleukin-2 (IL-2) gene were subcloned into corresponding restriction sites of retrovirus expression vector PLxSN. Mediated by liposome, the recombinant plasmid 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 osteosarcoma (OSC) cells were infected by the recombinant retrovirus, terming OSC/ScFv-IL-2. The virus titer was detected by using NIH3T3.②The transfected OSC9901 cells by ScFv-IL-2 gene were identified by polymerase chain reaction (PCR), reversed transcription-PCR and Western blotting. After the fusion protein was constructed, DNAssist and ANTHEPROT V5 softwares were used to analyze the amino acid sequence, the secondary structure, and the physico-chemical property of fusion protein. RESULTS: ①After the restriction enzyme and PCR identification, the pL(ScFv-IL-2)SN as a fusion protein expression vector, was constructed successfully, and high titer C26 cells were obtained; the expression of recombinant protein was confirmed by Western blotting.②On the fusion genes, the DNA sequence was analyzed with DNAssist nucleic acid sequence analysis software, and their secondary structure and physico-chemical property were analyzed with ANTHEPROT V5. CONCLUSION: The property of fusion protein can be analyzed and forecasted by means of bioinformatics network resources, and the approach may provide evidences for investigating single-chain bi-functional antibody gene.