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Construction of a Salmonella choleraesuis crp mutant
豬霍亂沙門氏桿菌 (Salmonella choleraesuis) 主要引起豬細菌性敗血症，由於其屬於細胞內寄生菌且具多重抗藥性，導致藥物治療極為不易，同時也成為公共衛生及疾病防疫上的一大隱憂。目前沙門氏桿菌症之防治，除加強衛生管理外，減毒活菌疫苗的使用亦為一有效之防疫策略，由於減毒活菌疫苗不僅可以引起宿主之體液性免疫反應，尚可激起細胞性免疫反應，而口服免疫的方式更可使腸道產生分泌型IgA抗體，大幅改善傳統死菌疫苗僅能引起體液性免疫反應之缺點。本實驗室利用自殺載體 (suicide vector; pGP704) 進行標誌基因之插入突變 (insertion mutation) ，以構築crp基因缺損株，並經PCR篩選、基因定序及Southern hybridization確認後，再利用生長曲線及Biolog GN2套組檢驗其表現型，結果發現基因缺損株之生長速度及碳源代謝之能力確實與parent strain不同。進一步於小鼠進行安全性試驗，結果發現基因缺損株經腹腔或口服攻毒後，其LD50分別提高為3.5×104 CFU及2.1×1010 CFU以上，較parent strain分別高出103及104倍以上，此外，經腹腔或口服免疫基因缺損株之小鼠，亦能承受高致死劑量之攻毒，顯示所構築之基因缺損株於小鼠的確具高安全性及免疫保護性。在病理學檢查方面，實驗小鼠以相同劑量之parent strain及基因缺損株攻毒5-6天後，經parent strain攻毒之小鼠已有典型之臨床症狀及組織病理學病變，但經基因缺損株攻毒之小鼠仍無相同之臨床症狀，且其肝臟及脾臟之組織病理學病變亦較parent strain攻毒之小鼠輕微或無任何變化。另外，小鼠以1×108 CFU之基因缺損株進行免疫後，其血清抗體力價有明顯提升之情形，但免疫小鼠之脾臟淋巴細胞經沙門氏桿菌抗原刺激後，卻無顯著之增殖，因此針對沙門氏桿菌所激起之細胞性免疫反應，仍須更進一步加以探討及研究。以上結果顯示本實驗室已建立豬霍亂沙門氏桿菌特定基因突變之技術，並成功構築一於小鼠之實驗動物模式下減毒且具免疫
The diarrheagenic disease Salmonellosis caused by Salmonella choleraesuis and S. typhimurium is among the most important bacterial swine diseases worldwide which featured with gastroenteritis as well as systemic septicemia. Because the bacteria reside intracellularly, it does not respond well to antibiotics and results in developing multi-drugs resistant strains. The live-attenuated vaccine recently has become an extraordinary strategy in preventing Salmonella infection in swine due to its ability to elicit both humoral and cellular immunity along with IgA secretion after oral immunization. The aim of this study is to construct a S. choleraesuis crp gene knockout mutant and to utilize it as a potential live attenuated vaccine strain. In this trial, the suicide vector pGP704 and double marker genes selection strategies via homologous recombination had been applied. The selected candidate mutant strains were further screened with PCR detection, sequencing and southern hybridization for genotype identification. Moreover, the Biolog GN2 Microplate assay was also applied for phenotype identification. The results showed that the crp mutant strain not only grew slowly but also reduced its carbon sources utilization in comparison with parent strain. In addition, the mutant strain showed less virulence in mice according to its LD50 displayed 103 and 104 times more than parent strain via intraperitoneal (3.5 104 CFU) and oral (2.11010 CFU) inoculation respectively. The immunized mice also showed highly resistant to lethal dose challenge with parent strain and induced less clinical signs and histopathological lesions in both liver and spleen. The serum antibody titers of mice increased dramatically after immunized with 1108 CFU of mutant strain, while no significant change of lymphocyte proliferation assay was noticed two weeks after each immunization. These results demonstrated that the crp mutant of S. choleraesuis had been successfully constructed and showed attenuation in mouse model, we suggested it could provide as a vaccine candidate for further trial in swine in the near future.
|Appears in Collections:||獸醫病理生物學所|
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