Construction of recombinant replication-defective adenovirus expressing heme oxygenase-1 gene in mouse

WANG Rong-qi; et al

doi:10.13406/j.cnki.cyxb.2010.12.004

您当前的位置：

首页 >

文章列表页 >

Construction of recombinant replication-defective adenovirus expressing heme oxygenase-1 gene in mouse

更新时间：2024-01-17

- Construction of recombinant replication-defective adenovirus expressing heme oxygenase-1 gene in mouse
- Journal of Chongqing Medical University Vol. 35, Issue 12, Pages: 1836-1839(2010)
- 作者机构：
  
  河北医科大学第三医院中西医结合肝病科
- 作者简介：
- 基金信息：
- DOI：10.13406/j.cnki.cyxb.2010.12.004
  CLC： R346
- Published：2010，
扫描看全文
[1]王荣琦,南月敏,吴文娟,赵素贤,韩芳,付娜.小鼠血红素氧合酶-1复制缺陷型重组腺病毒的构建[J].重庆医科大学学报,2010,35(12):1836-1839.

WANG Rong-qi, et al. Construction of recombinant replication-defective adenovirus expressing heme oxygenase-1 gene in mouse. [J]. Journal of Chongqing Medical University 35(12):1836-1839(2010)
[1]王荣琦,南月敏,吴文娟,赵素贤,韩芳,付娜.小鼠血红素氧合酶-1复制缺陷型重组腺病毒的构建[J].重庆医科大学学报,2010,35(12):1836-1839. DOI： 10.13406/j.cnki.cyxb.2010.12.004.

WANG Rong-qi, et al. Construction of recombinant replication-defective adenovirus expressing heme oxygenase-1 gene in mouse. [J]. Journal of Chongqing Medical University 35(12):1836-1839(2010) DOI： 10.13406/j.cnki.cyxb.2010.12.004.

摘要

目的:利用细菌内同源重组法构建含血红素氧合酶-1（Heme oxygenase-1

HO-1）基因的重组腺病毒（Adenovirus

Ad）。方法:采用逆转录聚合酶链反应（Reverse transcription polymerase chain reaction

RT-PCR）以小鼠成纤维细胞扩增目的基因HO-1

经BglⅡ和XhoⅠ双酶切后克隆至含绿色荧光蛋白（Green fluorescence protein

GFP）基因的pAdTrack-CMV穿梭载体

将之PmeⅠ酶切线性化后

在含有Ad骨架质粒pAdEasy-1的BJ5183大肠杆菌内进行同源重组

选取经鉴定含目的基因的重组体质粒

PacⅠ酶切后用脂质体转染人胚肾293细胞

包装成重组体Ad Ad-HO-1。采用RT-PCR对重组体Ad进行鉴定

利用穿梭质粒pAdTrack-CMV中带有的GFP报告基因

监测病毒滴度和感染效率。结果:PCR、序列测定、以及限制性酶切法证实HO-1基因正确插入Ad表达载体

并在人胚肾293细胞中包装出重组Ad

荧光显微镜下可观察到GFP表达

收获病毒的滴度为1.25×1011 pfu/ml。结论:成功构建pAd-HO-1重组Ad表达载体

且重组Ad在人胚肾293细胞中能有效转录

为非酒精性脂肪性肝炎及其它慢性肝病的基因治疗奠定了基础。

Abstract

Objective:To construct a recombinant adenovirus(Ad) vector expressing heme oxygenase-1(HO-1) gene by the homologous recombination in Escherichia coli strain BJ5183.Methods:HO-1 gene was amplified from embryonic fibroblast cell of mouse by RT-PCR.Digested with BglⅡand XhoⅠ

the HO-1 gene was cloned into shuttle vector pAdTrack-CMV that carried a green fluorescence protein(GFP) gene.PmeⅠlinearized shuttle plasmid was transformed into E.coli BJ5183 containing adenovirus backbone vector pAdEasy-1 to achieve the homologous recombination.The DNA of identified recombinant plasmid was digested with Pac I and transfected to HEK293 cells to package recombinant adenovirus particles Ad-HO-1.The target gene was detected by RT-PCR.Infection efficiency and titre of the Ad-HO-1 in 293 cells was measured through the expression of GFP.Results:It was confirmed by RT-PCR

sequencing and restriction enzyme analysis that the target gene was cloned correctly to the shuttle plasmid.The expression of GFP could be observed by fluorescence microscope.The viral titer purified adenovirus was 1.25×1011 pfu/ml.Conclusion:Recombinant adenovirus vector was constructed successfully and packed in HEK293 cells

which can serve as a good gene transfer vector for the study of gene therapy in chronic liver diseases

such as non-alcoholic steatohepatitis.

关键词

血红素氧合酶-1重组腺病毒基因治疗

Keywords

Heme oxygenase-1recombinant adenovirusgene therapy

references

Views

下载量

CSCD

Alert me when the article has been cited

提交

Tools

Publicity Resources

Construction of gene therapy vector rAd-preS1 and identification of liver cell targeting function

Effect of TIMP-1 siRNA on the expression of TIMP-1 and collagen in rats with liver fibrosis

Effect of autocrine IL-4 on killing activity of CTL induced by K₅₆₂

The influence of MDR1 gene therapy on the survival rate of few late hepatocarcinoma balb/c mouse

The expression of P-gp during the MDR1 gene transferring into the hematopoietic cells of the Balb/c mouse bone marrow

Related Author

ZHANG Li

ZHANG Mingman

ZHU Xiaomei

HE Xiaozhang

Zhang Yan

Lang Qing

Shi Xiaofeng

Liu Qi

Related Institution

Children Research Center，the Children’s Hospital，Chongqing Medical University

Department of Infectious Diseases,the Second Affiliated Hospital,Chongqing Medical University

Department of Immunology, College of Pediatrics

重庆医科大学儿科学院外科

Department of Surgery,College of Pediatrics,Chongqing Medical University

⁰