選殖和分析分離自台灣的豬霍亂沙門氏桿菌鞭毛素基因

Abstract

豬霍亂沙門氏桿菌﹙Salmonella enterica serovar Choleraesuis，簡寫成S. Choleraesuis﹚，屬於腸內細菌科﹙Enterobacteriaceae﹚兼氧性桿菌，為一種細胞內的病原菌，可在人體和豬隻產生疾病，但感染豬隻較為嚴重。它為帶有周身鞭毛的革蘭氏陰性菌，鞭毛為絲狀的細菌運動構造。鞭毛可以細分為三個部分 ： basal body， hook 和filament，filament為鞭毛素蛋白 (flagellin) 所構成，細菌鞭毛也被認為具有致病的能力。二相 (diphasic)沙門氏桿菌擁有兩種鞭毛素基因： fliC和fljB ， 可以交替表現出兩種不同抗原性的phase I (FliC) and phase II (FljB) 鞭毛素蛋白，此現象稱之為相位變異 (phase variation)。本實驗室之前已對S. Choleraesuis CH12440 之鞭毛素蛋白基因進行研究，並發現fliC基因多出36個核苷酸為其他菌株所缺失，本實驗則另外對S. Choleraesuis CH12440 FljB鞭毛素蛋白基因進行研究。此外，也利用PCR技術分析台中疾病管制局 (CDC) 收集自台灣各地豬隻和人體分離出來的199隻S. Choleraesuis菌株的鞭毛素基因。結果顯示，199隻菌株的fliC基因構造都與菌株CH12440的 fliC一樣，擁有完整功能且均有36個核苷酸插入序列，而198隻菌株的fljB基因和菌株CH12440的 fljB則都存在插入序列IS10 (insertion sequence 10)。最後也利用MTT (methythiazoletetrazolium)分析法研究表現的FljB蛋白對小鼠巨噬細胞株 (mouse macrophage cell lines) 存活率的影響，顯示和之前本實驗室研究的FliC蛋白一樣具有細胞毒殺能力。

Salmonella enterica serovar Choleraesuis (S. Choleraesuis) belonged to the family Enterobacteriaceae and was a host-adapted, facultative, intracellular pathogen. It could cause the diseases in human and pigs, but more serious in pigs. It was a gram-negative bacterium with peritrichous flagella. Flagellum, a hair-like appendage, was the bacterial locomotive structure. A flagellum could be subdivided into three parts: basal body, hook and filament which composed of flagellin protein. Flagella had also been recognized as one of the potential virulence factors of microorganisms. Diphasic Salmonella enterica strains possessed two nonallelic structural genes, fliC and fljB, encoding phase I and II flagellin, respectively, with different antigenecity. A phenomenon called phase variation that a bacterium expressed alternately phase I and II flagellin. Our lab had cloned and characterized the phase I fliC gene from S. Choleraesuis 12440 previously. There was an insert of 36 nucleotides absent in other phase I flagellin genes. In this study, phase II flagellin gene fljB was cloned and characterized from S. Choleraesuis 12440. One hundred and ninety-nine strains of S. Choleraesuis collected from Taiwan by Taizhong center for disease control (CDC) were analyzed using PCR technique. The data indicated all the fliC genes of 199 strains had the same structure as that of strain 12440 with a 36-bp insert. One hundred and ninety-eight strains and strain CH12440 had a fljB gene with a IS10 (insertion sequence 10). The fljB without transposon insert was cloned and expressed. The toxicity of expressed FljB protein on mouse macrophage cell lines was studied by MTT (methythiazoletetrazolium) assay. FljB protein displayed, as FliC , a cytotoxic effect on the cells.