Construction method of bocavirus small intestine epithelium organoid infection model

A construction method, Boca virus technology, applied in the biological field, can solve problems such as evaluating the infectivity of Boca virus

Active Publication Date: 2021-06-15
BAOXIN ASIA PACIFIC BIOTECH SHENZHEN CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] There is currently no robust in vitro model to assess bocavirus infectivity in humans

Method used

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  • Construction method of bocavirus small intestine epithelium organoid infection model
  • Construction method of bocavirus small intestine epithelium organoid infection model
  • Construction method of bocavirus small intestine epithelium organoid infection model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0114] Example 1 Culture of Small Intestinal Epithelial Organoids

[0115] (1) Wash the normal intestinal tissue, peel off the muscular layer with a sterile scalpel blade, cut the mucosal epithelial tissue into small pieces with a size of about 5 mm, and wash 10 times with ice-cold PBS.

[0116] (2) The tissue block was digested with Liberase (TH grade, Roche Life Science Company) at 37°C for 60 minutes, and was vigorously pipetted with a pipette every 15 minutes.

[0117] (3) Digest the remaining fragments and treat them with TrypLE Express (Invitrogen) at 37°C for 20 min.

[0118] (4) Collect the supernatant and centrifuge at 200 g for 3 min at 4°C. Cell pellets were resuspended in Matrigel (growth factor-reduced, BD Biosciences) and distributed in two 48-well culture plates (25 μL Matrigel / well, containing 500 crypts / fragments or 1000 single cells).

[0119] (5) Matrigel was polymerized at 37°C for 10 minutes, and 500ul of human intestinal organoid medium was added to eac...

Embodiment 2

[0130] Example 2 Organoid Infection Model—Bocavirus Infection of Small Intestinal Epithelial Organoids

[0131] The positive clinical specimens are directly subjected to virus challenge experiments, and real-time fluorescent quantitative PCR is used to detect the organoid culture supernatant and the bocavirus genome in the organoids, which is characterized in that it includes the following steps:

[0132] (1) Organoid infection model

[0133] For human intestinal organoids, clinical Boca virus-positive diarrhea specimens were used for infection, and real-time fluorescent quantitative PCR technology and Western blotting technology were used for virus infection identification.

[0134] (2) Virus challenge experiment

[0135] Experiments related to Boca virus infection were all carried out in a biosafety level 2 laboratory (BSL-2). Positive samples of Boca virus were selected, and 2 mL of prepared aliquoted and preserved samples were taken to incubate organoids for 2 hours. Sha...

Embodiment 3

[0163] Example 3 is an effect test on the model constructed by organoids.

[0164] Through the effect test of the model constructed by using antiviral drugs, the experimental results show that the antiviral drugs used can inhibit the replication of virus particles in cells, and different concentrations and different types of drugs show different inhibitory effects. This shows that the Boca virus in vitro infection model has been successfully constructed, and can be used for the screening and effect evaluation of anti-Boca virus drugs.

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PUM

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Abstract

The invention discloses a construction method of a bocavirus small intestine epithelium organoid infection model, which comprises the following steps: separating and culturing small intestine epithelium organoids; and infecting the small intestine epithelium organoids with bocavirus. The model solves the problem that no in-vitro separation and culture method for bocavirus exists, especially a digestive tract infection model, is used for rapidly evaluating the infectivity of the bocavirus, and provides a new experimental basis for the pathogenesis of bocavirus infected intestinal tracts.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for constructing a Boca virus small intestinal epithelial organoid infection model. Background technique [0002] Human bocavirus (HBoV) is a new parvovirus discovered by Swedish scholar Tobias Allander et al. through molecular screening. Its amino acid sequence shares 43% homology with that of canine minute virus (CMV). %, the homology with the bovine boca virus (bovine parvovirus, BPV) amino acid sequence is 42%. It belongs to the Parvoviridae family and belongs to the genus Bocavirus. It is an icosahedral non-enveloped single-stranded linear DNA virus with a genome length of 5.2kb and contains 3 open reading frames, which encode non-structural proteins NS1, NP1 and structural proteins respectively. The genome conservation of VP1 / VP2 and HBoV is relatively good, and the diversity of VP1 / VP2 genes is relatively large. [0003] Human bocavirus, as a newly discovered pathog...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N7/00C12R1/93
CPCC12N5/0679C12N7/00C12N2509/00C12N2509/10C12N2750/14021C12N2750/14051C12N2501/117C12N2501/119C12N2501/11C12N2500/32C12N2501/02C12N2501/415C12N2501/155C12N2500/38C12N2501/727C12N2501/15C12N2501/345Y02A50/30
Inventor 陈喆叶智浩
Owner BAOXIN ASIA PACIFIC BIOTECH SHENZHEN CO LTD
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