高温阴性细菌中的限制修饰现象及限制性内切酶TspAI的分离鉴定

RESTRICTION AND MODIFICATION IN A GRAM-NEGATIVE THERMOPHILIC BACTERIUM AND ISOLATION OF RESTRICTION ENDONUCLEASE TspA I

从对噬菌体P228有限制修饰作用的、带鞭毛的革兰氏阴性好热性细菌FD230中,分离出限制性内切酶TspAI.对λDNA,pBR322DNA和φX174RFDNA-HaeⅢ酶切片段进行酶切后,可以确认TspA 和EcoRⅡ是具有相同识别顺序的限制性内切酶,识别顺序均为CC_T^AGG.TspAⅠ易于分离纯化,热稳定性好,最适酶切温度为55～65℃。

A restriction endonuclease TspA I had been isolated from the Gram-negativeand flagellate thermophilic bacterium FD230. TspA I functions upon phage p228 interms of restriction and modification. By cleaving λDNA, pBR322DNA and φX174RFDNA-Hae III fragment, it had been identified as a restriction endonuclease posses-sing the same recognition sequence as that of EcoR II, i.e. the cleavage site isCC_T^AGG. TspA I could be easily isolated and purified. The enzyme was active over atemperature range of 30~80℃. Moreover, it was stable at 60℃ for as long as 30minutes.