Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/66535
標題: 家禽霍亂、雞傳染性鼻炎與水禽雷氏桿菌症之重組次單位疫苗研發
Development of Recombinant Subunit Vaccines for Fowl Cholera, Infectious Coryza and Riemerella Anatipestifer
作者: 張伯俊
關鍵字: 畜牧獸醫類
基礎研究
摘要: 家禽霍亂(fowl cholera)、雞傳染性鼻炎(infectious coryza)與水禽雷氏桿菌症(昔稱傳染性漿膜炎, infectious serositis),為台灣地區養禽產業所面臨之三種主要細菌性疾病。長久以來農民雖使用抗生素與菌苗來控制此三種疾病,但此三種疾病仍經常發生,並造成嚴重損失。本研究室先前曾成功研發以重組PlpE 蛋白,作為家禽霍亂之重組次單位疫苗,此次單位疫苗不但能保護雞隻對抗家禽霍亂,也能突破血清型之障礙,對於不同血清型之家禽霍亂皆有保護力,極具推廣價值。本計劃將以本研究室發展PlpE 疫苗之成功經驗為基礎,持續深耕家禽霍亂次單位疫苗之研發成果,並將成功經驗擴展至雞傳染性鼻炎及水禽雷氏桿菌症。本計劃為三年計劃,其中第一年度有兩個目標,目標一為探討家禽霍亂菌中另外兩個脂蛋白(PlpP 與PlpX),作為次單位疫苗之潛力。家禽霍亂之病原為Pasteurellamultocida (P. multocida),而在P. multocida 基因體中,plpP 與plpX 基因分別位於plpE基因之上、下游區域,且此三個基因所轉譯蛋白之胺基酸序列具有顯著之相似性,因此PlpE、PlpP 與PlpX 可能具有類似之功能。既然重組PlpE 次單位疫苗能提供動物有效之保護力,那麼PlpP 與PlpX 也應有成為次單位疫苗之潛力,因此本計劃第一年之目標一,是以E. coli 生產重組PlpP 與PlpX 蛋白,並進行小鼠與雞隻之免疫與攻毒試驗,以檢測PlpP 與PlpX 作為次單位疫苗之潛力。若結果為肯定,我們將以PlpE、PlpP 與PlpX 作各種配方之組合,以開發出最有效且價廉之家禽霍亂次單位疫苗。本計劃第一年度之目標二,則是測試重組PlpE 蛋白,除了家禽霍亂外,能否能作為豬、牛巴斯德桿菌症,如豬肺炎、豬萎縮性鼻炎、牛出血性敗血症等之次單位疫苗。研究方法為以重組PlpE 蛋白免疫小鼠,再分別以豬源及牛源之P. multocida 菌株進行攻毒試驗,以測試PlpE 是否有潛力作為P. multocida 所引起之豬、牛疾病之次單位疫苗。若答案為肯定,則可將PlpE 之應用範圍從家禽霍亂推廣至其它由P. multocida 所引起之豬、牛疾病。本計劃第二年度之目標,則是發展雞傳染性鼻炎之次單位疫苗,雞傳染性鼻炎是由Avibacterium paragallinarum (A. paragallinarum,昔稱Haemophilus paragallinarum)所引起,此菌目前仍無成功次單位疫苗之報導,但是A. paragallinarum 在分類上接近能引起人類幼童肺炎之病原菌Haemophilus influenzae (H. influenzae),而H. influenzae 目前在文獻上已有許多成功次單位疫苗之報導,其成份包括P4、P6、protein D 及largeadhesin 等。因此本研究將以H. influenzae 之成功經驗為模式,開發A. papadallinarum之重組P4、P6、protein D 及large adhesin 蛋白作為次單位疫苗。方法為利用生物資訊之技術,設計退化性PCR 引子(degenerated PCR primers),藉以從A. papadallinarum 中增幅並選殖出完整之P4、P6、protein D 及large adhesin 基因,並將上述基因選殖到E.coli 中大量表現與純化,再進行雞隻之免疫與攻毒試驗,以瞭解上述重組P4、P6、proteinD 及large adhesin 蛋白是否能成為A. paragallinarum 之次單位疫苗。本計劃第三年度之目標,則是研發由Riemerella anatipestifer (R. anatipestifer)感染所引起之水禽雷氏桿菌症之次單位疫苗。R. anatipestifer 目前在文獻中並無任何成功次單位疫苗之報導,但是R. anatipestifer 在分類上是屬於Flavobacteriaceae 科,在此科之菌種中,已有Flavobacterium psychrephilum 之ompH (P18) 與Ornithobacteriumrhinotracheale 之Or77 等,可作為次單位疫苗之報導。因此本研究將開發R. anatipestifer之重組OmpH (P18)、Or77 及一個稱為GldJ 之蛋白作為次單位疫苗,方法同樣是利用生物資訊的方法,設計退化性PCR 引子,進行R. anatipestifer 之ompH、or77、gldJ 基因之選殖,並以E. coli 系統進行上述基因之表現工作,藉以獲取重組蛋白,再進行鴨隻之免疫與攻毒試驗,以測試上述重組OmpH (P18)、Or77 及GldJ 蛋白,是否能成為有效之R. anatipestifer 次單位疫苗。總結而言,本計劃是以本研究室發展PlpE 次單位疫苗之成功經驗為基礎,發展FC、IC 與RA 之次單位疫苗,我們的策略皆是尋找文獻中已報導確實有效之次單位疫苗成份為標的,再以分子生物之方式進行FC、IC 與RA 菌中相對基因之選殖與表現,因此成功之機會將很高。由於FC、IC 與RA 三種疾病對台灣地區養禽產業具有相當大之重要性,而文獻中有關上述三種疾病之次單位疫苗報告也很少,因此本計劃兼具實用與學術價值,如能順利執行,將能對台灣地區養禽產業乃至生技產業之發展有所貢獻。
Fowl cholera, infectious coryza and infectious serositis are three majorbacterial diseases that lead to economic loss of the poultry industry inTaiwan. Although farmers have used antibiotics and bacterins to control thethree diseases, outbreaks of these diseases remain occurring and causingsignificant loss. Our laboratory successfully developed the PlpE subunitvaccine for fowl cholera. Chickens vaccinated with PlpE werecross-protected against the challenge with different serotypes of P.multocida. This research project is proposed based on this successfulexperience and extend this experience to the development of subunitvaccine against infectious coryza and infectious serositis.This research project is a three-year-project. In the first year, we willinvestigate whether or not PlpP and PlpX of P. multocida could serve as thesubunit vaccine for fowl cholera. The plpP and plpX genes are located at theclose vicinity of the 5'- and 3'- ends of the plpE gene and the proteinsequences of PlpP and PlpX have significant homology with PlpE. Thesefindings suggest that PlpP, PlpX and PlpE might have similar or coordinatedfunctions. Since PlpE is a good subunit vaccine, it is possible that PlpP andPlpX are also good vaccine candidates. Therefore, we will produce andpurify recombinant PlpP and PlpX using the E. coli system and investigatewhether or not recombinant PlpP and PlpX protect chickens and miceagainst challenge with P. multocida. If recombinant PlpP and PlpX do conferprotections, we will test different combinations of PlpP, PlpX and PlpE todevelop the most effective and economic vaccine for P. multocida. Thesecond goal of the first year study is to test whether or not recombinantPlpE protect animals against porcine and bovine diseases caused by P.multocida. Towards this goal, we will immunize mice with recombinant PlpEand challenge the mice with strains of P. multocida that cause bovinepneumonia, bovine atrophic rhinitis and porcine haemorrhagic septicaemia.Achievements of this study might extend the use of PlpE to control porcineand bovine diseases caused by P. multocida.The goal of the second year study is to develop subunit vaccine againstinfectious coryza caused by Avibacterium paragallinarum (also known asHaemophilus paragallinarum). To date no successful subunit vaccine hasbeen developed for A. paragallinarum; however, A. paragallinarum isclosely related to Haemophilus influenzae that causes pneumonia ofchildren; moreover, several subunit vaccines, including those containing theP4, P6, protein D, or the large adhesion were developed for H. influenzae.On basis of these results, we will design degenerated PCR primers toamplify P4, P6, protein D, and large adhesion genes from A. paragallinarum.Recombinant proteins encoded by these genes will be expressed in E. colisystem and their efficacy in protecting chickens against challenge of A.paragallinarum will be evaluated.The goal of the third year study is to develop subunit vaccine againstinfectious serositis caused by Riemerella anatipestifer (RA). To date nosuccessful subunit vaccine has been developed for R. anatipestifer. However,R. anatipestifer belongs to the family of Flavobacteriaceae and two subunitvaccines have been developed for control of diseases caused by bacteria ofthis family; one is the OmpH (P18) for Flavobacterium psychrephilum andthe other is Or77 for Ornithobacterium rhinotracheale. Based on theseresults, we use bioinformatic methods to design degenerated PCR primersto amplify ompH (P18), or77, gldJ genes from R. anatipestifer. Recombinantproteins encoded by these genes will be expressed in E. coli system andtheir efficacy in protecting ducks against challenge of R. anatipestifer will beevaluated.In summary, we proposed to develop subunit vaccines against FC, IC andRA based on our successful experience in developing the PlpE subunitvaccine. Our strategy is to search in the literature for genes encodingsuccessful subunit vaccines and then use molecular methods to clone andexpress the corresponding genes from P. multocida, A. paragallinarum andR. anatipestifer. We believe that this strategy shall work well. Because thecontrol of FC, IC and RA are very important for the poultry industry inTaiwan and only few reports are available on developing subunit vaccinesagainst the three diseases, this research project will be of both academicand practical values and will help the progress of poultry industry andbiotechnology industry in Taiwan.
URI: http://hdl.handle.net/11455/66535
其他識別: NSC97-2313-B005-008-MY3
文章連結: http://grbsearch.stpi.narl.org.tw/GRB/result.jsp?id=1666637&plan_no=NSC97-2313-B005-008-MY3&plan_year=97&projkey=PD9709-0222&target=plan&highStr=*&check=0&pnchDesc=%E5%AE%B6%E7%A6%BD%E9%9C%8D%E4%BA%82%E3%80%81%E9%9B%9E%E5%82%B3%E6%9F%93%E6%80%A7%E9%BC%BB%E7%82%8E%E8%88%87%E6%B0%B4%E7%A6%BD%E9%9B%B7%E6%B0%8F%E6%A1%BF%E8%8F%8C%E7%97%87%E4%B9%8B%E9%87%8D%E7%B5%84%E6%AC%A1%E5%96%AE%E4%BD%8D%E7%96%AB%E8%8B%97%E7%A0%94%E7%99%BC
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