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研究生:鄒行煦
研究生(外文):Hsin-Hsu Tsou
論文名稱:對蝦白斑病病毒結構蛋白基因VP28之選殖、表現及單源抗體的製備
論文名稱(外文):Cloning, expression and monoclonal antibody preparation for Penaeus white spot syndrome virus structure protein gene VP28
指導教授:郭村勇
指導教授(外文):Tsun-Yung Kuo
學位類別:碩士
校院名稱:國立宜蘭大學
系所名稱:生物技術研究所碩士班
學門:農業科學學門
學類:畜牧學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:60
中文關鍵詞:白斑病病毒重組蛋白VP28單株抗體定量聚合酶鏈反應
外文關鍵詞:White Spot Syndrome Virusrecombinant VP28monoclonal antibodyreal-time PCR
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中文摘要
本論文之主要目的,擬針對白斑病病毒(White Spot Syndrome Virus;WSSV)VP28基因進行選殖並將該基因送入大腸菌內以不同條件進行誘導表現,同時純化該重組蛋白製備單源抗體。此外,以Real-Time PCR (SYBR Green I)針對WSSV罹病蝦體組織建立一病毒核酸定量之檢測方法。
以PCR法增幅WSSV VP28基因,可獲得一629 bps之DNA片段,經限制酶切割後選殖入原核表現載體pET24a,經定序確認無誤後,轉型入大腸桿菌表現宿主BL21 codon plus;以1mM IPTG進行誘導測試,發現在31 kDa位置可表現出一基因重組蛋白質,將該重組蛋白以LC/MS/MS進行部份胺基序列鑑定,鑑定結果經資料庫比對後確認為WSSV VP28 (Protein Coverage 27.8%)。分別以0.1、0.5、1及3 mM IPTG以及22、30及37℃等條件進行誘導,分析重組蛋白的表現型態,結果顯示在37℃條件下,所表現出的重組蛋白VP28 (rVP28)大部分皆形成包函體,但在低溫條件(22、30℃)下,rVP28形成可溶態的比例有明顯增加。將rVP28可溶態部份以His-bind親合性管柱純化,並免疫BALB/c小鼠進行單源抗體的製備。經過細胞單株化篩選後,得到3株可穩定分泌抗體之融合瘤細胞株,以西方墨點法進行分析,顯示抗體除了能夠辨識rVP28,對於純化的病毒蛋白質亦具有特異性反應。
將白蝦病材組織液,萃取出核酸並序列稀釋成為10-1~10-8的待測樣品,再分別以Real-Time PCR及Nest-PCR進行檢測。結果顯示此兩種檢測方法之敏感度相似,在10-7樣品中皆可增幅出病毒核酸片段,而以標準曲線(Standard curve)推算10-7樣品起始分子數,大約為89 copy numbers。
Abstract
The main purposes of this research include cloning of WSSV VP28 gene and its expression under different induction conditions. The expressed recombinant proteins were used for monoclonal antibody production. In addition, the technique of absolute quantization using real-time PCR (SYBR Green I) was studied.
In PCR amplification of WSSV VP28 gene, a 629 bp DNA fragment was obtained, restriction enzyme digested, and ligated into prokaryotic expression vector pET24a. After sequence analysis confirmation for correctness, the recombinant DNA was transformed into expression host BL21 codon plus. After induction with 1mM IPTG, a 31 kDa protein was shown to be expressed and after analyzing partials of amino acid sequence by LC/MS/MS, the protein was confirmed to be WSSV VP28 (Protein coverage 27.8%). Different conditions with various temperatures of 22, 30, and 37°C, and different combinations of 0.1, 0.5, 1, and 3 mM IPTG concentrations for recombinant protein expressions were tested. It was found that regardless of the IPTG concentration, proteins expressed at 37°C were in the inclusion body state. Under lower temperature, a vast increase of soluble proteins were expressed. rVP28 soluable form proteins were purified by affinity column and used to immunize BALB/c mice. After limiting dilution and ELISA screening hybridomas, 3 different rVP28 monoclonal hybridomas were successfully produced. Western blot assay demonstrated that rVP28 monoclonal hybridoma’s cultured medium could recognized either purified rVP28 or WSSV protein.
Homogenization and extraction of WSSV infected tissue to prepare the viral DNA sample (10-1~10-8). Using both Real-time PCR and Nest-PCR technique to compare detection limite. The result showed that sensitivity of both method was similarity in viral 10-7 DNA sample. Calculation the starting quantity, is sensitive to about 89 copy number.
目錄

中文摘要.............................................................i
英文摘要 ...........................................................ii
目錄................................................................iv
表目錄.............................................................vii
圖目錄............................................................viii

第一章 緒言........................................................1

第二章 文獻探討......................................................2

一、 對蝦白斑病病毒(WSSV)簡述.......................................2
二、 WSSV VP28基因及其重組蛋白之研究................................4
三、 WSSV相關之分子診斷技術.........................................5

第三章 實驗架構......................................................7

第四章 材料與方法.....................................................9

一、 pET24a-VP28重組質體的構築.....................................9
1.1 PCR增幅WSSV VP28基因序列......................................9
1.1.1 瓊脂凝膠電泳分析..............................................9
1.1.2 PCR產物的純化.................................................9
1.1.3 核酸濃度測定.................................................10
1.2 中量質體DNA的純化............................................10
1.3 限制酵素切割與核酸接合反應.....................................11
1.4 大腸桿菌勝任細胞的製備........................................11
1.4.1 細胞轉型作用.................................................11
1.5 重組質體的確認...............................................12
1.5.1 核酸定序分析.................................................12
1.5.2 小量質體DNA的純化............................................12

二、 重組蛋白VP28的表現、胺基酸鑑定以及純化..........................13
2.1 重組蛋白VP28表現型態(Solubility)分析..........................13
2.1.1 全菌蛋白質(Total cell protein)比例分析........................13
2.1.2 可溶性蛋白質(Soluble protein)比例分析.........................13
2.1.3 SDS-PAGE分析................................................13
2.1.4 胺基酸鑑定分析...............................................14
2.2 重組蛋白VP28的純化...........................................14
2.2.1 全菌蛋白質溶液的均質及初步分離.................................14
2.2.2 不可溶態(Inclusion body)蛋白質分析...........................15
2.2.3 親合性管柱的純化.............................................15
2.2.4 蛋白質濃度測定...............................................15

三、 rVP28抗體的製備及抗體力價分析.................................16
3.1 兔抗rVP28高免血清製備........................................16
3.1.1 rVP28多株抗體力價測定(Western blot)..........................16
3.2 rVP28單株抗體的製備..........................................16
3.2.1 小鼠免疫策略.................................................16
3.2.2 細胞培養液製備...............................................17
3.2.3 細胞融合反應及融合瘤細胞的篩選.................................17
3.2.4 融合瘤細胞抗體分泌能力測定(ELISA)..............................17
3.2.5 融合瘤細胞之單株化............................................18
3.2.6 rVP28單株抗體力價測定(Western blot)..........................18

四、 WSSV病毒之檢測與定量分析......................................19
4.1 病材組織均質液的製備..........................................19
4.1.1 WSSV病毒的純化...............................................19
4.2 病毒核酸(DNA)萃取............................................19
4.3 Nest-PCR檢測................................................20
4.3.1 One step-PCR反應............................................20
4.3.2 Two step-PCR反應............................................20
4.4 WSSV病毒定量分析.............................................20
4.4.1 pms94-rbcTA質體的構築........................................20
4.4.2 標準分子量質體的製備..........................................21
4.4.3 Real-time PCR檢測...........................................21

第五章 結果.........................................................22

一、 pET24a-VP28 重組質體的構築...................................22
二、 重組蛋白VP28的表現、胺基酸鑑定以及純化..........................22
三、 rVP28抗體的製備及抗體力價分析.................................23
四、 WSSV病毒之檢測與定量分析......................................24

第六章 討論.........................................................25

附錄............................................................... 28

參考文獻............................................................43



表目錄


表一、本論文中所使用的引子列表.........................................28
表二、重組蛋白VP28之部分胺基酸序列鑑定(LC/MS/MS)........................29



圖目錄


圖一、PCR擴增WSSV VP28基因之電泳分析圖.................................30
圖二、 WSSV VP28基因選殖入原核載體pET24a後之定序分析結果................31
圖三、重組蛋白VP28表現型態(Solubility)分析(37℃).......................32
圖四、重組蛋白VP28表現型態(Solubility)分析(30℃).......................33
圖五、重組蛋白VP28表現型態(Solubility)分析(22℃).......................34
圖六、重組蛋白VP28純化的結果圖(15% SDS-PAGE)...........................35
圖七、rVP28多株抗體力價測定...........................................36
圖八、rVP28單株抗體力價測定...........................................37
圖九、Nest-PCR檢測敏感度分析(1.5% Agarose gel)........................38
圖十、Real-time PCR定量分析結果(標準分子量質體與負對照樣品)..............39
圖十一、Real-time PCR定量分析結果(病毒核酸樣品)........................40
圖十二、Real-time PCR定量分析之標準曲線圖(Ct值對應log SQ)...............41
圖十三、Real-time PCR檢測樣品之起始分子數(Starting Quantity)...........42
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