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標題: | 蝦白點症病毒蛋白激?基因之研究 Study of the protein kinase gene of shrimp white spot syndrome virus |
作者: | Wang-Jing Liu 劉宛菁 |
出版年 : | 2000 |
學位: | 碩士 |
摘要: | 本論文以蝦白點症病毒(WSSV)長3.4kb的基因體片段進行序列分析及基因鑑定。此片段含有五個開放譯讀區(open reading frames,ORFs),其中ORF1與蛋白激?(protein kinase,PK)酵素作用區(catalytic domain)有很高的相似性。ORF2可轉譯出含204個胺基酸的蛋白質,為已知的蝦白點症病毒膜蛋白VP28。ORF3、ORF4和ORF5分別可轉譯出193、200及229個胺基酸的蛋白質。利用反轉錄聚合?鏈反應進行基因轉錄時序性分析,結果可知ORF1、VP28及ORF3為早期表現的基因,ORF4及ORF5則屬於晚期表現的基因。由於pk基因在病毒的致病力上扮演重要的角色,因此本研究針對蝦白點症病毒pk1基因進行選殖、定序及其特性分析。經由北方雜合反應結果顯示ORF1之轉錄子為2.7kb,而ORF1在3.4kb的片段上僅1020個核?酸長,因此首先對蝦白點症病毒pk1完整基因進行選殖。經選殖及鑑定後可知蝦白點症病毒pk1基因全長共2193個核?酸序列,可轉譯出730個胺基酸,分子量為81kDa,由胺基酸序列特性分析可知pk1基因為serine/threonine的蛋白激?。蝦白點症病毒PK1胺基酸序列含有蛋白激?所特有之保守性區域,subdomain I與II與其他物種進行比對其相似度介於24%?41%,subdomain VI其相似度介於69%?92%之間,subdomain VII相似度介於44%?77%之間,subdomain VIII相似度介於45%?63%之間及subdomain IX相似度介於21%?51%之間。分析蝦白點症病毒pk1基因結構可發現兩個TATA box分別位於轉譯起點上游80至75及19至14個核?酸的位置,而在3'端具有加聚嘌呤信號(AATAAA,poly A signal)。經試管內轉錄及轉譯分析結果顯示蝦白點症病毒PK1分子量為87kDa與電腦預測值相近,並且試管內所得之87 kDa產物可被抗蝦白點症病毒PK1重組蛋白抗血清所辨認。 In the present study, a 3.4 kbp DNA fragment from white spot syndrome virus (WSSV) genomic HindIII library was studied. Following DNA sequencing and computer program prediction, five open reading frames (ORFs) were identified in this fragment. ORF1 has a high similarity to the protein kinase (PK) catalytic domain. ORF2 is identical to a previously reported WSSV envelope protein (VP28). Orf3, orf4 and orf5 potential encode for polypeptides of 193, 200 and 229 amino acids. Temporal analysis of the ORFs showed that ORF1, VP28 and ORF3 were first expressed at an early stage, but ORF4 and ORF5 were expressed at late stage. PK plays an important role in the pathogenicity of viruses, so this study focused on the cloning, sequencing, and characterization of WSSV pk1 gene. Northern blot analysis with an orf1-specific riboprobe detected a major transcript of 2.7 kb, which indicated that the 3.4 kbp fragment contained only part of WSSV pk1 gene. The full length WSSV pk1 gene was found in an 8421bp fragment from an EcoRI library. It contains 2193 nucleotides and encodes an 81-kDa protein of 730 amino acids. WSSV PK1 contains serine-threonine protein kinase consensus motifs I through XI. Compared to the PKs of other species, amino acid similarities are 24?41% for subdomains I and II; 69?92% for subdomain VI; 44?77% for subdomain VII; 45?63% for subdomain VIII; and 21?51% for subdomain IX. Analysis of WSSV pk1 gene structure indicated two TATA boxes at positions 75?80 and 14?19 nucleotides upstream of the translation start site, and a poly A signal (AATAAA) also located at the 3' terminal of the gene. Expression of WSSV PK1 with a rabbit reticulocyte generated an 87-kDa protein, which is in close agreement with the predicted size of 84-kDa from the amino acids sequence. This in vitro transcription and translation product was recognized by anti-WSSV PK1 recombinant protein antiserum. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75141 |
全文授權: | 未授權 |
顯示於系所單位: | 動物學研究所 |
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