产毒性大肠杆菌CFA/Ⅰ基因表达与DNA超螺旋状态的关系

STUDY ON RELATION BETWEEN DNA SUPERCOILING AND CFA/ I GENE EXPRESSION

将CFA／I结构基因(cfaABCE)克隆到载体pJRD184上构建了质粒pJGX15A,并将其转入大肠杆菌topA野生株E.coli HB101和topA缺陷株E.coli DM800中.Western blot测定的结果表明,在CFA／I的正调控基因cfaD基因缺失时,两株重组菌株都能够表达CFA／I抗原.分别抽提两个重组菌株对数前期和对数中期的质粒pJGX15A DNA,并对其进行氯喹琼脂糖凝胶电泳分析,结果显示E.coli DM800细胞中质粒pJGX15A的负超螺旋水平比野生型细胞E.coliHB101中的较低.同时,菌落免疫酶斑分析的结果显示DM800受体菌中CFA／I的表达水平比HB101受体菌中的表达水平高.实验结果表明,CFA／I结构基因的表达与DNA负超螺旋水平有明显的负相关性,与负超螺旋促进基因表达的看法不同.

A recombinant plasmid pJGX15A was constructed by cloning the structure gene cluster cfaABCE into she Sac I /Xho I site of the pasmid vector pJRD184. Two E. coli strains were used as host to express colonization factor antigen I (CFA/ I ), HB101 (topA+ ) and DM800(topA-). The results of Western blotting suggested that both of the two transformants expressed CFA/ I steadily, even without the positive regulating gene cfaD. The DNA samples of plasmid pJGX15A isolated from the two tranformants were taken on electrophoresis in the system containing chloroquine, and the results showed that pJGX15A from the DM800 transfotmant had lower levels of negative DNA supercoling; which was opposite to the common opinions. However, the DM800 transformant produced higher levels of CFA/ I by test with clony immanollot assay. The results of the experiment indicated that the expression of CFA/ I was significantly relative to the level of DNA supercoliling.