Garbe T R, Barathi J, Barnini S, Zhang Y, Abou-Zeid C, Tang D, Mukherjee R, Young D B
MRC Tuberculosis and Related Infections Unit, RPMS, Hammersmith Hospital, London, UK.
Microbiology (Reading). 1994 Jan;140 ( Pt 1):133-8. doi: 10.1099/13500872-140-1-133.
Electroporation with shuttle plasmids carrying a kanamycin resistance gene as a selectable marker failed to generate transformants in two mycobacterial species currently being used in human vaccine trials (Mycobacterium w and Mycobacterium vaccae). In contrast, efficient transformation [10(3)-10(5) transformants (micrograms DNA)-1] was obtained using novel vectors with selection based on expression of resistance to hygromycin. The hygromycin resistance vector was also found to be more efficient than kanamycin resistance vectors for transformation of Mycobacterium smegmatis and Mycobacterium bovis BCG. The hygromycin resistance vector was used to overexpress superoxide dismutase of Mycobacterium tuberculosis in M. vaccae in a form suitable for detailed structural analysis. The potential use of this approach for generation of novel recombinant mycobacterial vaccines is discussed.
携带卡那霉素抗性基因作为选择标记的穿梭质粒电穿孔法，未能在目前用于人类疫苗试验的两种分枝杆菌（结核分枝杆菌w和母牛分枝杆菌）中产生转化体。相比之下，使用基于潮霉素抗性表达进行选择的新型载体可实现高效转化[10(3)-10(5)个转化体/(微克DNA)]。还发现潮霉素抗性载体在耻垢分枝杆菌和卡介苗的转化中比卡那霉素抗性载体更有效。潮霉素抗性载体用于在母牛分枝杆菌中过表达结核分枝杆菌的超氧化物歧化酶，其形式适合进行详细的结构分析。本文讨论了这种方法在新型重组分枝杆菌疫苗生产中的潜在用途。
