VEGF165 and Ang-1 double-gene co-expression vector and application thereof

A co-expression vector, ang-1 technology, applied in application, gene therapy, genetic engineering, etc., can solve the problems of low gene transfer efficiency, difficult to coordinate and calculate the dose, and achieve the effect of avoiding angiogenesis

Inactive Publication Date: 2011-09-21
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the past combined gene therapy, two vectors carried two genes respectively, which were given sequentially or simultaneously. In the transgenic therapy, two gene transfections were required, and the doses of the two genes were difficult to coordinate and calculate when the two genes were administered separately, and the gene transfer efficiency was low.
Not yet seen: the same adenovirus vector carries two angiogenesis-promoting genes, and they are co-expressed in vascular endothelial progenitor cells (EPCs), combined with gene combined with stem cell transplantation to treat ischemic myocardium, promote angiogenesis in ischemic areas and improve heart function report

Method used

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  • VEGF165 and Ang-1 double-gene co-expression vector and application thereof
  • VEGF165 and Ang-1 double-gene co-expression vector and application thereof
  • VEGF165 and Ang-1 double-gene co-expression vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Construction and identification of VEGF165 and Ang-1 double-gene recombinant vector, using IRES-mediated VEGF165 and Ang-1 double-gene adenovirus co-expression mode, the schematic diagram is as follows figure 1 shown.

[0046] (1) Material

[0047] Bgl II, Sal I, Not I, Xho I and EcoR V restriction enzymes, T4 DNA ligase, DL2000marker, Taq DNA polymerase, dNTPmix, Oligo d(T) 18 Purchased from TaKaRa Company; Reverse transcriptase MMLV, λ-Hind III digest DNA marker and low molecular weight protein marker were purchased from MBI Company; daily small-scale plasmid extraction kit, PCR product cleaning kit, DNA gel recovery kit were all purchased from From Hangzhou Weitejie Biochemical Technology Co., Ltd.; pGEZ-Term plasmid, pSV-K3-VEGF165 plasmid, and Escherichia coli DH5α were donated by Professor Yang Jicheng from the Department of Cell and Molecular Biology, Soochow University School of Medicine; GFP-labeled pAdTrack-CMV transfer plasmid, adeno Viral backbo...

Embodiment 2

[0073] Example 2: Infection of peripheral blood vascular endothelial progenitor cells in vitro with VEGF165 and Ang-1 double-gene adenoviral vector obtained in Example 1

[0074] (1) Material

[0075] 8d chicken embryos were purchased from Suzhou Hatchery; Pingyangmycin (PYM) was purchased from the First Affiliated Hospital of Soochow University; 24-well and 6-well cell culture were purchased from CORNING Company; RPMI1640 was purchased from GIBCO Company; calf serum was purchased from Hangzhou Four Seasons Qing Company; human VEGF165 antibody [VEGF(A-20): sc-152-G], human Ang-1 antibody [Ang-1(C-19): sc-6320] were purchased from Santa Cruz Company; immunofluorescent staining reagents Box-anti-goat Cy3 was purchased from Beyontian Biotechnology Research Institute; human VEGF, Ang-1 ELISA detection kits were purchased from Jingmei Company.

[0076] (2) Activity detection of VEGF165 and Ang-1 recombinant adenovirus (CAM test)

[0077] Chicken embryos with good growth status af...

Embodiment 3

[0107] Example 3: The VEGF165 and Ang-1 double-gene modified peripheral blood vascular endothelial progenitor cells obtained in Example 2 promote angiogenesis in rabbit ischemic myocardium.

[0108] In order to observe the effect of VEGF165 and Ang-1 gene-modified EPCs obtained in Example 2 on promoting neovascularization in ischemic tissue in vivo, in this example, on a rabbit myocardial ischemia model, the EPCs were proliferated in vitro and passed VEGF165 and Ang-1 Gene-modified EPCs were transplanted from the arterial route to ischemic myocardium, and the effect of promoting angiogenesis in ischemic tissue was observed to evaluate the biological function of VEGF165 and Ang-1 double-gene co-expression vector and single-gene modified EPCs.

[0109] (1) Material

[0110] Clean-grade New Zealand white rabbits (male, weighing 2.5-3.1 kg) were selected from the Animal Experiment Center of Soochow University [license number: SCXK (Su) 2002-0008] and raised at a temperature of 18-...

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Abstract

The invention discloses a VEGF165 and Ang-1 double-gene co-expression vector. The double-gene co-expression vector contains a human gene VEGF165 and a human Ang-1 gene; the double-gene co-expression vector is pAdTrack-CMV-Ang-1-IRES-VEGF165; the double genes are recombined to transfer vectors; the double genes are in homologous recombination with the adenovirus framework plasmid pAdEasy-1 and then are packed and augmented in QB 1-293A cells so as to obtain an Ad-Ang-1-IRES-VEGF165 double-gene co-expression recombination adenovirus; and finally a double-gene recombination adenovirus carrying the VEGAF and the Ang-1 infects vessel endothelial progenitor cells so as to promote the angiogenesis of ischemic myocardium and improve functions of the heart.

Description

technical field [0001] The invention belongs to the field of recombinant vectors, and in particular relates to the application of VEGF165 and Ang-1 dual gene co-expression vectors in the preparation of drugs for promoting angiogenesis. Background technique [0002] It is well known that many cellular and chemical factors participate in the regulation of angiogenesis. At present, it is believed that: VEGF (vascular endothelial growth factor) has biological effects such as specifically promoting the proliferation of endothelial cells, inducing the growth of new blood vessels, and then forming collateral circulation, etc., and plays a role in the initiation and maintenance stages of angiogenesis; Cell growth factor) is mainly involved in the initial process; while Ang-1 (angiopoietin), IGF (insulin-like growth factor) and PDGF (platelet-derived growth factor) mainly affect the maturation and stability of blood vessels. Experimental studies have revealed that exogenous VEGF gen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/861C12N7/01A61K48/00A61P9/00C12R1/19
Inventor 吕海涛杨吉成杨向军谢宇锋韩莲花盛伟华李红霞周亚锋冯星
Owner SUZHOU UNIV
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