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Annexin V-FITC exosome capture affinity magnetic beads, preparation method thereof and method for extracting exosome by using affinity magnetic beads

A technology of pet-28b-annexinv and -pet-28b-annexinv is applied in the field of AnnexinV-FITC exosome capture affinity magnetic beads, which can solve problems such as difficult elution, time-consuming and laborious, and exosome damage, and achieve economical Convenience, high purity, and complete form

Active Publication Date: 2019-12-06
ZHENGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the methods for extracting exosomes mainly include ultracentrifugation, sucrose density gradient centrifugation, ultrafiltration, polymer precipitation, and immunomagnetic bead capture. However, these methods have their own advantages and disadvantages.
Ultracentrifugation is the most widely used and effective exosome extraction method reported in the literature. Although this method can obtain exosomes with high purity, the centrifugation steps are cumbersome, time-consuming and labor-intensive, and require expensive equipment.
Sucrose density gradient centrifugation, polymer precipitation and other methods are easy to operate and take a short time, but their purity cannot be guaranteed, and often contain a large amount of impurity proteins, which have a great impact on the subsequent application and research of exosomes
Although the exosomes extracted by the immunomagnetic bead method are relatively pure, this method based on antibody binding will be difficult to elute and cause damage to the exosomes

Method used

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  • Annexin V-FITC exosome capture affinity magnetic beads, preparation method thereof and method for extracting exosome by using affinity magnetic beads
  • Annexin V-FITC exosome capture affinity magnetic beads, preparation method thereof and method for extracting exosome by using affinity magnetic beads
  • Annexin V-FITC exosome capture affinity magnetic beads, preparation method thereof and method for extracting exosome by using affinity magnetic beads

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Experimental program
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Effect test

Embodiment 1

[0039] The preparation method of Annexin V-FITC exosome capture affinity magnetic beads is as follows:

[0040]Step 1-1: Annexin V prokaryotic expression: transfer the constructed pET-28b-Annexin V plasmid with His tag into E. coli expression strain BL21(DE3) to obtain BL21(DE3)-pET-28b-Annexin V strain (it is enough to use conventional technical means here, not the key point of the present invention, no more details), 37 ° C 200rpm culture to OD value 0.6-0.9, add IPTG (i.e. isopropylthiogalactopyranoside) to the above culture The final concentration of IPTG in the final system is 0.5-1mM, and the expression is induced overnight at 20°C;

[0041] Step 1-2: Protein extraction: collect the cells after induction of expression, add PBS to resuspend the cells; ultrasonically break for 0.5-2 hours in an ice bath, use a low-temperature centrifuge to centrifuge at 12,000g for 30 minutes at 4°C, and collect the supernatant. And use 0.45μm water filter membrane to carry out suction fi...

Embodiment 2

[0052] A method for extracting and separating cellular exosomes, comprising the following steps:

[0053] Step (1): Express and purify Annexin V, prepare Annexin V-FITC, and construct Annexin V-FITC exosome capture affinity magnetic beads. In this example, step (1) adopts the operation of Example 1 to obtain exosomes capture affinity beads;

[0054] Step (2): Separation and concentration of the cell culture supernatant, preferably in the following manner:

[0055] Step 2-1: Cell culture: Gastric cancer cell MGC 803 was cultured for 48 hours, then replaced with serum-free medium, and the supernatant was collected after 36 hours of culture, which was recorded as cell culture medium;

[0056] Step 2-2: Low-speed centrifugation: Use a low-temperature centrifuge to centrifuge the cell culture solution harvested in step 2-1 at 4000g for 30 minutes at 4°C. After centrifugation, transfer the supernatant in the centrifuge tube (referred to as the primary supernatant) to A new tube is...

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Abstract

The invention provides Annexin V-FITC exosome capture affinity magnetic beads, a preparation method thereof and an extraction method. The extraction method mainly comprises the following steps: (1) expression and purification of Annexin V, preparation of Annexin V-FITC, and construction of Annexin V-FITC exosome capture affinity magnetic beads; (2) cell culture and supernatant separation and concentration: separating out cell culture supernatant after the cell culture, and carrying out concentration treatment; (3) exosome extraction: co-incubating the cell culture supernatant obtained in the step (2) with the exosome capture affinity magnetic beads to obtain an affinity magnetic bead-exosome compound; and (4) exosome eluting: co-incubating the obtained affinity magnetic bead-exosome compound with an eluent, and collecting the eluent so as to obtain an extracted and separated exosome product. The exosome capture affinity magnetic beads disclosed by the invention can be repeatedly used and are convenient to extract, and the purity of the extracted exosome is high.

Description

technical field [0001] The invention belongs to the technical field of exosome extraction, and in particular relates to Annexin V-FITC exosome-capturing affinity magnetic beads, a preparation method thereof and a method for extracting exosomes using the same. Background technique [0002] Exosomes are nanoscale extracellular membrane vesicles produced by the fusion of intracellular multivesicular bodies and cell membranes, with a diameter of 30-150 nm and a density of 1.13-1.19 g / mL, containing lipids, proteins, mRNA and miRNA and other bioactive molecules, widely distributed in serum, urine, saliva and other biological fluids. As an important transfer carrier of intercellular communication and genetic material, exosomes can participate in various physiological and pathological processes by delivering their contents to recipient cells. [0003] At present, the methods for extracting exosomes mainly include ultracentrifugation, sucrose density gradient centrifugation, ultraf...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/09H01F1/00
CPCC12N5/0693H01F1/0018C12N2509/00
Inventor 刘宏民郑一超范琦琦赵丽娟李迎迎
Owner ZHENGZHOU UNIV
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