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研究生:王尉光
論文名稱:鴨B型肝炎的檢測與其DNA疫苗的研發
論文名稱(外文):Detection of duck hepatitis B virus and its DNA vaccine development
指導教授:張文興
學位類別:碩士
校院名稱:國立嘉義大學
系所名稱:農業生物技術研究所
學門:農業科學學門
學類:農業技術學類
論文種類:學術論文
論文出版年:2004
畢業學年度:94
語文別:中文
中文關鍵詞:鴨B型肝炎病毒Pre S基因DNA疫苗PCR快速檢測
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中文摘要

鴨B型性肝炎病毒 (Duck Hepatitis B Virus; DHBV)是禽類宿主中最先被鑑定出來的hepadnaviridae B virus,其基因體組成類似於人類的HBV。DHBV的傳染途徑也與於人類的B型性肝炎相似,DHBV能藉由血液、體液或糞便在鴨群間水平傳染,亦能藉由垂直感染的模式來將病毒傳遞給後代,所以DHBV是一種在鴨群間高度傳染的疾病;受到DHBV感染的鴨子其會有慢性的持續性感染,會導致肝臟產生病變,引發肝炎、肝硬化和肝炎的症狀產生。本研究於2005年在台灣省嘉義縣溪口的養鴨業者取得鴨隻的血液,藉由聚合酶鏈鎖反應(Polymerase Chain Reaction;PCR)的技術,增幅出DHBV的Pre S 基因序列,此Pre S 基因序列經由核酸定序與網路資料庫比對結果得知與大陸發佈的DHBV病毒株,其相似性有95%以上,此結果證明我們已經取得DHBV的Pre S基因序列。透過建立PCR的最佳化條件,可以用來檢測鴨隻身上是否帶有DHBV的病毒,其靈敏度可以檢測到picogram以下的病毒核酸量,此最佳化的PCR反應可以有利日後的檢測,而增幅出來的Pre S 基因序列也可以選殖到DNA的表現型載體(pVAC1-mcs)上,希望建構出來的DNA疫苗能防治鴨子的DHBV,降低養鴨業者的損失。

關鍵字: 鴨B型肝炎病毒、Pre S基因、DNA疫苗、PCR快速檢測
Abstract

Duck hepatitis B virus (DHBV) was the first identified heapadnaviridae B virus form the host of poultry. The genome composition of DHBV was similar to human’s HBV. The contagious pathway of DHBV is similar to human hepatitis B virus. DHBV could through horizontal route of transmission in duck groups via blood, body fluid, and fecal, moreover, it could transmit DHBV to their future generations via vertical route of transmission. Therefore, DHBV is a high contagious disease in ducks. Duck infect with DHBV will cause chronic and persistent infection, and cause the damage of liver such as liver inflammation and liver intumescences. This study was collected ducks blood form Xikou town, Chiayi county, Taiwan, in 2005. Using polymerase chain reaction (PCR) to amplified Pre S gene sequence of DHBV. Through the nucleic acid sequence analysis and compared in network database. This Pre S gene sequence has 95% similarity to China reported DHBV virus species. The result confirms that we have had Pre S sequence of DHBV. Through establish the PCR optimization condition, we can apply this technology to determine DHBV of ducks. It’s has great sensitivity which could determine the amount of virus nucleic acid to the level of pictogram, and useful for future determine. This Pre S sequence could clone to DNA pVAV1-mcs. To expect that the DNA vaccine design form this study could prevent DHBV of ducks and reduce cost of duck raising.
中文摘要-------------------------------------------------------------------------I
英文摘要-----------------------------------------------------------------------III
目錄---------------------------------------------------------------------------- IV
圖次--------------------------------------------------------------------------VIII
表次---------------------------------------------------------------------------XII
第一章 簡介鴨B型肝炎病-----------------------------------------------------1
第一節 鴨子--------------------------------------------------------------------1
第二節 鴨子B型肝炎------------------------------------------------------- 2
2-1 鴨B型肝炎之背景-----------------------------------------------------2
2-2 病毒分類與特性--------------------------------------------------------3
2-3 型態結構-----------------------------------------------------------------5
2-4 病毒複製-----------------------------------------------------------------6
2-5 病毒的傳染與症狀-----------------------------------------------------8
第三節 病毒的檢測方法----------------------------------------------------9
第四節 疫苗的開發---------------------------------------------------------11
第五節 本文研究目的------------------------------------------------------12
第六節 參考文獻------------------------------------------------------------13
第二章 鴨B型肝炎Pre S基因的選殖及其核酸序列分析------------23
第一節 前言------------------------------------------------------------------23
第二節 材料與方法---------------------------------------------------------25
2-1 病毒樣本的採樣-----------------------------------------------------25
2-2 鴨隻血液前處裡-----------------------------------------------------26
2-3 引子設計--------------------------------------------------------------26
2-4 建構最佳化的 DHBV PCR----------------------------------------27
2-5 利用pGEM-T Easy vector來選殖Pre S基因序列-----------30
2-5-1 利用Pfu聚合酵素來進行PCR增幅Pre S基因序列------30
2-5-2 PCR產物瓊脂膠Agarose電泳分析--------------------------31
2-5-3 電泳膠體純化DNA的膠體回收-------------------------------32
2-5-4 Pfu合成的PCR產物進行3’端加A--------------------------33
2-5-5 利用pGEM-T Easy vector來接合Pre S基因----------------34
2-5-6 細菌的轉殖作用---------------------------------------------------35
2-5-7 質體DNA的純化-------------------------------------------------36
2-5-8 利用限制酶酵素來進行重組質體的確認---------------------37
2-6 重組質體的DNA定序分析與資料庫比對---------------------38
2-6-1 重組質體DNA純化----------------------------------------------38
2-6-2 重組質體的核酸定序---------------------------------------------39
第三節 結果-----------------------------------------------------------------41
3-1 設計增幅鴨B型肝炎Pre S基因引子-----------------------------41
3-2 利用PCR增幅Pre S基因與膠體電泳分析----------------------41
3-3 使用限制酶酵素來進行重組質體的確認-------------------------42
3-4 重組質體的DNA定序-----------------------------------------------43
3-5 Pre S基因的DNA比對分析--------------------------------------44
3-6 Pre S基因的胺基酸分析-------------------------------------------45
第四節 討論 -----------------------------------------------------------------46
第五節 結論------------------------------------------------------------------49
第六節 參考文獻------------------------------------------------------------50
第三章 鴨B型肝炎病毒PCR與 nester PCR快速檢測法-----------85
第一節 前言------------------------------------------------------------------85
第二節 材料與方法---------------------------------------------------------86
2-1-1 病毒樣本的採樣----------------------------------------------------86
2-1-2 人工接DHBV-------------------------------------------------------86
2-2 鴨隻血液前處裡------------------------------------------------------87
2-3 PCR與nester PCR檢測-------------------------------------------88
2-4 瓊脂膠Agarose電泳分析-------------------------------------------91
第三節 結果------------------------------------------------------------------91
3-1 最佳化的PCR檢測條件--------------------------------------------91
3-2 利用PCR檢測鴨隻血清的DHBV病毒-------------------------92
第四節 討論 -----------------------------------------------------------------93
第五節 結論------------------------------------------------------------------96
第六節 參考文獻------------------------------------------------------------96
第四章 建構重組質體以發展DNA與次單位疫苗--------------------107
第一節 前言----------------------------------------------------------------107
第二節 材料與方法-------------------------------------------------------109
2-1 次選殖Pre S基因到表現質體(pVAC)--------------------------109
2-1-1 勝任細胞的製備--------------------------------------------------109
2-1-2 Vector(pVAC)與insert DNA(Pre S)的製備-------------------110
2-1-3 質體( pVAC)DNA與insert DNA(Pre S)的接合反應------111
2-1-4 質體(pVAC-Pre S)DNA的電穿孔轉殖法-------------------112
2-1-5 重組質體(pVAC-Pre S)的純化--------------------------------113
2-1-6 重組質體(pVAC-Pre S)的酵素切割確認--------------------114
2-1-7 重組質體(pVAC-Pre S)的DNA純化------------------------115
2-1-8 重組質體(pVAC-Pre S)的DNA定序與序列分析---------116
2-2 次選殖EGFP基因到重組質體(pVAC-Pre S)-----------------118
2-2-1 Vector(pVAC-Pre S)與insert DNA(EGFP)的製備----------118
2-2-2 質體pVAC-Pre S與EGFP的接合反應---------------------120
2-2-3 細菌(pVAC-EGFP-Pre S)的轉殖作用------------------------120
2-2-4 質體(pVAC-EGFP-Pre S)DNA的抽取-----------------------121
2-2-5 利用限制酶酵素來進行重組質體的確認-------------------122
2-2-6 重組質體(pVAC-EGFP-Pre S)DNA純化--------------------123
2-2-7 重組質體(pVAC-EGFP-Pre S)的核酸定序------------------124
2-3 重組質體(pVAC-EGFP-Pre S)的大量純化--------------------126
2-4 利用精子載體法來做基因轉殖鴨-------------------------------127
2-5 DNA疫苗的動物試驗---------------------------------------------128
第三節 結果--------------------------------------------------------------128
3-1 選殖Pre S基因到表現載體pVAC------------------------------128
3-2 平端選殖EGFP基因到表現載體(pVAC-Pre S)--------------129
3-3 檢測基因轉殖鴨的血液是否有EGFP的表現----------------130
3-4 PCR檢測基因轉質鴨是否轉殖成功----------------------------130
第四節 討論---------------------------------------------------------------131
第五節 結論---------------------------------------------------------------132
第六節 參考文獻---------------------------------------------------------133

圖次
第一章 鴨子與鴨B型肝炎----------------------------------------------------1
圖 1 鴨B型肝炎病毒的結構圖--------------------------------------------21
圖 2 鴨B型肝炎病毒的生活史--------------------------------------------22
第二章 鴨B型肝炎Pre S基因的選殖以及核酸序列分析------------23
圖 1 分析中國大陸的11株DHBV序列保守性(部分圖)------------52
圖 2 使用45℃的anneal溫度來尋求最佳化的鎂離子作用濃度---53
圖 3 調整60℃的anneal溫度來增加PCR產物的專一性-----------54
圖 4 使用稀釋的血清樣本來進行PCR反應---------------------------55
圖 5 PCR 產物(Pre S)進行3’端加A(腺螵呤)作用-------------------56
圖 6 重組質體(TA-Pre S)選殖株的酵素(EcoRI)切割確認圖--------57
圖 7 利用EcoRI確認重組質體(TA-Pre S切NCO I &Bam HI)
和重組質體(TA-Pre S切Bam HI & Pst I)----------------------58
圖 8 TA-Pre S(pfu) 8sp6-1定序圖----------------------------------------59
圖 9 TA-Pre S(pfu) 8sp6-2定序圖----------------------------------------60
圖 10 TA-Pre S(pfu) 8sp6-3定序圖--------------------------------------61
圖 11 TA-Pre S(pfu) 8T7-1定序圖---------------------------------------62
圖 12 TA-Pre S(pfu) 8T7-2定序圖---------------------------------------63
圖 13 TA-Pre S(pfu) 8T7-3定序圖---------------------------------------64
圖 14 (TA-Pre S切Nco I & Bam HI) M13F-1定序圖----------------65
圖 15 (TA-Pre S切Nco I & Bam HI) M13F-2定序圖----------------66
圖 16 (TA-Pre S切Nco I & Bam HI) M13F-3定序圖----------------67
圖 17 (TA-Pre S切Nco I & Bam HI) M13R-1定序圖----------------68
圖 18 (TA-Pre S切Nco I & Bam HI) M13R-2定序圖----------------69
圖 19 (TA-Pre S切Nco I & Bam HI) M13R-3定序圖----------------70
圖 20 (TA-Pre S切Bam HI&Pst I) M13F-1定序圖---------------------71
圖 21 (TA-Pre S切Bam HI&Pst I) M13F-2定序圖---------------------72
圖 22 (TA-Pre S切Bam HI&Pst I) M13F-3定序圖---------------------73
圖 23 (TA-Pre S切Bam HI&Pst I) M13R-1定序圖---------------------74
圖 24 (TA-Pre S切Bam HI&Pst I) M13R-2定序圖---------------------75
圖 25 (TA-Pre S切Bam HI&Pst I) M13R-3定序圖---------------------76
圖 26 分析選殖Pre S基因與31株病毒Pre S基因相似性-----------77
圖 27 Pre S基因的演化樹分析圖-----------------------------------------78
圖 28 分析Pre S 胺基酸與31株病毒Pre S胺基酸的比對分析----79
第三章 鴨B型肝炎病毒PCR與 nester PCR快速檢測法----------85
圖 1 使用低黏合溫度來調整鎂離子濃度--------------------------------98
圖 2 使用不同黏合溫度來進行PCR反應-------------------------------99
圖 3 連續稀釋質體(TA-Pre S)DNA來測試靈敏度--------------------100
圖 4 運用Pre S引子檢測嘉義縣溪口鄉養鴨戶的鴨隻血液---------101
圖 5 使用增幅全長病毒的引子來檢測鴨血清樣本--------------------102
圖 6 利用PCR與nested PCR來檢測人工接種DHBV陽性
血清約十五週後的成鴨----------------------------------------------103
圖 7 使用不同黏合溫度來進行PCR反應------------------------------104
圖 8 利用PCR來檢測人工接種DHBV陽性血清
兩週後的小鴨----------------------------------------------------------105
圖 9 利用PCR來檢測人工接種DHBV陽性血清
約十五週後的成鴨----------------------------------------------------106

第四章 建構重組質體pVAC-EGFP-Pre S來發展
DNA與次單位疫苗------------------------------------------------107
圖 1 利用限制酶酵素來製備insert DNA(Pre S)與
vector DNA(pVAC)----------------------------------------------------135
圖 2 使用限制酶酵素(EcoRI和EcoRV)來確認重組質體
(pVAC-Pre S)-----------------------------------------------------------136
圖 3 pVAC-Pre S 定序圖定Pre S-1------------------------------------137
圖 4 pVAC-Pre S 定序圖定Pre S-2------------------------------------138
圖 5 pVAC-Pre S 定序圖定Pre S-3------------------------------------139
圖 6 利用限制酶酵素來製備insert DNA(EGFP)與vector
DNA(pVAC-Pre S)---------------------------------------------------140

圖 7 利用限制酵素(Nco I)來進行重組質體
(pVAC-EGFP-Pre S)切割確認---------------------------------------141
圖 8 pVAC-EGFP-Pre S定EGFP-1-------------------------------------142
圖 9 pVAC-EGFP-Pre S定EGFP-2-------------------------------------143
圖 10 pVAC-EGFP-Pre S定EGFP-3------------------------------------144
圖 11 pVAC-EGFP-Pre S定Pre S-1-------------------------------------145
圖 12 pVAC-EGFP-Pre S定Pre S---------------------------------------146
圖 13 pVAC-EGFP-Pre S定Pre S-3------------------------------------147
圖 14 利用PCR檢測基因轉殖鴨是否成功--------------------------148

表次

Table 1 收集NCBI現階段完整的鸭B型肝炎病毒株-----------------80
Table 2 組合完整的Pre S基因---------------------------------------------81
Table 3 Pre S基因的序列比對----------------------------------------------82
Table 4 胺基酸序列比對表--------------------------------------------------84
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