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Exosome containing miR-135b-5p and application of exosome in rotavirus infection resistance

A mir-135b-5p, rnamir-135b-5p technology, applied in rotavirus exosomes and its application fields, can solve problems to be studied and the like

Active Publication Date: 2022-05-13
INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] As a double-stranded RNA virus, there is a complex interaction between rotavirus and host cells, but whether exosomes are secreted after rotavirus infection of host cells and the role of exosomes in the process of virus infection and the specific regulatory mechanism have not yet been established. Reported, to be studied

Method used

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  • Exosome containing miR-135b-5p and application of exosome in rotavirus infection resistance
  • Exosome containing miR-135b-5p and application of exosome in rotavirus infection resistance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1. Analysis of the biological properties of exosomes secreted by RV infection. After the wild-type rotavirus virulent strain ZTR-68 (G1[8] type) was infected with MA104 cells, the supernatant was collected and ultrafiltration centrifugation and Exosome isolation steps such as magnetic bead sorting are performed, hoping to isolate exosomes produced by rotavirus infection. Analyze the particle size of the exosomes collected by NTA to identify the particle size and concentration of the isolates; observe the size and shape of the isolates by electron microscopy; extract the protein of the exosome samples, and detect the exosome marker CD63 by WB , CD81, Alix and CD9 expression in the isolates.

[0028] The results of transmission electron microscopy of the isolate showed that the particle size of the isolate was about 140nm, with a membrane structure, which was consistent with the characteristics of exosomes ( figure 1 A). The purity and size of the isolate were a...

Embodiment 2

[0029] Example 2, the in vitro effect of exosomes in RV replication

[0030] After confirming that the isolates were RV-infected cells and the cells were able to release exosomes, the effect of exosomes on RV infection and replication was further analyzed. To analyze whether exosomes play a role in RV, we added exosomes and RV to MA104 cells at the same time. Rotavirus-specific immunofluorescence detection was performed 16 hours after the addition, and the results showed that, compared with the exosomes isolated from the control group, the protein expression of rotavirus was significantly decreased, suggesting that the exosomes isolated after RV infection may be It will prevent RV from entering the cell and inhibit the replication of rotavirus by directly binding to the virus or binding to the RV receptor on the cell ( image 3 C). Exosomes were added to MA104 cells after virus infection for 6 hours, and immunofluorescence detection was performed 16 hours after virus infecti...

Embodiment 3

[0033] Embodiment 3, step 1, cell culture

[0034] The cells used in this study were MA104 cells, and the culture conditions were MEM medium containing 8% newborn calf serum and 1% double antibody.

[0035] Step 2. Virus infection

[0036] (1) Take the RV virus liquid out of the -80°C refrigerator, place it at room temperature until the virus liquid is completely melted, add acetylated trypsin and calcium chloride, and bathe in 37°C water bath for 60 minutes to activate the virus;

[0037] (2) Gently wash the cell surface twice with serum-free cell culture medium, and then add the activated virus solution;

[0038](3) Place the cells inoculated with the virus solution on a shaker in a carbon dioxide incubator at 37°C, and slowly shake for 1 hour to absorb. Then the virus liquid was discarded, replaced with serum-free cell culture medium (containing trypsin at a final concentration of 1 μg / mL), and placed in a 37°C carbon dioxide incubator to continue culturing.

[0039] Ste...

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Abstract

The invention belongs to the field of biological medicine, and particularly relates to a rotavirus exosome and application thereof. At present, related reports about whether exosomes are secreted after rotavirus infects host cells or not and the regulation mechanism of the exosomes in the virus replication process do not exist. The invention discloses an exosome containing miR-135b-5p and an application of the exosome in inhibiting rotavirus infection, including an application in preparing a drug for treating rotavirus and a reagent for neutralizing rotavirus.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to rotavirus exosomes and applications thereof. Background technique [0002] Exosomes (Exosomes) are extracellular vesicles released by the fusion of multivesicular bodies (MVB) and the plasma membrane of the cell, originating from intraluminal vesicles during MVB formation. It has a typical lipid bilayer structure, vesicles with a diameter of about 30-120nm, and a floating density of about 1.13-1.19g / mL. The surface of exosomes secreted by different cells has some common marker proteins, such as heat shock protein 70 (HSC70), heat shock protein 90 (HSP90), and tetraspanins CD9, CD63, CD81, and CD82. Exosomes carry a variety of molecules, including specific protein families, functional mRNA, and miRNA. There are mainly four ways to play the role of information transmission between cells: (1) exosomes serve as signal complexes, directly stimulating receptor cells through cel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K35/22A61P31/14C12N5/10C12N15/113
CPCA61K35/22A61P31/14C12N5/0686C12N15/1131C12N2310/141
Inventor 周艳李鸿钧胡晓青陈蓉吴晋元
Owner INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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