从藤黄微球菌中进行复苏促进因子（Rpf）基因的PCR克隆、测序及在大肠杆菌中的表达
PCR cloning of the resuscitation-promoting factor (Rpf) gene from Micrococcus luteus, sequencing and expression in Escherichia coli.
作者信息
Matsuda M, Togo M, Kagawa S, Moore J E
机构信息
Laboratory of Molecular Biology, Graduate School of Environmental Health Sciences, Azabu University, Sagamihara, Japan.
出版信息
Microbios. 2001;104(407):55-61.
A polymerase chain reaction (PCR) cloning procedure was developed for the resuscitation-promoting factor (Rpf) gene of Micrococcus luteus using strains NCIMB 13267, JCM 1464T, JCM 3347, and JCM 3348. A PCR product of the Rpf gene fragment was ligated into a cloning vector pBluescript II KS (+) with the restriction endonucleases Eco RI and Bam HI. The ligation mixture was used to transform Escherichia coli DH5alpha. The DNA sequence of the Rpf gene cloned from strain JCM 1464T was 84% homologous with that of NCIMB 13267, and from strains JCM 3347 and JCM 3348 it was 100% and 86% homologous, respectively. Recombinant Rpf proteins of M. luteus NCIMB 13267 and JCM 1464T after expression in E. coli BL21 harbouring the pET-19b-Rpf plasmid, and after purification, were approximately 16 kD for both strains.
利用NCIMB 13267、JCM 1464T、JCM 3347和JCM 3348菌株，开发了一种用于藤黄微球菌复苏促进因子（Rpf）基因的聚合酶链反应（PCR）克隆程序。Rpf基因片段的PCR产物用限制性内切酶Eco RI和Bam HI连接到克隆载体pBluescript II KS（+）中。连接混合物用于转化大肠杆菌DH5α。从JCM 1464T菌株克隆的Rpf基因的DNA序列与NCIMB 13267的DNA序列同源性为84%，从JCM 3347和JCM 3348菌株克隆的Rpf基因的DNA序列同源性分别为100%和86%。藤黄微球菌NCIMB 13267和JCM 1464T的重组Rpf蛋白在含有pET-19b-Rpf质粒的大肠杆菌BL21中表达并纯化后，两种菌株的重组蛋白大小均约为16 kD。
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