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Establishing method and application of system for packing adenoviruses with mesenchymal stem cells

A MSC and adenovirus technology, applied in the field of viral vector construction, can solve the problems of naked virus attack and lack of immunogenicity of mesenchymal stem cells

Inactive Publication Date: 2019-11-26
黄映辉
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

(2) Mesenchymal stem cells lack immunogenicity, and using them to encapsulate recombinant adenoviruses can solve the problem of naked viruses being attacked by host defense mechanisms

Method used

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  • Establishing method and application of system for packing adenoviruses with mesenchymal stem cells
  • Establishing method and application of system for packing adenoviruses with mesenchymal stem cells
  • Establishing method and application of system for packing adenoviruses with mesenchymal stem cells

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Embodiment Construction

[0028] 1. Look up the E1A+E1B sequence of the Human adenovirus 5 gene on NCBI, specifically 560bp to 3509bp on the adenovirus genome, adding XbaⅠ and NotⅠ restriction sites at the beginning and the end, and send the sequence to Sangon Bioengineering (Shanghai) Co., Ltd. Perform full sequence synthesis.

[0029] 2. Digest 1 μg of the pCDH-CMV-MCS-EF1-Puro plasmid with 1 μl each of NEB’s XbaⅠ and NotⅠ enzymes, and use agarose gel to separate the digested vector fragments and use Tiangen Biochemical Technology (Beijing) Ltd.'s Agarose Gel Recovery Kit to recover the linearized vector after enzyme digestion.

[0030] 3. Use 1 μl NEB T4 DNA ligase to transfer 150ng of synthetic E1 gene and 50ng of linearized carrier DNA, transfer the ligation product into DH5α competent cells, and spread it on the LB solid culture dish with ampicillin resistance.

[0031] 4. Pick a single clone on the culture plate the next day for next-generation sequencing, save the strains whose sequencing resu...

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Abstract

The invention relates to an establishing method and application of a system for packing adenoviruses with mesenchymal stem cells, and belongs to the field of gene therapy. The method comprises the steps of reforming MSC cells as a carrier to obtain an MSC-E1 cell line for stable expression of E1 genes, enabling replication-defective adenoviruses having the effect of killing and damaging tumor cells to be infected with the cell line, and through intra-cellular assembling, preparing a cell system which can be packed and reproduce adenoviruses, wherein the cell system is used for performing caudal vein injection on a tumor model mouse, and the purpose of well killing and damaging tumor masses of the mouse is achieved. The established system can solve the three bottlenecks in tumor gene therapy, so that a bioremediation preparation is not attacked by a host defense mechanism, the bioremediation preparation can specially achieve the tumor parts, and therapeutic genes are in high-efficient expression in tumor tissues.

Description

technical field [0001] The invention relates to the construction of a virus vector, the in vitro transfection of cloned gene E1 of MSC cells, and the infection of MSC cells by recombinant adenovirus, which is applied to the treatment of breast cancer and belongs to the field of gene therapy. Background technique [0002] The means of gene therapy is to introduce tumor suppressor genes into tumor cells with a certain carrier, make them express and execute the killing effect on tumor cells. At present, many tumor suppressor genes have been discovered. Viral vectors are widely used in scientific research and clinical practice due to their high efficiency, and more than 80% of them use adenovirus and retrovirus. Retroviruses can integrate into the host genome, so insertion mutations may occur, causing safety problems. However, adenovirus can efficiently express the target gene without integrating into the host genome, so it is safer to use and has more development prospects. S...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N5/10A61K48/00A61P35/00
CPCC12N7/00C12N5/0662A61K48/0008A61P35/00C12N2710/10021C12N2710/10022C12N2510/00
Inventor 黄映辉张霖马羚
Owner 黄映辉
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