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尹俊, 扈庭茂, 李金泉, 张春兰, 郭志成, 周欢敏. 2004: 内蒙古绒山羊毛囊兴盛期皮肤cDNA文库的构建及KAP6-2 cDNA的克隆. 动物学研究, 25(2): 166-171.
引用本文: 尹俊, 扈庭茂, 李金泉, 张春兰, 郭志成, 周欢敏. 2004: 内蒙古绒山羊毛囊兴盛期皮肤cDNA文库的构建及KAP6-2 cDNA的克隆. 动物学研究, 25(2): 166-171.
YIN Jun, HU Tin-mao, LI Jin-quan, ZHANG Chun-lan, GUO Zhi-zheng, ZHOU Huan-min. 2004. Construction of a Skin cDNA Library of Cashmere Goat and Cloning of KAP6-2 Fulllength cDNA. Zoological Research, 25(2): 166-171.
Citation: YIN Jun, HU Tin-mao, LI Jin-quan, ZHANG Chun-lan, GUO Zhi-zheng, ZHOU Huan-min. 2004. Construction of a Skin cDNA Library of Cashmere Goat and Cloning of KAP6-2 Fulllength cDNA. Zoological Research, 25(2): 166-171.

内蒙古绒山羊毛囊兴盛期皮肤cDNA文库的构建及KAP6-2 cDNA的克隆

Construction of a Skin cDNA Library of Cashmere Goat and Cloning of KAP6-2 Fulllength cDNA

  • 摘要: 用SMART技术构建了绒山羊(Capra hircus)毛囊兴盛期皮肤组织的cDNA质粒文库。Trizol Reagent(GIBCO/BRL)分离RNA后,用Oligotex(QIAGEN)提取mRNA;以锚定引物反转录合成cDNA第一链作为模板,以2个锚定引物,用长链PCR扩增全长cDNA双链。CHROMA SPIN-400去除小片段后用SfiⅠ酶切,连接到SfiⅠ消化的pBluescript Ⅱ SK(带有SfiⅠA和B两个位点)质粒载体中,转化 E.Coli 5α,库容量为1.8×105clones。随机挑选克隆提取质粒,5′测定插入片段的核苷酸序列,与GenBank数据库比对后发现1个KAP6-2 cDNA,序列号为AY316158。绒山羊KAP6-2由675个核苷酸组成,编码96个氨基酸,具有高甘氨酸/酪氨酸HGTps与富含半胱胺酸蛋白之间的中间特征。

     

    Abstract: In order to understand the molecular mechanism of the characteristic of cashmere,we constructed a cDNA library using the SMART cDNA library construction kit (Clontech).Total RNA was isolated from the goat (Capra hircus) skin tissue with hair follicle angen.Oligotex (QIAGEN) was used to isolate mRNA from total RNA.The "anchor firststrand cDNA" synthesized by reverse transcription with the SMART technique.The LD-PCR was performed using a modified oligo (dT) primer and an anchor primer as the primer set,and anchor first-strand cDNA as the template to enrich the cDNA population for full-length sequences.After digestion with SfiⅠand size fractionation,SMART cDNA was ligated into the SfiⅠ -digested pBluescript Ⅱ SK (with SfiⅠA and B site).The ligation mixture was transformed into E.Coli 5α.The cDNA library contained 1.8×105 independent clones.Randomly select cDNA clones and sequence with the 5 prime end,a full-length KAP6-2 cDNA was found by compared with those in the NCBI database (nr) using the Blast-N programs,it showed 75.5% identity in amino acids with mouse KAP6-2 and the accession number in GenBank is AY316158.

     

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