贵宾犬α-干扰素基因的扩增、克隆与序列分析  

Amplification,Cloning and Sequencing of Interferon-α Gene from Poodle

作　　者：宋亚鹏[1] 陈红英[1] 崔保安[1] 贾艳艳[1] 郭显坡[1] 张蕾[1] 

机构地区：[1]河南农业大学牧医工程学院,河南郑州450002

出　　处：《江苏农业学报》2010年第2期339-343,共5页Jiangsu Journal of Agricultural Sciences

基　　金：国家农业科技成果转化基金项目(2007BAD07A06)

摘　　要：参照基因库中收录的犬α-干扰素基因序列设计1对引物,应用PCR技术直接从贵宾犬肝组织基因组DNA中扩增IFN-α基因,将回收得到的特异性片段克隆于pGEM-T Easy载体中,转化感受态细胞DH5α,经蓝白斑筛选,质粒PCR鉴定及酶切鉴定筛选阳性重组质粒,测定其核苷酸序列。测序结果表明,贵宾犬α-干扰素基因全长为564 bp,包含一个完整的开放阅读框,编码187个氨基酸的多肽,推导氨基酸序列有3个潜在的N-糖基化位点,有10个与二硫键形成有关的半胱氨酸。用DNAStar软件将所得序列与人和其他种动物的IFN-α基因进行核苷酸及氨基酸同源性比较,并通过绘制系统进化树可知,IFN-α基因存在种属的差异性,亲缘关系越近,同源性越高。One pair of primers was designed and synthesized according to the canine interferon-α（IFN-α） gene nucleic acid sequence published in the GenBank.The IFN-α gene was amplified from genome DNA of poodle liver by PCR.The purified PCR products were linked to pGEM-T Easy vector and then transformed into competent cells of DH5α.By identification of blue-white spot screening,plasmid PCR and enzyme digestion,positive strain was sent to TaKaRa for sequencing.The sequencing results showed that poodle IFN-α gene was obtained.The cloned poodle IFN-α gene span is 564 bp,which includes one open-reading frame encoding 187 amino acid residues.And there are three potential N-glycosalation sites and ten cysteines in the deduced amino acid sequence.Homology of nucleotide and amino aids of IFN-α gene between poodle and humans and other animals and the phylogenetic tree indicated that IFN-α gene varied in species,and the closer the relationship,the higher the homology.

关 键 词：犬 IFN-Α 扩增 基因克隆 序列分析 

分 类 号：S829.2[农业科学—畜牧学]