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Replication defect type recombination adenovirus

A recombinant adenovirus, replication-deficient technology, applied in the field of biomedicine, can solve problems such as weakening the therapeutic effect, and achieve the effect of wide clinical application value

Inactive Publication Date: 2008-05-28
THE THIRD AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIV OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] As the molecular basis of the body's resistance to various injuries, the DNA damage repair system plays a vital role in maintaining the stability and integrity of the genome. However, for radiotherapy and chemotherapy that kill cancer cells by damaging DNA, this process undoubtedly weakens the therapeutic effect. Effect

Method used

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  • Replication defect type recombination adenovirus
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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Construction and Identification of pDC316-EGFP-U6-APE1siRNA Adenovirus Shuttle Plasmid

[0043] according to us [4] The designed effective human APE1 siRNA sequence was analyzed and designed with the Ambion siRNA target sequence analysis system to scan the human APE1 gene cDNA coding sequence with the gene number NM_001641. 867-885 in the APE1 gene cDNA coding sequence has a specific siRNA target sequence of 19 nt in total, and the expression sequence of the human APE1 gene siRNA is designed, and the expression sequence contains the sense strand sequence, antisense strand sequence, connection The 9-base Loop loop sequence and terminal terminator sequence of the sense strand and the antisense strand, the sequence is as sequence 1 in the sequence list, and BamH I and Hind III enzyme cutting sites are introduced at both ends of the series 1, respectively. Synthesize two complementary single strands as follows:

[0044] Top strand:

[0045] 5′-GATCCGCTGGTACGACT...

Embodiment 2

[0049] Example 2 Ad5 / F35-APE1siRNA recombinant adenovirus packaging, amplification and purification 2.1 Ad5 / F35-APE1siRNA recombinant adenovirus packaging

[0050] Packaging principle and flow chart: The recombinant adenovirus is constructed using the adenovirus Ad5 / F35MaxTM packaging system of Benyuan Zhengyang Company. The shuttle plasmid pDC316-EGFP-U6-APE1siRNA carrying the human APE1 gene siRNA expression sequence and the adenovirus backbone plasmid pBHG-fiber5 / 35 were co-transfected into 293 cells. Plasmids are combined, and Cre / loxP system is used to perform site-directed recombination in 293 cells to produce Ad5 / F35-APE1 siRNA recombinant adenovirus with human APE1 gene siRNA expression sequence. The recombinant virus obtained in this way is a replication-deficient adenovirus with E1 deletion, and the virus can only realize the expression of foreign genes in cells that cannot provide the E1 region, but does not have the ability to proliferate. The packaging process of...

Embodiment 3

[0075] Example 3 Infection Efficiency of Ad5 / F35-APE1siRNA Recombinant Adenovirus to Colorectal Cancer Cells

[0076] 3.1 Detection of Infection Efficiency in Vitro

[0077] LOVO cells were cultured in an incubator containing 5% CO2 at 37°C, and the culture medium RPMI1640 contained 10% FCS, 1.0×10 5 U / L penicillin and streptomycin. The day before adenovirus infection, LOVO cells were seeded in a six-well plate, 5×10 per well 5 cells. Infect LOVO cells with adenovirus Ad5 / F35-APE1siRNA or control adenovirus Ad5 / F35-EGFP and Ad5-EGFP at a multiplicity of infection (MOI) of 0-20 for 90 min, then discard the culture medium and replace with complete medium After 24h, the expression of EGFP in LOVO cells was observed under a fluorescent inverted microscope and photographed. Digest the cells with 0.25% trypsin, centrifuge at 500g for 5min at room temperature, discard the supernatant, resuspend the cells in 0.01M PBS, centrifuge at 500g for 5min at room temperature, collect the c...

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Abstract

The invention relates to plication-deficient recombinant adenovirus which contains the expression sequence of siGNA of the gene APE1 of human being. The sequence of siGNA of the gene APE1 of human being at which the two ends are provided with restriction site is chemically synthesized and inserted under the prompter U6 of the shuttle plasmid of adenovirus after restriction enzyme, then shuttle plasmid pDC316-EGFP-U6-APE1siRNA is constructed, and then shuttle plasmid pDC316-EGFP-U6-APE1siRNA and the bone plasmid of adenovirus pBHG-fiber5 / 35 are simultaneously cotransfected into cell 293, consequently the recombinant adenovirus Ad5 / F35-APE1siRNA with the expression sequence of siGNA of the gene APE1 of human being is achieved through the fixed-point recombination produced by the system function of Cre / loxP. The recombinant adenovirus is capable of effectively preventing the expression of the gene APE1 and effectively strengthening the sensibility of radiotheraphy and chemotherapy of tumour cell.

Description

technical field [0001] The present invention relates to the field of biomedical technology, in particular to a replication-deficient recombinant adenovirus containing exogenous DNA fragments constructed by using Ad5 / F35 adenovirus vector and AdMax adenovirus packaging system, the recombinant adenovirus can effectively inhibit tumor cell APE1 Gene expression can effectively enhance the sensitivity of tumor cells to radiotherapy and chemotherapy. Background technique [0002] As the molecular basis of the body's resistance to various injuries, the DNA damage repair system plays a vital role in maintaining the stability and integrity of the genome. However, for radiotherapy and chemotherapy that kill cancer cells by damaging DNA, this process undoubtedly weakens the therapeutic effect. Effect. Therefore, gene therapy targeting DNA damage repair genes is expected to effectively enhance the sensitivity of tumor cells to radiotherapy and cytotoxic chemotherapy drugs. [0003] AP...

Claims

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Application Information

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IPC IPC(8): C12N15/861C12N15/12
Inventor 王东向德兵陈正堂谢家印李梦侠仲召阳
Owner THE THIRD AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIV OF PLA
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