豬胸膜肺炎放線桿菌第1 型細胞毒素之定性

將豬胸膜肺炎放線桿菌（Actinobacillus pleuropneumoniae, Ap）培養上清液以50%硫酸銨濃縮後，利用液相層析法（gel filtration）純化105 kDa的第1型細胞毒素（Apx I）。經硫酸銨濃縮後測得的溶血力價減少的99.75%，層析純化後溶血能力則完全消失。以感染豬恢復後血清利用斑點免疫法（blot assay）分析層析後的收集液，發現主要之抗原－抗體反應位於4個峰中的第1、2個峰的收集液，由分子量標的物（molecular weight marker）推算，分子量大於440 kDa。利用SDS-PAGE電泳和免疫轉漬試驗（immunoblot）分析第1、2個峰的收集液，發現分子量105 kDa的第1型細胞毒素（Apx I）是反應抗原。因此在濃縮、純化過程中第1型細胞毒素會互相結合而形成聚合物，可能因而造成溶血力價的降低。利用純化細胞毒素抗原免疫兔子所得之高免抗血清以雙向免疫擴散試驗，測得之抗體力價為1:64遠高於恢復豬血清的1:4，但是中和抗體力價只有1:512，低於恢復血清的1 :4096。因此細胞毒素聚合物的形成也會影響中和抗體的產生。

關鍵字

胸膜肺炎放線桿菌；第1型細胞毒素

並列摘要

Purification of Actinobacillus pleuropneumoniae (AP) RTX-toxin I (Apx I) from culture supernatant of Ap serotype 1 was done by gel filtration. However, hemolytic activities of purified Apx I showed significant decrease after purification. The culture supernatant antigens were detected in the first and second peak of fractions by dot blot assay with convalescent serum. The molecular weight of these antigens were greater than 440 kDa. However, the Apx I was the main component of antigens of peak 1 and 2 when analyzed by SDS-P AGE and immunoblotted with convalescent serum. The results suggested that Apx I formed aggregates during the process of purification. Titers of rabbit hyperimmune serum against purified Apx I was 1:64 higher than convalescent serum (1:4) detectd by double immuno-diffusion test. Contrarily, hemolytic neutralization titer of hyperimmune serum against Apx I was 1:512 much lower than convalescent serum (1:4096). Passive immunization with antiserum against purified Apx I could protect mice from challenging with virulent Ap serotype 1 but its efficacy was lower than convalescent serum. It was concluded that aggregation of ApxI during purification would affect protect efficacy.

並列關鍵字

Actinobacillus pleuropneumoniae ； Apx I

國際替代計量

豬胸膜肺炎放線桿菌第1 型細胞毒素之定性

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