| The objectives of this study is to research the recombination expression,purification and biological function of cyclotide,which was found from the Chinese herb Viola yedoensis and named 241 b.This paper mainly containing three parts,the first part contained the soluble protein expression and enrichment,relesesed soluble 241 b from the inclusion bodies and optimized the purfication process of cyclotides.The second part applied the motheds that involved in bacteriostatic test,cell experiment and C.elegans nematode to test the biological functions of 241 b.The last part was monitor for the fermentation process,and we used 16 S r DNA to identify the two mixed strain,and C.elegans nematode model was used to test them effects on cyclotide 241 b.The results as follows:1.We optimized the express process that based on the methods our laboratory,which used to induce the soluble cyclotides 241 b.Based on experiment results,a suite of technological conditions were proposed as below.We was used 8mol/L urea to dissolve the inclusion bodies,and the dissolved proteins were dialyzed in renaturing solution containing 1 mmol/L GSH,0.1 mmol /L GSSG,and 0.8 mmol/L L-Arg at 25°C.The finally reduction rate of cyclotides is 1.4 mg from 1 g inclusion bodies.2.We used 70% ACN to elute the solution cyclotides that affinitied by C18 SPE.The cyclotides that were dissolved in 70% ACN purfied in RP-HPLC by the programs: 0-5 min(88% A phase and 12% B phase),5.01-10 min(40%A phase and 60% B phase).10.01-30(10% A phase and 90% B phase),30-30.10(88% A phase and 12% B phase),30.10-40(88% A phase and 12% B phase),(A phase is ultrapure water that containing 0.05% TFA,B phase is 90% ACN hybrid with 0.05% TFA).The purfied cyclotides structure was determined by MOLDI-TOF-MS.3.We determined the 241 b has inhibition to two G-bactariels,that named Escherichia coli and Pseudomonas aeruginosa,and two G+ bactariel which named Bacillus subtilis and Staphylococcus aureus.The MIC of them are 0.2,1,0.2,0.2 mg/m L respectively.We used the CCK-8 to test the effect of 241 b on the cell viability of MCF-7 cell line and got there had opposite effects that the concentrates of 241 b is 0.03 mg/m L will accelerate the proliferation of MCF-7 to 38.44%,while is 1 mg/m L will appear inhititory active up to 95%.And the IC50 is 0.225 mg/m L.We also determined the influences of the combination of 241 b and Cd Cl2 to MCF-7.The results tells us under the appropriate concentrations,the 241 b will combinate with the Cd2+ inhance the viability of the MCF-7.While we seed the L1 C.elegans by 241 b lonely for 48 h,there were no adults when the concentrate of 241 b is 0.5 mg/m L and there had nobody alive at the 1mg/m L.of the C.elegans was reduced by 36.36% while we seed 0.05 mg/m L 241 b to the young adults,and 90.9% was reduced while the concentrate of 241 b is 2 mg/m L.It means that the cyclotides of which named 241 b has strongly inhibitory effects to the C.elegans.4.To determined the influence of the shake flsk and stirring fermenter on the cyclotides production,we did some experiments under the optimized conditions that 5% seed solution to the LB media,which containing 10 g/L glucose,and IPTG induced the bacterial for 8 h when they were cultured to OD600 at 1.2.At the finally,the biomass in the fermenter is 5.38 g/L,enhanced by 34.76% than the shake flsk for biomass is 3.5 g/L.At the same time,the cycotides in the inclusion bodies reduced 30.43%.The quality of 241 b is 68.57 mg by 1L fermentation fluid,while the shake flask is 44.91 mg,increased by 34.5%.These results reviewed that enlarge cultivation the 241 b based on the original expression process will enhance the quality of 241 b,which laid a foundation for the industrialization production.5.Two strains of serratia marcescens that is pollution of 241 b preserve fungus,was identified by the technology named 16 S r DNA.The C.elegans model was used to test the effects of these two bacteria stains on the bioactivities of 241 b,that named 241b-5 and 241b-7.We found that seed C.elegans 241b-5 or 241b-7,they were have side effects on the behavior that growth rate,lifespan and body bends.While they were seed combinated with 241 b,the side effect on the growth rate and body bends was decreased.When the 241b-5 combination with 241 b,there had no side effect on lifespan,but the 241b-7 will cut lifespan for 16%.Sothese results tell us the clean experimental environment is important to the quality of the fermentation products.In summary,we had acquired the bioactivities cyclotides 241 b by the recombination technology.This will provide much practice experience for our laboratory to construct the mutant library. |
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