Screening Of Recombinant Human Collagen Was Highly Expressed Strains And Research Oxidation Resistance

Collagens are extracellular most important fibrin, is an extracellular matrix of the skeleton, so that the cell has a good tension and elasticity and in cell development plays an important role in. With the development of science, people for collagen also had a more profound and extensive knowledge. Collagen as an important natural protein resources, by virtue of its unique structure and properties, has been infiltrated into various fields, both in the biomedical, food industry and textile industry, cosmetics industry has broad application prospects. But using the traditional method of collagen extraction not only extraction methods have many shortages,extraction of collagen also exists virus hidden trouble and allogeneic immune rejection. In recent years, with the development of biological technology, using gene engineering method to obtain the target protein, by virtue of its low immunogenicity, no virus hidden trouble and other advantages, has been gradually pay attention to and should be used in the industrial production. In this paper, using Escherichia coli as the host bacteria, through screening, using four different engineering strain of expression rate higher strains, and the use of add inducer after induction time, IPTG concentration, liquid inoculum and pH on recombinant Escherichia coli culture conditions were optimized, resulting in high expression strain, and then using affinity chromatography method for recombinant Escherichia coli and purified, the aim is to get higher purity of protein And by in vitro method for the determination of the lipid oxidation inhibition rate, the scavenging rate of hydroxyl free radicals and superoxide anion in the autoxidation reaction to the determination of its antioxidant propertie to provide reference data for future research..First of all, using the PCR amplification techniques will get encoding human collagen DNA fragments into the E. coli BL21, BL21(DE3), DE3, Rosetta (DE3), and screening of positive clones, using induction agent IPTG on its induced expression, through the SDS-PAGE electrophoresis proved recombinant collagen molecular weight of46kDa and from screening a high expression amount of strains used as engineering bacteria, the plasmid stability analysis.Useing of the ultrasound screening recombinant E. coli cells were broken, and after to12000rpm centrifugal at4℃, supernatants were collected and sedimentation analysis of soluble recombinant protein, to the small batch flask fermentation expression based onengineering strain the production of collagen culture conditions. The pH of the medium, the bacteria inoculum size, adding the inducer IPTG induction time and inducer at a final concentration of the culture conditions were optimized, and expression products were purified by affinity chromatography. Finally, the inhibition rate of lipid oxidation, hydroxyl radical scavenging rate and superoxide anion, since the oxidation and other aspects of the determination to analyze the expression product of the oxidation resistance of E. coli recombinant strain for the follow-up study like collagenthe application provides a certain reference.Collagen gene CoL6A2engineering of recombinant E. coli bacteria, the expression of the fusion protein molecular weight of about46kDa. The experimental results show that the reorganization of the collagen can be stabilized in the expression of the engineered bacteria, and most of the soluble form. Fusion protein in optimal expression conditions:bacteria inoculated in liquid LB containing Amp by5%of the inoculum sizemedium, the culture around2.5h to the OD value of0.7, adding a final concentration of0.1mol/L inducer IPTG-induced expression of7h, when the highest amount of recombinant protein expressed in E. coli, the total bacterial proteinabout28.1%. Optimized E. coli by affinity chromatography purification, the collagen protein analysis, the purity can reach more than90%. Use of antioxidant properties of the recombinant protein and its reaction. Recombinant proteins on lipid oxidation inhibition rate of79.3%, hydroxyl radical scavenging rate of75.05%and significant inhibition of superoxide anion to hydrogen peroxide.Conclusion:The recombinant protein using genetic engineering techniques, and effective solution to traditional methods of the non-water-soluble extract of animal collagen, and is conducive to the purpose of high-purity protein affinity chromatography. Combination Optimization of Culture Conditions, and laid the foundation for the next step of high-density fermentation, and new ways to find a suitable for the production of human collagen. The antioxidant properties carried out detailed research and analysis, and found that collagen has a very good antioxidant properties, the collagen used in cosmetics, play a role in delaying aging and for the reorganization of collagen for the recombinant protein in the beauty provide the basis of the application of the products.