沙門氏腸炎桿菌是一株人畜共通的病原菌，最常經由污染的雞肉及雞蛋而感染嬰幼兒、老年人或免疫力差的成人。Egg-associated Salmonellosis的感染案例逐年上升，最主要是因為許多Salmonella菌株都產生多重抗藥性。因此,對於疫苗的開發及尚未發現之致病機轉的研究，相對地就顯的非常重要。故本實驗室積極的希望能從S.enteritidis的基因庫中篩選出與免疫保護及感染致病相關的重要因子，以期望能發展成疫苗或研究未知之致病機轉。
之前實驗室由構築好的S. enteritidis的基因庫中，利用綿羊血培養基去進行致病因子的篩選，共篩選出兩種具有溶血活性的基因，分別命名為outer membrane protein p1 (ompp1) 及hemolytic activity gene (hly)。OMP暴露在菌體的表面，可以被抗體所認識而成為一個好的免疫原。經由Cell fractionation的分析，證實此OMPP1蛋白質是位於outer membrane fraction，因此認為它可能與寄主的immune response有關；而hemolysin-like protein具有一段signal peptide，會將此蛋白質鑲嵌在膜上或分泌至細胞外，所以也具有成為免疫原的潛力。於是進行此二段基因的構築及表現，並進一步以動物實驗模式觀察主動免疫的保護效果，初步發現外膜蛋白P1（OMPP1）約具有60%的保護效果，而溶血活性蛋白（HLY）則只有10％的保護效果。另外在被動免疫上，先利用腹腔注射的方法打入175 c.f.u.的S.enteritidis，經3小時後使細菌循環到全身，再利用尾靜脈注射打入200ug的抗體，做緊急的保護中和效果，初步發現外膜蛋白P1（OMPP1）約具80%的治療效果，而溶血活性蛋白（HLY）則沒有治療效果。因此我們認為外膜蛋白P1（OMPP1）蛋白質對動物體有良好的保護(60％)及治療(80％)效果，具有發展成為疫苗的潛力。
為了使我們能更瞭解ompp1 gene在Salmonella致病能力上所扮演的角色，於是我們針對ompp1 gene，利用同源染色體重組之特性進行基因剔除(gene knockout)，並由 PCR及Western blotting的結果確認無誤後，再以細胞培養實驗去證實，將Salmonella 的ompp1 gene 破壞之後，此突變株對HeLa cell 的adhesion抑制達70％，對invasion的抑制達73％；另外再以BALB/C小鼠測試突變株與野生株半致死率(LD50)的差異，由結果顯示，ompp1 gene在Salmonella的致病感染上應該有其重要性。

Salmonella enteritidis is a facultative intracellular pathogen that can cause disease in humans and animals. Poultry meat and eggs are considered to be the major source of infection for infant, elderly, and those with impaired immune system. Egg associated salmonellosis have been raised because many strains of salmonella have become resistant to several of the antibiotics traditionally used to treat it. Today, modern chemotherapy must consider the treatment of antibiotics resistance and target factors that are required for the pathogenesis of bacterial infection. The purpose of this research is to characterize the virulence-associate factors and elucidate their roles in the pathogenesis of S.enteritidis.
In our previous study, two hemolytic activity genes have been cloned from genomic library of S.enteritidis (Sal-5). One gene encodes outer membrane protein P1 (OMPP1), and the other encodes hemolysin like protein (HLY). OMP are exposed on the surface of the bacterial cell and they can serve as phage receptors and react with antibodies. In this study, the results of cell fraction experiment indicated that OMPP1 is an outer membrane associated protein. HLY seems to have a cleavable N-terminal signal peptide and may be anchored in the membrane or secreted to the medium by prediction of computer. With their physical and biological characteristics, OMPP1 and HLY may be used successfully as vaccine antigens and good immunogens.
To evaluate whether OMPP1 and HLY could confer protective immunity against S.enteritidis infection or not, active and passive immunization experiments were performed. Mice immunized with OMPP1 showed 60％ protection and immunized with HLY showed 20％protection against challenged with 373 c.f.u. of S.enteritidis, compared to a 90％ mortality in mice immunized with BSA. For passive immunization experiment, mice challenged intra-peritoneally (I.P.) with 175 c.f.u. of S.enteritidis were intra-venous (I.V.) treated with 200ul anti-OMPP1 or anti-HLY antibody one times a day for five consecutive days. Antibody treatment resulted in a survival rate of 80％ for using anti-OMPP1 antibody and 10％ for using anti-HLY antibody, compared to a 90％ mortality in mice treated with pre-immune antibody. These results indicated that OMPP1 is a good immunogen and can provide the protective effects in both active and passive immunization.
In order to study the role of OMPP1 in the pathogenesis of Salmonella infection, ompp1-deficient strain was constructed by using integrated plasmid to disrupt the original ompp1 gene. In vitro studies of bacterial adhesion and invasion on intestinal epithelial cell lines and epithelial monolayer were performed. Compared to wild-type strain, the ompp1 mutant exhibited a 70％ decrease in adhesion ability and a 73％decrease in invasion ability. These results indicated that OMPP1 is involved in cellular adhesion and invasion, and plays an important role of Salmonella in natural infection route.

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