CS3纤毛抗原表达调控机理的研究

Studies on the Expression Regulation of the CS3 Fimbriae Antigen Gene of Enterotoxigenic Escherichia coli

ＣＳ３是某些肠毒素大肠杆菌菌体表面上的多聚物，它能使病原菌粘附于宿主的小肠上皮细胞上，是致病的重要因素．为了探索ＣＳ３菌毛抗原基因的表达调控机制，根据ＣＳ３亚基结构基因的核苷酸序列分析表明，在其翻译起始位点的上游存在着ｒｂｓ位点及原核启动子的－１０区和－３５区ＤＮＡ序列．采用基因重组技术将ＣＳ３结构基因上游１２０ｂｐ的ＤＮＡ片段亚克隆进缺乏启动子而只含报告基因ｌａｃＺ的质粒ｐＣＢ２６７中．凝胶滞留和启动报告基因表达的实验证明了ＣＳ３亚基结构基因具有自身的启动子（Ｐｓ）．将该启动子上游区域不同长度的核苷酸片段克隆进ｐＣＢ２６７中，报告基因表达结果表明ＣＳ３结构基因的表达受其上游区域的抑制．核苷酸序列分析发现，在Ｐｓ－３５区上游５５０ｂｐ和８４０ｂｐ处各存在一个富Ａ－Ｔ簇．结合原核启动子的一般作用规律推知，ＣＳ３的表达可能受ＤＮＡ结合蛋白型的正向调节因子的作用．用ＣＦＡ／１菌毛抗原基因的正向调节基因ｃｆａＤ对ＣＳ３基因进行的互补表达试验表明ｃｆａＤ基因不仅可消除上游区对Ｐｓ的抑制，而且可大幅度地提高Ｐｓ的启动能力．在分析表达调控的基础上获得ＣＳ３重组高效表达．同时提出了其表达调控模型．

CS3 is a multimeric surface protein surrounding certain enterotoxigenic Escherichia coli (ETEC) strains.The ability of ETEC to adhere to the intestinal epithelium of the host is an important virulence determinant,and the adhesion is mediated by the proteinaceous surface antigens.The expression reuglation of the CS3 fimbriae antigen gene was explored.According to the nucleotide sequence of the CS3 subunit gene,the gene seems to have a typical ribosome binding site(rbs),and -10 as well as -35 sequences of prokaryotic promoter in the CS3 gene upstream from the translation start site.A DNA fragment of 120 bp upstream from the translation start site was subcloned into the plasmid pCB267 containing a reporter gene lacZ without promoter.Based on the results of gel retardation and expression of the reporter gene,the CS3 structural gene was under the control of this promoter.The different sizes of DNA fragments upstream from the promoter were cloned into the pCB267.Results of expression of lacZ gene showed that the promoter of the CS3 gene could be inhibited by its upstream sequence.The sequence analyses also revealed that there existed two regions of (A T) rich sequence at 550 bp and 840 bp upstream from -35 region.From these results,it was inferred that expression of the CS3 gene might be regulated by a DNA binding protein which was a positive regulator.Complementation analyses showed that the cfaD,a positive regulator of the CFA/1 opreron could not only eliminate the inhibition caused by the upstream region,but also greatly enhance the expression from the promoter of the CS3 gene.A model for the regulation of expression of the CS3 gene was proposed.