Study on function of phospholipase C gene binding protein from Clostridium perfringens

产气荚膜梭菌磷脂酶C基因结合蛋白的功能研究

基本信息

批准号：
08670308
负责人：
OKABE Akinobu
金额：
$ 1.6万
依托单位：
Kagawa Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

项目摘要

Phospholipase C (PLC) is one of the major virulence factors of Clostridium perfringens. The expression of a PLC-encoding gene (plc) is regulated by bent DNA locating upstream of a plc promoter and also by plc-binding protein, which can bind to the coding region of the gene. The plc-binding protein was partially purified from C.perfringens NCTC 8237, a PLC high-producing strain. One polypeptide, of which molecular mass was estimated to be 56 kDa by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) , bound to a fragment within the plc coding region. Its N-terminal amino acid sequence was determined and PCR product was generated using degenerate primers corresponding to the sequence. A 6-kb EcoRI fragment from the NCTC 8237 chromosome, which hybridized with the PCR product, was cloned into pUC19. Nucleotide sequencing of this fragment and similarity search of the predicted amino acid sequence revealed that the gene encoding for the 56 K polypeptide is hemD-ctyGC and other open reading frames found in the fragment are all related to the enzymes for heme biosynthesis. When the 6-kb fragment was cloned into a shuttle-vector pJIR418 and C.perfringens strain 13 was transformed with the plasmid, othe hemD-cysGC-encoding gene was expressed in the transformant. However, extract from the transformant did not show a positive gel retardation to the plc gene. It may be possible that the plc-binding protein comigrates with the 56 K polypeptide on SDS-PAGE.Study was also conducted on a role of the bent DNA locating upstream of the plc promoter. It has been shown to play a role in the regulation of the plc gene expression in response to alteration of temperature.

磷脂酶C（PLC）是灌注梭状芽胞杆菌的主要毒力因子之一。 PLC编码基因（PLC）的表达受PLC启动子上游以及PLC结合蛋白的弯曲DNA调节，该蛋白可以与基因的编码区域结合。 PLC结合蛋白从C.Perfringens NCTC 8237（PLC高生产菌株）部分纯化。十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）估计分子量为56 kDa，其中一个多肽是一个多肽，与PLC编码区域内的碎片结合。确定其N末端氨基酸序列，并使用与序列相对应的退化引物生成PCR产物。与PCR产物杂交的NCTC 8237染色体中的6 kb Ecori片段被克隆到PUC19中。该片段的核苷酸测序和对预测的氨基酸序列的相似性搜索表明，编码56 K多肽的基因是hemd-ctygc，片段中发现的其他开放式阅读框都与血红素生物合成的酶有关。当将6-kb片段克隆到穿梭矢量pjir418中，并用质粒转化perfringens菌株13时，在转化剂中表达了othe hemd-cysGC编码基因。但是，转化剂中的提取物并未显示出对PLC基因的阳性凝胶延迟。 SDS-PAGE上的PLC结合蛋白可能与56 K多肽漫画。还在PLC启动子上游的弯曲DNA的作用上进行了研究。已经显示，它在响应温度改变时在PLC基因表达的调节中起作用。