Study On The Recombinant Spider Silk Protein As Scaffold Material For Tissue Engineering

Spider silk is a special fibrin. It has high strength, elasticity, flexility, elongation and tensile strength, along with lightness, the endurance of ultraviolet radiation, biodegradation, and so on. Spider silk is a new generation of the natural macromolecule fibrous biomaterials. Especially, the spider dragline silk is of the best mechanical properties. 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. This paper established the high-density fermentation of the engineering strain pNSR-16/BL21(DE3)pLysS and the large-scale purification technics of the recombinant spider silk protein. Based on it, we prepared the fibrin film and the porous scaffold, and studied the degradable behaviour and the cytotoxicity in vitro.Based on the optimum culture of the engineering strain pNSR-16/BL21(DE3)pLysS in shake-bottle, we carried out the high-density fermentation in 10L fermental jar. The end cell density OD600 of the engineering strain pNSR-16/BL21(DE3)pLysS was 60-90, and the expression of the recombinant spider silk protein was up to the 11-12% of the total protein by the Do-stat fed-batch culture.The small-scale purification of the recombinant spider silk protein was carried out through the His-Bind Affinity Chromatography. The iso-electric point was 6.45 by IEF. The purified protein was farther proved as the recombinant spider silk protein using Western Blot, ultrasonating the cell, adjusting pH 9.10, 60℃ heating for 8-10 mins, 15% (NH4)2SO4 precipitation, We can achieve the recombinant spider silk protein with the purity 90% at least.This paper reported the preparation of biomaterial from the recombinant spider silk protein and the part mechanical properties of the biomaterial for the first time. The silk fibrous protein solution was prepared by dissolving the recombinant spider silk protein in 98% formic acid after dialyzed and lyophilized. The film can be made through scraping the protein solution on the plastic disk. NaCl as porogen, the porous scaffold can be manufactured by salt-leaching. The mechanical properties of the scaffold material can be improved by mixing the polymer and the fibroin protein. The effect of many factor on the structure and mechanical of scaffold was discussed.