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研究生:陳麗瑄
研究生(外文):CHEN, LEE-HSUAN
論文名稱:以多瘤性病毒外套蛋白作為基因載體之研究
論文名稱(外文):Studies of Gene Transfer by Polyoma Virus Major Capsid Protein VP1
指導教授:楊雅雯楊雅雯引用關係王萬波蘇慕寰蘇慕寰引用關係
指導教授(外文):Yang Ya-WunWang Wan-PoSu Mu-Hwang
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:藥學系
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
論文出版年:1997
畢業學年度:85
語文別:中文
論文頁數:83
中文關鍵詞:基因治療多瘤性病毒外套蛋白非病毒性載體基因轉染磷酸鈣共沉澱法直接注射法
外文關鍵詞:gene therapypolyoma virus major capsid protein VP1nonviral vectorgene transfercalcium phosphat coprecipitationdirect injection
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本實驗以非病毒性載體-多瘤性病毒外套蛋白 VP1 作為基因轉染
的載體.其來源有購得之 Pseudofect 及 基因工程將 PyVP1 選殖在 E.
coli 經表現而得. 結果顯示 Pseudofect 具有包裹質體 DNA 的
能力而 E. coli VP1 則否. 活體外實驗顯示 Pseudofect 與 E. coli
VP1 的基因轉染效果較磷酸鈣共沉澱法差,約只有其 30~50 %.活體內結果
顯示單純質體 DNA 即可成功達到基因轉染的目的, poly-L-lysine 及 E.
coli VP1 則約增加基因轉染效率 1.5 倍. 由活體外與活體內之
實驗結果顯示 Pseudofect 與 E. coli VP1 其基因轉染的效率並無顯著
差異.

The biological requirements for successful somatic gene
therapy include the ability to efficiently introduce recombinant
genes into the target cells.Two general strategies have been
proven to be useful, including both viral vectors and the
formulated DNA expression vectors. Recombinant viral vectorshave
been less desired in the development of gene tranfer strategies
due to the concerns of safety. In the present study, delivery of
the recombinant genes by the nonvirial strategies utilizing the
polyomavirus (Py) major capsid protein VP1 as a gene carrier was
attempted. The results obtained were compared withthose obtained
by Pseudofect,(a recombinant PyVP1 expressed in baculovirus)
andby the calcium phosphate coprecipitation method. The
polymavirus VP1 pseudocapsids generated from recombinant
baculovirus, have been shown to be able to effectively transfer
the exogenous DNA into several mammalian cell lines in vitro.
Experiments were carried out in thisstudy to investigate the
feasibility of transferring foreign DNA into therecipient host
by PyVP1 purifed from recombinant E.coli,both in vitro and in
vivo. The reporter genes employed were pCMVb, containing the
lacZ gene under thecytomegalovirus promoter, and pPyMT-1,
containing the polyomavirus middle Tantigen. To prepare
the E.coli VP1/DNA complex, purified E.coli VP1 was sonicated,
mixed with CsCl - purified DNA and then incubated with an
appropriate amountof poly-L-lysine (MW30000 - 70000) at room
temperature. The NaCl concentrationwas then adjusted to 35 mM,
followed by sonication before use. The level of DNA
protected by VP1 was first examined by incubating DNAwith VP1,
followed by DNase digestion. The experimental results indicated
that exogenous DNA was not protected by E.coli VP1, whereas
about 5-10% of the inputDNA was protected from DNase treatment
by VP1 Pseudofect. NIH3T3, a polyomavirus permissive cell
line, and FR3T3, a polyomavirustransformation competent cell
line were transfected by pCMVb and pPyMT-1,respectively.In vitro
DNA transfection was carried out by treating the cells withE.
coli VP1,Psesudofect, and the calcium phosphate coprecipitation
methods.Expression of b-galactosidase or PyMT-1 reporter genes
was examined by X-galstaining, exzyme-linked immunosorbent assay
(ELISA), and Western blot followedby PhosphorImager analysis.
The results obtained showed that although expression of b-gal
was detected in several cells at 48 hours post-transfection, the
efficiency of gene transfer by E.coli was about 30-50% as
compared to the calciumphosphate method. VP1 Pseudofect,
generated from baculovirus, on the other hand,showed a lower
transfection efficiency than those reported by others.
Nostatisticall significant difference on the in vitro
transfection efficiency was observed for PyVP1 obtained from two
different sources. Parallel experiments were performed in
vivo by injecting pCMVb or pPyMT-1 plasmid DNA/E.coli VP1
mixture into the livers of Wistar rats. Liverswere harvested 72
hours after direct gent transfer. Expression of the reporter
genes were analyzed by X-gal staining, ELISA, and Western blot
analysis, andthe results were compared with those obtained by
direct injection of naked DNAsolution and DNA-poly-L-lysine
complex suspensions. Direct injection of plasmid DNA into liver
exhibited sufficient gene expression. Complexation withpoly-L-
lysine was found to increase about 1.5 fold of the reporter
geneexpression. Microscopic examination of liver sections
revealed intracellularblue granules consisten with b-gal
activity. The majority of transfected hepatocytes were found to
be localized in the vicinity of the injection sites.Semi-
quantitative analysis of b-gal and PyMT-1 gene expression by
ELISA andWestern blot analysis suggested that PyVP1 obtained
either from baculovirus orE.coli exhibits no significant effect
on the efficiency of gene transfer in vivo.

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