普氏立克次体14kD蛋白基因的扩增与克隆

AMPLIFICATION AND CLONING OF THE GENE ENCODING THE 14kD PROTEIN OF R.PROWAZEKII VIRULENT STRALN BREINL

根据普氏立克次体弱毒株－Ｅ株１４ｋＤ表面蛋白基因的ＤＮＡ序列设计合成了一对寡核苷酸引物，二引物的５＇端分别加上了限制性内切酶ＥｃｏＲＩ和ＨｉｎｄⅢ酶切位点。采用此引物对成功地扩增了普氏立克次体强毒株－－Ｂｒｅｉｎｌ株（国际标准株）的１４ｋＤ表面蛋白基因，基因的分子大小为０．７２ｋｂ。扩增获得的基因ＤＮＡ经限制性内切酶ＨｉｎｄⅢ和ＥｃｏＲＩ酶切后与经相同酶切的质粒载体ｐＵＣ１９连接，转化受体大肠杆菌工程菌ＪＭ１０３，经酶切和ＤＮＡ斑点杂交鉴定，成功地克隆了扩增的普氏立克次体强毒株（Ｂｒｅｉｎｌ株）的１４ｋＤ表面蛋白基因。

ccording to the DNA sequence of R.prowazekii avirulent strain E14kD protein gene,a pair of primers weredesigned,the restriction enzyme EcoR Ⅰ,Hind Ⅲ recognization sequences were added in to the 5'ends of the primers. The 14kD protein gene of R. prowazekii virulent strain Breinl was amplified by PCR with the primers,the size of the 14kD protein gene DNA was 0.72kb.The amplified 14kD protein gene DNA was digested with re-striction enzymes Hind Ⅲ and EcoR Ⅰ,then ligated to plasmid vector pUc19 which had been digested with the same restriction enzymes.The recombinant plasmid was transfered to the competent E.coli JM 103.Identified by enzyme digestion and DNA hybridization ,the gene encoding the 14kD protein of R.prowazekii virulent strainBreinl has been successfully cloned.