Segada L M, Lesse A J
Department of Microbiology, State University of New York at Buffalo, New York, USA.
Gene. 1997 Dec 19;204(1-2):185-94. doi: 10.1016/s0378-1119(97)00545-3.
Haemophilus influenzae biogroup aegyptius, the causative agent of Brazilian purpuric fever (BPF), expresses a heat-modifiable 48 kDa outer membrane protein, P1, which is conserved in most Brazilian case-clone isolates. To study the role of P1 in pathogenesis of BPF we constructed via homologous recombination an isogenic P1-deficient mutant of H. influenzae biogroup aegyptius. The procedure involved a modification of Hererot's method for development of competence. Modifications included variations in the growth conditions, use of cAMP, specific characteristics of the donor DNA, and antibiotic selection. P1-deficient mutants were confirmed by SDS-PAGE, loss of reactivity with a specific monoclonal antibody on Western blot, restriction analysis and Southern blot. Our results establish the first successful transformation of homologous DNA into H. influenzae biogroup aegyptius.
埃及生物群流感嗜血杆菌是巴西紫癜热（BPF）的病原体，它表达一种热可变的48 kDa外膜蛋白P1，该蛋白在大多数巴西病例克隆分离株中是保守的。为了研究P1在BPF发病机制中的作用，我们通过同源重组构建了埃及生物群流感嗜血杆菌的P1基因缺失同基因突变体。该过程涉及对赫勒罗特感受态诱导方法的改进。改进包括生长条件的变化、cAMP的使用、供体DNA的特定特性以及抗生素选择。通过SDS-PAGE、Western印迹上与特异性单克隆抗体反应性的丧失、限制性分析和Southern印迹证实了P1基因缺失突变体。我们的结果首次成功地将同源DNA转化到埃及生物群流感嗜血杆菌中。
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