豬霍亂沙門氏桿菌鞭毛蛋白之免疫力分析

Immunogenicity Analysis of Flagellin of Swine Salmonella enterica Serovar Choleraesuis

謝耀清(Yao-Ching Hsieh) ； 黃彥霖(Yen-Lin Huang) ； 蔡耿宇(Keng-Yu Tsai) ； 吳文鑾(Wen-Luan Wu)

《臺灣獸醫學雜誌》 36卷2期 (2010/06) Pp. 155-165

https://doi.org/10.7009/TVJ.201006.0155

摘要

豬霍亂沙門氏桿菌(Salmonella enterica serovar Choleraesuis)長期潛伏於豬場，且抗藥性菌株產生，造成養豬產業的重大經濟損失。本研究以引子對FliC-F和FliC-R增幅鞭毛蛋白基因(fliC)，並比較標準菌株及近4年16株野外分離株的鞭毛蛋白之氨基酸序列，顯示菌株間之鞭毛蛋白序列無顯著變異。將標準菌株之fliC基因構築於pGEX4T-1載體，轉型至E.coli BL21以大量表現鞭毛蛋白，將純化的鞭毛蛋白以10、20、50μg不同濃度以腹腔接種於BALB/c小鼠兩次，間隔二週。小鼠每週固定採集眼窩血分離血清進行酵素連結免疫分析法(enzyme-linked immunosorbent assay; ELISA)檢測抗體，二次免疫50μg組與陽性對照組（市售減毒疫苗，Suisaloral(上標 ®)）抗體均可達160X。此外，在添加刀豆蛋白(con A)條件下培養免疫接種小鼠的脾臟細胞，利用ELISA檢測上清液內IL-2、IFN-γ和TNF-α三種細胞激素含量，二次免疫50μg組的小鼠脾臟細胞分泌量分別為529、849及392pg/mL，顯著高於其他組別，僅略低於陽性對照組。在攻毒試驗中，二次免疫50μg組存活率為100%且無臨床症狀。綜合上述實驗結果，豬霍亂沙門氏桿菌的鞭毛蛋白具有激起宿主體液性和細胞性免疫反應的能力，可有效抵抗豬霍亂沙門氏桿菌感染，顯示鞭毛蛋白可當作次單位疫苗或免疫佐劑的選擇，進而應用於防治豬霍亂沙門氏桿菌。

關鍵字

豬霍亂沙門氏桿菌 ； 鞭毛蛋白 ； 免疫力

並列摘要

Salmonella enterica serovar Choleraesuis is one of the predominant pathogens in farms of Taiwan. Typical clinical manifestations include septicemia, diarrhea, weight loss, growth retardation, and even death. This pathogen causes a huge economic loss due to the emergence of antibiotics-resistant strains. It is important to develop a safe and more effective vaccine. In this study, FliC-F and FIiC-R were used as primers to amplify flagellin gene (fliC). The flagellin sequences of the standard strain and those of 16 recently isolated strains were aligned, showing that the fligellin sequences of these strains were similar. The recombinant flagellin was expressed in F. coli BL21, purified, and used to immunize BALB/c mice at 10, 20 or 50 μg via intraperitoneal route for twice at a 2-week interval. Blood samples were weekly collected by orbital bleeding and antibody responses were determined by enzyme-linked immunosorbent assay (ELISA). The mice immunized with 50 μg twice had the same magnitude of antibody response (160X) as those immunized with the commercialized attenuated vaccine, Suisaloral(superscript ®). Spleen cells of immunized mice were cultured and the amounts of cytokines such as IL-2, IFN-γ, and TNF-α in the supernatants were determined by ELISA. The levels of IL-2, IFN-γ and TNF-α were 529, 849 and 392 pg/mL, respectively, in mice immunized twice with 50 μg of flagellin, which was comparable with the result of the group immunized with the attenuated vaccine. Immunized mice were challenged with Salmonella enterica serovar Choleraesuis. The survival rate was 100% in mice immunized twice with either 50 μg of flagellin or the attenuated vaccine. The results suggested that the flagellin could provide protection against Salmonella enterica serovar Choleraesuis and had the potential as a subunit vaccine against the infection of Salmonella enterica serovar Choleraesuis.

並列關鍵字

Salmonella enterica serovar Cholersesuis ； flagellin ； antigenicity