用酵母双杂交技术筛选与HSPC016相互作用的蛋白

中华皮肤科杂志 ›› 2011, Vol. 44 ›› Issue (1): 44-46.

用酵母双杂交技术筛选与HSPC016相互作用的蛋白

宋志强¹,孙丽华²,郝飞³

1. 第三军医大学西南医院皮肤科
2. 第三军医大学第一附属医院西南医院
3. 重庆第三军医大学西南医院皮肤科

收稿日期:2010-02-25 修回日期:2010-08-20 出版日期:2011-01-15 发布日期:2011-01-10
通讯作者: 郝飞 E-mail:haofei62@medmail.com.cn
基金资助:
HSPC016蛋白调控毛乳头细胞生长和功能的分子机制研究

Screening of proteins interacting with HSPC016 by yeast two-hybridization technique

Zhiqiang Song,孙丽华 SUN Li-Hua,Hao Fei

Received:2010-02-25 Revised:2010-08-20 Online:2011-01-15 Published:2011-01-10
Contact: Hao Fei E-mail:haofei62@medmail.com.cn

摘要/Abstract

摘要： 目的采用酵母双杂交技术筛选毛乳头细胞中与造血干细胞（hematopoietic stem/progenitor cells, HSPC）分化相关基因HSPC016相互作用的蛋白，了解其参与调控毛乳头细胞凝集性生长的分子机制。方法应用酵母双杂交系统，将构建的pGBKT7-HSPC016诱饵质粒与含原代人毛乳头细胞cDNA文库质粒的酵母Y187 进行配合，筛选与HSPC016相互作用的蛋白，通过回复性杂交试验验证其可靠性，并对阳性克隆进行测序和生物信息学分析。结果筛选出4个与HSPC016有相互作用的蛋白，包括转录因子叉头框蛋白O1（forkhead box O1，FOXO1）、丝裂原活化蛋白激酶11、磷酸次黄苷酸激酶3调节亚单位3（PIK3R3）、肝X受体，它们均与细胞内能量代谢、转录调节相关。结论 HSPC016可能通过参与细胞内活性氧水平调节，并与细胞内参与能量和转录调节的相关信号分子相互作用，调节毛乳头细胞的凝集性生长状态。

关键词: 酵母双杂交

Abstract: Objective To screen and identify proteins interacting with hematopoietic stem/progenitor cell differentiation-related gene HSPC016, and to explore the molecular mechanism involved in the regulation by HSPC016 on the aggregative behavior of dermal papilla cells. Methods By using yeast two-hybridization, HSPC016 gene was sub-cloned into pGBKT7 to construct the bait plasmid （named as pGBKT7-HSPC016） in yeast AH109. The cDNA yeast expression library of human dermal papillae cells in yeast Y187 was screened with the bait plasmid and the proteins interacting with HSPC016 were identified. Yeast two-hybridization retransformation experiment was conducted to exclude the false positive clones and verify the interactions, then, the positive clones were sequenced and analyzed by using bioinformatic methods. Results The bait plasmid pGBKT7-HSPC016 was constructed successfully and there was no self-activation in or toxicity against yeast AH109. Four proteins，including forkhead family of transcription factors （FOXO1）, mitogen-activated protein kinase 11 （MAPK 11）, phosphoinositide-3-kinase （PIK3R3） and liver X receptor were screened and identified. Bioinformatic analysis revealed that these proteins had close relationship with intracellular energy metabolism and translational regulation. Conclusions HSPC016 may regulate the aggregative behavior of DPCs by regulating the levels of intracellular reactive oxygen species （ROS） and interacting with signaling molecules involved in intracellular energy metabolism and translational regulation.

Key words: Yeast two-hybrid

宋志强孙丽华郝飞. 用酵母双杂交技术筛选与HSPC016相互作用的蛋白[J]. 中华皮肤科杂志, 2011, 44(1): 44-46.

Zhiqiang Song 孙丽华 SUN Li-Hua Hao Fei. Screening of proteins interacting with HSPC016 by yeast two-hybridization technique[J]. Chinese Journal of Dermatology, 2011, 44(1): 44-46.

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