| 朱松明张捷丁新德陈治泉高敏.大鼠过氧化物酶体增生因子激活受体γ基因慢病毒表达
载体的构建与鉴定[J].,2011,11(2):259-261 |
| 大鼠过氧化物酶体增生因子激活受体γ基因慢病毒表达
载体的构建与鉴定 |
| Construction and Detection of Lentiviral Expression Vector of RatPPAR-γGene |
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| DOI： |
| 中文关键词: 过氧化物酶体增生因子激活受体γ 慢病毒载体 肝纤维化 |
| 英文关键词: Peroxisome proliferator-activated receptor gamma Lentviral Hepatic fibrosis |
| 基金项目: |
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| 中文摘要: |
| 目的：构建大鼠过氧化物酶体增生因子激活受体γ(peroxisome proliferator-activated receptor gamma)基因慢病毒表达载体，
获得可供转染的滴度，为进一步研究该基因在肝星状细胞活化（Hepatic stellate cells, HSC）及肝纤维化中的作用机制提供物质基
础。方法：大鼠PPAR-γ 基因序列进行PCR 扩增，与经AgeI 酶切后的pGC-FU-3FLAG 载体连接产生慢病毒载体表达质粒
pGC-fu-3flag-PPARG，转化DH5α，PCR 筛选阳性克隆，测序并转入293T 细胞Western blot 鉴定，而后将pGC-fu-3flag-PPARG，
pHelper 1.0，pHelper 2.0 三质粒共转染293T 细胞，包装成慢病毒，收集上清浓缩病毒测定病毒滴度。结果：DNA 测序及Western
blot鉴定证实构建的大鼠PPAR-γ基因慢病毒表达载体pGC-fu-3flag-PPARG正确，浓缩慢病毒悬液的滴度为2×108TU /ml。结
论：成功构建携带大鼠PPAR-γ 基因的重组慢病毒表达载体。 |
| 英文摘要: |
| Objective: To construction of recombinant lentiviral expression vector carrying rat peroxisome proliferator-activated
receptor gamma (PPAR-γ)gene, and to obtain the titer of the lentiviral stock for investigating the mechanism of PPAR-γexpression in
the activation of HSC cells and hepatic fibrosis. Methods：Rat PPAR-γencoding sequence was amplified, then was ligated with
pGC-FU-3FLAG lentivirol vector to construct the lentiviral expression vector named pGC-fu-3flag- PPARG. It was identified by PCR,
DNA sequencing and Western blot . Virus packaging cells (293T cells) were co-transfected with lentivirol vector pGC-fu-3flag- PPARG,
pHelper 1.0 and Helper 2.0 to construct lentiviral. The titers of recombinant viruses were determined by Real time qPCR. Results：
Lentiviral expression vector of rat PPAR-γgene proved by sequencing and Western blot was successfully constructed. The titer of
recombinant viruses was 2 ×108TU /ml. Conclusion：Rat PPAR-γlentiviral expression vector was constructed successfully. |
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