犬瘟熱病毒Hemagglutinin基因之親源分析及其表現

Phylogenetic Analysis and Expression of the Hemagglutinin Gene of Canine Distemper Virus

何志祥(Chin-Hsiang Ho)

指導教授 ： 林茂勇

國立屏東科技大學/農學院/獸醫學系所/碩士(2008年)

https://doi.org/10.6346/NPUST.2008.00062

摘要

犬瘟熱 ( Canine Distemper；CD ) 是由負向單股RNA之副黏液病毒科 ( Paramyxoviridae ) 的麻疹病毒屬 ( Morbillivirus ) 之CD病毒 ( CDV ) 所引起的好發於3-6月齡幼犬之高傳染性呼吸道性、消化道性、神經性及皮膚性致死性疾病。CDV之H蛋白為具高度抗原變異性之誘發病毒吸附宿主細胞的具免疫保護力抗原。為探索台灣野外CDV-H基因的變異情形，本研究以具高度敏感性和高特異性之反轉錄聚合酶鏈反應( RT-PCR ) 增幅14株野外分離株與1株疫苗株之H全長基因 ( 1877 bp )，經選殖和定序後以DNASTAR及Mega 4軟體分析，並與GenBank中其他28株CDV之H基因序列進行其親源關係分析，依目前的CDV可分成六大群，而台灣分離株歸屬於Asia-1群。本實驗所採集本地分離株CDV之H蛋白全長基因經與疫苗株比對結果在核苷酸序列上相差為8.1-11.1 ％，在胺基酸序列之差異為8.7-13.2 ％，顯示台灣分離CDV之H基因在核苷酸與胺基酸序列上與傳統疫苗株間的變異度頗大。本試驗另以增幅出之全長H基因以PCR- restriction fragment length polymorphism ( RFLP ) 的方式進行犬隻病毒感染之研究，發現以Sma I限制酶可用於區分疫苗株及野外株。本研究選PT15/TW05本地分離株以真核表現系統可表達出約68 KDa之H 重組蛋白，且利用西方轉漬法 ( Western blotting ) 可初步確認表現蛋白之正確性。該次單位蛋白或可供將來之CDV分子生物學的研究應用。

關鍵字

H蛋白 ； 親源性分析 ； PCR-RFLP ； 西方轉漬法

並列摘要

Canine distemper virus ( CDV ) is a negative-stranded RNA virus belonging to genus Morbillivirus within the family Paramyxoviridae. Canine distemper ( CD ) is a highly contagious disease in pupies, particularly in 3 to 6 months of age, with high morbidity and mortality. CD in dogs may be manifested by a combination of respiratory, ocular, gastrointestinal, neurologic and cutaneous signs. CDV-Hemagglutinin ( H ) protein encoded by H gene provides a highly antigenic variation and works as the immunoprotective protein in initiating the cellular attachment to the host cells. In order to study the variation of nucleotide sequence on CDV-H gene, a pairs of primers were designed for reverse transcription-polymerase chain reaction ( RT-PCR ) amplification the full length H gene ( 1877 bp ) of 14 strains of Taiwanese isolates and 1 vaccine strain with high sensitivity and specificity. The amplified products were then cloned, sequenced and phylogenetic analysed with other 28 strains of CDVs collected from the GenBank by the software of DNASTAR and Mega 4.
The Taiwanese CDVs was classified into one ( Asia-1 ) genotype among the current existing 6 types. The difference on nucleotide sequence and amino acid of the H gene between the Taiwanese isolates and the vaccine strain was 8.1-11.1 ％and 8.7-13.2 ％respectively. Taiwanese CDV isolates of CDV provides the highest genetic variation on the H gene from the vaccine strain among the 5 CDV genetypes. In addition, a strain of Taiwanese isolate ( PT15/TW05 ) was chosed for eukaryotic expression of the CDV-H recombinant protein with the yield of 68 KDa protein further verified by Western blotting. The recombinant H protein might be good for use in the future molecular biological study of CDV.

並列關鍵字

H protein ； Phylogenetic Analysis ； PCR-RFLP ； Western blotting

參考文獻

黃宣憲。犬瘟熱感染犬隻臨床檢體之病毒核酸偵測、定量及H基因之序列分析。台灣大學碩士論文。台北。台灣。2005。

Alldinger, S., W. Baumgärtner, P. van Moll and C. Orvell（1993）In vivo and in vitro expression of canine distemper viral proteins in dogs and non-domestic carnivores. Arch. Virol. 132:421-8.

Alleman, A. R., M. M. Christopher and D. A. Steiner（1992）Identification of intracytoplasmic inclusion bodies in mononuclear cells from the cerebrospinal fluid of a dog with canine distemper. Vet. Pathol. 29: 84-85.

Appel, M. J. and B. A Summers（1995）Pathogenicity of morbilliviruses for terrestrial carnivores. Vet. Microbiol. 44: 187-91.

Appel, M. J., C. Reggiardo, B. Summers, A. S. Pearce-Kelling, C. J. Mare, T. H. Noon, R. E. Reed, J. N. Shively and C. Orvell（1991）Canine distemper virus infection and encephalitis in javelinas (collared peccaries). Arch. Virol. 119: 147-52.