牛β-酪蛋白基因上游调控序列PCR扩增及克隆  

PCR Amplifing and Cloning of Upstream Regulative Sequence of the Bovine βCasein Gene

作　　者：刘松财[1] 张玉静[1] 欧阳红生[1] 张永亮[1] 刘万臣[1] 黎诚耀[1] 赵建军[1] 金扩世[1] 汪玉松[1] 

机构地区：[1]解放军农牧大学

出　　处：《中国兽医学报》1998年第1期42-44,共3页Chinese Journal of Veterinary Science

基　　金：军队医药卫生青年基金

摘　　要：采用蛋白酶Ｋ法从母牛肝中提取染色体ＤＮＡ，将其纯化后作为ＰＣＲ扩增模板。以引物设计软件从牛β－酪蛋白基因上游６００ｂｐ至第１个外显子设计引物，引物长度１９ｎｔ，跨度６３７ｂｐ。扩增产物电泳结果显示，在分子量Ｍａｒｋｅｒ６５７ｂｐ带附近，出现一明显亮带，这一结果与设计产物大小基本一致。用低温冷冻法纯化ＰＣＲ产物，煮沸裂解法提取载体ｐＢｌｕｅｓｃｒｉｐｔⅡＫｓ＋质粒，ＥｃｏＲＶ酶切质粒ＤＮＡ，Ｔ４ＤＮＡ连接酶连接ＰＣＲ扩增片段和质粒ＤＮＡ。将重组质粒ＤＮＡ转化到ＪＭ１０１大肠杆菌中，经筛选、酶切、测序鉴定，结果表明所克隆的片段为牛β－酪蛋白基因上游调控序列。The bovine chromosome DNA was extracted from bovine liver by proteinase K method. The DNA was used as template of PCR. The PCR primers were designed between exon Ⅰ and 5′ upstream sequence 600 bp of bovine βcasein gene. The PCR products were electrophoresed and a band similar to 657 bp band of the molecular weight Marker was observed under ultraviolet light. The pBluescript Ⅱ Ks+ plasmids were extracted by boiling method and then cut by Eco RV. The PCR products and prepared plasmids were linked by T4 DNA ligase. Through transferring, screening, enzymolysis and sequencing, it was demonstrated that the partly regulative sequence of 5′ upstream of bovine βcasein gene has gotten.

关 键 词：Β-酪蛋白 调控序列 PCR 克隆 牛 

分 类 号：S852.2[农业科学—基础兽医学]