摘要
X-box 结合蛋白 1 是一种重要的转录因子,参与体内多项信号转导过程. 为进一步研究 XBP1 的生物学功能,运用酵母双杂交技术在肝细胞文库中筛选 XBP1 的结合蛋白. 首先运用 PCR 技术扩增获得 XBP1 的编码序列,克隆至 pGEM-T 载体,经测序鉴定后,亚克隆至诱饵载体 pGBKT7 中,转化酵母 AH109(a type). 免疫印迹检测诱饵质粒 pGBKT7-XBP1 在AH109 酵母中的表达之后,含有诱饵质粒的酵母 AH109 与含有肝细胞 cDNA 文库质粒 pACT2 的酵母 Y187(αtype)配合,配合后的二倍体酵母生长在含有 X-α-gal 的营养缺陷型培养基上 (SD/-Trp-Leu-His-Ade) 进行选择和筛选,经测序和序列比对确定阳性克隆的开放读码框 ORF,得到 7 种不同的蛋白质. 为了进一步验证这些筛选蛋白质与 XBP1 的相互作用,克隆其中一种蛋白质 MT1E,并运用 GST pulldown 和免疫共沉淀技术成功检测了 MT1E 和 XBP1 的相互作用(体外 / 体内),结果提示,MT1E 可能是 XBP1 的一个新的调节蛋白. 通过酵母双杂交技术筛选得到的 7 种蛋白质分别与肝细胞基础代谢、蛋白质的合成与运输、细胞的增殖与凋亡密切相关. 上述结果有助于揭示 XBP1 的生物学功能,为进一步探讨 XBP1 的表达和调控机制提供新线索.
X-box binding proteinl （XBP1） is an important transcription factor, which participates in many signal transduction procession.To investigate the biological function of XBP1, yeast two-hybrid system to screen proteins interacting with XBP1 in hepatocytes was performed. The XBP1 coding sequence was amplified by polymerase chain reaction （PCR） method, and was cloned in pGEM-T vector. After the target region was sequenced, it was subcloned into the bait plasmid pGBKT7, then was transformed into yeast AH109（a type）. After the expression of bait plasmid pGBKT7-XBP1 in AH109 yeast strains were proved by Western blot. The transformed yeast AH109 was mated with yeast Y187（α type） containing hepatocyte cDNA library plasmids pACT2 in 2 xYPDA medium. Diploid yeast was plated on synthetic dropout nutrient medilium （SD/-Trp-Leu-His-Ade） containing X-α-gal for selection and screening. After sequencing and verifying ORF of positive colonies,7 different kinds of proteins were obtained.In order to further testify the interaction between the screened proteins and XBP1, one of positive colonies MT1E was cloned.The interaction between MTIE and XBP1 in vitro/in vivo were examined successfully with GST pulldown and coimmunoprecipitations.lt was shown that MTIE would be a new regulatory protein of XBP1. These screened proteins by yeast two-hybird system were closely correlated with liver fundamental metabolism,protein synthesis and transport, cell proliferation and apoptosis. The results mentioned above contributed to reveal the XBP1 biological function, and brought some new clues for further exploration of the expressing and regulating mechanism of XBP1.
出处
《生物化学与生物物理进展》
SCIE
CAS
CSCD
北大核心
2006年第8期745-753,共9页
Progress In Biochemistry and Biophysics
基金
国家自然科学基金资助项目(30371479).~~
关键词
X-box结合蛋白1
酵母双杂交
转录因子
诱饵
X-box binding protein 1, yeast two-hybrid system, transcription factor, bait
