口腔链球菌丙酮酸氧化酶基因缺陷型变异株的构建

Construction of the Pyruvate Oxidase Gene Deficiency Variant Strain of Streptococcus oralis

目的 :利用口腔链球菌丙酮酸氧化酶基因 (sopox)的克隆序列 ,重组构建新的不能产生H2 O2 的口腔链球菌变异株。方法 :口腔链球菌株ATCC10 5 5 7经培养后用酚—氯仿法抽提细菌染色体基因组DNA ,经PCR扩增sopox基因 ,用BamHI进行限制酶切 ;参照Chris方法进行电转化 ,挑选阳性菌落测定其上清液中H2 O2 的含量 ;将细菌传 3～4代后再次重复上述检测。结果 :转化子经筛选后得到 1株阳性菌落 ,测定上清液中H2 O2 含量 ,第 1次检测表明变异株产生H2 O2 的量仅有所下降 (介于阳性对照ATCC 10 5 5 7和阴性对照大肠杆菌JM10 9之间 ) ,经 3～ 4次传代后变异株中上清液H2 O2 量已经明显低于阴性对照。结论

Objective: The purpose of this study is to construct a pyruvate oxidase gene deficiency variant strain of Streptococcus oralis (S. oralis).Methods: The sopox gene, which was got using polymerase chain reaction (PCR), and the 130_basepair segment of which was cut down with endonuclease BamHI, and transferred into S. oralis (ATCC10557) by using electrotransformation. The authors obtained a variant strain of S. oralis, and then the catalase activity of the first culture and 3_4 subcultures was examined.Results: The authors obtained a pyruvate oxidase gene deficiency variant strain of S. orlis. The catalase activity examination showed that the ability of producing H 2O 2 of the variant strain of S. orlis declined , whose catalase activity was between those of the positive control (ATCC10557) and the negative control (Escherichia coli, JM109). But the produced H 2O 2 quantity of their subcultures was less than that of the negative control.Conclusion:The construction of the pyruvate oxidase gene deficiency variant strain of Streptococcus oralis is successful.