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
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Examples
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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Application Information
- IPC
- C12N15/66; C12N15/861; C12N15/53; A61K48/00; A61P35/00
- Inventors
- 滕理送; 毛晨宇