人苯丙氨酸羟化酶基因克隆及其原核表达质粒的构建

 [目的]克隆健康人苯丙氨酸羟化酶基因全长cDNA序列,构建并鉴定其原核表达质粒pTrcHisB/PAH。[方法]采用RT-PCR方法从人肝组织中扩增PAH基因全长cDNA,T/A克隆到pMD18-T载体中,双酶切后将cDA亚克隆入pTrcHisB载体,构建pTrcHisB/PAH质粒,经限制性内切酶鉴定并测序。[结果]人PAH基因cDNA正确克隆入pTrcHisB表达载体,与Genebank中PAH基因序列一致。[结论]成功构建pTrcHisB/PAH表达质粒,为进一步进行研究PAH蛋白表达和重组蛋白活性鉴定奠定基础。

Cloning of Human Phenylalanine Hydroxylase Gene and Construction of Prokaryotic Expression Plasmid

Objective To clone human phenylalanine hydroxylase (PAH) gene and construct a prokaryotic expression plasmid. Methods The cDNA of human PAH Gene amplified with RT-PCR from health human liver tissue was cloned into pMD18-T vector by T/A Ligation. After double digested,PAH cDNA fragment was subcloned into pTrcHisB vector to construct a prokaryotic expression plasmid. Restriction endonucleases analysis and sequencing were used to conform the recombinant plasmid. Results The full length PAH cDNA was correctly inserted into prokaryotic expression vector,and its sequencing was consistent with reported sequence derived from Genebank. Conclusion The successful construction of prokaryotic expression plasmid provides a basis for further studies on the expression,recombined protein biological activities.