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標題: 雞傳染性鼻炎重組次單位疫苗之研發
Development of recombinant subunit vaccines for infectious coryza
作者: 潘彥江
Pan, Yen-Chiang
關鍵字: Avibacterium paragallinarum
infectious coryza
RTX toxins
HA protein
RTX 毒素
出版社: 微生物暨公共衛生學研究所
引用: 林茂勇,宋華聰。2006。禽病診治(第二版)。藝軒圖書出版社。台北市。中華民國。2,177-180。 陳秉誼。2009。雞傳染性鼻炎菌莢膜生合成基因之分析與重組次單位疫苗之研究。國立中興大學獸醫微生物學研究所。 Abascal, E.N., Guerra, A.C., Vazquez, A.S., Tenorio, V.R., Cruz, C.V., Zenteno, E., Contreras, G.P., Pacheco, S.V., 2009, Identification of iron-acquisition proteins of Avibacterium paragallinarum. Avian Pathol 38, 209-213. Barnard, T.G., Van Heerden, E., Bragg, R.R., Albertyn, J., 2008, Haemophilus paragallinarum haemagglutinin: role in adhesion, serotyping and pathogenicity. Onderstepoort J Vet Res 75, 11-16. Bisgaard, M., Christensen, J.P., Bojesen, A.M., Christensen, H., 2007, Avibacterium endocarditidis sp. nov., isolated from valvular endocarditis in chickens. Int J Syst Evol Microbiol 57, 1729-1734. Blackall, L.L., 1995, Vaccines against infectious coryza. Blackall, P.J., 1991, An Evaluation of the Cross-Protection Afforded by Inactivated Infectious Coryza Vaccines. Aust Vet J 68, 266-267. 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摘要: 雞傳染性鼻炎 (infectious coryza) 係由Avibacterium paragallinarum感染雞隻造成上呼吸道疾病,常導致肉雞淘汰率增加與蛋雞產蛋性能下降,造成業界嚴重經濟損失。Av. paragallinarum目前可分為3種血清型,臺灣的田間感染以死菌疫苗免疫來控制,目前台灣地區每年所使用之疫苗數目皆在億劑以上。本研究可分為兩部分,第一部份是以新世代基因體定序技術,對血清型C型標準株H18株進行全基因體定序。以定序分析結果發現,在H18基因體序列中,存在多種與毒素合成相關的基因,其中RTX toxins (repeats in toxin) 合成相關基因為目前巴斯德桿菌科中最重要的毒力因子之一,可做為次單位疫苗成份。以PCR進行分析,發現不同之Av. paragallinarum菌株中,皆存在RTX toxins之基因。本研究為首度在Av. paragallinarum中找到RTX toxins基因之報導。H18株中所找到之 RTX 毒素操縱子 (operon) 基因群,命名為avxIABD,其中avxIA為主要之毒素基因,而avxIB及avxID則與毒素之分泌相關基因。AvxIA之基因全長約6.8Kb,可表現出大小為2286胺基酸之蛋白。以Pfam程式分析發現,AvxIA蛋白序列中存在12個Hemolysin type calcium-binding repeats與1個RTX C-terminal domain,此特性與其它RTX毒素類似。另一方面,AvxIA蛋白中也存在1個Peptidase_S8 domain,以及1個Proprotein convertase P-domain,此二結構區 (domains) 在其他RTX毒素中並未被發現過。為瞭解其中Peptidase_S8及Proprotein convertase P-domain之功能與抗原性,本研究將能表現含此二結構區之基因片段,選殖至原核表現系統中表現出85 kDa大小的重組蛋白,本研究以Western blot進行分析,結果顯示此蛋白可被標準株H18的高免血清辨認,未來將以此段蛋白研究生物功能與進行動物試驗,評估是否能做為疫苗抗原使用。本研究之第二部份,是針對先前已發表之Av. paragallinarum HA蛋白rF5片段進行分析,結果發現rF蛋白之主要抗原決定區,是位於271-462 bp位置,由此位置所表現之重組蛋白具有做為次單位疫苗之潛力。
Infectious coryza (IC) is an acute upper-respiratory disease caused by infection with Avibacterium paragallinarum. The disease is distributed worldwide and causes significant economic loss of poultry industry. In Taiwan, more than one hundred million doses of IC vaccine have been used every year to control the disease. The goal of this study is twofold. The first is to use the next generation sequencing technology to conduct sequence analyses of the complete genome of Av. paragallinarum strain H18, which is a serovar C reference strain. The result showed that H18 contains a number of genes involved in the biosynthesis of toxins. Among these toxins, the RTX toxin (repeat in toxin) is an important virulence factor of bacteria in the family Pasturellaceae. The RTX toxin has been used as a component of subunit vaccine. PCR analyses showed that the RTX gene is present in many strains of Av. paragallinarum. The gene encoding the RTX toxin is located at an operon named avxIABD, in which avxIA encodes the toxin while avxIB and avxID encode proteins required for secretion of the RTX toxin. The avxIA gene is 6861bp in size and has the potential to encode a protein of 2286 amino acid. Pfam analyses showed that AvxIA contains 12 copies of the hemolysin type calcium-binding domain and one copy of RTX C-terminal domain. These two domains have been found in other RTX toxins. However, AvxIA contains a peptidase_S8 domain and proprotein convertase P-domain, which are not found in other RTX toxins. To investigate the antigenicity and function of the peptidase_S8 domain and proprotein convertase P-domain, an 85 kDa recombinant protein containing these two domains were expressed in E. coli and purified with a Ni-affinity column. Western blot analyses showed that this recombinant protein reacted with hyper-immune serum against the whole-cell antigen of strain H18. The biological function of this recombinant and potential of using this protein as a vaccine antigen will be evaluated. The second part of this study is to investigate the antigenictity of the rF5 fragment from the HA protein of Av. paragallinarum. The result showed that the major antigenic determinant sites of rF5 is located at the 271-462 bp of the HA protein. Recombinant protein contain this region might have potential to be used as the antigens for subunit vaccines against Av. paragallinarum.
其他識別: U0005-1408201115564500
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