关键词: 角质细胞生长因子1； 截短突变；sf-9细胞；杆状病毒系统

Abstract:

Abstract:Objective To explore the feasible of the expression of recombinant truncated human keratinocyte growth factor 1 (rhKGF1_d23)  in  insect cells and to provide a new method to product rhKGF1_d23.Methods The truncated gene of kgf1_d23 optimized  by insect prefer codon was amplified and subcloned into pFastBac vector.With the help of helper plasmid,the Tn7 section of pFastBac-kgf1_d23 was transposed to  the mini-attTn7 site of Bacmid DNA to obtain  the recombinant Bacmid-kgf1_d23.Bacmid-kgf1d23 shuttle vector was used to transfect sf-9 cells by lipofection kit to produce recombinant baculovirus P1.The  baculoviral stock was amplified and the virus titer was increased until P3 stock.The cells were broken by sonic and the lysate liquid and purified by heparin affinity chromatography and cation exchange; MTT method was used to detect the  rhKGF1_d23mitogen activity.Results  kgf1d23 gene was optimized by insect bias codon and the recombinant  pFastBac-kgfd23 and Bacmid-kgf1d23 had been successfully constructed.The SDS-PAGE results showed that  the rhKGF1D23 expressed mainly in cells and until 60 h post-infection,and the optimal harvest time was  in 84-96 h.Heparin affinity and SP cation exchange could remove 90% noisy proteins.The MTT results demonstrated that the purified rhKGF1_d23protein with 0.3-25.0 μg•L^-1  could significantly stimulate HaCat cells to proliferate in a dose-dependent manner.Conclusion The rhKGF1_d23expression is obtained in sf-9 cells with heparin binding characteristics,as well as immunogenic and cell biology activity.

Key words: keratinocyte growth factor　1, truncated mutation, sf-9 cells, baculovirial expression system