摘要
采用原核表达载体pet-32-a+构建了羊驼皮肤组织cDNA文库。双链cDNA采用Universal Ribo Clonec DNA Synthesis System合成,并经过琼脂糖分级胶回收,去除了小于400 bp的片断;通过酶切载体的去磷酸化和插入片断的磷酸化,提高了重组率,蓝白斑筛选结果全部为阳性;库扩增后检测容量达2.25×107。随机挑取阳性克隆进行单引物测序分析,七个测序样品中获得两个与毛发生长相关的EST(expression sequence tag),分别为S100钙离子结合蛋白A3基因(S100 calcium binding protein A3,S100A3)的部分序列和高硫角蛋白关联蛋白基因(high sulfur keratin-associated protein,KRTAP3-3)的部分序列,说明所构建的文库具有较高的代表性。
A cDNA library from the skin of alpaca in china was constructed using the prokaryotic expression vector pet- 32-a^＋. All the cDNA fragments are normalized by 1% agarose gel, and the fragments below 400 bp were discarded. The digested vector was processed by dephosphorylase and the cDNA fragments were processed by phosphorylase. The positive recombination rate was 100 % examined by blue/white colony. The library capacity was 2.25 × 10^7. Randomly selected 7 positive clones were sequenced by Shaihai Sangon bio-engineering company using T7 terminator primer. Two ESTs related to the wool growth were obtained, which are S100 calcium binding A3 gene and high sulfur keratin-associated protein gene.
出处
《激光生物学报》
CAS
CSCD
2007年第2期222-225,共4页
Acta Laser Biology Sinica
基金
国家自然科科学基金资助(30571070)
