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研究生：	劉卜慈 Liu, Pu-Sin
論文名稱：	利用假性狂犬病毒攜帶腺病毒相關病毒Rep和Cap基因的方法產生腺病毒相關病毒載体 Development of a new method using pseudorabies virus carrying AAV Rep and Cap genes for production of AAV vector
指導教授：	楊啟瑞 Yang, Chi-Rei 楊文宏 Yang, Wen-Cho 蔡宗欣 Tsai, Cho-Chi 吳昭良 Wu, Chao-Liang
學位類別：	碩士 Master
系所名稱：	醫學院 - 生物化學研究所 Department of Biochemistry
論文出版年：	2002
畢業學年度：	90
語文別：	英文
論文頁數：	62
中文關鍵詞：	重組腺病毒相關病毒、缺陷性病毒、重組腺病毒相關病毒、載体、重組腺病毒相關病毒
外文關鍵詞：	AAV, rAAV, vector, PRV
相關次數：	點閱：97 下載：0
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所謂基因治療就是將特定基因送入體內，以改善或彌補體內基因的缺陷。目前最重要的是發展如何有效地將基因傳送入體內。基因傳送載体主要可以區分為病毒和非病毒性載体。而腺病毒相關病毒 (adeno-associated virus; AAV) 載体是最近被廣泛注意用於人類基因治療的傳送載体。本研究目的著重於發展腺病毒相關病毒載体。發展腺病毒相關病毒當作基因傳送載體，主要是根據其生物特性。腺病毒相關病毒是一種具有單股DNA的極小病毐 (parvovirus)，其可以感染多種類細胞和組織，不論是分裂或未分裂中細胞，且對人無致病性。而且其缺少野生種病毒基因，所以能減少對細胞的毒性和免疫性，利用此病毒載体優點是：其為一種缺陷性病毒須要有其它幫助病毒 (helper virus) 協助下才能組裝成具有感染性病毒顆粒，否則感染細胞時只會嵌入細胞染色体，而造成潛伏狀態，也因此能長期表現其所攜帶之治療基因。然而目前有很多用來產生重組腺病毒相關病毒載体方法，在本研究中，我們發展新的方法去產生腺病毒相關病毒載体。為了有效產生腺病毒相關病毒載体，利用假性狂犬病毒攜帶腺病毒相關病毒的Rep和Cap基因，去產生腺病毒相關病毒載体。此假性狂犬病毒攜帶腺病毒相關病毒的Rep和Cap基因的病毒將之命名為“ PS ”病毒。此從假性狂犬病毒所衍生出來的gD、gE和TK突變的缺陷性PS病毒，具有輔助和複製功能，可用來幫助重組腺病毒相關病毒。當將pSub201/EGFP和PS病毒共同轉染293細胞時，可產生重組腺病毒相關病毒。為了能每次皆能成功地產生重組腺病毒相關病毒，我們針對其操作歩驟中: pSub20l/EGFP的轉入效率和所須加入PS病毒濃度做定量。找出其理想濃度來順利產生重組腺病毒相關病毒。然而利用此標準操作方式成功產出攜帶前胸腺素(prothymosin a)基因的重組腺病毒相關病毒，更證明其在細胞內具有功能，且將其感染小鼠肌肉，也會表現此蛋白。總之，在未來我們可以利用此方法來產生帶有任何基因之重組腺病毒相關病毒應用在基因治療上。

A number of vector systems have been developed for the purpose of gene transfer including both viral and non-viral delivery vehicles. Adeno-associated virus (AAV) attracts wide attention as a potential human gene therapy vector. The advantages of this vector system are that it is naturally defective; AAV infects a wide variety of cell and tissue types and it readily integrates into the target cell’s genome and is considered to be nonpathogenic. The absence of all wild-type coding proteins bypasses the cellular toxicity often observed with other viral vectors and has potential for long-term therapeutic gene expression. There are several methods to generate recombinant AAV (rAAV) vectors. In this study, we report a novel method to produce rAAV vectors. In order to facilitate the production of rAAV vector, pseudorabies virus (PrV) was engineered to express the AAV Rep and Cap genes, yielding PS virus. The PS virus, a gD/gE/TK triple mutant derived from a recombinant PrV, was used as a helper and packaging virus for generating rAAV stocks. Cotransfection of the PS virus together with pSub201/EGFP into 293 cells resulted in the production rAAV expressing GFP. We determined the optimal conditions of pSub201/EGFP DNA transfection efficiency and multiplicity of infection of the PS virus for rAAV production. Furthermore, we produced rAAV expressing prothymsin α (ProTα) protein using the same method. Expressions of proTα protein in vitro and in vivo through rAAV/ProTα gene delivery were detectable by immunohistochemistry, demonstrating that this system worked. In the future, other genes of interest can be successfuliy constructed into rAAV by this method.

Chinese abstract                                           9
English abstract                                          10
Introduction                                              11
Gene therapy and vector desig                             11
AAV as a viral vector                                     12
Production of rAAV.                                       13
Pseudorabies virus (PrV)                                  15
PrV vector.                                               16
Our strategy of producing rAAV                            17
Sources of experimentalreagents.                          18
Bacterial sources                                         19
Plasmids.                                                 19
Primers.                                                  19
Solutions                                                 20
Cell lines and viruses.                                   21
Methods                                                   21
Miniprep of plasmid DNA.                                  22
Maxiprep of plasmid DNA.                                  22
Preparation of competent cells.                           23
DNA transformation of competent cells.                    24
Spectrophotometric determination of the amount of DNA  24
Cell culture                                              25
Subculture of cells.
Cell counts using hahemacytometer                         25
Transfection                                              26
Lipofectintransfection
Calcium phosphate transfection                            26
Construction of pDgD-rep/cap plasmid.
Construction of PS virus
Construction of pSub201/EGFP and pSub201/ProTa            27
plasmid
Isolation of viral DNA                                    28
Detection of AAV rep gene in PS virus by PCR              29
Determination of TCID50 of PS virus.                      29
Production of recombinant rAAV-EGFP by co-transfecting
pSub201/EGFP with PS virus                                29
Production of rAAV/EGFP by amplifying rAAV/EGFP via PS virus infection
Titration of rAAV/EGFP by the fluorescent cell assay      30
Immunohistochemistry                                      31
Functional assay (cell proliferation assay)               31
Detection of ProTa in skeletal muscle of mice injected with rAAV/ProTa                                                32
Determination of rAAV/ProTa titer by TCID50               32
Results                                                   33
Construction of pDgD-AAVrep/cap plasmid                   33
Generation and characterization of PS virus               34
Production of rAAV/EGFP by co-transfecting pSub201/EGFP
with PS virus                                             34
Effect of packaging conditions on rAAV yield              35
Influence of the transfection efficiency of pSub201/EGFP plasmid on rAAV/EGFP yield                                35
Influence of PS viral titer (TCID50) on rAAV yield        35
The titer of PS virus influenced rAAV amplification       36
ProTa expression in NIH3T3 cells infected with rAAV/ProTa 36
Cell proliferation assay for ProTa                        36
Transgene expression in the skeletal muscle of mice injected
with rAAV/ProTa                                           36
Discussion                                                38
Reference                                                 57
Figure
1.Traditional methods for production of rAAV.              41
2.Our strategy for production of rAAV/gene x by co-transfection of pAAV/gene x and PS virus.                  42
3.Construction of pDgD-AAVrep/cap plasmid.                 43
4.Generation of PS virus, a recombinant PrV carrying AAV’s Rep and Cap genes (rPrVAAVrep-cap), using a simple two-step method.                                                    44
5.Construction of AAVpSub201/EGFP plasmid.                 45
6.Construction of pSub201/ProTa plasmid.                   46
7.Our strategy for production of rAAV/gene x by co-        transfection of pAAV/gene x and PS virus.                  47
8.Transfection of 293 cells with pSub201/EGFP followed by super-infection with PS virus resulted in rescue of infectious AAV/EGFP                                                 48
9.Effect of packaging conditions on rAAV yield.            49
10.Detection of AAV-Rep gene from PS virus by PCR
analysis.                                                  50
11.Influence of the transfection efficiency of pSub201/EGFP
plasmid on rAAV/EGFP yield.                                51
12.Determination of the optimal amount of PS virus required
for successful production of rAAV/EGFP.                     52
13.Determination of the optimal amount of PS virus required
for successful production of rAAV/EGFP.                     53
14.The outline of production of rAAV/ProTα by
co-transfection of pSub201/ProTα and PS virus and
ProTα expression was detected by IHC.                       54
15.Cell proliferation assay for ProTa.                      55
16.Transgene expression in the skeletal muscle of mice injected with rAAV/ProTa.                                                56
                                    

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周錦洪。人類前胸腺激素單株抗體的製備和應用。國立成功大學生物化學研究所碩士論文中華民國86年6月.

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