牛α-s1酪蛋白基因3'端DNA的PCR扩增、克隆及鉴定

PCR Amplification,Cloning and Identification of the 3＇ End of the Bovine Alpha-s1 Casein Gene DNA

本研究以牛血总ＤＮＡ为模板，用ＰＣＲ技术扩增牛α－ｓ１酪蛋白基因的５＇端３．６ｋｂＤＮＡ片段和３＇端１．５ｋｂＤＮＡ片段的调控区序列，得到了相应的特异性扩增片段．用Ｋｌｅｎｏｗ处理后，将两个扩增片段分别与经Ｓｍａｌ酶切的ｐＵＣ１８质粒载体用Ｔ４ＤＮＡ连接酶连接，转化大肠杆菌ＪＭ１０９．在含有Ｘ－ｇａｌ，ＩＰＴＧ和Ａｍｐ的选择培养基上培养．从白色菌落中提取质粒ＤＮＡ，进行限制酶切鉴定．结果得到一个插入有１．３ｋｂＤＮＡ片段的阳性克隆．对其进行部分序列分析表明，该片段为５＇端缺失约１７０ｂｐ的牛α－ｓ１酪蛋白基因３＇端及下游区序列．

The 3. 6 kb of 5'-flanking and its upstream fragment and the 1. 5 kb of 3'-flanking and itsdownstream fragment of bovine α-s1 casein gene were obtained by PCR amplification from totalgenomic DNA of obovine blood. The PCR fragments were treated with the Klenow fragments toproduce flush double-stranded ends and then were inserted into pUC18 vectors digested withSmaI. The E. coli JM109 were transformed with the recombinant plasmids and incubated on LBmedium containing X-gal,IPTG and Amp. The plasmid DNAs isolated from the white colonieswere identified by digesting with restriction endonucleases. A positive clone containing 1. 3 kb ofinsert were obtained. Partial sequencing the 1. 3 kb of DNA demonstrated that the insert is the 3'-flanking DNA fragment lacking 170 hp from 5'end of the 1. 5 kb of PCR fragment of bovine α-s1casein gene.