Frey J
Institute for Veterinary Bacteriology, University of Berne, Switzerland.
Res Microbiol. 1992 Mar-Apr;143(3):263-9. doi: 10.1016/0923-2508(92)90018-j.
We have constructed a pair of broad host range expression vectors, pJFF224-NX and pJFF224-XN, based on plasmid RSF1010, which enable cloning and efficient expression of genes in Actinobacillus pleuropneumoniae and Pasteurella haemolytica and in Escherichia coli. The vectors consist of the minimal autonomous replicon of the broad host range plasmid RSF1010 and a type II chloramphenicol acetyl transferase gene for chloramphenicol resistance selection. In addition, they contain a gene expression cassette based on the E. coli bacteriophage T4 gene 32 promoter region and a transcription stop signal, which are separated by a segment of multiple cloning sites in both orientations. Electroporation and subsequent selection for chloramphenicol resistance was used for the introduction of the vectors in A. pleuropneumoniae and P. haemolytica. A promoterless xy/E gene from the Pseudomonas putida TOL plasmid was cloned onto pJFF224-NX. This plasmid enabled efficient expression of active catechol2,3oxygenase in A. pleuropneumoniae and P. haemolytica. It was stably maintained in A. pleuropneumoniae without antibiotic selection, showing less than 0.1% loss after 100 generations, while native RSF1010 and other RSF1010-based vectors were unstable in this host.
我们基于质粒RSF1010构建了一对广宿主范围表达载体pJFF224-NX和pJFF224-XN，它们能够在胸膜肺炎放线杆菌、溶血巴斯德氏菌以及大肠杆菌中进行基因的克隆和高效表达。这些载体由广宿主范围质粒RSF1010的最小自主复制子和一个用于氯霉素抗性选择的II型氯霉素乙酰转移酶基因组成。此外，它们还包含一个基于大肠杆菌噬菌体T4基因32启动子区域和一个转录终止信号的基因表达盒，二者在两个方向上均由一段多克隆位点隔开。通过电穿孔法并随后选择氯霉素抗性，将这些载体导入胸膜肺炎放线杆菌和溶血巴斯德氏菌中。来自恶臭假单胞菌TOL质粒的无启动子xy/E基因被克隆到pJFF224-NX上。该质粒能够在胸膜肺炎放线杆菌和溶血巴斯德氏菌中高效表达活性儿茶酚2,3双加氧酶。它在胸膜肺炎放线杆菌中无需抗生素选择即可稳定维持，在100代后损失率低于0.1%，而天然RSF1010和其他基于RSF1010的载体在该宿主中则不稳定。
