Application of Ewing sarcoma (EWS) or up-regulator thereof in preparing medicament for treating diabetes and preventing and treating tumorigenesis of diabetic individuals
A technology for diabetes, diabetic blood vessels, applied in the field of biomedicine
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[0081] 1. Method
[0082] 1. Viral vector construction for overexpressing EWS (named: E-OE) or Fbxw7 (named: F-OE) protein
[0083](1) Amplify the nucleotide sequences of EWS and Fbxw7 by PCR reaction (the sequence of EWS is shown in SEQ ID NO: 1, and the sequence of Fbxw7 is shown in SEQ ID NO: 2), and the two ends are connected with EcoRI and BamHI restriction sites point;
[0084] (2) double restriction digestion of PCR products and purification;
[0085] (3) Also double digest the H201pAdeno-MCMV-EGFP-3FLAG vector plasmid (Shanghai Heyuan Biotechnology Co., Ltd.), recover and purify the large fragment, and insert the EWS or Fbxw7 gene fragment in step (2) using molecular cloning technology instead of EGFP to the EcoRI and BamHI restriction sites of the shuttle plasmid;
[0086] (4) Colony PCR screens transformants, and the screened positive clones are sequenced and verified;
[0087] (5) Sequencing verifies the correct clones, and extracts high-purity plasmids;
[008...
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