小鼠STAT4、STAT6基因克隆及其真核表达质粒的构建

Cloning of mouse signal transducers and activators of transcription-4/6 and construction of eukaryotic expression plasimid

目的:分别克隆小鼠信号传导及转录活化因子-4(Signal transducers and activators of transcription-4,STAT4)、信号传导及转录活化因子-6(Signal transducers and activators of transcription-6,STAT6)基因全长cDNA序列,构建并鉴定其真核表达质粒pIRES2-EGFP-STAT4/STAT6。方法:采用RT-PCR方法从BALB/c小鼠脾脏组织中扩增STAT4/STAT6基因全长cDNA,T/A克隆到pMD19-T载体中,双酶切后将cDNA亚克隆入pIRES2-EGFP载体,构建pIRES2-EGFP-STAT4/STAT6质粒,经限制性内切酶鉴定并测序。结果:小鼠STAT4/STAT6基因cDNA正确克隆入pIRES2-EGFP表达载体,与Genebank中STAT4/STAT6基因序列一致。结论:成功构建pIRES2-EGFP-STAT4/STAT6表达质粒,为进一步研究STAT4/STAT6蛋白表达和相互作用奠定了基础。

Objective：To clone mouse signal transducers and activators of transcription（STAT） 4 and STAT6 gene and construct a eukaryotic expression plasimid. Methods：The cDNA of mouse STAT4 and STAT6 gene amplified with RT-PCR from normal mouse spleen tissue were cloned into pMD19-T vector by T/A ligation. After double digested,STAT4/STAT6 cDNA fragment was suhcloned into pIRES2-EGFP vector to construct a eukaryotic expression plasimid. Restriction endonucleases analysis and sequencing were used to comform the recomhinant plasimid. Results：The full length STAT4/STAT6 cDNA was correctly inserted into eukaryotic expression plasimid,and its sequencing was consistent with reported sequence derived from Genebank. Conclusion：The successful construction of eukaryotic expression plasimid provides a basis for futher studies on the infection of STAT4/STAT6 interaction to the downstream cytokines.