79 results about "CD63" patented technology

The invention discloses an EV membrane protein single molecule detection method based on droplet microfluidics and application thereof. The method comprises the following steps: firstly, constructing an Ab-oligo compound which is formed by sequentially connecting an antibody, Linker, Linker connection DNA, RNA capable of being specifically cut by RNA enzyme and template DNA for starting signal amplification; then capturing the extracellular vesicles in the plasma by using the CD9 / CD63 / CD81 functionalized magnetic beads; the method comprises the following steps: adding an Ab-oligo compound and a sealing buffer solution into extracellular vesicles, incubating, removing the uncombined Ab-oligo compound, adding RNA enzyme for incubating and enzyme digestion, collecting template DNA for starting signal amplification, and performing microdroplet digital PCR analysis to realize single molecule analysis of EV membrane protein. According to the invention, the one-to-one correspondence relationship between the antibody and oligonucleotide is established through Linker for the first time, single protein information is converted into single nucleic acid chain information, and EV membrane protein single molecule detection is realized. The method is simple to operate, can qualitatively and quantitatively detect the single EV membrane protein, and is beneficial to further research on the relationship between the EV membrane protein and diseases.

The application discloses a separation-free plasma tumor-derived extracellular vesicle detection method, relates to the technical field of biology, and comprises CD63, HER2, GPC1 or / and EpCAM; probe sequences are shown in SEQ ID NO.1-SEQ ID NO.4; the probe is applied to the extracellular vesicle detection reagent in the separation-free plasma, single EVs imaging analysis, detection of the image of a single EV after direct dilution of plasma, dynamic immune detection, and statistics of the number of specifically combined EVs. The application applies the separation-free extracellular vesicle detection probe to the immune detection reagent, avoids the separation process of plasma EVs, does not need to use fluorescence detection, can characterize the single EVs binding kinetics, and can detect 1 target EVs in 350 background particles.

The invention discloses a chimeric antigen receptor exosome targeting a CD8T cell, a CAR-T cell and a construction method and application of the chimeric antigen receptor exosome, and relates to the technical field of biomedicine, and the construction method comprises the following steps: co-transfecting three plasmids, namely BRD-PTK-CD8 < + >-Lamp2b, pcDNA3.1-CD63-L7Ae and pcDNA3.1-N6 (SP)-Nb60-T20-C / D box, into a 293F cell, incubating, and purifying to obtain the exosome capable of targeting the CD8T cell. The exosome can deliver CAR-mRNA to T cells and convert CD8 + cells into CAR-T cells, and blood of an infected person does not need to be drawn; cAR molecules aiming at in-vivo malignant cells can be prepared in vivo; chemotherapy and radiotherapy are not needed, and side effects are avoided; and universality is achieved.

The invention discloses an amniotic epithelial cell-derived exosome for treating gestational hypertension and application thereof, and belongs to the technical field of biological medicines. The exosome (hAECs-exos) is obtained by secreting and extracting human amniotic epithelial cells (hAECs), the particle size distribution of the exosome (hAECs-exos) is 30-150 nm, and the exosome (hAECs-exos) expresses exosome specific markers CD9, CD63 and CD81. The invention also provides a preparation method of the exosome. The preparation method comprises the following steps: isolated culture of hAECs, and ultracentrifugal extraction and identification of the exosome. Animal experiments prove that when the exosome is applied to a gestational hypertension disease model animal in an intravenous injection mode, the blood pressure and urine protein level of the gestational hypertension disease model animal can be remarkably reduced, and the fetal rat dysplasia condition is improved. The invention provides a brand-new and effective biological preparation candidate scheme for the treatment of the gestational hypertension disease.

Disclosed are a specific marker combination of mesenchymal stem cell-derived small extracellular vesicles and the use thereof. The characteristic marker combination comprises aminopeptidase N (CD13), integrin β-1 (CD29), membrane glycoprotein THY-1 (CD90), and markers CD9, CD63 and CD81. By using the characteristic marker combination of mesenchymal stem cell-derived small extracellular vesicles for nano-flow cytometry analysis at the single-vesicle level, mesenchymal stem cell-derived small extracellular vesicles can be identified. On the basis of the provided characteristic marker combination, specific modification and engineering on the basis of small extracellular vesicle membrane proteins can further be realized.

The application provides an engineered exosome loaded with a CD63 transmembrane domain-matrix metalloproteinase fusion protein and a preparation method and application thereof. The engineered exosome is secreted by a genetically engineered mesenchymal stem cell, has a CD63 derived transmembrane domain anchored on the membrane, and has a matrix metalloproteinase (MMP) loaded on the inner side of the membrane. The fusion protein comprises, from N-terminus to C-terminus, a fourth transmembrane domain and a C-terminal cytoplasmic tail of a human CD63 protein, a flexible linker peptide, and a human MMP without a natural signal peptide. The application realizes efficient and directional loading of MMPs by using a natural biological generation path of the exosome, shields the inhibition of MMPs by metalloproteinase tissue inhibitors in a scar tissue by using the membrane of the exosome, realizes specific activation of MMPs in a target cell, degrades abnormally deposited collagen, adjusts the ratio of type I and type III collagens, and plays an anti-fibrosis and anti-scar formation role. The application also provides a preparation method of the engineered exosome and application thereof in preparation of a medicine for preventing or treating pathological scars.

The present application provides a fusion protein comprising an anti-CD3 single-domain antibody, an exosome protein CD63 and an RNA binding protein, and uses thereof. The fusion protein of the present application achieves the effects of treating cancer, immune regulation and activating immune cells through various efficacy experiments.

The application discloses an exosome-based protein delivery system and application thereof. The exosome-based protein delivery system comprises a loading subsystem and / or an unloading subsystem, the polypeptide peptide segment expressed by the nucleotide sequence of the loading subsystem comprises CD63, a connecting peptide, an enterokinase cleavage site and a delivered protein or polypeptide, and the polypeptide peptide segment expressed by the nucleotide sequence of the unloading subsystem comprises CD63, a connecting peptide, enterokinase or enterokinase light chain. The application also constructs a human umbilical cord mesenchymal stem cell strain hUC-MSC CDNF‑exo on November 13, 2024 in the Guangdong Provincial Microbial Culture Collection Center, and the preservation number is GDMCC NO:65478. The human umbilical cord mesenchymal stem cell strain hUC-MSC CDNF‑exo The purpose of delivering CDNF into the cytoplasm of target cells is achieved.

The application discloses a nucleic acid free extraction reagent of plasma exosome and a direct PCR amplification kit. The nucleic acid free extraction reagent comprises 0.001-0.1% polidocanol and 1-100 mM Tris-HCl in mass volume concentration, and the solvent is water. The direct PCR amplification kit provided by the application comprises the nucleic acid free extraction reagent and an immunomagnetic bead suspension, a washing liquid, a free extraction reagent and a PCR Mix solution; the surface of the immunomagnetic bead is simultaneously coated with three exosome specific antibodies of CD9, CD63 and CD81, so that the recovery rate of the exosome can be greatly improved during the extraction and purification of the plasma exosome; the PCR Mix solution contains an anti-inhibitory component, can effectively neutralize the inhibitors such as proteins in the PCR system, and ensures the amplification efficiency of the PCR. The nucleic acid free extraction reagent of the application can realize the amplification detection of the exosome nucleic acid free extraction, and simplifies the operation process of the PCR detection.

The invention belongs to the field of biomedicine, and particularly relates to a bifunctional modified magnetic bead and an extraction method of exosomes in cerebrospinal fluid. Aiming at the defects of an existing magnetic bead capture method in the aspect of removing impure protein, the invention firstly provides a bifunctional modified magnetic bead, a CD63 antibody and a sulphobetaine type zwitterionic polymer are modified on the surface of the bifunctional modified magnetic bead, specific capture of the exosome is realized on the basis of antigen-antibody specific binding of CD63 and the exosome, and meanwhile, the adsorption effect on the impure protein is reduced. The magnetic beads are applied to extraction of exosomes in cerebrospinal fluid, and the technical defects that the sample size of the cerebrospinal fluid is small, and the recovery rate of a traditional method is not high are effectively overcome.

The invention provides methods for prognosing an increased risk of early onset preeclampsia (EOPE) in a pregnant subject before the disease onset. The methods comprises quantifying small extracellular vesicles that express pairs of biomarkers selected from CD10, CD63 and placental alkaline phosphatase (PLAP) in a sample obtained from the subject.

The application discloses a kind of targeted exosome chimeric antigen receptor molecules for treating HIV infection and its application, belong to the field of biological medicine technology.The application is packaged into targeted exosome by coding anti-HIV CAR mRNA directionally.The nano antibody sequence of targeted CD3, CD4, CD8+T receptor on T cell is fused to the N-terminal of LAMP-2B to construct specific targeting plasmid, so that the targeted peptide is expressed on the surface of exosome membrane, the expression plasmid containing RNA binding protein L7A and exosome tag protein CD63 is constructed, and the exosome loaded with CAR mRNA and targeted to T cell is obtained by three-plasmid co-transfection of HEK293F cells containing C / D box CAR plasmid.The exosome can construct human CAR-T cell when incubated with PBMC in vitro or back into humanized mouse in vivo, and has significant killing virus infected cell activity.

This invention broadly relates to the field of bioassay technology, and more specifically, to a method for enriching exosomes and the conjugates used in this method. The conjugates are formed by conjugating a CD63 antibody or its antigen-binding fragment with a matrix support; wherein the heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3 of the CD63 antibody or its antigen-binding fragment are shown in SEQ ID NO: 1-3, and the light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3 of the antibody or its antigen-binding fragment are shown in SEQ ID NO: 4-6. The conjugates provided by this invention have high affinity and high purification efficiency, and show great promise for application in exosome purification.

The multifunctional antimonene-gold nano composite material (AM-Au NCs) is rapidly, simply and conveniently prepared and is used for multi-analyte fluorescence sensing (such as metal ions, CD63 and tumor-derived exosomes), molecular information coding and protection. Sodium dodecyl benzene sulfonate, AM, Au < 3 + > and NaBH4 are mixed by using different formulas at room temperature to prepare AM-Au NCs with an adjustable gold nanostructure and characteristics. And interestingness is that AM-Au NCs doped with the gold nanoparticles show wide fluorescence quenching capability on the nanosheets, and the fluorescence quenching capability is remarkably different in different dyes and DNA (deoxyribonucleic acid) consisting of different basic groups. A fluorescent sensing platform constructed by AM-Au NCs is combined with a dye or an aptamer and is used for detecting metal ions, CD63 and tumor-derived exosomes. In real samples, through binary or digital coding of selective response and diversified DNA sequences of sensing systems, a series of molecular encryption and hiding technologies are provided and applied to realize molecular-level protection of special text information.

The application discloses a preparation method and application of an engineered exosome, and relates to the technical field of biology.The application provides a preparation method of an engineered exosome, and a carrier construction step is provided for constructing CD63-VEGF into a FUGW carrier, and replacing an Ubc promoter in the FUGW carrier with EF1a to obtain FUGW(EF1a)-CD63-VEGF, wherein the sequence of the FUGW(EF1a)-CD63-VEGF is shown as SEQ ID NO:5.The application successfully develops the engineered exosome capable of carrying VEGF by using synthetic biology technology, and the application provides a train of thought for exosome drugs and drug development with the exosome as a delivery carrier.The engineered exosome carrying VEGF has a remarkable effect on promoting hair growth.

The present invention relates to a method for production of viruses, viral derivatives, extracellular vesicles (EVs), such as exosomes or microvesicles, or recombinant viral vectors in a cell line, comprising the step of overexpressing SNAP47, VPS37B, SMPD3A / SNAP47, or CD63 in combination with VPS37B, SNAP47 or SMPD3A in said cell line, as well as to a cell line stably overexpressing at least one of these genes.

The invention discloses an extraction method and application of membranous small vesicles. The extraction method comprises the following steps: collecting a biological sample, and selecting at least one of blood, urine and cell culture supernatant; centrifuging the sample at 10,000 xg for 20 minutes, and removing cell debris and apoptotic bodies to obtain a pretreated sample; adding 20% by volume of sample separation liquid and CD63 / CD81 antibody modified magnetic beads into the pretreated sample, and incubating and capturing target vesicles; freezing the captured vesicles with liquid nitrogen for 15-35 minutes, and dissolving the vesicles with a 33-43 DEG C water bath; and cracking the dissolved vesicles with 0.5 W / cm < 2 > ultrasonic waves to finally obtain the intact vesicles of 30-70 nm and soluble protein factors. Original plasma or urine can be directly treated, dilution is not needed, and loss of a target object is avoided; a low-cost and degradable biomimetic polymer is developed to replace an imported kit, and the cost is reduced by 75%; the whole process lt; the time is 2.5 hours, which is superior to that of an ultracentrifugation method; and the extracted membranous small vesicles have good integrity and are suitable for liquid biopsy and drug carrier construction.

The present invention pertains to combination comprising a CD63-binding molecule and a drug and / or a tracer for use in a method of therapy and / or in a method of in-vivo diagnosis, wherein the drug and / or the tracer is delivered to a myofibroblast.

The invention relates to the technical field of biological medicines, and provides a high VEGF protein / miR-210 extracellular vesicle induced by a serum-free combined culture medium and application of the high VEGF protein / miR-210 extracellular vesicle. According to the invention, high VEGF protein / miR-210 extracellular vesicles are obtained based on induction of a serum-free culture medium; the serum-free culture medium comprises a basic culture medium component combination: sugar-free RPMI1640 and MCDB131 in a volume ratio of 4: 1; the metabolic regulation additive is prepared by synergistically adding sodium pyruvate, cysteine and a proline hydroxylase inhibitor and taking alanine glutamine as an energy substitute; the growth factor is a combination of polyvinyl alcohol, 4-hydroxyethylpiperazine ethanesulfonic acid, ITS-X, epidermal growth factors and basic fibroblast growth factors, wherein the ITS-X, the epidermal growth factors and the basic fibroblast growth factors are added before use. CD63 and CD81 are specifically expressed on the surface of the prepared high VEGF protein / miR-210 extracellular vesicle, the high VEGF protein / miR-210 extracellular vesicle is rich in VEGF protein and miR-210, and the high VEGF protein / miR-210 extracellular vesicle is applied to targeted therapy of allergic rhinitis by inhibiting Th2 type immunoreaction.

This invention discloses a high-BNIP3-loaded exosome, its preparation method, and its applications, relating to the field of biomedical engineering technology. The high-BNIP3-loaded exosomes are obtained by pre-treating human bone marrow mesenchymal stem cells transfected with a recombinant expression vector carrying the BNIP3 gene, followed by 24 hours of hypoxia at 1% oxygen concentration. The exosomes have a particle size of 40-130 nm and highly express BNIP3 and exosome markers HSP70, CD63, and TSG101. The preparation method includes constructing a pcDNA3.1-BNIP3 recombinant vector, transfecting and screening stable-expressing engineered cells, hypoxia pre-treatment, and purification by gradient centrifugation combined with ultracentrifugation. This invention achieves efficient loading and targeted delivery of BNIP3 through a synergistic strategy of genetic engineering and microenvironment simulation, significantly improving the endocytosis efficiency of nucleus pulposus cells, activating mitophagy, restoring mitochondrial function, and promoting extracellular matrix synthesis. It can be used to prepare drugs for treating intervertebral disc degeneration and has excellent clinical application prospects.

The present invention relates to a method for production of viruses, viral derivatives, extracellular vesicles (EVs), such as exosomes or microvesicles, or recombinant viral vectors in a cell line, comprising the step of overexpressing SNAP47, VPS37B, SMPD3A / SNAP47, or CD63 in combination with VPS37B, SNAP47 or SMPD3A in said cell line, as well as to a cell line stably overexpressing at least one of these genes.

The invention discloses a method for detecting in-situ release of RNA by exosome based on rolling circle replication amplification, and belongs to the technical field of exosome detection. The invention designs and develops a detection probe taking a sulfydryl hairpin probe and a Padlock probe as core components, the sulfydryl hairpin probe can target an exosome membrane protein CD63, and after substances such as the Padlock probe and Phi 29 DNA polymerase are added, an RCA amplification reaction is initiated. Triton X-100 can punch an exosome membrane so that the exosome membrane can release miRNA located in the exosome membrane, and the miRNA is combined with an amplification chain to release a hidden G4 chain body sequence. The added Hemin is combined with the G4 to form a G4 conjoined compound, and the compound can display peroxidase catalytic activity and can catalyze H2O2 to be decomposed to generate oxygen free radicals, so that a detectable signal is generated. The detection method provided by the invention can be used for carrying out high-sensitivity detection on the low-concentration exosome miRNA.

The invention discloses composite hydrogel as well as a preparation method and application thereof. The preparation method comprises the following steps: immersing a reticular metal substrate into a first pre-polymerization solution, taking out the reticular metal substrate, and performing cross-linking polymerization; immersing into a second pre-polymerization solution, taking out, and carrying out cross-linking polymerization; dissolving and removing the reticular metal substrate to obtain double-network hydrogel; a double-network hydrogel is modified by adopting a DNA long chain to obtain a composite hydrogel, the 5'terminal amino group of the DNA long chain is modified, the DNA long chain is grafted on the double-network hydrogel through amidation reaction, and the DNA long chain comprises a plurality of repeated CD63 aptamer sequences. According to the preparation method, the reticular metal substrate is taken as a template, polymerization is initiated in sequence to form the double-network hydrogel, and an internal regular three-dimensional macroporous structure is ensured, so that exosomes are easy to diffuse into the material; comprising a plurality of repeated CD63 aptamer sequences and carboxyl groups of the double-network hydrogel, so that abundant capture sites are formed, and specific efficient separation and enrichment of exosomes in a sample can be realized.

The application belongs to the technical field of biomedical engineering, and particularly relates to a multi-channel paper chip device and application thereof in detection of small extracellular vesicle membrane proteins. The core component of the multi-channel paper chip device disclosed by the application is a paper chip co-modified by a nucleic acid aptamer and a metal organic framework (MOF). The device is to grow the MOF material on the paper chip in situ to provide an amino modification site of the small extracellular vesicle membrane protein aptamer, reduce non-specific adsorption of the paper to impurity proteins, and serve as a reference for ratio type detection. Then, the aptamer is modified on the MOF to capture small extracellular vesicles expressing corresponding proteins through immunological affinity. Meanwhile, a detection probe DP composed of a small extracellular vesicle membrane protein CD63 aptamer and a G-quadruplex is developed to detect the content of the captured small extracellular vesicles by colorimetry. The device can be used to detect the small extracellular vesicle membrane proteins in multiple channels, and realize precise diagnosis and typing of cancers.

The present invention relates to a composition for promoting cell regeneration and its application. Specifically, a composition for promoting cell regeneration is provided, comprising L-tryptophanol oxalate, isosorbide-2-acetate, and glabridin. The composition is used to prepare a cell regeneration agent and construct a cell model. The composition can effectively enhance cell proliferation and CD63 expression levels in cells. The present invention not only effectively enhances cell regeneration capacity, but also is simple to operate and low in cost. It can be applied to the preparation of cosmetics, skin care products, wound repair materials or drugs, and skin repair.

EVs are being recognized as vectors for drug delivery. In particular. EV loading with targeting and therapeutic agents brings along an interesting opportunity to translate EVs into a bio-mimetic selective delivery system. Indeed. EVs constitute a physiological carrier being potentially less immunogenic than artificial delivery vehicles. The inventors now developed a novel method to control the loading of a cargo into EVs on demand. These EVs are equipped, if necessary, with non-viral fusogen, therefore enhancing EV-cargo delivery into acceptor cells. To acutely measure this process, they follow the fate of a luciferase-tagged cargo. Cargo loading was enabled through a drug-reversible inducible dimerization system. Briefly, donor cells were transfected with plasmids encoding for FKBP-tagged CD63, a classical membrane EV marker, and FRB-Nanoluciferase (NLuc) that is normally cytosolic. Upon addition of the dimerizing drug. FRB-Nluc interacts with FKBP-CD63 and is recruited into secreted EVs. This is accompanied by an enhanced delivery into acceptor cells. This phenomenon can be further enhanced when EVs are equipped with syncitin1, a mammalian fusogenic protein that trigger fusion between EV membrane and the plasma membrane of acceptor cells. Using this novel process, the inventors further demonstrated that the catalytic domain of the Diphteria toxin (DTA), that is responsible for protein synthesis inhibition and ultimately cell death, can be delivered to acceptor cells via functionalized EVs. This led to protein synthesis inhibition and death of acceptor cells. This novel method and the derived applications promise to open new doors in precision care medicine, especially when EVs will be equipped with antibodies raised against cell specific antigens.

The application discloses a kind of based on microfluidic and AI image recognition basophil activation detection method and system.The method comprises: the peripheral blood sample to be measured is injected into the microfluidic chip device containing conical microporous filter membrane and enriched basophil;In reaction cavity, pre-mixed dry CD63-FITC and CD203c-PE fluorescently labeled antibody and allergen stimulant are added;After incubation at 37 DEG C, 512×512 pixel high-resolution image is obtained by digital microscopic imaging system;Improved target detection model is used to identify activated cells, the model uses lightweight FasterNet backbone network and dynamic attention mechanism, generates cell activation heat map by Gaussian kernel density estimation, combines fluorescence intensity weighted calculation activation index and outputs visual report.System includes microfluidic chip module, fluorescently labeled module, temperature control incubation module, image acquisition module, AI analysis module and man-machine interaction module.The application realizes high sensitivity, high specificity and automatic detection, and is suitable for clinical diagnosis of allergic diseases and blood diseases.

The invention discloses a biomarker detection kit and a detection method for prognosis evaluation of multiple myeloma, and relates to the technical field of biological medicine detection.According to the kit, circMYC and circSETD2 bicyclic RNA combination in serum exosomes serves as a core prognosis marker, and the kit contains an exosome solid phase capture reagent, sulfydryl modified silicon dioxide magnetic beads and the like; the isothermal amplification detection reagent contains a specific SDA primer pair and the like; a double quality control reagent containing a CD63 peptide fragment marker and the like; positive control and negative control are provided; the serum exosome bicyclic RNA is selected as a core prognostic marker, a complete detection system is constructed, an exclusive reagent efficiently enriches and purifies the exosome, the detection specificity and accuracy are improved, a reliable basis is provided for prognostic layering, integrated operation is achieved, the process is simplified, clinical application is achieved, quantitative analysis is matched with a clear interpretation standard, and the detection efficiency is improved. The reagent is stable and convenient, the result is comparable, and the clinical application value is improved.

The invention provides a targeting exosome for hair regeneration and a preparation method thereof. The method comprises the following steps: separating dermal mastoid cells with a cell marker CD133 from a hair follicle strain of a healthy donor, and culturing the dermal mastoid cells in a laboratory conforming to a Taiwan GTP cell preparation factory domain in China in a heterologous and serum-free environment. Then, a culture solution containing exosomes is collected from the cell culture solution, and the multiple exosomes have surface markers such as CD9, CD63 and CD81; and finally, separating, purifying and concentrating the exosome from the culture solution by utilizing a tangential flow filtration system method or a size exclusion chromatography method and other technologies to obtain the final targeting exosome freeze-dried powder product. The invention also provides a liquid hair growth preparation prepared from the exosome product.

The invention belongs to the technical field of biology, and particularly relates to a cell exosome and a separation method and application of an animal cell exosome based on two-stage filtration and multi-stage centrifugation. The separation method comprises the steps of cell culture, primary centrifugation, secondary centrifugation, two-stage filtration, two times of ultracentrifugation, resuspension identification and the like. Optimized parameters are as follows: inoculating 1 * 10 < 6 >-3 * 10 < 6 > cells, culturing for 48-52 hours by using 10% exosome-deficient serum, carrying out gradient centrifugation of 600-700g and 12000-15000g, carrying out two-stage filtration of 0.45 [mu] m and 0.22 [mu] m, carrying out two times of ultracentrifugation of 110000-12000g, and carrying out PBS (Phosphate Buffer Solution) resuspension. The obtained exosome contains TSG101 and CD63 markers, is high in purity and complete in structure, can be used for preparing tumor diagnosis markers or delivery carriers, and has the advantages of being low in cost, high in universality and capable of being standardized.