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A self-clearing adenovirus vector and its preparation method

An adenovirus and vector system technology, applied in the field of self-clearing adenovirus and its preparation, can solve the problems of helper virus contamination and complex preparation process, and achieve the effect of reducing the number and reducing the impact

Inactive Publication Date: 2016-08-17
THE SECOND AFFILIATED HOSPITAL ARMY MEDICAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Adenoviral vector is one of the main vectors used in the process of foreign gene introduction, but adenoviral vector itself will bring many problems, these problems have not been well resolved so far, the most difficult to solve is: After infecting 293 cells or other somatic cells, the gene encoding the E1 region can be regained to produce replication competent adenoviruses (RCAs)
This problem is most prominent in the first-generation adenovirus; although the second-generation adenovirus has removed the E2a and / or E4 region coding genes on the basis of the first generation, this problem has only been improved to a certain extent, but it has not yet been confirmed. It can be completely solved; the third-generation adenovirus only retains the terminal repeat sequence ITRs at both ends of the adenovirus genome and the packaging sequence at the 5' end. In theory, this improvement of the third-generation adenovirus can completely eliminate the production of RCAs; However, in practical applications, the preparation process of the third-generation adenovirus is very complicated and can only be completed with the help of helper virus, which leads to the troublesome problem of helper virus contamination

Method used

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  • A self-clearing adenovirus vector and its preparation method
  • A self-clearing adenovirus vector and its preparation method
  • A self-clearing adenovirus vector and its preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Construction and in vitro verification of the self-clearing adenovirus of the present invention: choose AdEasy TM The adenoviral vector system was used as the basis for its transformation.

[0044] 1) Cloning the corresponding genes from pBR322 prokaryotic expression plasmids carrying rtetR-VP16 and TRE-Pmin CMV-Cre genes by PCR method. rtetR-VP16 upstream primer: 5'-GGG GTA CCA TGT CTA GAC TGG ACA A-3', downstream primer: 5'-GGG GTA CCC TAT AGT TCT AGA GGC T-3'; TRE-PminCMV-Cre gene upstream primer: 5'-CCC TCG AGC TCG AGT TTA CCA-3', downstream primer: 5'-CCC TCG AGC TAA TCG CCA T-3'. PCR reaction program: After preheating, the main cycle of PCR reaction for rtetR-VP16 is 57°C for 1min, 70°C for 30sec; the main cycle for PCR reaction of TRE-PminCMV-Cre gene is 56°C for 1min, 70°C for 30sec. Extend at 70°C for 5 minutes;

[0045] 2) Digest the pShuttle shuttle plasmid with kpnI single enzyme (1ul of kpnI enzyme, 10ul of pShuttle, 5ul of 10×Buffer, 2.5ul of BSA, add w...

Embodiment 2

[0061] Construction of self-clearing recombinant adenovirus and AdEasy expressing BMP-4 and VEGF TM Recombinant adenovirus, and infected rat bone marrow mesenchymal stem cells:

[0062] 1) Cloning the corresponding genes from pBR322 prokaryotic expression plasmids containing bone morphogenetic protein-4 (bone morphogenetic protein4, BMP-4) and vascular endothelial growth factor (vascular endothelial growth factor, VEGF) genes respectively. Among them, the upstream primer of BMP-4: 5′-TGA TTC CTG GTA ACC GAA TGC T-3′, the downstream primer: 5′-GGC ACC CAC ATC CCT CTA CTA-3′; the main cycle of PCR reaction is 56°C 1min, 71°C 30sec; VEGF upstream primer 5′-CCA AGC TTA TGA ACT TTC TGC TG-3′, downstream primer 5′-CCA AGC TTT CAC CGC CT-3′, PCR reaction main cycle is 54°C 1min, 71°C 30sec, main cycle After completion, all were extended at 71°C for 5 minutes. The sequence of BMP-4 is shown in SEQ ID NO.4, and the sequence of VEGF is shown in SEQ ID NO.5.

[0063] 2) Digest the rec...

Embodiment 3

[0070] Verification of the working efficiency of recombinant adenovirus:

[0071] ① Take the above-mentioned recombinant self-clearing adenovirus AdEasyN-BMP-4 and AdEasyN-VEGF and the traditional AdEasy-based TM The constructed AdEasy-BMP-4 and AdEasy-VEGF were used to infect rat BMSCs; 48h later, Dox was added to make the final concentration of Dox in the medium to be 10umol / L;

[0072] ②After 5 days, observe whether the recombinant self-clearing adenovirus AdEasyN-BMP-4 and AdEasyN-VEGF added with Dox and the traditional AdEasy-based TM The green fluorescence expression of AdEasy-BMP-4 and AdEasy-VEGF constructed to evaluate the degradation efficiency of self-clearing recombinant adenovirus;

[0073] ③The levels of cytokines BMP-4 and VEGF in the cell culture medium of the four groups were detected according to the method steps described in the ELISA kit instructions;

[0074] ④According to the method steps described in the instructions of the real-time quantitative RT-PC...

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Abstract

The present invention provides a self-clearing adenovirus, which is transformed based on the AdEasyTM adenovirus vector system, and the shuttle plasmid used is pShuttle-CMV, wherein the transformation refers to the insertion of the rtetR-VP16 gene sequence into the shuttle plasmid pShuttle-CMV , TRE‑PminCMV‑Cre gene sequence and two LoxP sites in the same direction. The present invention can induce the degradation and death of adenovirus at any time, and eliminate foreign genes and adenovirus vector itself before the seed cells required for tissue engineering are applied in the body; it can greatly reduce the amount of adenovirus entering the body, thereby reducing the impact of the vector itself on the body , to provide a safer and more reliable carrier for tissue engineering products.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a self-clearing adenovirus and a preparation method thereof. Background technique [0002] Exogenous gene introduction technology is a common technology in the field of tissue engineering. Researchers often use this technology to introduce genes encoding bone morphogenetic protein (Bone Morphogenetic Protein, BMP) 2, 4, 7, etc. into bone marrow mesenchymal stem cells (Bone Marrow Stem Cells, BMCs) and adipose stem cells to repair bone defects ; Introduce the gene encoding vascular endothelial growth factor (VEGF) into bone marrow mesenchymal stem cells for vascular reconstruction; and carry siRNA to knock down the expression of the host cell's own genes, etc. These studies have achieved good results. [0003] Adenoviral vector is one of the main vectors used in the process of foreign gene introduction, but adenoviral vector itself will bring many problems, these problems have not bee...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/861C12N7/01
Inventor 李元朝何威王儒鹏周春丽张斌
Owner THE SECOND AFFILIATED HOSPITAL ARMY MEDICAL UNIV
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