Extraction method and application of exosome

An extraction method and exosome technology, applied in the field of exosome extraction, can solve the problems of low extraction purity, high cost, and long processing time

Pending Publication Date: 2021-12-10
光武惠文生物科技(北京)有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the above methods either have low extraction purity, or take too long to process, or have specific requirements for the amount of sample extracted, or involve the use of large instruments, or the cost of extraction is too high, so they cannot be widely promoted and applied.

Method used

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  • Extraction method and application of exosome
  • Extraction method and application of exosome
  • Extraction method and application of exosome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1 LDLR affinity chromatography pretreatment of samples

[0070] Take 400 μl of normal human serum / plasma (sodium citrate blood collection tube), and dilute it with PBS solution 1:1 for later use. Wash the LDLR column 3 times with its buffer solution, and let it fall by natural gravity. Add the reserve solution to the LDLR column several times in batches, drop by gravity, and collect the liquid after passing through the column. The schematic diagram of LDLR chromatography column processing samples is shown in the appendix figure 1 .

[0071] It is verified that the LDLR column has a certain ability to remove LDL protein, which can achieve the effect of removing lipoprotein and purifying exosomes. As the volume of the solution becomes larger after passing through the column, it is extremely important to select a subsequent enrichment method for exosomes. Concentrating the exosome solution can improve the detection sensitivity and facilitate subsequent downstrea...

Embodiment 2

[0072] The oxide enrichment concentration of embodiment 2 eluent

[0073] Take 8mg of oxide, add 200μl PBS solution to wash, spin for 10s, and discard the supernatant. Repeat this step twice. The oxides were formulated into a 0.05mg / μl suspension in advance and dispersed by ultrasonic for 1h. Since the initial serum volume is 400 μl, take 160 μl of the oxide suspension and centrifuge for 10 seconds, discard the supernatant, add PBS to wash twice, centrifuge and discard the supernatant.

[0074] Add the liquid after passing through the column to the oxide precipitate, mix well, and incubate with shaking for 30min. After the incubation, centrifuge for 10 seconds, discard the supernatant and add PBS solution to wash twice, then discard the supernatant immediately. Add PBS to mix and precipitate, which is the combination of exosomes and oxides.

Embodiment 3

[0075] Determination of the evaluation markers of the exosomes extracted in Example 3

[0076] 5S and U6 are excluded from the reference genes suggested by genomics. Based on genomic analysis, it is recommended to select as internal reference genes including miR-221, miR-128, miR-451, let-7a, miR-221, miR-16, miR -181c, miR-26a.

[0077] Therefore, based on the characteristics of high content and stable expression in exosomes, we currently select miR181a-5p as the quantitative standard of exosomes and the subsequent selection of internal reference genes. For the content of some miRNAs in human blood exosomes, see the attached figure 2 .

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Abstract

The invention discloses an extraction method of exosomes. The method mainly comprises the operation steps: passing serum/plasma (a sodium citrate blood collection tube) through an LDLR affinity chromatography column, and enriching and concentrating the collected liquid with oxide. According to the invention, LDLR affinity chromatography is adopted to remove low-density lipoprotein in serum/plasma, and oxide is adopted to concentrate a collected buffer solution, so that a method for purifying exosome from serum is established. According to the invention, particle number is detected through nanoflow, exosome surface markers are detected through western blot, the transmission electron microscope detection results show that yield of the extracted exosome is high, the extracted exosome is further combined with a splitting method to release the internal substances of the exosome, and nucleic acid and protein can be subjected to the subsequent research. The method has advantages of simple operation steps, short extraction time, high purity of prepared exosomes, suitability for small samples and high cost performance, and is suitable for popularization and application in the field of in vitro diagnosis.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for extracting exosomes and its application. Background technique [0002] Compared with traditional tissue biopsy techniques, liquid biopsy, as a detection method for cancer, has many significant advantages such as non-invasiveness, high accuracy, easy sampling, and low price. Liquid biopsy performed by taking blood is less invasive, so it has become a hot technology in the field of cancer diagnosis. [0003] Among them, the use of exosomes in liquid biopsy has more advantages, such as comprehensive feedback information, less trauma, higher specificity, and high stability. [0004] Exosomes, when used as a source of quantitative and qualitative information, can inform the presence of malignancy and tumor burden. Exosome liquid biopsy test samples can be blood, urine, saliva or cerebrospinal fluid and other body fluids with tumor-related biomarkers. Cancer cell-derived ex...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/00C12N15/113C12Q1/6851
CPCC12N5/00C12N15/113C12Q1/6851C12N2509/10C12N2509/00C12N2310/141
Inventor 付洁王伊李华柏李雪洁
Owner 光武惠文生物科技(北京)有限公司
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