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Establishment method of novel coronavirus-induced lung injury animal model and mouse model thereof

A coronavirus and animal model technology, which is applied in animal husbandry and other fields, can solve the problems of non-specificity and incomplete development of COVID-19 animal models, and achieve the effect of simple process, low cost and easy operation

Active Publication Date: 2021-10-08
HUZHOU CENT HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to address the deficiencies in the prior art, so a method for establishing an animal model of lung injury caused by a novel coronavirus and a mouse model thereof are provided to solve Incomplete and non-specific problems in the development of COVID-19 animal models in the prior art

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  • Establishment method of novel coronavirus-induced lung injury animal model and mouse model thereof
  • Establishment method of novel coronavirus-induced lung injury animal model and mouse model thereof
  • Establishment method of novel coronavirus-induced lung injury animal model and mouse model thereof

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Embodiment 1

[0047] Search the two amino acid sequences of SARS-COV and SARS-COV-2 in NCBI Conserved Domains (NP_828851.1, YP_009724390.1), the RBD region of SARS-COV corresponds to the 318-569 segment, and the RBD region of SARS-COV-2 Corresponding to section 331-583. Continue to use the BioEdit software to compare the RBD region sequences, and the five important sites 125, 155, 163, 171, and 175 in the RBD region of SARS-CoV correspond to 125, 156, and 164 in the RBD region of SARS-CoV-2 , 172, and 176 positions (a glycine G is inserted into position 152 of SARS-CoV-2). At the same time, we also found that although the RBD domain is a relatively conserved region, the first 4 of the 5 important sites have undergone significant changes. The amino acid at position 125 was changed from tyrosine Y to leucine L, and at Leucine L at position 1 is converted to phenylalanine F, aspartic acid N at position 163 is converted to glutamine Q, and threonine T at position 171 is converted to asparagine...

Embodiment 2

[0049]Clone the full-length S-6His Tag gene (positions 125, 152, 155, 163, and 171 of the RBD region are respectively leucine L, glycine G, phenylalanine F, glutamine Q, and asparagine N) to In the eukaryotic expression vector pCDNA3.4, the recombinant pCDNA3.4-S-6His Tag vector is extracted by transforming E.coilDH5α and removing endotoxin with the plasmid extraction kit. The recombinant plasmid was transfected into suspended HEK-293 cells through PEI transient transfection, and the target protein S-6His Tag was expressed, and then the S protein expressed in the cell supernatant was detected by using His 6FF chromatography filler. Purified to obtain the His-tagged S fusion protein, which was passed through mice (BALB / c male mice, 7 weeks old, 20-28g, provided by Zhejiang Academy of Medical Sciences [animal use license number: SYXK (Zhejiang) 2019- 0001]) tail vein injection of 120ug / 300uL (phosphate buffered saline), the experimental animals maintained a light and dark cycle ...

Embodiment 3

[0056] 30 BALB / c male mice (7 weeks old, 20-28g, provided by Zhejiang Academy of Medical Sciences [animal use license number: SYXK (Zhejiang) 2019-0001]) were selected and raised in clean, constant temperature, and constant humidity in separate cages. 1. Avoid the strong light environment, and carry out the experiment after one week of adaptive feeding with standard feed. According to the random number table method, they were divided into 3 groups (n=10): phosphate buffer saline blank control group C, tail vein injection of recombinant RBD protein SRBD group and tail vein injection of recombinant albumin control group RA.

[0057] Group C tail vein injection of PBS solution 300uL, S RBD Group RA was injected with recombinant RBD protein (purchased from Raybiotech, 230-01103, Gene ID: QHD43416, purity>80%) 120ug / 300uL into tail vein, and RA group was injected with recombinant albumin (purchased from Raybiotech, 268-11133-1, Gene ID: 213) 120ug / 300uL, sacrificed after 24h.

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Abstract

The invention relates to the field of experimental animal models, in particular to an establishment method of a novel coronavirus-induced lung injury animal model and a mouse model thereof. The establishment method comprises the following steps: (1) constructing plasmids by using a COVID-19 spike protein S gene sequence; (2) performing plasmid transfection or prokaryotic expression; (3) preparing a COVID-19 lung injury animal model; (4) evaluating the COVID-19 lung injury animal model, wherein the No.152 site of the RBD region in the gene sequence of the COVID-19 spike protein S is glycine G. According to the method, plasmid construction is carried out by using the No.152 site of the RBD region as a COVID-19 spike protein S gene sequence of glycine G, and the establishment method for preparing the novel coronavirus-induced lung injury animal model is directly and specifically provided from the gene sequence level, so that the content is increased for basic research and animal experiment research; and more specific animal model selection is provided for further pathological research, medicine and vaccine development and the like.

Description

technical field [0001] The invention belongs to the field of experimental animal models, and in particular relates to a method for establishing an animal model of lung injury caused by a novel coronavirus and a mouse model thereof. Background technique [0002] 2019 Novel Coronavirus Pneumonia (CoronaVirus Disease 2019, COVID-19) is an infectious disease caused by a new betacoronavirus-Novel Coronavirus (Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2). All patients have different degrees of lung injury, and severe cases can rapidly progress to acute respiratory distress syndrome (severe acute respiratory syndrome, SARS) and acute fatal lung failure, advanced multiple complicated organ dysfunction syndrome (MODS) and multiple organ dysfunction syndrome (MODS). Organ failure (MOF) is an important factor leading to the death of COVID-19 patients. The lung pathology of patients with COVID-19 showed diffuse alveolar damage and hyaline membrane formation, which were ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01K67/027
CPCA01K67/027A01K2207/10A01K2267/0337
Inventor 孟志鹏刘静童飞胡四平何焕钟
Owner HUZHOU CENT HOSPITAL