摘要
目的为证实调控ｉｐａＢＣＤ表达除侵袭质粒上的两个正性调控基因ｖｉｒＦ和ｉｎｖＥ外，染色体上也可能存在与ｉｎｖＥ共同参与促进ｉｐａ基因表达的因子。方法应用低拷贝克隆载体ｐＡ－ＣＹＣ１８４，在大肠杆菌构建福氏志贺氏菌染色体基因文库。经半乳糖苷酶活性测定、分子杂交等筛选，获得一株能增进上皮细胞侵袭性基因ｉｐａＢ表达的ＤＮＡ分子克隆ｐＱ４４５。结果该片段约为４．３ｋｂ长，ＤＮＡ序列分析表明有两个完整的开放阅读框架（ＯＲＦ），经插入缺失突变发现，其中一个ＯＲＦ的表达产物能增加ｉｐａＢ的活性，为ｉｐａ正性调节因子，定名为ｃｒｉＡ。ｃｒｉＡ基因位于细菌染色体８７．３分处，志贺菌属大多菌群都携带该基因。ｃｒｉＡ约１４３８ｂｐ，编码４７６个氨基酸残基的酸性蛋白，分子量约５２６００。结论ＣｒｉＡ蛋白可能凭借其２６８～２９８位和３２０～３４１位两处的氨基酸残基位点亮氨酸拉链区结构。
Objective The expression of invasion plasmid antigen genes (ipaBCD) of Shigella flexneri is positively regulated by genes of virF and invE at the same invasion plasmid. The study here is to defini a factor on the chromosome of Shigella spp, which could be effective to promote ipa expression in cooperation with invE. Methods A 4.3kb DNA fragment (pQ445) cloned from S. flexneri mutant (HW 1002) into the low copy expression vector pACYC184 was selected by using β galactosidase assay and Western blot with convalescent serum. It showed the enhancement of ipaB expression.The regulation region located at 87.3 min was confirmed by restriction mapping and DNA hybridization with a probe of Pst1 DNA fragment of pQ445. Results An ORF (orfB, criA) within this region was identified as a positive regulator gene and its complete nucleotide sequence was determined. Furthermore, the probe did react with genomic DNA from other Shigella spp. and E.coli strain. Conclusions cirA protein predicted (476AA,52.6kDa) from criA gene (1438bp) contains the leucine zipper motifs of amino acid residues of 268 AA 298 AA and 320AA 341AA which appear to have regulatory activity for InvE protein.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
1998年第5期347-352,共6页
Chinese Journal of Microbiology and Immunology
关键词
痢疾杆菌
基因表达调控
分子克隆
Shigella spp
Gene expression regulation
Molecular cloning
Genes
bacterial
