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研究生:曾喜暖
研究生(外文):HsiNuan Tseng
論文名稱:以大腸桿菌表現並純化日本腦炎病毒之核心蛋白
論文名稱(外文):Expression and purification of the capsid protein of Japanese encephalitis virus in E. coli
指導教授:王孟亮
學位類別:碩士
校院名稱:國立中興大學
系所名稱:獸醫微生物學研究所
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:49
中文關鍵詞:日本腦炎病毒核心蛋白之選殖表現和純化
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摘要
日本腦炎病毒(Japanese Encephalitis Virus;JEV)屬黃病毒科、黃病毒屬(Flavivirus),為單股正向(positive-sense) 具封套之 RNA病毒。在病毒的結構蛋白中,核心蛋白為分子量最小(約11 kD)且具高鹼性,它將genomic RNA包裹之後形成ribonucleoprotein complex。本實驗以日本腦炎病毒減毒疫苗株為材料,進行reverse transcriptase-polymerase chain reaction (RT-PCR)選殖核心蛋白基因(84 — 476 bp),將其接至載體pET32a後轉至大腸桿菌BL21(DE3),經IPTG (isopropylβ-D-thiogalactopyranoside)誘導重組蛋白表現。將定序後的序列輸入GenBank置換成胺基酸序列,發現其鹼性胺基酸lysine和arginine佔21﹪(26/126)。比較黃病毒屬中各病毒的胺基酸序列發現有三段保留的區域,第一段為自基因體RNA 5’起算第二個轉譯起始密碼的位置,序列為Asn- Met-Leu-Lys- Arg五個胺基酸;第二段是位於核蛋白的C端共14個胺基酸組成,這一段序列是黃病毒屬中保留性最高的一段,其序列為WLASLAVVIAYA GA;而第三段保留序列則發現在核蛋白的胺基酸序列自N端起第42個胺基酸後連續的16個胺基酸組成的疏水區,這段序列在JEV為FVLATITFFKFTALA PT 16個胺基酸。全長的日本腦炎核蛋白基因在E.coli經IPTG誘導後表現量不高,進而針對胺基酸序列上的兩段主要的疏水性胺基酸的片段,分別在相對應的基因序列上以限制酵素切除,其表現產物利用聚丙醯烯胺電泳分析,發現融合蛋白(fusion protein)的表現明顯增加。以PCR增幅日本腦炎5’端非轉譯區和部份的核蛋白基因(242 bp),將這段DNA與純化的核心蛋白進行gel mobility shift assay,研究兩者之結合性質,結果發現以E. coli表現的核心蛋白可以與5’端非轉譯區的DNA結合,使DNA在膠體上泳動的速度延遲,另外加入mouse anti-JEV coreDD抗體之後可以使DNA在膠體泳動的速度減緩。

Abstract
Japanese encephalitis virus (JEV) is a single-stranded, enveloped RNA virus belongs to the family Flaviviridae. The genomic RNA of JEV is approximately 11 kb long and has positive polarity. The nucleocapsid (core) protein, the smallest structural protein (11 kD), forms the viral nucleocapsid. In this study, the cDNA of the viral core protein gene was amplified by RT-PCR (reverse transcriptase-polymerase chain reaction) and the products were cloned into the pET32a expression vector. Then the core protein expression plasmids were transformated into the E. coli strain BL21 (DE3) and the expression clones were induced by IPTG(isopropyl-β-D-thiogalactopyr- anoside). Sequence analysis of amino acid (26/126) shows that core protein is rich in basic amino acid residues (~ 21%). A comparison of different flavivirus C proteins reveals that there are three conserved regions in the core amino acid sequence. First, there is a conserved sequence box of five amino acids (Asn-Met-Leu-Lys-Arg) at the second start codon downstream of the first start codon. Second, the most significantly conserved characterictics are the hydrophobic sequence (WLASLAV VIAYAGA) at the carboxy-terminus. Third, the conserved hydrophobic region located from F42 to T57 (FVLATI TFFKFTALA PT). In order to increase the production of core protein, two hydrophobic regions truncated core expression vectors were constructed. The full length and deletion constructions induced by IPTG, and the products were analysis by SDS-PAGE. The yields of expressed proteins of hydrophobic deletion mutants were better than that of full-length core protein. In addition, we purified the bacterially expressed core protein, and examined its binding to the DNA fragment of 5’-NCR and partial core gene of JEV. The results of mobility shift analysis demonstrated that the recombinant core protein could bind to the DNA fragment.

目次
中文摘要…………………………………………………………………I
英文摘要…………………………………………………………………II
目次………………………………………………………………………IV
圖次………………………………………………………………………VI
第一章 緒言……………………………………………………………1
第二章 文獻探討………………………………………………………3
2-1 JEV的一般理化特性………………………………………….3
2-2 JEV之生態學……………………………………..……………3
2-3黃病毒屬的分類………………………………………………..5
2-4 JEV基因體的研究……………………………………………..6
2-5 JEV蛋白的研究………………………………………………..7
2-6核蛋白的特性…………………………………………………..8
2-7 pET之蛋白質表現系統……………………………………….10
第三章 材料與方法……………………………………………………11
3-1 JEV核心蛋白表現質體的構築……………………………….11
3-2 JEV核心蛋白deletion mutants質體之構築……………….11
3-3質體DNA的製備……………………………………………....12
3-4勝任細胞的製備與轉型作用………………………………….12
3-5菌株之篩選…………………………………………………….13
3-6 JEV重組核心蛋白之表現…………………………………….13
3-7蛋白質膠體電泳分析(SDS-PAGE)………………………….14
3-8 JEV核心蛋白的純化………………………………………….15
3-9 JEV核心蛋白多株抗體的製備……………………………….16
3-10西方墨點法(Western blotting)…………………………16
3-11 DNA片段的製備………………………………………………17
3-12 Mobility shift assay and mobility supershift assay17
第四章 結果……………………………………………………………19
4-1 JEV核心蛋白表現質體之構築…………………………………19
4-2 JEV結構蛋白基因之選殖………………………………………19
4-3 JEV核心蛋白表現質體之確認…………………………………19
4-4 JEV全長核心蛋白基因和胺基酸定序…………………………20
4-5 JEV核心蛋白之hydrophobic plots分析…………………….20
4-6 JEV核心蛋白的表現和純化…………………………………….21
4-7以西方墨點法確認重組的JEV核心蛋白………………………..21
4-8以自備mouse anti-JEV core DD polyclonal antibody偵測被
JEV感染細胞中的病毒核心蛋白………………………………21
4-9 Gel mobility shift and supershift assay…………….22
第五章 討論…………………………………………………………….36
第六章 參考文獻……………………………………………………….40
圖次
圖一:JEV structural protein gene 構築示意圖…………………23
圖二:JEV core gene表現載體構築示意圖………………………….24
圖三a:JEV結構蛋白RT-PCR結果………………………………………25
b:JEV結構蛋白RT-PCR產物選殖入pGEM載體結果……………......25
圖四:三種不同長度的JEV core gene選殖入pGEM載體結果……….26
圖五a:DNA序列…………………………………………………………27
b:胺基酸序列……………………………………………….………..27
圖六a:JEV core DD pET32a蛋白經過胺基酸N端定序後的結果……28
b:已知pET32b Trx-Tag thioredoxin胺基酸序列………………….28
圖七:JEV core protein之hydrophobic plots………………….…29
圖八:JEV core gene經IPTG誘導表現之SDS-PAGE分析圖譜……….30
圖九:JEV core fusion protein經純化後以SDS-PAGE分析圖譜….31
圖十a:純化後之fusion core protein之western Blotting………32
b:自行製備的mouse anti-core DD Ab偵測JEV感染老鼠腦組
織的病毒之western Blotting………………………………….…..32
圖十一:JEV core D protein和 5‘NCR and partial core gene DNA片段結合之gel mobility shift結果……………………………………33
圖十二:JEV core D protein、5‘NCR and partial core gene DNA片段和mouse-anti JEV polyclonal Ab結合之gel mobility supershift.34
圖十三:JEV core DD protein、5'NCR and partial core gene DNA片段和mouse-anti JEV polyclonal Ab結合之gel mobility supershift.35

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