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Recombined human bFGF and PDGF-B duplicate adenovirus carrier and uses thereof

A PDGF-B and PDGF-BB technology, applied in the field of adenovirus bivalent vectors, can solve the problems of impossible to control the infection efficiency of two adenoviruses, difficult to control the expression amount of two genes, difficult to control the expression amount, etc. The effect of reducing the frequency of medication, improving the treatment effect, and reducing the cost of production and use

Active Publication Date: 2008-12-10
GUANGXI WUZHOU PHARMA GRP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, the gene therapy program using adenovirus as a carrier is basically single gene therapy, that is, using adenovirus as a carrier to introduce a single therapeutic gene, including a marker gene or a therapeutic gene, into the body. The gene therapy program on the carrier enters the clinic
Moreover, in preclinical studies, the introduction of adenovirus double genes is mainly through the insertion of an internal ribosome entry site (internal ribosome entry site, IRES) between the two genes, sharing the same promoter to achieve simultaneous activation of the two genes. For the purpose of expression, the expression efficiency of the second gene is affected by the expression of the first gene, and the expression level is difficult to control
Alternatively, use two adenoviruses to carry two different genes at the same time, but it is almost impossible to control the infection efficiency of the two adenoviruses in vivo studies, and the expression of the two genes is also difficult to control
At present, there is no report on the gene therapy program using bFGF (basic Fibroblast Growth Factor, basic fibroblast growth factor) and PDGF-BB (platelet derived growth factor, platelet-derived growth factor) at the same time

Method used

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  • Recombined human bFGF and PDGF-B duplicate adenovirus carrier and uses thereof
  • Recombined human bFGF and PDGF-B duplicate adenovirus carrier and uses thereof
  • Recombined human bFGF and PDGF-B duplicate adenovirus carrier and uses thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0041] Example 1 Construction and screening of recombinant bFGF and PDGF-B double gene adenovirus of the present invention

[0042] See the detailed construction process of recombinant bFGF and PDGF-B double gene adenovirus figure 1 In order to obtain an expression vector containing two independent expression frames (including different promoters, multiple cloning sites, and tailing signals) in the adenovirus, the bFGF gene was first inserted into the eukaryotic expression vector pSI (purchased from Promega), and then used The restriction site on the vector cuts out the sequence containing the complete bFGF expression framework (SV40 promoter-secreted bFGF-SV40 tailing signal), and inserts it into the modified shuttle vector pAdenoVator-CMV5△B362 (pAdenoVator-CMV5 purchased from Qbiogen Company), the bFGF shuttle plasmids inserted in the same direction or reverse direction—pAdv-F10 and pAdv-F5, respectively; insert the PDGF-B cDNA fragment into the recombinant bFGF shuttle vector...

Embodiment 2

[0068] Example 2 Construction and screening of recombinant bFGF adenovirus and recombinant PDGF-B adenovirus

[0069] The detailed construction process of the recombinant bFGF adenovirus is as follows: It is carried out on the basis of the recombinant bFGF shuttle vector pAdv-F5 and pAdv-F10 in Example 1, respectively, with the circular plasmid containing the entire adenovirus genome (E1, E3 deletion) pAdenoVator△E1E3 was electroporated into BJ5183 to obtain pAd-F10 and pAd-F5; the pAd-F5 and pAd-F10 linearized by Pac I were co-transfected with lipofectamine 2000 using conventional co-transfection methods to transfect 293 cells, and the results were obtained by screening Recombinant adenovirus Ad-F5 and Ad-F10. After extracting the viral DNA from the primary virus seed solution, PCR amplifies the inserted bFGF DNA fragment (PF1+PF2) to identify whether it is a recombinant adenovirus, and at the same time amplifies the E2B region fragment (PB5+PB6) that identifies the characteristi...

Embodiment 3

[0072] Example 3 Mass amplification and purification of recombinant adenovirus of the present invention

[0073] After the different recombinant adenoviruses constructed in the above examples were amplified by 293 cells, the recombinant adenovirus-Ad-F, Ad-P and Ad-FP were purified by two-step CsCl density gradient ultracentrifugation. The first step was discontinuous The CsCl gradient removes most of the cell debris and defective virus particles (that is, virus particles that do not have infectious activity). In the second step, a continuous CsCl density gradient is used to completely separate infectious virus particles from defective virus particles, and finally Desalting by dialysis, remove CsCl, and exchange to the required preservation solution.

[0074] See the results of the virus discontinuous CsCl density gradient centrifugation Picture 11 -A, the two white bands formed by the light-visible virus particles, the upper band is weaker and more diffuse, which are virus parti...

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Abstract

The invention belongs to the gene therapy field. The technical problem to be solved is to provide a new gene therapy product capable of effectively treating diseases of a cardiovascular system. The invention particularly relates to an expression vector containing recombinant human bFGF and PDGF-B double genes, and a preparation method and an application thereof. The vector which contains the gene capable of encoding bFGF protein and the gene capable of encoding PDGF-B protein can simultaneously express the bFGF protein and the PDGF-BB protein in a eukaryotic cell. An optimal proposal is to make the gene capable of encoding the bFGF protein and the gene capable of encoding the PDGF-B protein be expressed respectively under the control of different promoters. Experiments show that: the recombinant vector has good application prospect for treating myocardial ischemia of coronary heart disease and provides a new choice for treating the diseases of the cardiovascular system.

Description

Technical field [0001] The invention belongs to the field of gene therapy, and specifically relates to an adenovirus bivalent vector recombined with human bFGF and human PDGF-B and its use Background technique [0002] Cardiovascular diseases are the main diseases that seriously threaten human health and cause death in modern society. Coronary heart disease is the most common cardiovascular disease, with extremely high mortality and disability rates. The most commonly used methods for the treatment of coronary heart disease are interventional coronary artery bypass surgery and intramyocardial laser drilling. Although these methods have played a positive role in the treatment of coronary heart disease, they have their own limitations. At present, foreign scholars are trying to use angiogenesis therapy to eliminate or alleviate myocardial ischemia. Angiogenesis is the application of angiogenesis factors to stimulate the growth of small blood vessels in the ischemic area of ​​myocar...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/861C12N15/85C12N5/10A61K48/00A61P9/10
Inventor 杨莉魏于全
Owner GUANGXI WUZHOU PHARMA GRP
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