Respiratory tract organoid culture system and method for constructing bocavirus infection model

A culture method and technology of the culture system, applied in the direction of virus, culture process, tissue culture, etc., can solve the problem of evaluating the infectivity of Boca virus

Pending Publication Date: 2021-04-09
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 validated in vitro model to assess bocavirus infectivity in humans

Method used

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  • Respiratory tract organoid culture system and method for constructing bocavirus infection model
  • Respiratory tract organoid culture system and method for constructing bocavirus infection model
  • Respiratory tract organoid culture system and method for constructing bocavirus infection model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] Embodiment 1 human lung organoid culture

[0094] (1) Human tissue collection

[0095] Human lung tissue is the normal lung tissue removed from the hospital through bronchoscopy or surgery. For the resection of normal tissues next to proliferative lesions (including nodules, benign and malignant tumors, etc.), a distance of more than 3 cm should be kept from the proliferative lesions. Rinse normal bronchial mucosal tissue and alveolar tissue. Tissue was cut into pieces approximately 5 mm in diameter and further rinsed with cold PBS. For endoscopic biopsy specimens, at least 500 biopsy samples were collected.

[0096] (2) Tissue separation and cell dissociation

[0097] Tissue fragments and 2mmol / L EDTA cold chelation buffer (5.6mmol / L Na in deionized water 2 HPO 4 ,8.0 mmol / L KH 2 PO 4 , 96.2mmol / L NaCl, 1.6mmol / L KCl, 43.4mmol / L sucrose, 54.9mmol / L D-sorbitol, 0.5mmol / LDL-dithiothreitol) and incubated on ice for 30min. After removing the EDTA buffer, the tissu...

Embodiment 2

[0110] Embodiment 2 organoid infection model

[0111] For human lung organoids, clinical Boca virus-positive samples were used for infection, and real-time fluorescent quantitative PCR technology and Western blotting technology were used for virus infection identification.

[0112] Virus challenge experiment

[0113] 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 2ml of prepared aliquoted and preserved samples were taken to incubate organoids for 2 hours. Shake the culture plate to facilitate virus adsorption to host cells. Then the virus liquid was discarded, the cells were washed 3 times with 1×PBS, and the respiratory organoid medium containing 2% FBS was added to continue the culture. Collect organoid culture supernatant and organoid lysate at different times.

[0114] Real-time quantitative PCR and whole genome sequence detection of bocavirus genome in organoi...

Embodiment 3

[0143] Example 3 Detection of Changes in Inflammatory Factors

[0144] After the infection of the organoid Boca virus infection model, the changes of inflammatory factors were detected to determine the inflammatory response of virus infection and the interaction with cells

[0145] Preparation of tissue culture supernatant:

[0146] Centrifuge samples to immediately remove debris and store at -20°C. Avoid multiple (>2) freeze / thaw cycles.

[0147] Tissue culture supernatants may need to be diluted, and tissue / cell extractions should be performed in neutral buffers containing reagents and conditions that do not affect assay performance. Detergents, salts, denaturant concentrations that are too high, pH values ​​that are too high or too low, etc. can negatively affect the assay. Organic solvents should be avoided. Tissue / cell extraction samples should be free of particles such as cells or tissue debris.

[0148] (1) Add 200 μl of washing buffer to each well. Seal and stir ...

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Abstract

The invention discloses a respiratory tract organoid culture system, a respiratory tract organoid culture method and a method for constructing a bocavirus human respiratory tract organoid infection model. The establishment of the model firstly solves the problem that no in-vitro isolated culture method is available for bocavirus at present, and meanwhile, the model is used for quickly evaluating the infectivity of novel respiratory tract viruses and provides a new experimental basis for the pathogenesis of respiratory tract infection caused by bocavirus.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for constructing a respiratory organoid culture system and a Boca virus 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 has 43% homology with that of canine minute virus (CMV). , and the amino acid sequence homology of bovine boca virus (bovine parvovirus, BPV) is 42%. Belonging to the Parvoviridae family and Bocavirus genus, it is an icosahedral non-enveloped single-stranded linear DNA virus with a genome length of 5.2kb, containing 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 pathogen, is an important...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N7/00
CPCC12N5/0688C12N7/00C12N2501/415C12N2501/117C12N2501/119C12N2501/11C12N2501/345C12N2500/32C12N2501/02C12N2501/195C12N2501/15C12N2501/405C12N2500/38C12N2750/14052Y02A50/30
Inventor 陈喆叶智浩
Owner BAOXIN ASIA PACIFIC BIOTECH SHENZHEN CO LTD
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