Molecular genetical study of low-temperature growth in Listeria monocytogenes

单增李斯特菌低温生长的分子遗传学研究

基本信息

批准号：
06670314
负责人：
MAKINO Sou-ichi
金额：
$ 1.41万
依托单位：
Obihiro University of Agricuruture and Veterinary Medicine (1995)国立公衆衛生院 (1994)
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06670314/
关键词：
Listeri monocytogenes Virulence NaCl PCR flagella detection 低温増殖能鞭毛病原性食品

项目摘要

Molecular genetical analysis of the growth at low temperature and at high concentration of NaCl in Listeria monocytogenes have been studied. We focused on the formation of flagella at low temperature. Similtaneously, we have established the direct detection system of L.monocytogenes DNA from food by PCR.1.Anti-fkagella antisurum : Using flagella protein isolated from SDS-polyacrylamidgel elecrtophoresis, We have made anti-rabbit serum raised against flagellin (anti-fla).2.Cloning of flagella-related genes by anti-fla : In E.coli, we have isolated a several clones positively reacted on the anti-fla, followed by the genetical analysis and immunological analysis. Although one clone was the structural gene for flagella-formation, other 2 clones were new genes different from the flagella gene. But the both clones, clone 1 and clone 2, produced their gene products reacted against anti-fla by western blotting analysis.3.fla-minus mutant : We constructed fla-minus mutant, followed by invasion tests in the epithelial cells and infection tests to mouse. But, we could not detect any defference between the mutant and its parent strain.4.PCR method : Using the nucleotide sequence of clone 1, we constructed a set of primer for Listeria species. By PCR,we established the direct detection method from foods.5.NaCl resistance : The relationship between the growth of L.monocutogenes and the NaCl concentration in broth was studied. At high concentration of NaCl, the growth was inhibited, but the virulence of L.monocytogenes for mouse mostly same. At the venetral injection, we observed the change of virulence.

对单核细胞增生李斯特氏菌在低温和高浓度氯化钠条件下生长的分子遗传学分析进行了研究。我们重点关注低温下鞭毛的形成。同时，我们还建立了食品中单增李斯特氏菌DNA的PCR直接检测体系。 1.抗fkagella抗血清：利用SDS-聚丙烯酰胺凝胶电泳分离鞭毛蛋白，制备了针对鞭毛蛋白的抗兔血清（anti-fla ).2.抗fla克隆鞭毛相关基因：在大肠杆菌中，我们分离到了几个呈阳性反应的克隆关于抗fla，其次是遗传分析和免疫学分析。虽然1个克隆是鞭毛形成的结构基因，但另外2个克隆是与鞭毛基因不同的新基因。但是通过蛋白质印迹分析，克隆1和克隆2这两个克隆都产生了抗fla反应的基因产物。 3.fla-minus突变体：我们构建了fla-minus突变体，然后进行了上皮细胞的侵袭测试和感染测试到鼠标。但是，我们没有检测到突变体与其亲本菌株之间有任何差异。4.PCR方法：利用克隆1的核苷酸序列，我们构建了一组李斯特菌引物。通过PCR建立了从食品中直接检测的方法。5.耐盐性：研究了单切李斯特菌的生长与肉汤中氯化钠浓度的关系。高浓度的NaCl时，生长受到抑制，但单增李斯特菌对小鼠的毒力基本相同。在腹腔注射时，我们观察到毒力的变化。