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研究生:吳沛宇
研究生(外文):Pei-Yu Wu
論文名稱:以BAC載體承接假性狂犬病毒TNL株全長基因體
論文名稱(外文):Construction of the full-length pseudorabies virus TNL strain genome as the bacterial artificial chromosome
指導教授:張天傑
指導教授(外文):Tien-Jey Chang
學位類別:碩士
校院名稱:國立中興大學
系所名稱:獸醫學系
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:70
中文關鍵詞:假性狂犬病毒
外文關鍵詞:bacterial artificial chromosomepseudorabies virusPRVherpesBACvirus vectorrecombinationgenome
相關次數:
  • 被引用被引用:1
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細菌人工染色體(Bacterial Artificial Chromosome,BAC) 是以E. coli的mini F-plasmid為基礎所建構的載體,能夠承載近300 kb的大分子DNA片段,複製並穩定的保存。近年來有越來越多的病毒,尤其是herpesvirus,其基因體被轉接到BAC作為infectious clone來應用,並在E.coli中進行基因的置換與修飾,以快速的建構出研究所需要的突變病毒株。本實驗參考Smith等(2000)的做法來建立PRV-TNL株的BAC全長基因體。首先將eGFP螢光基因表現序列前後各接上loxP site並置於病毒11 k、28 k基因序列中,以superinfection的方式使其與病毒基因體置換,將螢光基因嵌入,而後篩選產生螢光病毒斑之突變病毒株TNL-GFP。經過十五代的篩選,再以supertransfection的方式利用cre表現載體產製cre酵素而將螢光病毒基因體中的eGFP表現序列切除,而後篩選出無法發光的病毒株TNL-loxP。將此病毒基因體經PCR增幅後進行定序,證明此病毒基因體於11 k、28 k片段之中已引入了34個base的loxP序列。為了再次利用cre- loxP重組將BAC載體引入病毒中,故將pCC1TM(Epicentre)載體嵌入GFP表現卡匣,並利用supertransfection的方式與TNL-loxP病毒基因體進行重組,並挑選產生螢光病毒斑之突變病毒株TNL- BAC。上述載體可經由L-Arabinose誘導而提高BAC載體的複製數目以方便日後實驗進行。以plaque assay評估上述三種突變病毒株力價,結果顯示,eGFP表現序列嵌入11 k與28 k基因片段中間會導致病毒力價下降約100倍,而由cre酵素截除eGFP表現序列後則回復其病毒力價。然而再次引入10 kb的BAC載體序列後,不但毒力下降1000倍且病毒斑大小也縮小。此外,定序結果顯示病毒基因體可能有產生序列改變,致使無法於預估位置夾出螢光卡匣,但以線性化後的pCC1載體為探針進行Dot-Blot仍可證實其基因體內的確含有BAC載體片段。最後將感染TNL-BAC細胞的DNA以電穿孔送入E. coli後,長出的菌落經抽取質體分析,除了以pCC1載體為探針進行Dot-Blot能偵測到訊號外,經PCR卻無法證實有無PRV的序列。

Construction of full-length infectious clones of virus genome is useful for the study of viral pathogenesis and the application of vaccine development. We used the recently developed bacterial artificial chromosome (BAC) as a vector to carry 150 kb full-length genome of the pseudorabies TNL strain (a Taiwan local strain). A green fluorescent protein (eGFP) expression cassette flanked by two 34 base loxP sites was inserted into the segment of 11 k and 28 k genes for the homologous recombination. After 15 passages, green plaques were cloned and named TNL-GFP. By using the method of cre-loxP site-specific recombination, dark plaques, the TNL-loxP, were picked and proved to have the loxP site insertion of PRV genome by sequencing. We modified pCC1TM BAC vector from Epicentre to be inserted with another GFP cassette. This BAC vector containing a loxP site and the GFP expression cassette was integrated into PRV genome again by cre-loxP recombination thus resulted in green fluorescent plaques, named PRV-BAC. By plaque assay, we found the insertion of a GFP cassette between 11 k and 28 k genes would reduce the viral titer. Moreover, another large insertion as BAC vector could decrease the plaque size of PRV。By sequencing of the PCR fragment, we sugessted there might be rearrangements in TNL-BAC genome, thus the inserted GFP fragment could not be detected by PCR. Therefor the insertion of BAC vector was still identified by the dot-blot hybridization with linearised pCC1 as probe。After transforming E. coli with TNL-BAC infected cell DNA by electroporation, the plasmids from chloramphenicol resistant colonies were purified. However, these plasmids can only be detected to have the pCC1 sequence by dot-blot hybridization, but they didn’t show any detectable PRV-TNL sequence by PCR.

目錄
目錄------------------------------------------------------------------------------------------------------- I
圖次 ---------------------------------------------------------------------------------------------------- IV
表次--------------------------------------------------------------------------------------------------- - - V
摘要 ---------------------------------------------------------------------------------------------------- V I
Abstract ----------------------------------------------------------------------------------------------- VII
前言 ------------------------------------------------------------------------------------------------------ 1
文獻探討 ------------------------------------------------------------------------------------------------ 2
1. 細菌人工染色體Bacterial Artificial Chromosome (BAC) -------------------------------- 2
1-1. 命名 -------------------------------------------------------------------------------------------- 2
1-2. Conjugation與F plasmid --------------------------------------------------------------------- 3
1-3. F plasmid與Hfr cell -------------------------------------------------------------------------- 5
1-4. F plasmid的結構 - - ---------------------------------------------------------------------------6
1-5. mini-F plasmid --------------------------------------------------------------------------------- 7
1-6. Bacterial Artificial Chromosome的建構及演進 ---------------------------------------- 8
1-7. BAC的應用與HGP ------------------------------------------------------------------------ 11
1-8. BAC與病毒 ---------------------------------------------------------------------------------- 12
2. 研究疱疹病毒的新工具 ----------------------------------------------------------------------14
2-1. Herpesvirus mutagenesis的歷史---------------------------------------------------------- 14
A. 化學突變 ------------------------------------------------------------------------------------ 15
B. 同源重組所導致的定點突變 (site-directed mutagenesis) ------------------------- 15
C. 由互相重疊的cosmid株產製突變病毒----------------------------------------------- 15
D. BAC載體--------------------------------------------------------------------------------------15
2-2. Herpesvirus 的BAC mutagenesis ---------------------------------------------------------16
A. Allelic exchange ------------------------------------------------------------------------------16
B. Linear DNA 互換 --------------------------------------------------------------------------- 17
C. Transposon mutagenesis--------------------------------------------------------------------- 17
D. cre-loxP recombination ----------------------------------------------------------------------17
E. FRT-FLP recombination ---------------------------------------------------------------------18
3.假性狂犬病毒與BAC -------------------------------------------------------------------------- 18
3-1.PRV BAC的建構 ---------------------------------------------------------------------------- 19
3-2. Self-excisable PRV-BAC -------------------------------------------------------------------19
3-3. PRV BAC的相關應用 --------------------------------------------------------------------- 20
3-3. 建構假性狂犬病毒TNL株BAC全長基因體 --------------------------------------- 21
材料與方法 -------------------------------------------------------------------------------------------22
1. 重組載體的製備 -------------------------------------------------------------------------------22
1-1. 大量質體抽取 - - - - -------------------------------------------------------------------------- 22
1-2. 線性化重組質體 --------------------------------------------------------------------------- 22
2. 重組病毒的建構 -------------------------------------------------------------------------------22
2-1. 細胞培養 -------------------------------------------------------------------------------------22
2-2. 病毒接種 -------------------------------------------------------------------------------------23
2-3. 挑取病毒斑 --------------------------------------------------------------------------------- 23
2-4. 病毒斑純化(plaque purification) --------------------------------------------------------- 23
2-5. DNA轉染,Qiagen EffectenceTM套組 ----------------------------------------------------23
2-6. DNA轉染,Qiagen poly-FectTM套組 ----------------------------------------------------- 24
2-7. DNA轉染,電穿孔-------------------------------------------------------------------------- 24
3. 重組病毒的分析 ------------------------------------------------------------------------------- 24
3-1. Plaque assay ----------------------------------------------------------------------------------- 24
3-2. 小量病毒基因體的抽取 -----------------------------------------------------------------25
3-3. PCR 確認-------------------------------------------------------------------------------------25
3-4. Dot blot確認 ---------------------------------------------------------------------------------26
4. BAC載體的建構 --------------------------------------------------------------------------------26
5. E. coli.的轉型 ------------------------------------------------------------------------------------27
5-1. 抽取infected cell DNA(1) -----------------------------------------------------------------27
5-2. 抽取infected cell DNA(2) -----------------------------------------------------------------27
5-3. 電穿孔轉型E. coli - ------------------------------------------------------------------------27
6. 重組病毒的建構 ------------------------------------------------------------------------------- 28
6-1. TNL-GFP的構築 ----------------------------------------------------------------------------28
6-2. TNL-loxP的構築 ----------------------------------------------------------------------------28
6-3. TNL-BAC的構築 --------------------------------------------------------------------------- 29
結果----------------------------------------------------------------------------------------------------- 31
1. TNL-GFP的構築-------------------------------------------------------------------------------- 31
2. TNL-loxP的構築-------------------------------------------------------------------------------- 31
3. BAC載體的構築 --------------------------------------------------------------------------------32
4. 引入BAC載體至病毒基因體 ---------------------------------------------------------------32
5. 病毒特性的分析 -------------------------------------------------------------------------------33
5-1. Plaque assay - -----------------------------------------------------------------------------------33
5-2. PCR確認TNL-BAC病毒基因體 - - - -----------------------------------------------------33
5-3. Dot Blot確認TNL-BAC病毒基因體 --------------------------------------------------- 34
6. 將TNL-BAC轉型至E. coli ------------------------------------------------------------------- 34
討論----------------------------------------------------------------------------------------------------- 55
參考文獻------------------------------------------------------------------------------------------- - - -60

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