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SARS-CoV-2 (severe acute respiratory syndrome-corona virus disease-2) inhibitor and application thereof

A sars-cov-2 and sars-cov-2-s technology, applied in the field of neutralizing antibodies against SARS-CoV-2, can solve problems such as limited sources, mismatch of donor and recipient blood types, etc.

Inactive Publication Date: 2020-06-26
JIANGSU PROVINCIAL CENT FOR DISEASE CONTROL & PREVENTION PUBLIC HEALTH RES INST OF JIANGSU PROVINCE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the source of polyantibody plasma is not only limited, but its clinical application is also limited by conditions such as difficult quality control, donor-recipient blood type mismatch, and potential infectious factors. Human monoclonal antibodies can effectively overcome the above problems

Method used

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  • SARS-CoV-2 (severe acute respiratory syndrome-corona virus disease-2) inhibitor and application thereof
  • SARS-CoV-2 (severe acute respiratory syndrome-corona virus disease-2) inhibitor and application thereof
  • SARS-CoV-2 (severe acute respiratory syndrome-corona virus disease-2) inhibitor and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] The cultivation and material preparation of embodiment 1 virus

[0076] The SARS-CoV-2 virus was isolated by the patent applicant from the peripheral blood of a Jiangsu patient in the acute phase in 2020. After the virus was inoculated into Vero cells, at 37°C, 5% CO 2 Cultured for 5 days under certain conditions, collected the supernatant and determined the 50% tissue infection dose (50% tissue culture infectious dose, TCID 50 ). All manipulations are performed in BSL-3 laboratories.

[0077] Recombinant S protein (SARS-CoV-2-S), commercially purchased, the sequence is shown in SEQ ID NO.1.

Embodiment 2

[0078] Example 2 Construction of ScFv human antibody library and screening of anti-SARS-CoV-2-S protein single chain antibody

[0079] 2.1 Materials

[0080] Primers: Family-specific light chain (Vκ and Vλ), IgG heavy chain (VH) and overlap-PCR primers were designed according to the book "Phage Display", including Vκ12 pair, Vλ24 pair, VH 6 pair, and overlap-PCR1 pair.

[0081] Primers for constructing human scFv library:

[0082] Vκforward primers:

[0083] 5'GGGCCCAGGCGGCCGAGCTCCAGATGACCCAGTCTCC 3' (SEQ ID NO.66)

[0084] 5'GGGCCCAGGCGGCCGAGCTCGTGATGACYCAGTCTCC 3' (SEQ ID NO. 67)

[0085] 5'GGGCCCAGGCGGCCGAGCTCGTGWTGACRCAGCTCC 3' (SEQ ID NO. 68)

[0086] Vκreverse primers:

[0087] 5'GGAAGATCTAGAGGAACCACCTTTTGATYTCCACCTTGGTCCC 3' (SEQ ID NO. 69)

[0088] 5'GGAAGATCTAGAGGAACCACCTTTTGATCTCCAGCTTGGTCCC 3' (SEQ ID NO. 70)

[0089] 5'GGAAGATCTAGAGGAACCACCTTTAATCTCCAGTCGTGTCCC 3' (SEQ ID NO. 71)

[0090] 5'GGAAGATCTAGAGGAACCACCTTTGATATCCACTTTGGTCCC 3' (SEQ ID NO. 72)

[0...

Embodiment 3

[0134] Example 3 Candidate scFv monoclonal microneutralization experiment (all operations were carried out in BSL-3 laboratory)

[0135] 3.1 Preparation of phage antibody

[0136] Pick 15 single colonies that were positive in the Phage-ELISA test and place them in 2ml 2×YT medium (containing 100 μg / mL ampicillin, 12.5 μg / mL tetracycline, and 1 g / mL glucose), culture them overnight at 37°C with shaking, and the next day 1:10 were inoculated into 10ml of 2×YT medium (containing 100 μg / mL ampicillin and 30 μg / mL tetracycline) and cultured with shaking at 37°C for 6 hours, and the helper phage VCSM13 (final concentration of 1×10 9 PFU / mL), incubate at 37°C for 1 h, add kanamycin (final concentration is 50 μg / mL), shake at 30°C overnight, centrifuge at 900 g for 30 minutes, discard the precipitate, add 5×PEG / NaCl to the supernatant, mix Put it on ice for 6 hours, centrifuge at 900g for 30min, discard the supernatant, resuspend the pellet with 1ml PBS, centrifuge at 900g for 30min,...

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Abstract

The invention relates to an SARS-CoV-2 (severe acute respiratory syndrome-corona virus disease-2) inhibitor and application thereof and in particularly relates to a neutralizing antibody for SARS-CoV-2 and application of the neutralizing antibody. According to the antibody, a phage display technology is adopted to construct a high-capacity human immunity phage antibody library, and an SARS-CoV-2 protein is adopted a target, human antibody single-chain antibody fragments are screened, and an antibody with a good neutralizing function on SARS-CoV-2 viruses is obtained. The antibody provided by the invention can be used for treating diseases caused by infection of novel corona viruses, and has significant clinical application value.

Description

technical field [0001] The invention belongs to the field of cellular immunology, and in particular relates to a neutralizing antibody against SARS-CoV-2 and application thereof. Background technique [0002] Corona Virus Disease 2019 (COVID-19), referred to as "New Coronary Pneumonia", is a pneumonia caused by SARS-CoV-2 virus infection. Respiratory droplet transmission is the main route of transmission. It grows in a relatively closed environment. There is a possibility of aerosol transmission when exposed to high-concentration aerosols for a long time, and other transmission routes have yet to be clarified; the crowd is generally susceptible, and the incubation period is 1 to 14 days, generally 3 to 7 days. The clinical types include asymptomatic infection, mild, common, severe, and critical. The prognosis of the elderly and those with underlying diseases is poor, and the symptoms of children are relatively mild. As of 11:00 on February 27, 2020, a total of 78,630 confir...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13G01N33/569A61K39/42A61P31/14
CPCA61K2039/505A61P31/14C07K16/10C07K2317/56C07K2317/565G01N33/56983G01N2333/165
Inventor 张文帅郭喜玲焦永军曾晓燕朱凤才朱宝立
Owner JIANGSU PROVINCIAL CENT FOR DISEASE CONTROL & PREVENTION PUBLIC HEALTH RES INST OF JIANGSU PROVINCE
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