Optimization Of The Conditions Of High Cell Density Fermentation For The Engineering Bacterial Strain With RGD-spider Silk Protein Gene And Study On Cell Biocompatibility Of The Scaffold

The study on the spider silk has an aroused motive in biomaterials and tissue engineering. Our laboratory staff synthesized the gene of the recombinant spider dragline silk protein with RGD motif which involved the cell adhesion site by the gene engineering method, and constructed the engineering strain pNSR-16/BL21(DE3)pLysS. Based on the optimum shake culture and high-density fermentation of the engineering strain, factors such as pH, cultivation temperature, inducer, and the concentration of Gly/Ala were selected and tested at three levels, respectively. The optimum conditions of the parameters were established by the Do-stat fed-batch culture.The scaffold can be obtained when spider protein mixed with other polymerizers such as PVA. The endotoxin can be released when the targeted protein was obtained in the purification process, as RGD-16-multimer was cloned into the prokaryotic expressing vector. Phisicial and chemical methods were attempted to remove endotoxin which has toxicity in tissue engineering. Biomaterials with up to standard were obtained.The tissue engineering porous scaffold can be used in biomedicine domain. The cytotoxicity experiment of recombinant spider silk protein pNSR-16 and natural polymer pNSR-Z were discussed by culturing NIH3T3 cell with extracellular matrix. The leaching liquor and the scaffold had no restraint effort in NIH3T3 cell growing. Cells adhesived and grew on these scaffolds. It proved the scaffolds had better biocompatibility.