Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/15215
標題: 巴氏桿菌毒素次單位DNA疫苗之免疫效力評估
Efficacy study of recombinant Pasteurella multocida toxin (PMT) DNA vaccine
作者: 吳誌銘
Wu, Chi-Ming
關鍵字: Pasteurella multocida toxin
巴氏桿菌毒素
PMT
DNA vaccine
vaccine
DNA疫苗
次單位疫苗
出版社: 獸醫病理學研究所
摘要: 已知Pasteurella multocida產生的毒素(P. multocida toxin;PMT)是引起豬萎縮性鼻炎(atrophic rhinitis;AR)的主要致病因子。本實驗構築能表現完整與次單位PMT重組蛋白質之真核表現載體,並進一步藉由DNA疫苗免疫後,偵測特異性抗體及其分泌細胞被激活之情形來評估防治AR與日後應用於其他疾病之可行性。首先利用承載human cytomegalovirus promoter/ enhancer region的真核細胞表現載體,選殖PMT基因的各個片段而分別構築成pcDNA4.0/Tox1、Tox2、Tox7與完整PMT等四個選殖株;重組載體以細胞轉殖作用(Transfection)送入Vero cell、HeLa cell、PK-15 cell與Cos-7 cell等不同種類細胞株中,並成功誘發轉殖細胞表現PMT或次單位重組蛋白質,被轉殖之細胞同時可被特異性單株抗體所辨識,且重組蛋白質在各細胞內表現後並不會造成細胞型態改變或產生任何細胞病變。在動物試驗上,首先以各重組載體DNA進行BALB/c小白鼠後肢肌肉之免疫注射;結果顯示經免疫兩次後即能以ELISA(Enzyme-Linked Immunosorbant Assay)偵測到血清中特異性抗體之產生;此外,為進一步評估免疫動物其脾臟中特異性抗體分泌細胞(antibody secreting cells;ASC)數量之多寡與抗原之記憶性,亦建立以in vitro ELISPOT assay之方法來進行免疫效力評估,發現免疫組之脾臟細胞中特異性ASC數目明顯多於對照組之小白鼠,而再經攻毒試驗後ASC數目更大幅增加;經由兩種評估方式皆證實subunit PMT DNA vaccine的確可誘發小白鼠產生特異性免疫反應。而在攻毒試驗中,小白鼠經免疫三次後,所有試驗組皆能耐過1×與5×LD50的PMT challenge(1×LD50:1.3 μg PMT),且pcDNA/Tox2與pcDNA/Tox7免疫組甚至能抵抗10×LD50 PMT之攻擊,故本試驗證實DNA疫苗在動物模式中的確可誘發特異性免疫反應並保護小白鼠抵抗PMT攻擊;將進一步進行DNA疫苗對豬隻之免疫效力評估。
Pasteurella multocida toxin (PMT) is the major virulence factor associated with progressive atrophic rhinitis (AR), an important disease of upper respiratory system in swine. The purpose of this study is to construct the eukaryotic expression system for the recombinant subunit PMT and to evaluate the efficacy of this DNA vaccine in animal model. The recombinant plasmid vectors containing complete and partial length of PMT gene, including pcDNA4.0/PMT (nt. 216~4058), Tox1 (nt. 216~1666), Tox2 (nt. 1666~3168) and Tox7 (nt. 3168~4058), could successfully express the recombinant subunit PMT in Vero, HeLa, PK-15 and Cos-7 cells respectively. The transfected cells did not exhibit any morphologic change or specific nodule-like cytopathic effect (CPE) and could be identified by immunofluorescent staining and flow cytometry analysis. In animal model, the BALB/c mice were immunized intramuscularly with different recombinant plasmid vectors three times at 3 week intervals respectively, and finally challenged with 5 or 10 LD50 PMT (LD50: 1.3g PMT). After the second vaccination, antibody titers from all immunized mice increased obviously as tested by ELISA. Moreover, the number of antibody secreting cells (ASCs) isolated from the spleen of the immunized mice also increased markedly when cells were stimulated with subunit PMT protein in vitro and detected by ELISPOT analysis. After 5LD50 PMT challenge, all vaccinated mice could mount a good protective immunity except that pcDNA/PMT vaccinated group showing only 40% survivors. However, the pcDNA/Tox2 and pcDNA/Tox7 immunized mice could survive after 10LD50 PMT challenge. All these results demonstrated that the subunit PMT DNA vaccines provided good protective immunity against PMT in mice.
URI: http://hdl.handle.net/11455/15215
Appears in Collections:獸醫病理生物學所

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