Escherichia coli containing recombinant adenovirus plasmids and applications of recombinant adenovirus plasmids

A technology of recombinant adenovirus and Escherichia coli, which is applied in the direction of virus/bacteriophage, application, bacteria, etc., and can solve the problems of low transfection and expression efficiency

Active Publication Date: 2014-02-26
SUZHOU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The current bottleneck of gene therapy is the

Method used

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  • Escherichia coli containing recombinant adenovirus plasmids and applications of recombinant adenovirus plasmids
  • Escherichia coli containing recombinant adenovirus plasmids and applications of recombinant adenovirus plasmids
  • Escherichia coli containing recombinant adenovirus plasmids and applications of recombinant adenovirus plasmids

Examples

Experimental program
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Embodiment 1

[0122] The construction of embodiment 1 transfer vector

[0123] Material:

[0124] NotI, XbaI, XhoI, PmeI, PacI restriction enzyme, T4DNA ligase, DL2000marker, DreamTaq TM Green PCR Master Mix, RevertAid TM s First Strand cDNASynthesis Kit was purchased from Fermentas Company; daily small-scale plasmid extraction kit, PCR product cleaning kit, and DNA gel recovery kit were purchased from Hangzhou Weitejie Biochemical Technology Co., Ltd.; pAdTrack-CMV-PTEN, pAdTrack-CMV- ING4-PolyA-promoter and pAdTrack-CMV-PolyA-promoter were previously constructed in our laboratory [Zhang Haifeng, Yang Jicheng et al. Effect of adenovirus-mediated PTEN on the growth of A549 lung cancer cells in vitro and in vivo. Chinese Journal of Cancer Biotherapy, 2007, 14 (2), 173-178; Sheng Weihua, Yang Jicheng et al. Construction and expression of Ad-ING4-PolyA-Promoter-IL-24 double gene co-expression vector, Chinese Journal of Microbiology and Immunology, 30 (8): 695-703]; pAd-Easy-1 backbone pl...

Embodiment 2

[0133] Example 2 Construction and Identification of Homologous Recombination Adenovirus Plasmid

[0134] Construction and identification of ING4 or / and PTEN single gene, double gene co-expression homologous recombination adenoviral plasmid:

[0135] Recombination of pAdTrack-CMV-PolyA-promoter, pAdTrack-CMV-ING4-PolyA-promoter and pAdTrack-CMV-PolyA-promoter-PTEN, pAdTrack-CMV-ING4-PolyA-promoter-PTEN constructed above in our laboratory After the transfer plasmid was linearized with Pme I at 37°C for 2 hours, it was homologously recombined with the pAdEasy-1 (RGD) adenovirus backbone plasmid modified by RGD-4C, and BJ5183 competent bacteria were co-transformed by the calcium chloride method. Kana (50μg / ml) resistance selection of homologous recombination plasmids, positive clones were picked and plasmids were extracted for agarose gel electrophoresis, and pAdEasy-1(RGD)-pAdTrack-CMV-polyA- promoter (pAd.RGD-GFP for short), pAdEasy-1(RGD)-pAdTrack-CMV-ING4-polyA-promoter (pAd....

Embodiment 3

[0137] Example 3 Packaging and amplification of homologous recombinant adenovirus

[0138]The constructed pAd.RGD-GFP, pAd.RGD-ING4, pAd.RGD-PTEN, pAd.RGD-ING4-PTEN homologous recombinant adenoviral plasmids were digested with PacI and linearized, followed by agarose gel electrophoresis and gel slicing To recover large fragments, press Lipofectamin TM 2000 Liposome Operation Instructions The liposomes were transfected into QBI-293A human embryonic kidney cells for packaging. After transfection, GFP fluorescence was observed under a fluorescence microscope, and the expression of GFP could be seen under a fluorescence microscope 12 hours later, and the fluorescence intensity gradually increased with time after transfection. The first-generation recombinant adenovirus crude extracts of Ad.RGD, Ad.RGD-PTEN, Ad.RGD-ING4, and Ad.RGD-ING4-PTEN were collected 10 days after transfection. After secondary infection of QBI-293A packaging cells, fluorescence can be observed under an inve...

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Abstract

The invention relates to the technical field of biology, and especially relates to recombinant plasmids, recombinant adenovirus plasmids, and applications of recombinant adenovirus. The invention provides obtained escherichia coli (preservation number CCTCC No.2013212) containing a double-gene co-expressed homologous recombinant plasmid pAd.Easy-1(RGD)-pAdTrack-CMV-ING4-polyA-promoter-PTEN and recombinant adenovirus Ad.(RGD)--IG4-PTEN, and the escherichia coli and recombinant adenovirus can be used for preparation of drugs for treating malignant tumor and/or leukemia and chemotherapy sensitizers.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to Escherichia coli containing an RGD-modified double-gene co-expression recombinant adenovirus plasmid and the application of the RGD-modified double-gene co-expression recombinant adenovirus. Background technique [0002] Tumors have replaced cardiovascular and cerebrovascular diseases as the world's number one killer. According to the latest global cancer statistics report published by the American Cancer Society (ACS), in 2008, there were about 12.7 million new cancer cases and 7.6 million cancer deaths worldwide; Among them, developing countries accounted for 56% and 64% of new cancer cases and deaths, respectively. In China, the incidence of malignant tumors is rising particularly rapidly, with 2.2 million new cancer cases and 1.6 million cancer deaths each year. It is estimated that the number of morbidity and death in 2020 will increase by 60% compared with 2002. At present, th...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/861C12N7/01A61K48/00A61P35/00A61P35/02A61P35/04C12R1/19
Inventor 杨吉成缪竞诚刘济生
Owner SUZHOU UNIV
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