pEGFP-N2-CASK表达质粒的构建及鉴定

Objective To construct the expression plasmid of rat CASK protein. Methods Designed a pair of parimers used to amplify the coding sequence of the rat CASK, the cDNA was reverse transcriptased form total RNA extracted from the rat pancreatic islet beta cells Ins-1 as a template, after amplification the target gene fragment was inserted into the vector of pEGFP-N2, and transfected with competent cells DH5α, the positive clones were identified by agar plate containing kanamycin, and the recombinant plasmids were extracted and confirmed by complete sequencing. Transfect the recombinant plasmids confirmed by sequencing that the inserted fragment and design sequence is exactly the same into INS-1 cells，and identify if the expression plasmid is successful by western blot. Results Recombinant plasmid by single enzyme digestion and sequencing confirmed the insertion sequence is exactly the same, fusion protein pEGFP-N2-CASK was successfully expressed in INS-1 cells. Conclusion The expression plasmid pEGFP-N2-CASK of rat CASK protein is successfully constructed.