Burioni R, Plaisant P, Riccio M L, Rossolini G M, Satta G
Istituto di Microbiologia, Università Cattolica del S. Cuore, Rome, Italy.
New Microbiol. 1995 Apr;18(2):201-6.
A new plasmid cloning vector for Escherichia coli (pPhoR) suitable for direct detection of recombinant clones has been constructed. The plasmid is a multicopy of a vector which carries a pUC-derived origin of replication and beta-lactamase gene, and the phoN acid phosphatase-encoding gene from Providencia stuartii. Foreign DNA fragments can be cloned into unique restriction sites located within the phoN gene causing a loss of the acid phosphatase activity which is normally overproduced by E. coli strains carrying pPhoR. Since PhoN production can be easily detected by a plate histochemical assay, recombinant clones carrying foreign DNA fragments inserted in the phoN gene can be easily detected as PhoN-negative clolonies on the above medium. The efficiency of the pPhoR-based cloning system for direct cloning of PCR amplimers of a variable region of the HIV-1 genome was comparable to that of conventional cloning systems for direct detection of recombinant based on beta-galactosidase inactivation. Advantage of the pPho-R-based system include reduced costs for histochemical assays and the possibility of being used with any E. coli host.
构建了一种适用于直接检测重组克隆的新型大肠杆菌质粒克隆载体（pPhoR）。该质粒是一种载体的多拷贝形式，其携带源自pUC的复制起点和β-内酰胺酶基因，以及来自斯氏普罗威登斯菌的phoN酸性磷酸酶编码基因。外源DNA片段可克隆到位于phoN基因内的独特限制性酶切位点，导致酸性磷酸酶活性丧失，而携带pPhoR的大肠杆菌菌株通常会过量产生这种活性。由于通过平板组织化学检测可以轻松检测到PhoN的产生，因此在上述培养基上，携带插入phoN基因的外源DNA片段的重组克隆可以很容易地被检测为PhoN阴性菌落。基于pPhoR的克隆系统直接克隆HIV-1基因组可变区PCR扩增子的效率与基于β-半乳糖苷酶失活直接检测重组体的传统克隆系统相当。基于pPho-R的系统的优点包括组织化学检测成本降低，以及可与任何大肠杆菌宿主一起使用的可能性。
