Crasta Karen C, Chua Kim-Lee, Subramaniam Sumathi, Frey Joachim, Loh Hilda, Tan Hai-Meng
Institute of Molecular Agrobiology, National University of Singapore, Singapore.
J Bacteriol. 2002 Apr;184(7):1932-9. doi: 10.1128/JB.184.7.1932-1939.2002.
Riemerella anatipestifer is responsible for exudative septicemia in ducks. The genetic determinant of the CAMP cohemolysin, cam, from a strain of R. anatipestifer was cloned and expressed in Escherichia coli. Chromosomal DNA from serotype 19 strain 30/90 was used to construct a gene library in pBluescript II SK(-) vector in E. coli XL-1-Blue strain. The clones containing recombinant plasmids were screened for the CAMP reaction with Staphylococcus aureus. Those that showed cohemolysis were chosen for further analysis by sequencing. One of these clones, JFRA8, was subcloned to identify the smallest possible DNA fragment containing the CAMP cohemolysin determinant, which was located on a 3,566-bp BamHI-BstXI fragment which specified a 1,026-bp open reading frame. Clones containing recombinant plasmids carrying cam obtained by PCR cloning into E. coli M15 strain secreted an active CAMP cohemolysin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analyses confirmed that the recombinant strain expressed a protein with a molecular mass of 37 kDa and that strains from serotypes 1, 2, 3, 5, 6, and 19 expressed the cohemolysin. The deduced amino acid sequence showed high homology to those of O-sialoglycoprotein endopeptidases. Hydrolysis of radioiodinated glycophorin A confirmed that Cam is a sialoglycoprotease.
鸭疫里默氏菌可导致鸭的渗出性败血症。从一株鸭疫里默氏菌中克隆了CAMP协同溶血素的基因决定簇cam，并在大肠杆菌中进行表达。使用血清型19菌株30/90的染色体DNA构建基因文库，该文库存在于大肠杆菌XL-1-Blue菌株的pBluescript II SK(-)载体中。通过与金黄色葡萄球菌进行CAMP反应筛选含有重组质粒的克隆。选择那些显示协同溶血的克隆进行测序进一步分析。其中一个克隆JFRA8被亚克隆以鉴定包含CAMP协同溶血素决定簇的最小可能DNA片段，该片段位于一个3566 bp的BamHI - BstXI片段上，该片段指定了一个1026 bp的开放阅读框。通过PCR克隆到大肠杆菌M15菌株中获得的含有携带cam的重组质粒的克隆分泌出活性CAMP协同溶血素。十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳和蛋白质印迹分析证实重组菌株表达了一种分子量为37 kDa的蛋白质，并且血清型1、2、3、5、6和19的菌株表达了该协同溶血素。推导的氨基酸序列与O - 唾液酸糖蛋白内肽酶的序列具有高度同源性。放射性碘化血型糖蛋白A的水解证实Cam是一种唾液酸糖蛋白酶。
