原代培养人毛乳头细胞cDNA酵母表达文库的构建和鉴定

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

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CloneMiner^TM 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7.0×10^6 cfu/ml,文库总容量为1.4×10^7 cfu（colony-forming unit）.cDNA酵母表达文库的平均滴度为5.5×10^6 cfu/ml,文库总容量为1.1×10^7 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.

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 CloneMiner^TM cDNA Library Construction kit to construct primary cDNA library and yeast cDNA expression libary.Results The average titer and total clones were 7.0×10^6 colony forming units（cfu）/ml and 1.4×10^7 cfu respectively in the primary library,5.5×10^6 cfu/ml and 1.1×10^7 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.