Dmitriev A V, Smola J, Shalen K, Golubkov V I, Totolian A A
Zh Mikrobiol Epidemiol Immunobiol. 1994 Jul-Aug(4):24-8.
In a number of consecutive gene engineering operations a DNA fragment having a size of about 2.8 kb was cloned in Escherichia coli by means of Blue-script II SK+ used as vector. The insert contained pld gene coding the synthesis of phospholipase D, one of the key factors of C. pseudotuberculosis virulence. The stable and active expression of this gene in E.coli was achieved. High phospholipase A activity was accumulated in the periplasmatic space. The molecular weight of the synthesized protein was 31 kD. The product obtained by gene engineering methods was found to possess the biological activity of the natural product: it induced the hemolysis of sheep red blood cells in the presence of equi factor of Rhinococcus equi and inhibited the hemolytic activity of Staphylococcus aureus beta-hemolysin (phospholipase C). The pld gene cloned in these experiments differed from that of another C.pseudotuberculosis strain. Further research is underway with a view of searching for the limits of pls gene.
在一系列连续的基因工程操作中，通过使用作为载体的蓝白斑载体II SK+，将大小约为2.8 kb的DNA片段克隆到大肠杆菌中。插入片段包含编码磷脂酶D合成的pld基因，磷脂酶D是伪结核棒状杆菌毒力的关键因素之一。该基因在大肠杆菌中实现了稳定且活跃的表达。高磷脂酶A活性在周质空间中积累。合成蛋白的分子量为31 kD。通过基因工程方法获得的产物被发现具有天然产物的生物活性：在存在马红球菌等因子的情况下，它诱导绵羊红细胞溶血，并抑制金黄色葡萄球菌β-溶血素（磷脂酶C）的溶血活性。在这些实验中克隆的pld基因与另一株伪结核棒状杆菌的基因不同。目前正在进行进一步的研究，以寻找pls基因的界限。
