STREPTOCOCCUS-ESCHERICHIA-COLI SHUTTLE VECTOR PSA3 AND ITS USE IN THE CLONING OF STREPTOCOCCAL GENES
STREPTOCOCCUS-ESCHERICHIA-COLI SHUTTLE VECTOR PSA3 AND ITS USE IN THE CLONING OF STREPTOCOCCAL GENES
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DOI：
10.1128/aem.49.1.115-119.1985
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发表时间：
1985-01-01
影响因子：
4.4
通讯作者：
FERRETTI, JJ
中科院分区：
文献类型：
--
作者：
DAO, ML;FERRETTI, JJ
一种可在链球菌中复制的穿梭载体。而大肠杆菌pACYC184(氯霉素和四环素抗性)与链球菌pGB305(红霉素抗性)连接而成。该嵌合质粒命名为pSA3(氯霉素、红霉素和四环素抗性)，具有7个独特的限制性内切酶切点：EcoRI、EcoRV、BamHI、SalI、XbaI、NruI和SphI。克隆到EcoRI或EcoRV位点导致氯霉素耐药性失活，克隆到BamHI、SalI或SphI位点导致四环素耐药性失活。在红霉素存在下，pSA3在血链球菌和变形链球菌中转化并稳定存在。利用pSA3构建了变形链球菌GS5基因组文库，并用变形链球菌抗血清证实了该异源宿主表面抗原的表达。将先前克隆的链激酶决定簇亚克隆到pSA3中，该重组载体在选择性压力下稳定存在，并在大肠杆菌、血链球菌和变形链球菌中表达了链激酶活性。
A shuttle vector that can replicate in Streptococcus spp. and E. coli was 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 7 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 S. sanguis and S. mutans in the presence of erythromycin. One used pSA3 to construct a library of the S. mutans GS5 genome in E. coli and expression of surface antigens in this heterologous host was 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.