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Vector for genetic drug carrying of targeting exosome and establishment method and application thereof

A technology of exosomes and gene medicines, applied in the field of genetic engineering and biology, can solve the problems of low conversion rate, small selection of exogenous genes, and low efficiency, and achieve the effect of increasing capacity and widening selection

Active Publication Date: 2018-11-06
上海生博生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although viral vectors have been used as gene drug delivery tools and have successful clinical trials, the application of viral vectors is limited due to the risk of viral vectors integrating into the entire genome and mutating back to wild-type viruses.
Nanomaterials such as liposomes and nanoparticles are increasingly used in drug delivery systems, but there are still disadvantages such as low biocompatibility and poor stability. Therefore, exosomes, as a kind of vesicles secreted by cells, It is the best choice as a gene drug delivery carrier
[0005] Existing problems: At present, the most widely used is to use the markers of exosomes, namely tetraspanins CD63, CD81, and CD9, as guides, and to express other target genes or fluorescent proteins with marker proteins, so that the expressed Exogenous gene guide tape into exosomes
However, the use of such markers has the following disadvantages: first, the gene itself is relatively large and limited by the carrier, making the selection of foreign genes relatively small; second, the targeting of such marker proteins is relatively poor, and in The organelle membrane is also rich in such proteins, so the conversion rate is relatively small and the efficiency is low

Method used

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  • Vector for genetic drug carrying of targeting exosome and establishment method and application thereof
  • Vector for genetic drug carrying of targeting exosome and establishment method and application thereof
  • Vector for genetic drug carrying of targeting exosome and establishment method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1 vector construction

[0041] In the PLVX-CMV7-MCS-EF1-Puro vector (the vector was purchased from Shiao (Shanghai) Biomedical Technology Co., Ltd., the vector structure diagram is shown in figure 1 ) on the basis of constructing pLVX-CMV-EGFP-F5 / 8 typeC-EF1a-Puro vector. The PLVX-CMV7-MCS-EF1-Puro vector has a CMV7 promoter, a multiple cloning site MCS, a selective marker Puromycin, an ampicillin resistance gene, etc., forming a 7.4kb DNA fragment. The EGFP-F5 / 8 typeC sequence was added to the multiple cloning site between the CMV7 promoter and Puromycin to construct the vector PLVX-CMV7-EGFP-F5 / 8 typeC-EF1-Puro. The specific construction process of the carrier is:

[0042] 1. Design EcoRI-MFGE8(1-23aa)-EGFP-linker-RS1 according to the gene information of the CDS sequence on the mRNA of the human MFGE8 gene, the CDS sequence on the mRNA of the human RS1 gene, the EGFP tag protein sequence, and the linker tag sequence in GeneBank (59-224aa)-XhoI gene DNA ...

Embodiment 2

[0071] Example 2 Plasmid transfection, fluorescence observation

[0072] Cell preparation: 293T cells preserved in a liquid nitrogen tank (purchased from the Cell Bank of the Chinese Academy of Sciences), quickly revived in a water bath at 37°C, and after 1-2 minutes of revival, pour out all the cells and freezing solution in the freezing tube Into 10cm 2 In the culture dish, configure DMEM+10%FBS, add about 8mL of complete medium into the culture dish, observe the state of the cells under the microscope after 24 hours, when the confluence of the cells is greater than or equal to 80%, the cells need to be passaged. When the cell coverage reaches about 70%, it can be used for transfection.

[0073] Plasmid transfection: One day before transfection, select HEEK293T cells with good cell state, digest the cells with trypsin, and inoculate the cells with 4x10E6 cells in 10cm 2In the culture dish, add 10 mL of 10% FBS DMEM to the culture dish. After 24 hours, check the cells in th...

Embodiment 3

[0074] Example 3 Supernatant collection, exosome extraction and identification

[0075] Take a sterile 25ml centrifuge tube in the ultra-clean bench, draw the cell culture supernatant through an electric pipette gun, transfer it to a 25ml centrifuge tube, put the centrifuge tube symmetrically into the centrifuge, centrifuge at 3000g, 4°C for 20 minutes , put the centrifuge tube back into the ultra-clean bench steadily, absorb the supernatant and transfer it to a new 25mL centrifuge tube. Take 5mL supernatant and add 1ml ExoPerfect TM MUexosome separation and purification solution (purchased from Shiao (Shanghai) Biomedical Technology Co., Ltd.), mixed thoroughly with a pipette gun, and mixed at 4°C overnight or >14 hours. After mixing, centrifuge at 1500g at 4°C for 30 minutes, remove the supernatant with a pipette gun, then centrifuge at 1500g at 4°C for 30 minutes to remove the residual solution, add 50ul sterile PBS or deionized water to resuspend the exosome pellet, resu...

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Abstract

The invention discloses a vector for genetic drug carrying of a targeting exosome. The vector comprises a CMV7 promoter, a selective marker Puromycin and a resistance gene. An EGFP-F5 / 8 type C sequence shown as SEQ ID NO.1 is added at a multiple cloning site between the CMV7 promoter, a selective marker Puromycin polyclonal site as is added between the CMV7 promoter and the selective Puromycin marker. In addition, the invention also discloses an establishment method and application of the vector. The vector makes the exosome carry exogenous genes to the maximum extent for delivery of gene drugs. The gene sequence of the vector is smaller, the carrying capacity of the gene can be improved, and the selection range of exogenous genes is widened. The fluorescence effect of the vector is stronger, and obvious and intuitive observation can be achieved.

Description

technical field [0001] The present invention belongs to the field of genetic engineering and biotechnology, and in particular relates to a carrier, in particular to a carrier targeting exosomes for gene loading; in addition, the present invention also relates to the construction method and application of the carrier. Background technique [0002] Exosomes are extracellular vesicles secreted by cells. Almost all cells in the human body can produce exosomes, such as B cells, T cells, blood cells, tumor cells, etc., and exosomes exist in all body fluids, such as urine , blood, cerebrospinal fluid, etc. The diameter of exosomes is about 30nm to 100nm. Exosomes are mainly derived from the multivesicular bodies formed by the invagination of intracellular lysosomal particles, and are released into the extracellular matrix after the fusion of the multivesicular outer membrane and the cell membrane. [0003] Various substances contained in exosomes are derived from their cells, suc...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/66A61K48/00
CPCA61K48/00C12N15/66C12N15/86C12N2740/15043
Inventor 唐凡吴小江顾莉萍余虹
Owner 上海生博生物医药科技有限公司
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