Study On The Preparation Of The Recombimation RGD-containing Peptide From Wild Silk Fibroin And Their Cell Adhesion And Proliferation

Silk fibroin is a kind of nature protein, which mainly composed of fibroin and sericin. It has excellent mechanical properties and biocompatibility. Which is widely used in biological materials, especially in tissue engineering applications. Most of the studies mainly concentrated in the Bombyx mori silk fibroin and those studies have shown that Bombyx mori silk fibroin not only have good mechanical properties and biodegradable,but also support cell proliferation, adhesion, spreading, and induce differentiation of stem cells. However, the study of wild silkworms(Antheraea pernyi and Antheraea yamamai) involves slightly less. Highly repetitive RGD peptide distributes in the amino acid sequence of Antheraea pernyi and Antheraea yamamai silk fibroin. RGD peptide can be used as recognition sites of integrin and ligands protein. Which promote integrin binding with ligand, then the extracellular signals pass into cells and activate the relevant functions of cells. The application of wild silk worms in biomaterials will be greatly expended if the biology function of RGD peptide from Antheraea pernyi and Antheraea yamamai is cleared. In this article, the RGD peptide from wild silkworms(Antheraea pernyi and Antheraea yamamai) was designed by the method of gene engineering and expressed in E.coli. then purification protein obtain target protein.We carried on the quantitative and qualitative analysis for target protein, Besides, the biocompatibility of short RGD peptide was done for a further study. This thesis mainly included the following work:Firstly, we has been built to contain RGD peptides gene expression vector p ET-30a(+)and p GEX-KG, then the expression vector were transferred in E.coli 21 and induction by IPTG. Verfied the expression of recombinant protein by SDS-PAGE. Compared to p ET-30a(+)expression vector, the p GEX-KG expression vector can efficient expression recombinant protein and obtain soluble protein.Secondly, this thesis emphasis study four times( [-RGD-]4)and eight times([-RGD-]8)of RGD peptides insert into p GEX-KG expression vector,expression by IPTG induction.The fusion protein were marked as GST-[-RGD-]4 and GST-[-RGD-]8.We research optimized expression conditions of two fusion protein. It’s including different initial density, different inducer concentration and different induction time. The results revealed that the fusion protein GST-[-RGD-]4 optimized expression conditions :initial density OD600=1.5, 0.4 mmol/L IPTG concentration, induced 2 hours. The fusion protein GST-[-RGD-]8 optimized expression conditions :initial density OD600=0.9, 0.4 mmol/L IPTG concentration, induced 3 hours.Thirdly, the SDS-PAGE and MODI-TOF results show we obtain high purity fusion protein by using the GST affinity chromatography column.The protein concentration was measured by BCA protein concentration determination.The results showed that the two fusion protein GST-[-RGD-]4 and GST-[-RGD-]8 proteins production were about 55mg/L, 52.5mg/L respectively.Fourthly, the target peptides [-RGD-]4 and [-RGD-]8 were obtained by using thrombin protease enzyme digestion fusion protein GST-[-RGD-]4 and GST-[-RGD-]8. The SDS-PAGE and MODI-TOF results reveal that the measured molecular weigh of the target peptides were consistent with the theoretical value. This results also shows that the design of the peptides sequence have be right expression.Fifthly, we research the isoelectric points of fusion protein and target protein, the results show the measured isoelectric points of fusion protein and target protein were consistent with the theoretical value.The amino acid composition analysis of fusion protein GST-[-RGD-]4 and GST-[-RGD-]8 also show the recombinant protein have be right expression in E.coli 21. The preliminary exploration on the secondary structure of target peptides [-RGD-]4 and [-RGD-]8 were measured by Circular Dichroism,the results showed that the two target peptides were mainly α-helix structure.At last, explore the cell compatibility of two target peptides, the results reveal that compared with chemical synthesis RGD peptides, the target peptides [-RGD-]4 and [-RGD-]8 were conducive to rapid cell adhesion and spreading. While it’s no significant difference for cell proliferation.