Construction and application of multiple target-point anti-cancer adenoviruses

A technology of adenovirus and recombinant adenovirus, applied in virus/bacteriophage, applications, anti-tumor drugs, etc., can solve the problem of no siRNA combined use, achieve obvious gene therapy effect, improve gene transduction efficiency, and facilitate application

Inactive Publication Date: 2009-10-28
GENERAL HOSPITAL OF TIANJIN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there are eukaryotic expression vectors expressing siRNA and adenovirus shuttle plasmid vectors commercially available separately, but there is no combination of the three siRNAs in the same vector to achieve systematic RNAi experiments at the cell and whole animal levels Research

Method used

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  • Construction and application of multiple target-point anti-cancer adenoviruses
  • Construction and application of multiple target-point anti-cancer adenoviruses
  • Construction and application of multiple target-point anti-cancer adenoviruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Construction of eukaryotic expression plasmids targeting tumor cell AKT1, PI3K p85 subunit and COX-2

[0040] 1. Synthesize the annealing connection of single-stranded target gene fragments:

[0041] 2. Ligation of the diluted annealed fragments with the linearized plasmids PEGFP6-1, PGENESIL-2, and PEGFP6-4 vectors:

[0042] 3. Take 5ul each of the ligation products to transform competent cells DH5a, and spread them on Kana-containing r On LB plates with resistance (final concentration: 30ug / ml), cultivate overnight in a constant temperature incubator at 37°C.

[0043] 4. Pick 3 monoclonal colonies from each petri dish and inoculate them in 3ml containing Kana r In LB medium with resistance (final concentration: 30ug / ml), cultivate overnight in a constant temperature shaker at 37°C.

[0044] 5. Use a kit to extract a small amount of plasmid (Zhongding Company), and use SacI to perform enzyme digestion identification (see figure 2).

[0045] 6. Pick and ...

Embodiment 2

[0058] Example 2 Recombinant adenovirus construction experiment

[0059] 1. Subclone the COX2, AKT1 and P85 shRNA expression cassettes from pGenesil-2 into the pGSadeno adenovirus expression vector through LR in vitro homologous recombination: LR in vitro homologous recombination, extract the plasmid with The extracted plasmid was identified by PI-SceI / I-CeuI double enzyme digestion and electrophoresed on 1% Agarose gel.

[0060] 2. Packaging the recombinant adenovirus: Digest the recombinant adenovirus plasmid with PacI, transfect the adenovirus DNA linearized with PacI into HEK 293, infect the lysed supernatant from the previous step into HEK 293 again, and amplify the culture.

[0061] 3. Validation of Recombinant Adenovirus Production

[0062] Confirm by fluorescence:

[0063] Objective There is fluorescent protein eukaryotic expression cassette in the recombinant adenovirus, and the expression of fluorescent protein is obvious after 24 hours of transfection, indicating ...

Embodiment 3

[0064] Example 3 Antisense Akt1, PI3K p85 subunit and COX-2 mediated by recombinant adenovirus inhibit the growth of glioblastoma U251 cells, gastric adenocarcinoma SGC7901 and breast cancer MCF-7 in vitro

[0065] 1. Cell culture and transfection: Culture medium and culture conditions: DMEM medium containing 10% FBS, the total volume of the medium is about 1 / 10 of the volume of the culture bottle, and the culture conditions are 37°C, 5% CO2 humidified culture. The cells were divided into 3 groups, namely control group, nonsense sequence group and antisense COX-2, Akt1, PI3K p85 subunit adenovirus transfection group. The transfection method is carried out according to MOI=100, and the transduction takes 48-72 hours.

[0066] 2. RNA extraction and RT-PCR method to detect the expression levels of Akt1, PI3K p85 subunit and COX-2 after transfection:

[0067] 1) RNA extraction: 48-72 hours after transfection of siRNA, add 0.5ml Trizol to the 6-well culture plate, pipette several ...

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Abstract

The invention discloses a construction proposal that adopts a genetic engineering method to obtain the target knockdown expression of mRNA in cells, recombinant adenoviruses obtained by the proposal and a detailed application of eukaryotic expression plasmid to treating cancers. In the invention, U6 promoters which respectively aim at human beings and mice are inserted at the downstream of the multiple clone site; antisense Akt1, PI3K, p85 subunits and COX-2 insertion sequence segments are connected with the downstream of the U6 promoters; the obtained recombinant adenoviruses and the eukaryotic expression plasmid can be reproduced or multiplied in tumor cells of glioma, gastric cancer, mammary cancer, lung cancer and the like; the cDNA segment of the inserted antisense Akt1, PI3K, p85 subunits and COX-2 can realize high-efficiency expression in tumor cells and restrain the expression of Akt1, PI3K, p85 subunits and COX-2 of tumor cells, thus further affecting the expression of downstream genes (such as Mmps, Nfkb, p53, bax, caspases and the like) and having strong bystander effect against tumor cells. The adenoviruses and the eukaryotic expression plasmid have unique practicality in preparing tumor treating medicines and radiotherapy hypersensitivity.

Description

【Technical field】: [0001] The invention belongs to the technical field of biomedicine technology and malignant tumor gene therapy, and relates to a recombinant adenovirus and a eukaryotic expression plasmid, specifically anti-cancer recombinant adenovirus and a Construction of eukaryotic expression plasmids and inhibition of growth of human glioma, breast cancer and gastric cancer, and radiosensitization effect on the above tumors. 【Background technique】: [0002] Tumor is essentially a multi-gene abnormal disease, involving the overexpression of oncogenes and the mutation and deletion of tumor suppressor genes and other multi-link and multi-pathway abnormalities. The effects of traditional treatment methods (including surgery, chemotherapy and radiotherapy) are very limited, and the rise of biological targeting technology provides new ideas for the treatment of tumors. [0003] Studies in recent years have found that some small double-stranded RNA (double-stranded RNA, dsR...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/861A61K48/00A61P35/00C12R1/93
Inventor 张庆瑜康春生浦佩玉张春智付彦超张靖王涛
Owner GENERAL HOSPITAL OF TIANJIN MEDICAL UNIV
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