Research On The Cloning, Expression & Biological Activities Of HIL-17F

Objective: To clone and express ML-17F gene in E.coli and construct hIL-17F-transgenetic cell stably expressing hIL-17F protein and to study the biological activities of recombinant hIL-17F protein.Methods: To design and synthesize one pair of primers according to declared hIL-17F cDNA sequence from GenBank. The cDNA fragment encoding hIL-17F was amplified by RT-PCR from the total RNA extracted from PHA-activated peripheral blood mononuclear cells. It was cloned into pUCm-T vector and sequenced. Then recombinant prokaryotic expression vector pGEX-5X-3/hIL-17F and recombinant eukaryotic expression vector pSIV-1/hIL-17F were constructed and identified. The pGEX-5X-3/hIL-17F was transfected into E.coli BL21 and the inclusion bodies(IB) fusion protein was induced by IPTG The purified GST-hIL-17F fusion protein was identified by Western blot. The proliferation of ECV304 cells was observed by incubating them with soluble GST-hIL-17F fusion protein by MTT assay. The concentrations of IL-6, IFN-γ and TNF- α in the supernatants of ECV304 cells were determined by ELISA. The effect of GST-hIL-17F on the angiogenesis ofthe chick chorioallantoic membrane was assessed by CAM assay. The recombinant retrovirus vector pSIV-l/hIL-17F together with its two helper virus vectors pHIT456 and pHIT60 cotransfected into the package cell 293T by Liposome to produce mature recombinant retrovirus, then which was used to infect SMMC-7721 cell. The SMMC-7721 cell line stably expressing ML-17F protein was selected in the presence of G418 and identified by RT-PCR and Western blot.Results: A 41kD fusion protein was effciently induced in E.coli BL21 by IPTG, accounting for about 55% of the total bacterial protein. The purified GST-hIL-17F fusion protein was identified by Western blot. GST-hIL-17F fusion protein had obvious biological activities to inhibit the proliferation of ECV304 cells and enhance IL-6 secretion. GST-hIL-17F had a marked antiangiogenic activity. Results of RT-PCR and Western blot indicated that hIL-17F-transgenetic SMMC-7721 cell could stably express ML-17F target protein.Conclusion: ML-17F was effciently expressed in E.coli and the recombinant ML-17F protein had a marked antiangiogenic activity and had obvious biological activities to inhibit the proliferation of ECV304 cells and enhance IL-6 secretion, and the successful construction of hIL-17F-transgenetic SMMC-7721 cell stably expressing ML-17F protein by infection of retrovirus vector, which lays a foundation forfuture research on the mechanism of antiangiogenesis and tumor therapy by antiangiogenesis of recombinant hIL-17F protein.