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Application of NK cell exosome and related miRNA in preparation of COVID-19 virus inhibitor

A COVID-19, virus inhibitor technology, applied in DNA/RNA fragments, antiviral agents, recombinant DNA technology, etc., can solve problems such as difficulty, high failure rate, long R&D cycle, etc., to achieve low cost and low side effects , fast effect

Active Publication Date: 2020-08-07
SHANGHAI JIAKANG BIOLOGICAL ENG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the anti-RNA virus is based on the mechanism that the virus replication process relies on the host cell’s RNA polymerase and other mechanisms to develop specific RNA polymerase inhibitors (small molecule compounds, remdesivir), and the development cycle of such inhibitors is long, The high failure rate makes it very difficult to develop specific inhibitors against reverse transcriptase. In addition, the RNA of the COVID-19 virus can directly guide protein synthesis, and RNA polymerase inhibitors are not effective at all

Method used

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  • Application of NK cell exosome and related miRNA in preparation of COVID-19 virus inhibitor
  • Application of NK cell exosome and related miRNA in preparation of COVID-19 virus inhibitor
  • Application of NK cell exosome and related miRNA in preparation of COVID-19 virus inhibitor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Acquisition of aNK exosomes and their structural characteristics

[0023] 1.1 Collect IL-21 culture medium for 18 days of NK cells to obtain activated NK cells aNK. Flow cytometry detects that the purity of aNK cells reaches more than 95%, and the culture medium is free of bacteria and mycoplasma contamination.

[0024] 1.2 The aNK cell culture solution was centrifuged at 400 g for 5 min in a bench-top low-speed centrifuge to remove the cell pellet, and the supernatant was collected and stored at 4°C for later use.

[0025] 1.3 Use the hollow fiber tangential filtration system (Spectrum Laboratories KrosFlo ResearchII TFF System) to purify the exosomes in the culture medium. First, a 0.45μm mPES hollow fiber filter column (P-S02-E45U-10-N) was used to remove cell debris in the cell culture fluid; the filtrate was further passed through an mPES hollow fiber filter column with a molecular weight cutoff of 300-kDa (S02- E300-05-N) was concentrated to obtain crude exos...

Embodiment 2

[0030] Example 2: aNK exosomal miRNA sequencing and analysis

[0031] According to the aNK exosomes obtained in Example 1, Nanjing Shihe Gene Biotechnology Co., Ltd. was entrusted to sequence the miRNA in the aNK exosomes. Use NEBNext Multiplex Small RNA Library Prep Set for Illumina (NEB, USA) to build the library, and add sequencing adapters to the samples to be sequenced. The samples were sequenced using the Illumina X-ten PE150 platform, with at least 300 M reads per sample. In order to reduce false positives, independent sequences with a signal-to-noise ratio greater than 100 are included in the calculation of miRDeep log-odds score, and the range of miRDeep log-odds score is set to -10~10. In order to discover new miRNAs in aNK exosomes, use miRDeep Score=0 is regarded as the cut-off value, and all miRNAs exceeding the cut-off value are regarded as brand new miRNAs. Sequencing results showed that there was a large amount of miRNA expression (43.92%) in aNK cell exosomes, ...

Embodiment 3

[0033] Example 3: Initial NK cells, aNK cells, aNK exosomes, miRNAs in aNK exosomes and miRNAs (M) after base replacement inhibit the expression of COVID-19 virus S (Spike) protein

[0034] The COVID-19 virus S protein exists on the surface of the virus and is the core protein for the COVID-19 virus to enter human cells. If the virus loses the S protein, it will no longer be able to further infect human cells, so the S protein is the target of a variety of inhibitor designs. In order to determine whether the exosomes secreted by the initial NK cells, aNK cells, and aNK cells have the ability to resist the COVID-19 virus, we first used the COVID-19 virus S protein overexpression system as a model to observe the initial NK cells, aNK cells, and aNK Exosomes inhibit the replication of virus S protein. We expressed the COVID-19 virus S protein particle on HEK-293 cells as the test object. The initial NK cells (1M / ml), the aNK cells (1M / ml) obtained by activation according to Exampl...

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Abstract

The invention discloses an application of an NK cell exosome (EXO) and related miRNA (micro ribonucleic acid) in inhibition of a COVID-19 virus (new coronavirus), and provides a new thought for inhibition of the COVID-19 virus. The method has the advantages that: (1) the method is different from anti-retroviral inhibitors such as protease inhibitors and reverse transcriptase inhibitors, and miRNAhas high specificity and low side effects; (2) the inhibition effect can be enhanced by combining with a conventional treatment scheme; (3) the miRNA can be artificially synthesized, so that the costis low, and large-scale production can be realized; and (4) the compound has a remarkable inhibition effect on the duplication of the COVID-19 virus.

Description

Technical field [0001] The present invention relates to the application of exosomes (EXO) secreted by NK cells activated by IL-21 and related miRNAs in the preparation of COVID-19 virus inhibitors. Background technique [0002] NK cells are the main effector cells in the innate immune system, with natural anti-bacterial, anti-virus and ability to eliminate cancerous cells. In terms of the effect mechanism, NK cells kill cells infected by bacteria or viruses by releasing active substances such as perforin and granzyme. [0003] NK cells have a broad spectrum of viruses. They inhibit the virus based on the release of their interleukin factors and are non-specific. Therefore, although there have been speculations about NK cells improving the immunity of patients to resist the COVID-19 virus, their actual effect is very small. It has not been practically applied. [0004] In addition, NK cells can release exosomes. Exosomes are a type of nano-scale membranous vesicles secreted by cell...

Claims

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Application Information

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IPC IPC(8): A61K35/17A61K31/7088A61P31/14C12N15/113
CPCA61K31/7088A61K35/17A61P31/14C12N15/113C12N2310/141A61K2300/00
Inventor 徐以兵寿鑫朱珍芳
Owner SHANGHAI JIAKANG BIOLOGICAL ENG CO LTD
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