| Based on the molecular cloning and recombination techniques, 4 immunity-related or growth-related genes (IGF-I, hep-IGF, CXC-S457 and CC-KC70) from turbot were cloned to the pGEX-4T-1 vector and then transformed into E.coli BL21(DE3)plysS. Induced by IPTG, the recombinant proteins expressed and then were purified with GSTrap FF affinity chromatograph. The biological activities of the proteins were also analyzed with different methods.1. Cloning, recombinant expression and bioactivity analysis of Turbot IGF-IInsulin-like growth factor-I (IGF-I) is a conserved peptide expressed ubiquitously, which shows diverse effects on development, growth, and metabolism. With RT-PCR, the fragment encoding the Turbot (Scophthalmus maximus) mature IGF-I peptide was amplified. It was predicted that the mature peptide was composed of 70 amino acids including 6 cysteines which could form 3 disulfide bonds. The target fragment was successfully subcloned into the expression vector pGEX-4T-1, after induction with IPTG, the fusion proteins highly expressed in E.coli BL21(DE3)plysS. The result of SDS-PAGE showed that the fusion protein expressed in the form of inclusion bodies with molecular weight of 34 kD and maximally amounted to 59 % of the whole protein in the E.coli cell 4hours post induction. The western blotting indicated that recombinant protein had the antigenicity to anti-GST antibody. The inclusion bodies were dissolved in 6 mol/L guanidine chloride followed by pulse renaturation in refolding buffer containing 0.5 mol/L L-Arginine, 1.0 mol/L GSH and 0.2 mol/L GSSG. Then the renatured recombinant protein was purified by GSTrap FF affinity chromatography. The effect of purified GST-IGF-I on turbot kidney cells was analysised, and it indicated that the recombinant GST-IGF-I can stimulate the proliferation of the cells. 2. Recombinant expression and bioactivity analysis of Turbot hep-IGF-IThe hep-IGF recombinant gene was amplified using a pair of specific primers. The PCR products were cloned to pGEX-4T-1 vector and then the plasmid was transformed into E.coli BL21(DE3)plysS. After induced by IPTG for different time courses, the expression of GST-hep-IGF was determined by SDS-PAGE. The result shows that the expression amont can raised to the peak 7 hours post induction, maximally amounted to 55 % of the total protein in the E.coli cell. Western blotting indicated that recombinant protein had the antigenicity to anti-GST antibody. After ultrasonic disruption process, the supernatant was collected and pumped into GSTrap FF affinity Collumn. The antibacterial bioactivity of purified recombinant proteins to E.coli (ATCC25922) was analyzed with liquid growth inhibition method, and the different antibacterial effects were detacted in 8 hours post treatments.3. Recombinant expression and bioactivity analysis of CXC chemokine S457 and CC chemokine KC70 from TurbotThe S457 and KC70 mature genes were obtained with PCR using turbot liver cDNA as template. Sequence blast showed that the two chemokines shared low identity and similarity, that is about 30~60% identities with other chemokines of several animals. The amplified segments were subcloned to the pGEX-4T-1 vector respectively. After transformation and induction, the expression of recombinant proteins were detected with SDS-PAGE and the molecular weights of GST-S47 and GST-KC70 recombinant proteins were about 38kD and 36kD. The result of time course experiment showed the amount of GST-S47 and GST-KC70 reached the maximums 4 hours post induction, accounted to 47% and 46%, respectively. The western blotting indicated that recombinant protein had the antigenicity to anti-GST antibody. After ultrasonic disruption process, the supernatant is collected and pumped into GSTrap FF affinity Column. Then a chemotaxis assay was carried out, which is the definitive assay for chemokine activity. This assay showed migration of peripheral blood leukocytes across a membrane with 5um pores towards GST-S47 and GST-KC70 at the optimal concentration of 1ug/mL and 10ug/mL respectively.
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