Cellular Localization, qurification, and function of plasmud-determinedrirulence proteins of Salmonella

我们以前的研究表明，从败血症猪分离的霍乱沙门氏菌RF-1株含有50kb的毒力质粒pKDSC50，pKDSC50的6.4kb的Sali-EcoRI区包含spvR、spva、spvB和spvC四个SPV基因，是引起猪和小鼠全身疾病所必需的。SpvR基因是SpvA、SpvB和spvC三个基因转录的正激活基因。本研究的目的是获得了解沙门氏菌系统性感染所需的SPV蛋白的基本知识。SpvR蛋白的调节功能SpvR是SpvR自身基因表达的正调控蛋白，SpvA和SpvB是SpvR基因表达的负调控蛋白。结论：1.spvR基因在细菌生长的稳定期以rpos依赖的方式表达。SPV蛋白的分离、纯化和鉴定从霍乱沙门氏菌RF-1菌株中部分纯化了SpvR、SpVA、SpvB和SpvC四种SPV蛋白。经SDS-PAGE分析，纯化的Spv蛋白的相对分子质量为SpvR为31 kDa，SpVA为26 kDa，SpvB为73 kDa，SpvC为24 kDa，说明SpvB是一种疏水性蛋白，SpvC是一种碱性蛋白。SPV蛋白的免疫电子显微镜定位建立了SPV蛋白的免疫电子显微镜定位方法。霍乱沙门氏菌RF-1株冷冻超薄切片经多聚甲醛-裂解素固定后，显示SpvC为细胞质和外膜蛋白。SpvR、SpVA和SpvB蛋白的定位正在研究中。

Our previous studies showed that S.Choleraesuis strain RF-1 isolated from septicemic pig harbors 50kb-virulence plasmid designated pKDSC50, and the 6.4kb SalI-EcoRI region of pKDSC50 encompassing four spv genes, spvR,spvA,spvB,and spvC,is essential to produce systemic disease in pigs and mice. The spvR gene was characterized as a positive activator gene for transcription of the following three genes, spvA,spvB,and spvC.The aim of the present study is to obtain the basic knowledges of Spv proteins which are requird for understanding the Salmonella systemic infection.1. Regulatory function of Spv proteinsSpvR is a positive regulatory protein for the expression of its own gene, spvR.SpvA and SpvBare negative regulatory proteins for the expression of spvR gene. spvR gene is expressed in RpoS-dependent manner at the stationary phase of bacterial growth.2. Isolation, purification, and characterization of Spv proteinsThe four Spv proteins, spvR,spvA,spvB,and spvC were partially purified from S.Choleraesuis strain RF-1. Molecular weight of the purified Spv proteins by SDS-PAGE was determinded as 31 kDa for SpvR,26 kDa for SpvA,73 kDa for SpvB,and 24 kDa for SpvC.It was demonstrated that SpvB is a hydrophobic protein, and SpvC is a basic protein.3. Immunoelectron microscopic localization of Spv proteinsA method for the localizatiom of Spv proteins by immunoelectron microscopy was established. By using cryo ultra-thin section of S.Choleraesuis strain RF-1 fixed with periodete lysin-paraformaldehyde, SpvC was revealed to be a cytoplasmic and outer menbrane protein. The localization of SpvR,SpvA,and SpvB proteins is under investigation.