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目的:利用真核表达系统所获得的重组葡萄糖激酶蛋白建立葡萄糖激酶激活剂体外筛选方法。方法:将人源肝脏葡萄糖激酶基因插入到真核表达载体pPIC9K中,重组质粒经酶切和测序鉴定无误后转化毕赤酵母,用甲醇诱导表达。表达的蛋白进行SDS-PAGE鉴定,并对表达的蛋白进行活性测定,建立葡萄糖激酶激活剂体外筛选方法。利用该方法对大量化合物进行了筛选,并发现一先导化合物。结果:利用体外重组技术得到具有活性的葡萄糖激酶蛋白,并用于体外激活剂筛选方法的建立。结论:成功建立了葡萄糖激酶激活剂体外筛选方法。
Objective: To establish an in vitro screening method of glucokinase activator by using recombinant glucokinase protein obtained by eukaryotic expression system. Methods: Human liver glucokinase gene was inserted into eukaryotic expression vector pPIC9K. The recombinant plasmid was identified by restriction enzyme digestion and sequencing. The recombinant plasmid was transformed into Pichia pastoris and induced with methanol. The expressed protein was identified by SDS-PAGE, and the activity of the expressed protein was measured to establish glucokinase activator in vitro screening method. A large number of compounds were screened by this method and a lead compound was found. Results: The active glucokinase protein was obtained by in vitro recombination technique and used to establish the in vitro activator screening method. Conclusion: The in vitro screening of glucokinase activator has been successfully established.