Filtration Of Transgenic Sheep Skin Fibroblasts With Polymerized Spider Dragline Silk Protein Gene

The spider silk is known as yet to be the best fiber with many high performances in comparison with other natural materials or synthetic material. However there was a great difficulty in gaining large quantity of the spider silk. Due to the behavior and the characteristics of weaving web, the spider is unable to be cultured on a large scale. This research gained a good deal of enlightenment from the research that sheep can produce the wool consisting of keratin. In our previous studies, spider silk protein dimer and sheep hair keratin promoter B2C have been synthetized. In this research, reporter gene was connected with B2C, and then test its biological activity. Sheep hair keratin promoter was connected with PcDNA3.1to construct its eukaryotic expressing vector and the vector was connected with spider silk dragline protein dimer, then it is transfected into sheep skin fibroblast. After the resistance screening, the cells were enriched to establish spider silk dragline protein dimer sheep fibroblast cell lines. It laid the foundation of the transgenic Sheep that have clothing hair exprssing spider dragline silk protein. The main conclusions were as follows:(1)Biological activity of sheep hair keratin promoter B2C:The sheep hair keratin promoter was connected with the reporter gene GFP to construct recombinant plasmid B2C-GFP. B2C-GFP was transfected into mianyang skin fibroblasts. Specific bright green flurescene was observed around the cells under fluorescent microscope by blue light. This result indicated that the promoter B2C have good activity.(2)Construction of eukaryotic expressing vector: With CMV promoter be removed, pcDNA3.1was connected to the promoter B2C digested by the same enzyme and then to spider silk protein dimer2S. The eukaryotic expressing vector pcDNA3.1-B2C-2S was constructed.(3)Selection of transgenic positive cells:Sheep skin fibroblast were transfected with linear pcDNA3.1-B2C-2S and then selected with the suitable concentration of G418, best growing transgenic cells were gained.(4)Evaluation of transgenic cells:The enomic DNA of transgenic cells was extracted and detected by PCR. The result indicated that pcDNA3.1-B2C-2S was linked into the enomic DNA of sheep skin fibroblast. The cells can regain consciousness and stored in liquid nitrogen. After regain consciousness, the cells grew better.Based on the results as above, spider dragline protein dimer sheep fibroblast cell lines was obtained by the research.