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目的 通过构建霍乱弧菌toxR基因缺失株来研究toxR基因对霍乱弧菌减毒菌株IEM10 1和高产毒株 5 6 9B毒力表达的调控作用。方法 采用自杀性质粒和接合转移技术 ,将 2个中间含有四环素基因的toxR基因分别与霍乱弧菌减毒株IEM10 1和高产毒株 5 6 9B染色体toxR基因重组 ,从而获得toxR基因缺失株IEM10 1 4和 5 6 9B 43 ,并对 2个toxR基因缺失株和其原出发菌株的霍乱肠毒素的产率和主要外膜蛋白图谱进行比较。结果 采用GM1 ELISA检测受测菌CT基因表达 ,toxR基因缺失株 5 6 9B 43的P/N值为 1 82 ,而其原出发菌株 5 6 9B的P/N为 4 5 2 ,而IEM10 1和其toxR基因缺失株的P/N值均低于 2。采用SDS PAGE对受试菌外膜蛋白进行分析 ,toxR基因缺失株 5 6 9B和IEM10 1的外膜蛋白图谱相比 ,均多出 2条相对分子质量 (Mr)为 40× 10 3 和 43× 10 3 外膜蛋白区带。结论 toxR蛋白是霍乱肠毒素基因ctx表达的正调控因子 ,是霍乱弧菌主要外膜蛋白 (Mr 为 40× 10 3 和43× 10 3)编码基因的负调控因子。
OBJECTIVE: To study the regulatory effect of toxR gene on the virulence of V. cholerae attenuated strain IEM10 1 and the highly productive strain 56B9 by constructing the toxR gene deletion strain of V. cholerae. METHODS: Two toxR genes containing the tetracycline gene were recombined with the toxR gene of V. cholerae attenuated strain IEM10 1 and the high-yield strain 56B9B gene using suicide plasmid and conjugation transfer technique to obtain the toxR gene deletion strain IEM10 1 4 and 5 6 9B 43, and compared the yield and main outer membrane protein profiles of two toxR gene deletion strains and their original strains. Results The CTx gene expression of tested bacteria was detected by GM1 ELISA. The P / N value of toxR gene deletion strain 5 6 9B 43 was 1 82, whereas the original strain 5 6 9B had P 5 N 4 5 2 while the IEM10 1 and The toxR gene deletion strains had P / N values below 2. The outer membrane protein of the tested bacteria was analyzed by SDS PAGE. Compared with the outer membrane protein profile of the toxR gene deletion strain 5 6 9B and the IEM 10 1, two relative molecular masses (Mr) of 40 × 10 3 and 43 × 10 3 Outer membrane protein zone. Conclusion toxR protein is a positive regulator of ctx expression in cholera enterotoxin gene and is a negative regulator of the gene encoding major outer membrane proteins (Mr 40 × 10 3 and 43 × 10 3) of V. cholerae.