BIOCHEMICAL AND GENETIC MECHANISMS OF OBLIGATE INTRACELLULAR PARASITISM

专性细胞内寄生的生化和遗传机制

• 批准号: 3821979
• 负责人: J C WILLIAMS
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3821979
• 关键词: Escherichia coli; Q fever; autoradiography; bacterial DNA; bacterial genetics; bacterial polysaccharides; biological polymorphism; chromatography; communicable disease diagnosis; communicable diseases; electron microscopy; gene expression; genetic library; genetic manipulation; genetic strain; histochemistry /cytochemistry; host organism interaction; intracellular parasitism; lipopolysaccharides; microorganism culture; molecular cloning; nucleic acid sequence; nucleic acid structure; plasmids; silver impregnation; ultracentrifugation; virulence

The objective of this project was the cloning, expression and sequencing of a major antigen gene and to further chemically characterize the lipopolysaccharide (LPS) of Coxiella burnetii, the etiological agent of Q fever. A. Gene cloning. A gene library from the DNA of C. burnetii has been constructed in the cosmid vector pHC79. An immunoreactive gene product from clone pJB196 was identified as a 62 kilodalton (Kd) polypeptide both by Western blot and an in vitro transcription, translation system. Expression of the 62 Kd and a 14 Kd polypeptide was under the control of a heat shock promoter (HSP) in recombinant E. coli. Subcloning of pJB196 and sequencing a 3 Kb stretch of that DNA revealed two open reading frames, encoding a polypeptide of 58.9 Kd, and 10.5 Kd along with two appropriately spaced ribosomal binding sites. A transcriptional control element observed on the 5' side of the initiation codon resembled a HSP (at -452). Termination of this bicistronic mRNA synthesis may be under control of a rho-independent terminator with double dyad symmetry and delta G of -28.0 and 43.5 K cal, respectively. Four sequences were highly homologous to the 62 Kd protein from C. burnetii (greater than or equal to 50%). Three are from Mycobacteria and represent the immunodominant antigen of this species. The other is from Escherichia coli, detected as a gene that complements or suppresses a temperature-sensitive RNAase activity. Therefore this protein is conserved in phylogenetically distant bacterial genera. B. LPS Structure. The core structure of C. burnetii LPS consists of (1-3)-mannoheptose backbone with every residue (1-2) branched with a single mannose moiety. Lipid C (the counterpart of Salmonella lipid A) is quite unusual in that in lacks carbohydrate and is small in molecular weight. C. Significance. A heat shock operon in C. burnetii produces a major antigen highly homologous to a protein in both Mycobacteria and Escherichia coli. The core structure of LPS was defined and the lipid C molecule is significantly different from Salmonella lipid A.

该项目的目的是克隆，表达和 主要抗原基因的测序并进一步化学 表征Coxiella burnetii的脂多糖（LPS）， Q发烧的病因。 A.基因克隆。基因库 从C. burnetii的DNA中已构建在宇宙中 向量PHC79。 来自克隆的免疫反应基因产物 PJB196被确定为62千达尔顿（KD）多肽 蛋白质印迹和体外转录，翻译系统。 62 kd和14 kD多肽的表达在 重组大肠杆菌中热激启动子（HSP）的控制。 PJB196的亚克隆并测序3 kb的DNA 揭示了两个开放式阅读框，编码了58.9的多肽 KD和10.5 KD以及两个适当间隔的核糖体 绑定位点。 在 启动密码子的5'侧类似于HSP（AT -452）。 这种双科式mRNA合成的终止可能不在 控制双二元的Rho独立终结器 对称和三角洲G分别为-28.0和43.5 K Cal。 四个 序列与C的62 kD蛋白高度同源。 burnetii（大于或等于50％）。 三个来自 分枝杆菌，代表这种免疫主导抗原 物种。 另一个来自被检测为基因的大肠杆菌 这补充或抑制了对温度敏感的RNAase 活动。 因此，该蛋白在系统发育中保守 遥远的细菌属。 B. LPS结构。 的核心结构 C. burnetii lps由（1-3） - 甘露蛋白骨架与 每个残留物（1-2）都用一个甘露糖部分分支。 脂质 c（沙门氏菌脂质的对应物a）非常不寻常 缺乏碳水化合物，分子量很小。 C 意义。 C. burnetii中的热量冲击操纵子产生主要 抗原与分枝杆菌和蛋白质高度同源 大肠杆菌。 LPS的核心结构被定义了 脂质C分子与沙门氏菌脂质A显着不同。