Purification Of RhIGF-Ⅰ And Localization And Functional Study Of Potential Nonstructural Proteins Of SARS-CoV

Insulin-like growth factor-I(IGF-I) is a kind of active peptide that can promote many types of cells to grow. It plays an important regulatory role in the proliferation, differentiation , apoptosis of tissue cell and tumorigeneisis. However the industrial production is impeded for the complicated protein purification operation,the demanding condition and the low productivity. So it is very helpful to advance the research and application of IGF-I to establish a relatively simple proficient efficient purification route.The E.coli BL21(DE3) was transformed with the constructed recombinant plasmid pExSecl-IGF-I. The engineering bacteria were induced to express IGF-I by IPTG at a low temperature of 16℃ for 16h. Then bacteria were collected by centrifugation and split by ultrasonic method. The purified products were analyzed by Tris-Tricine SDS-PAGE. The immuno-activity of the recombinant proteins were analyzed by western blot. The target protein was soluble in the supernatant. By two-step chromatography, rhIGF-I was purified to 90% of purity. Western blot indicated that the recombinant protein could react with antibodies against hIGF-I.Bioinformatic analysis forecaste there are putative uncharacterized protein(1-5) that have no significant BLAST matches to known protein in SARS-CoV. Some research indicated nonstructural proteins play an important role in the virus replication and pathogenesis. Nonstructural proteins can be pathogen-related proteins.We constructed the vectors that respectively express the fusion protein of X1,X2 and green(GFP) or red fluorescent protein(RFP) in mammalian cells by recombinant PCR. The recombinant plasmids were transiently transfected into 293, PC12, COS7 and Vero cells and observed with laser scanning confocal microscope(LSCM). LSCM showed that fluorescence light was spread in all of cells transfected with pEGFP-N1 or pDsRedl-N1,but fluorescence light distributed in the cytoplasm in cells transfected with pEGFP-N1-X1or pDsRed 1-N1-X1, and fluorescence light was focused on the nucleus of cells transfected with pEGFP-N1-X2. Staining of cellular organelles and extraction of membrane protein indicated X1 localized in plasma membrane, endoplasmic reticulum , Golgi apparatus and et al.Flow cytomettry show X1 and X2 can induce the cell cycle block and the inhibition of proliferation in 293 and Vero cells. AnnexinV-PE and 7-AAD dual dye and flow cytometry were used to examine apotosis in COX7 cells. Results showed X2 protein could induce apotosis in COS7 cells. So X2 may be related with the cell apotosis.