中华皮肤科杂志 ›› 2010, Vol. 43 ›› Issue (6): 408-410.

• 论著 • 上一篇    下一篇

原代培养人毛乳头细胞cDNA酵母表达文库的构建和鉴定

宋志强1,孙丽华1,翟志芳2,郝飞3   

  1. 1. 第三军医大学西南医院皮肤科
    2. 重庆市第三军医大学西南医院皮肤科
    3. 重庆第三军医大学西南医院皮肤科
  • 收稿日期:2009-08-25 修回日期:2009-09-03 出版日期:2010-06-15 发布日期:2010-06-08
  • 通讯作者: 宋志强 E-mail:drsongzq@hotmail.com
  • 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

Construction and identification of yeast cDNA expression library of human dermal papillae cells in primary culture

  • Received:2009-08-25 Revised:2009-09-03 Online:2010-06-15 Published:2010-06-08
  • Contact: Zhiqiang Song E-mail:drsongzq@hotmail.com

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摘要：

目的 构建原代培养人毛乳头细胞cDNA酵母表达文库。方法 采用二步酶消化法分离正常人毛乳头细胞并置于DMEM培养基中培养；待细胞出现凝集性生长特性后，收集细胞并提取总RNA，采用CloneMinerTM cDNA Library Construction Kit试剂盒构建初级cDNA文库及cDNA酵母表达文库。结果 初级文库的平均滴度为7.0 × 106 cfu/ml，文库总容量为1.4 × 107 cfu（colony-forming unit）。cDNA酵母表达文库的平均滴度为5.5 × 106 cfu/ml，文库总容量为1.1 × 107 cfu；菌落PCR验证鉴定平均插入片段大于1.2 kb，重组率大于95%。结论 成功构建原代人毛乳头细胞cDNA酵母表达文库，为应用酵母双杂交筛选毛乳头细胞中与HSPC016相互作用的蛋白奠定基础。

关键词: 毛乳头细胞, 凝集性生长特性, 酵母双杂交, 文库

Abstract:

Objective To construct yeast cDNA expression library of human dermal papillae cells（DPCs） in primary culture. Methods Human dermal papilla cells （DPCs） were isolated by two-step digestion method and cultured in DMEM medium. Total RNA was extracted from primary DPCs that exhibited an aggregative behavior in culture, then, cDNA was synthesized and amplified by using CloneMinerTM cDNA Library Construction kit to construct primary cDNA library and yeast cDNA expression libary. Results The average titer and total clones were 7.0 × 106 colony forming units （cfu）/ml and 1.4 × 107 cfu respectively in the primary library, 5.5 × 106 cfu/ml and 1.1 × 107 cfu respectively in the yeast expression library. The average insert size was 1.2 kb and the recombination rate was above 95%. Conclusions The yeast cDNA expression library of DPCs in primary culture has been successfully constructed, which will lay a foundation for screening proteins interacting with HSPC016 gene in DPCs with yeast two-hybrid system.

Key words: dermal papilla cell, aggregative behavior, yeast two hybrid, cDNA library