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High-efficiency recombined adenovirus containing ndrg2 gene and pharmic purpose thereof

A recombinant adenovirus, v5-dest-ndrg2 technology, applied in the field of medicine and biology, can solve problems such as no reports or literature, and achieve the effects of low pathogenicity, obvious drug effect, and high transgenic efficiency.

Inactive Publication Date: 2009-10-14
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Through the present invention project team's search on the Internet and retrieval of domestic and foreign patent documents and published periodical papers, no report or document identical with the present invention has been seen yet.

Method used

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  • High-efficiency recombined adenovirus containing ndrg2 gene and pharmic purpose thereof
  • High-efficiency recombined adenovirus containing ndrg2 gene and pharmic purpose thereof
  • High-efficiency recombined adenovirus containing ndrg2 gene and pharmic purpose thereof

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Embodiment 1

[0042] The invention is a high-efficiency recombinant adenovirus containing ndrg2 gene, and the adenovirus preparation contains ndrg2 gene. Gateway system and adenovirus expression system ViraPower were used in the construction process TM The AdenoviralExpression System, uses an in vitro recombination method to package adenoviruses.

[0043] The construction process specifically includes, first, ndrg2 is cloned into the entry vector pENTR2B (see figure 1 ) to obtain the entry clone pENTR2B-NDRG2, and then use Invitrogen's recombinase LR Clonase TM IIEnzyme Mix for entry cloning with the expression vector pAd-CMV / V5-DEST (see figure 2 ) to obtain the expression clone pAd-CMV / V5-DEST-NDRG2, the expression clone was identified by PCR and linearized with the restriction endonuclease PacI, and then transfected into HEK293A packaging cells to obtain the recombinant adenovirus, which was named Ad- CMV / V5-DEST-ndrg2; TCID50 method was used to detect virus titer, and western blot...

Embodiment 2

[0044] Embodiment 2: Construction of ndrg2 recombinant adenovirus

[0045] 1. Materials

[0046] PUC19 plasmid containing two subtypes of NDRG2 gene length and short, Invitrogen's adenovirus expression system ViraPower TM The Adenoviral Expression System was provided by the Department of Chemistry, Fourth Military Medical University. DH5α-competent bacteria were preserved by the Department of Chemistry, Fourth Military Medical University. HEK293A packaging cells were preserved by the Department of Chemistry, Fourth Military Medical University. Ampicillin was purchased from Guangzhou Baotai Company; T4 Ligase and DL2000 Marker were products of Fermentas; restriction endonucleases EcoRI, XhoI, EcoRV, Taq enzymes, small-dose plasmid extraction kits and gel purification recovery kits were from Bao Bioengineering (Dalian) ) Co., Ltd., Lipofectamine2000 kit was purchased from Gibco BRL, PCR primers were synthesized by Beijing Aoke Biotechnology Co., Ltd., sequencing primers and ...

Embodiment 3

[0065] see Figure 9 , the high-efficiency recombinant adenovirus preparation containing ndrg2 gene was applied to glioma cell U251, and its growth inhibitory effect on glioma cell U251 was observed. At the same time, other control groups were also tested. Figure 9 Series 1 in the figure is the Ad-NDRG2L infection curve, series 2 is the Ad-NDRG2S infection curve, series 3 is the empty vector virus infection curve, and series 4 is the normal control group curve. It can be clearly seen in the figure that there is a large difference in the absorbance value of each group as time goes by. Ad-NDRG2L infection group (the 1st day compared with the 4th day, the cell number proliferation rate was 39.5%), Ad-NDRG2S infection group (the 1st day compared with the 4th day, the cell number proliferation rate was 22.4%) and Compared with the empty vector virus infection group (the 1st day compared with the 4th day, the cell number proliferation rate was 47.6%) and the normal control group (...

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Abstract

The invention provides a high-efficiency recombined adenovirus containing an ndrg2 gene and the pharmic purpose thereof. The recombined adenovirus contains the ndrg2 gene, a Gateway system and a Virapower Adenoviral Expression system are utilized in the construction process, and the adenovirus is packed by a recombination method in vitro. After being cloned to an introduction carrier, a target gene can be efficiently and quickly cloned to other carriers of a receptor by specific recombination sites on the carriers and recombinase without depending on a restriction enzyme. The package of the adenovirus in vitro simplifies the experiment steps, shortens the operation time, and has high multiplication efficiency, high transgene efficiency and high virus yield efficiency as well as the transduction efficiency as high as 100 percent. The invention solves the technical problem of recombination and package of the ndrg2 gene in vitro as well as probes and determines the lethal effect of the high-efficiency recombined adenovirus to brain glioma cells and cells of lung cancer, liver cancer, gastric cancer and colon cancer. The high-efficiency recombined adenovirus can be used as a medicament for treating brain glioma, long cancer, liver cancer, gastric cancer and colon cancer.

Description

technical field [0001] The invention belongs to the field of medical biotechnology, relates to the construction of a high-efficiency recombinant adenovirus containing ndrg2 gene, and also relates to the application of the high-efficiency recombinant adenovirus in the treatment of brain glioma, lung cancer, liver cancer, gastric cancer and colon cancer. Background technique [0002] Cancer is the first fatal disease of human beings. According to WHO statistics, 6.9 million people worldwide die from cancer every year, and about 8.7 million new cases occur. According to statistics released by the Ministry of Health: In 2007, the mortality rate of malignant tumors among urban residents in my country was 94.40 / 100,000, becoming the first fatal disease for the fifth consecutive year. Common tumors include lung cancer, liver cancer, gastric cancer, colon cancer and glioma. [0003] Glioma is the most common malignant tumor of the nervous system. It has high morbidity, disability ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01A61K48/00A61P35/00C12R1/93
Inventor 邓艳春秦娜
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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