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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。
OBJECTIVE: To clone the CDS sequences of signal transducer and activator of transcription-4 (STAT4) and signal transducers and activators of transcription-6 (STAT6) in mice and to construct and identify the yeast expression plasmids pGADT7-STAT4 and pGADT7- STAT6. Methods: The CDS sequences of STAT4 and STAT6 genes were amplified by PCR and subcloned into pMD19-T simple vector. The target genes STAT4 and STAT6 were obtained by restriction enzyme digestion and cloned into the yeast expression vector pGADT7 which had been digested by double digestion and CIAP dephosphorylation. The pGADT7-STAT4 and pGADT7-STAT6 were transformed into yeast AH109 cells. The protein expression of STAT4 and STAT6 were analyzed by Western blot. The cytotoxicity and self-activation of STAT4 and STAT6 were detected. Results: CDS sequences of STAT4 and STAT6 genes were successfully amplified and successfully cloned into pMD19-T simple vector and pGADT7 respectively. The sequencing results were satisfactory. The yeast expression plasmids pGADT7-STAT4 and pGADT7-STAT6 were successfully transformed into yeast AH109 cells without toxicity and self-activation. Western blot results confirmed that the yeast cells overexpressed the STAT4 and STAT6 fusion proteins. Conclusion: The yeast expression plasmids pGADT7-STAT4 and pGADT7-STAT6 were constructed successfully, which laid the foundation for further study of the interaction between STAT4 and STAT6 proteins and other proteins.