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Method for regulating and controlling adenovirus protein by double promoter and use thereof

A dual-promoter, promoter technology, applied in the directions of botanical equipment and methods, biochemical equipment and methods, applications, etc., can solve problems such as insufficient scope of application and large side effects

Inactive Publication Date: 2008-02-27
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The effect of radiotherapy in the treatment of malignant tumors is limited to several types of malignant tumors, the scope of application is not wide enough, and there are relatively large side effects

Method used

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  • Method for regulating and controlling adenovirus protein by double promoter and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1: Cloning of human telomerase catalytic subunit gene promoter and cyclooxygenase-2 gene promoter.

[0018] The adenovirus pXC1 vector was purchased from Canada Microbix Biosystem Inc. (Toronto), and pXC1 contains the sequence bp22-5790 of type 5 adenovirus.

[0019] Genomic DNA was extracted from fresh normal human blood, and the promoter of human telomerase catalytic subunit and the promoter of cyclooxygenase-2 gene were amplified by polymerase chain reaction (Polymerase Chain Reaction, PCR) technology. Add Age I restriction sites to the 5' end and 3' end of the enzyme catalytic subunit promoter, and add Eag I restriction sites to the 5' end and 3' end of the cyclooxygenase-2 gene promoter.

[0020] Primer 1: 5'-GCG GGA TGT GAC CAG ATG TT-3' (SEQ ID NO: 1)

[0021] Primer 2: 5'-TCT CCG CAT GTC GCT GGT T-3' (SEQ ID NO: 2)

[0022] Primer 3: 5'-TAT CAC CGG TAC AGA CGC CCA GGA CCG CGC T-3' (SEQ ID NO: 3)

[0023] Primer 4: 5'-ATT AAC CGG TAG GGC GGG GCC GCG GA...

Embodiment 2

[0035] Example 2: Construction, amplification and purification of specific proliferative adenoviruses dually regulated by human telomerase catalytic subunit promoter and cyclooxygenase-2 promoter.

[0036] 293 cells are formed by transforming human embryonic kidney cells with sheared type 5 adenovirus DNA, which contains and expresses type 5 adenovirus E1 region, and adenovirus DNA has a high transfection rate for it. The PBHGE3 adenovirus vector was purchased from Microbix Biosystem Inc. (Toronto), Canada, and it contains the right arm of adenovirus type 5 and has an E3 region. The modified adenovirus vector pXC1 / H181+C409 and PBHGE3 containing the left arm of type 5 adenovirus were co-transfected into 293 cells, and infectious adenovirus could be produced by homologous recombination. The virus is a specific proliferative adenovirus in which E1A is regulated by the human telomerase catalytic subunit promoter and E1B is regulated by the cyclooxygenase-2 promoter, and the rest ...

Embodiment 3

[0038] Example 3: The killing effect of Ad5-HC on tumor cells and normal cells

[0039] The primary fibroblasts isolated from normal epithelial tissue were negative for telomerase activity and did not express cyclooxygenase-2, so they were used as a normal cell for control. Tumor cell lines include: liver cancer cell line HepG2, SMMC7721, gastric cancer cell line BGC823, breast cancer cell line Bcap37, colon cancer cell line SW480, cervical cancer cell line Hela, all of the above cell lines were tested in the center of Run Run Shaw Hospital Affiliated to Zhejiang University School of Medicine The above tumor cell lines were all positively expressed in telomerase and cyclooxygenase-2. The above cell lines were divided into 10 4 Spread each well on a 96-well plate, and after culturing in a 37°C, 5% CO2 incubator for 24 hours, use the recombinant virus with a multiplicity of infection of 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78, and 0.39, respectively To infect the cells, s...

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Abstract

The invention provides a method for regulating and controlling adenovirus protein with double promoter, which comprises the following steps: inserting the human end granule enzyme catalysis subunit promoter and cyclooxygenase 2 promoter to E1A and E1B front promoter domain of adenovirus vector Pxc1 by cutting with Agel single enzyme and Eagl single enzyme and the vector of an adenovirus Pxc1; regulating and controlling the early gene E1A and E1B of adenovirus. The method is fit for the application of tumor gene medicine for regulating and controlling the early gene E1A and E1B of adenovirus. The method has the rational design, and the experiment indicates that the restructuring adenovirus can express the breeding and the killing of the adenovirus in the adenovirus of the end granule enzyme and the cyclooxygenase 2, but the adenovirus doesn't breed in the normal cell.

Description

field of invention [0001] The invention belongs to the field of life sciences, and specifically relates to a method and application for regulating the early protein of adenovirus by using a human telomerase catalytic subunit promoter and a cyclooxygenase 2 promoter, and using the two promoters to regulate the adenovirus respectively Early genes E1A and E1B enable this recombinant adenovirus to proliferate specifically in tumor cells that have telomerase activity and express cyclooxygenase 2, but basically do not proliferate in non-tumor cells. Background technique [0002] Malignant tumors have seriously endangered human health. In industrialized countries, one out of every five people dies from cancer-related diseases. At present, the methods for treating malignant tumors mainly focus on the three conventional means of surgery, chemotherapy and radiotherapy, but these methods are not very effective in treating many malignant tumors. Surgical treatment can only be aimed at ...

Claims

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Application Information

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IPC IPC(8): C12N15/66C12N15/861C12N15/53A61K48/00A61P35/00
Inventor 滕理送毛晨宇王浩浩华汉巨曹江
Owner ZHEJIANG UNIV
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