Analysis of susceptibility to staphylococcal exfoliative toxin and gene expression of plakoglobin in a newborn and an eight-day-old mouse

用LA-PCR体外克隆方法克隆了ETA底物的编码基因，并测定了该280bp片段的核苷酸序列。该序列与β-catenin基因3‘端非编码区的核苷酸序列高度同源(匹配128个，占96%)。小鼠β-连环蛋白与桥粒细胞内斑块蛋白有56%的同源性。用逆转录聚合酶链式反应比较新生小鼠和8日龄小鼠组织中β-连环蛋白和白蛋白基因的表达，发现β-连环蛋白(长度为264bp)和白蛋白(长度为254bp)在新生小鼠组织中的表达显著增加，而β-连环蛋白片段在出生8天的小鼠中未检测到表达。此外，在8天大的小鼠中，白蛋白的表达要么完全缺失，要么存在于非常低的水平。在小鼠发育的各个阶段，包括胚胎阶段、出生时和出生后8天，桥粒中都检测到桥粒中的桥粒蛋白、桥粒粘连蛋白、纤维凝集素、桥粒粘连蛋白和桥粒粘连蛋白。与未注射ETA的对照小鼠皮肤组织制备的相应组分相比(免疫印迹法)，注射ETA的新生小鼠皮肤组织制备的水不溶部分中的白蛋白含量较低。而注射ETA的新生小鼠皮肤组织的水不溶部分中DSG 1的含量与未注射ETA的对照小鼠皮肤组织中的DSG 1水平相同。采用双抗体夹心法建立了两种酶联免疫吸附试验(ELISA)来检测DSG-1和白蛋白。本研究建立的酶联免疫吸附试验方法可能为今后检测葡萄球菌性烫伤皮肤综合征患者临床标本中的白蛋白和DSG1提供一种灵敏的方法。

The gene coding the ETA substrate was cloned using the LA PCR in vitro cloning method, and the nucleotide sequence of the 280-bp fragment amplified by PCR was determined. This sequence showed a high degree of homology with the nucleotide sequence of the 3'-non-coding region of the β-catenin gene (128 of 133 nucleotids matched, 96%). Mouse β-catenin protein and plakoglobin, an intracellular plaque protein of desmosomes, are 56% identical. A comparison of β-catenin and plakoglobin gene expression using reverse transcriptase polymerase chain reaction(RT-PCR) in tissue from a newborn mouse and an eight-day old mouse revealed a significantly greater expression of β-catenin (264 bp in length) and plakoglobin (254 bp in length) in the newborn mouse, whereas expression of the β-catenin fragment was undetectable in the eight-day-old mouse. In addition, the expression of plakoglobin was either completely absent or present at very low levels in the eight-day-old mouse. Desmogleins, desmocollins, plectin, desmoyokin, and desmoplakin in Desmosomes have been detected at all stages of development in mice, including the embryonic stages, at birth, and 8 days after birth. Plakoglobin in the water-insoluble fraction prepared from ETA-injected newborn mouse skin tissues was present at low levels when compared (using immunoblotting) with that in a corresponding fraction prepared from control ETA-noninjected mouse skin tissues. However, the level of Dsg 1 in the water-insoluble fraction prepared from ETA-injected newborn mouse skin tissues was the same as that in the control ETA-noninjected newborn mouse skin tissues. Two enzyme-linked immunosorbent assays(ELISAs) were developed to detect Dsg 1 and plakoglobin using a double-antibody sandwich protocol. The ELISA procedures presented in this report may provide a sensitive method for the future detection of plakoglobin and Dsg 1 in clinical samples from patients with Staphylococcal scalded skin syndrome.