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A cell membrane particle expressing fusion membrane protein and its preparation and application

A cell membrane and membrane protein technology, applied in the field of synthetic biology, can solve the problems of inapplicable batch preparation, time-consuming and labor-intensive biological safety, low transfer efficiency, etc., and achieve the effect of improving transfer efficiency, improving efficiency, and morphological integrity

Active Publication Date: 2019-01-15
SHANTOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have found that mitochondria can spontaneously transfer between cells through membrane nanotubes (membrane nanotubes), and can restore the aerobic respiration function of mitochondria-deficient cells; mitochondrial microinjection can also achieve mitochondrial transfer, but strict operating requirements and Special instruments and equipment are not suitable for batch preparation; in addition, the use of actinomycin D (actinomycinD) can make cells functionally "enucleated" and become cytoplasts containing mitochondria, but some cells are not affected by drugs and still retain nuclei. function; recent studies have reported that the use of cell-penetrating peptide (CPP) can also effectively deliver mitochondria isolated in vitro
But the transmission efficiency is relatively low
[0005] Due to the problems of low delivery efficiency, time-consuming and labor-intensive delivery and biological safety in the current delivery system for delivering biomacromolecules and organelles, it is of great significance to establish a method for efficiently delivering biomacromolecules and organelles for the treatment of various diseases

Method used

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  • A cell membrane particle expressing fusion membrane protein and its preparation and application
  • A cell membrane particle expressing fusion membrane protein and its preparation and application
  • A cell membrane particle expressing fusion membrane protein and its preparation and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] A preparation of membrane granules (PMVs) expressing fusion membrane proteins. per 1x10 6 Ad293 cells were transfected with 1 mg of a plasmid expressing vesicular stomatitis virus protein (pLP-VSVG).

[0042] Preparation methods such as figure 1 Shown: Add 1μg of pLP-VSVG plasmid to a 1.5ml centrifuge tube, add 50μl of serum-free culture medium, mix well; add 3μl of Polyjet transfection reagent and 50ml of serum-free culture medium into a new 1.5ml centrifuge tube, And add it to the plasmid solution, mix well to get the transfection mixture, let it stand at room temperature for 5-10min, absorb the culture medium in the culture plate cells, and add 1ml of fresh culture medium, the transfection mixture Immediately add to cells and shake gently. After 12 hours, the culture medium was replaced with a new one, and after 48 hours, the cell suspension was digested and centrifuged at 800 g for 3 minutes. Under normal circumstances, in order to obtain more cells, a maximum o...

Embodiment 2

[0048] Characterization of cell membrane granules expressing fusin.

[0049] Ad293 cells were transfected with 1 ug pEGFP-N1 (a plasmid expressing green fluorescent protein), pMito-EGFP, and pH2B-EGFP (a plasmid expressing nucleoprotein H2B and green fluorescent protein fusion protein), 48 hours after transfection, and The cell membrane particles obtained by the extrusion method adopted in the above-mentioned Example 1 were observed under a confocal microscope. In addition, DNA separation technology, real-time PCR technology, western blotting and other technologies were used for characterization. Cell membrane granules obtained from cells transfected with pEGFP-N1, pMito-EGFP, pH2B-EGFP, these three plasmids mark cell cytoplasm, cell mitochondria and nucleus respectively. from figure 2 It can be seen that membrane granules contain mitochondrial DNA, cellular RNA and miRNA, cytoplasmic proteins and membrane proteins, but not nuclear DNA, nuclear proteins. Membrane particles...

Embodiment 3

[0051] Monitoring and functional validation of proteins delivered by membrane granules expressing fusin.

[0052] Ad293 cells were co-transfected with 1 mg pLP-VSVG and 1 ug pDsRed-Expressed (plasmid expressing red fluorescent protein). 48 hours after transfection, VSV-G cell membrane particles were obtained by extrusion in Example 1 above, and added to 1x10 4 Remove the Ad293 cells, digest the cells after 5h, put them on a 35mm confocal culture dish, stain the nuclei with DAPI at 2h and 12h, and observe under a confocal microscope. Ad293 cells were co-transfected with 1mg pLP-VSVG and 1mg pCMV (CMC promoter)-CreER (Cre recombinase and estrogen receptor fusion protein), and 48h after transfection, the VSV-G cell membrane particles were obtained by the above extrusion membrane, and join to 1x10 4 In pCMV-DsRed / loxP2 / DsRed (reporter gene of the Cre-Loxp system) recipient cells, 10 mM 4-hydroxytamoxifen (4-HT) was added to the culture medium. 48h for fluorescence microscope ob...

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Abstract

The invention relates to a cell membrane particle expressing parafusin and preparation and application of the particle. VSVG is expressed on the cell membrane particle. Biomacromolecules and organelles are wrapped in the cell membrane particle. The cell membrane particle does not have a cell nucleus. The preparation method includes the steps that transient transfection is conducted on Ad293 cells through expression vesicular stomatits virus protein plasmids; the cells are digested through pancreatic enzymes 48 hours after transfection is carried out, and then centrifugation is carried out for 3 min; an obtained cell suspension is placed in a syringe, the syringe is installed in a syringe filter provided with a polycarbonate membrane, the syringe is pushed so that the cell suspension can be pushed from one side of the filter to the other side of the filter, and the cell membrane particle expressing VSVG is obtained. The cell membrane particle expressing parafusin is applied to biomacromolecule and organelle transfer. The membrane particle is larger, can wrap biomacromolecules and mitochondria, and transfers protein and the mitochondria in the aspect of function. Extra chemical reagents are not added, activity of VSVG is effectively maintained, mitochondrion release efficiency of the membrane particle is improved after the membrane particle enters a body, and accordingly mitochondrion transfer efficiency is improved.

Description

technical field [0001] The invention relates to synthetic biology technology, in particular to a fusion vesicular stomatitis virus glycoprotein membrane particle and its preparation and application in delivering biological macromolecules and organelles. Background technique [0002] Studies have found that some diseases, such as phenylketonuria, have abnormal symptoms due to the lack of related proteases; while other diseases, such as neurodegenerative Alzheimer's disease, commonly known as Alzheimer's disease, are related to mitochondrial dysfunction There is a great relationship. Although significant progress has been made in the use of drug delivery systems for therapeutic purposes, however, these systems have not been able to effectively solve existing problems. [0003] A variety of drug delivery systems have been established. For example, nanoparticles can deliver therapeutic proteins and peptides through packaging or conjugation. However, complex production processes...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N15/85C12N15/63
Inventor 魏炽炬林浩鹏郑德锦
Owner SHANTOU UNIV
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