基因重组人葡萄糖激酶及其激活剂体外筛选方法的建立

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,并对表达的蛋门进行活性测定,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.

A method in vitro for discovering glucokinase activator

Objective:Establishment of glucokinase activator screening method using heterologous expression of eukaryon system.Methods:The pPIC9K vector was inserted a fragment of human liver glucokinase.The plasmid of pPIC9KGK was identified by restriction enzyme and sequencing.Then the plasmid was transfected to pichia pastoris via methanol induction.They produced the human liver recombinant GK protein,which was further detected by SDS-PAGE.The recombinant GK protein had enzymatic activity and was used in GK activator screening method.Using this method,lots of compounds to find GK activators were screened and a lead-compound LGK-01 was found.Results:Human liver recombinant GK protein was expressed in pichia pastoris,and the protein had enzymatic activity.Conclusion:GK activator screening method was established successfully in vitro.