小鼠STAT4和STAT6基因酵母双杂交表达载体的构建与鉴定

Construction and identification of yeast expression plasmids containing mouse signal transducers and activators of transcription-4/6

目的:分别克隆小鼠信号传导和转录活化因子-4(STAT4)、信号传导和转录活化因子-6(STAT6)基因编码序列(CDS序列),构建并鉴定其酵母表达质粒pGADT7-STAT4和pGADT7-STAT6。方法:PCR扩增STAT4和STAT6基因CDS序列,T-A亚克隆到pMD19-T simple载体中,酶切获取目的基因STAT4和STAT6,克隆入已经过双酶切和CIAP脱磷酸处理的酵母表达载体pGADT7,酶切鉴定并测序;转化pGADT7-STAT4和pGADT7-STAT6到酵母AH109细胞中,Western blot分析STAT4和STAT6的蛋白表达情况,同时检测其毒性和自激活作用。结果:成功扩增了STAT4和STAT6基因CDS序列,并分别成功克隆到pMD19-T simple载体和pGADT7中,测序结果符合要求。酵母表达质粒pGADT7-STAT4和pGADT7-STAT6成功转化到酵母AH109细胞中,无毒性和自激活作用,Western blot结果证实酵母细胞高表达融合蛋白STAT4和STAT6。结论:成功构建了酵母表达质粒pGADT7-STAT4和pGADT7-STAT6,为进一步研究STAT4和STAT6蛋白与其他蛋白质的相互作用奠定了基础。

AIM：To construct yeast expression plasmids containing mouse STAT4/6 gene for further study of their interaction with other proteins in yeast two-hybrid system.METHODS：Mouse STAT4/6 genes were amplified by PCR and T-A was cloned with pMD19-T simple vector and then was cloned into yeast expression vector pGADT7 cut with incision enzymes and treated with CIAP.The yeast expression vector pGADT7 was identified by enzyme cutting and sequencing.The yeast expression plasmids pGADT7-STAT4/6 were transformed into AH109 yeast cells and the expression of the STAT4/6 fusion proteins was detected by Western blot.Their toxicity and self-activation were also detected.RESULTS：Mouse STAT4/6 genes were successfully amplified and cloned into pMD19-T simple vector and pGADT7.Sequencing analysis revealed that both plasmids met the design of the study.The yeast expression plasmids pGADT7-STAT4/6 were successfully transformed into AH109 yeast cells,without toxicity or self-activation.The expression of STAT4/6 fusion proteins was confirmed by Western blot.CONCLUSION：The yeast expression plasmids pGADT7-STAT4/6 are successfully constructed and can be applied in the detection of their interaction with other proteins.