Construction Of Fine Wool Sheep Follicle-specific Expression Vector Of Spider Dragline Silk Protein Gene And The Foundation Of Transgenic Cell Lines

Spider silk is one of the most tenacious natural bio fiber found in nature which is mainly composed of spider silk protein.lt is difficult to produce or collect lots of spider silk protein artificially.However,it is feasible to transform spider silk protein gene into heterologous host.In this study,we used spider silk protein expression vector pK-2S to transfected merino fine wool sheep shin fibroblasts,then we screened transgenic cell lines and detected expression at DNA and RNA levels.This study laid the foundation of fosterring new sheep breed which can express spider dragline silk gene in hair follicles specifically by transgenic technology.1.Synthesized spider dragline silk protein gene monomer artificially according to the published cDNA fragments of Nephila clavata spider,then got the diploid (2S).2.1solated and cultured merino fine wool fibroblasts.The morphologies of cells were normal.The activity of cells were great through growth curve,and the karyotype were normal.3.Cloned sheep keratin-binding protein Kap6,1promoter,Constructed the eukaryotic expression vector pKap-EGFP and identify the promoter activity by cells transfected. The transfection experiments showed that Kap can start EGFP expression in the fetal sheep skin fibroblasts.4.we connected sheep keratin-binding protein Kap6.1promoter with2S,than constructed hair follicles specific expression vector pK-2S using PEGFP-N1as original vector.5.After linearizing plasmid pK-2S,we transfected fine wool merino sheep fetal skin fibroblasts by lipofectamine.Then screened transgenic cell lins through600μg/mL G418.Through detecting part of positive clones by PCR of cell lines’whole genome,we got some positive clones.Then selected part of clones to carry out the RT-PCR,the results showed that the target gene had expressed at RNA level.