Dao M L, Ferretti J J
Appl Environ Microbiol. 1985 Jan;49(1):115-9. doi: 10.1128/aem.49.1.115-119.1985.
A shuttle vector that can replicate in both Streptococcus spp. and Escherichia coli has been constructed by joining the E. coli plasmid pACYC184 (chloramphenicol and tetracycline resistance) to the streptococcal plasmid pGB305 (erythromycin resistance). The resulting chimeric plasmid is designated pSA3 (chloramphenicol, erythromycin, and tetracycline resistance) and has seven unique restriction sites: EcoRI, EcoRV, BamHI, SalI, XbaI, NruI, and SphI. Molecular cloning into the EcoRI or EcoRV site results in inactivation of chloramphenicol resistance, and cloning into the BamHI, SalI, or SphI site results in inactivation of tetracycline resistance in E. coli. pSA3 was transformed and was stable in Streptococcus sanguis and Streptococcus mutans in the presence of erythromycin. We have used pSA3 to construct a library of the S. mutans GS5 genome in E. coli, and expression of surface antigens in this heterologous host has been confirmed with S. mutans antiserum. A previously cloned determinant that specifies streptokinase was subcloned into pSA3, and this recombinant plasmid was stable in the presence of a selective pressure and expressed streptokinase activity in E. coli, S. sanguis (Challis), and S. mutans.
通过将大肠杆菌质粒pACYC184（氯霉素和四环素抗性）与链球菌质粒pGB305（红霉素抗性）连接，构建了一种可在链球菌属和大肠杆菌中复制的穿梭载体。所得的嵌合质粒命名为pSA3（氯霉素、红霉素和四环素抗性），具有七个独特的限制性酶切位点：EcoRI、EcoRV、BamHI、SalI、XbaI、NruI和SphI。克隆到EcoRI或EcoRV位点会导致氯霉素抗性失活，而克隆到BamHI、SalI或SphI位点会导致大肠杆菌中的四环素抗性失活。pSA3被转化后，在红霉素存在的情况下，在血链球菌和变形链球菌中稳定存在。我们已使用pSA3在大肠杆菌中构建了变形链球菌GS5基因组文库，并且用变形链球菌抗血清证实了该异源宿主中表面抗原的表达。一个先前克隆的指定链激酶的决定簇被亚克隆到pSA3中，该重组质粒在选择性压力存在的情况下稳定，并在大肠杆菌、血链球菌（Challis）和变形链球菌中表达链激酶活性。
