Schultz A J, McCardell B A
Division of Microbiology, Food and Drug Administration, Washington, D.C. 20204.
J Clin Microbiol. 1988 Jan;26(1):57-61. doi: 10.1128/jcm.26.1.57-61.1988.
DNA colony hybridization with three 18- to 20-base-long synthetic oligonucleotide probes for cholera toxin (CT) was used to screen 12 clinical isolates of Aeromonas hydrophila. Under stringent hybridizing (overnight at 40 degrees C) and washing (1 h at 50 degrees C) conditions, nine strains reacted with the 32P-labeled CT probes. Concentrated (10x) cell-free supernatants or lysates from eight cultures, heated at 56 degrees C for 20 min, produced cytotonic effects in Y-1 mouse adrenal cells and Chinese hamster ovary (CHO) cells and caused a 1.5- to 22-fold increase in production of cyclic AMP in CHO cells. Preincubation with anti-CT reduced the CHO cell titer of cell lysates by 10-fold. In the GM1 ganglioside enzyme-linked immunosorbent assay, heated supernatants and lysates gave readings equivalent to 3.5 to 100 ng of CT. Three proteins with molecular weights of 89,900, 37,000, and 11,000 reacted with anti-CT on immunoblots of cell lysates from sodium dodecyl sulfate-polyacrylamide gels. These results suggest that there is DNA homology and immunological cross-reactivity between CT and the A. hydrophila cytotonic toxin.
采用霍乱毒素(CT)的三种18至20个碱基长的合成寡核苷酸探针进行DNA菌落杂交,以筛选12株嗜水气单胞菌临床分离株。在严格的杂交(40℃过夜)和洗涤(50℃1小时)条件下,9株菌株与32P标记的CT探针发生反应。来自8种培养物的浓缩(10倍)无细胞上清液或裂解物,在56℃加热20分钟后,在Y-1小鼠肾上腺细胞和中国仓鼠卵巢(CHO)细胞中产生细胞紧张效应,并使CHO细胞中环磷酸腺苷的产生增加1.5至22倍。用抗CT预孵育可使细胞裂解物的CHO细胞效价降低10倍。在GM1神经节苷脂酶联免疫吸附试验中,加热的上清液和裂解物的读数相当于3.5至100 ng的CT。在十二烷基硫酸钠-聚丙烯酰胺凝胶的细胞裂解物免疫印迹上,三种分子量分别为89,900、37,000和11,000的蛋白质与抗CT发生反应。这些结果表明,CT与嗜水气单胞菌细胞紧张毒素之间存在DNA同源性和免疫交叉反应性。