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Neutralizing nano antibody for resisting novel coronavirus SARS-CoV-2 and application thereof

A nanobody and coronavirus technology, applied in the direction of antiviral agent, virus/bacteriophage, antiviral immunoglobulin, etc., can solve the problem that there is no report of neutralizing nanobody against the new coronavirus SARS-CoV-2

Active Publication Date: 2021-10-29
INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Currently, there are no reports of neutralizing nanobodies against the novel coronavirus SARS-CoV-2

Method used

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  • Neutralizing nano antibody for resisting novel coronavirus SARS-CoV-2 and application thereof
  • Neutralizing nano antibody for resisting novel coronavirus SARS-CoV-2 and application thereof
  • Neutralizing nano antibody for resisting novel coronavirus SARS-CoV-2 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Example 1: Screening of Nanobodies Recognizing the RBD Domain of the SARS-CoV-2 Spike Protein

[0080] Step 1: Rescue the phage surface-displayed nanobody library

[0081] The fully synthetic nanobody library is stored in the host bacteria in the form of phagemids, and before the panning process begins, the library is rescued and made into a phage-displayed antibody library. The specific method is as follows:

[0082] Take 1mL (OD 600 =100) Antibody library glycerol bacteria, inoculated into 5 bottles of 200mL 2TY-CARB medium (OD 600 = 0.1), 37°C, shake at 250rpm to OD 600 = about 0.5; add 1.6 × 10 to each bottle of bacterial liquid 12 PFU M13KO7, stand at 37°C for 30 minutes, shake at 37°C, 200rpm for 30 minutes; transfer the bacterial solution to a centrifuge bottle, centrifuge at 2200g for 15 minutes, resuspend the cell pellet with 400mL 2TY-CARB-KAN, 30°C, 250rpm Shake the bacteria for 14-16 hours; put the bacterial liquid into centrifuge bottles (200mL per bot...

Embodiment 2

[0091] Example 2: Nanobody and Fc protein fusion expression

[0092] Step 1: Construction of recombinant expression vector

[0093] Positive cloned genes were amplified using the upstream primer shown in SEQ ID No. 8 and the downstream primer shown in SEQ ID No. 9.

[0094] SEQ ID No. 8: 5'-ggcgctagccaagttcaattggttgaat-3'

[0095] SEQ ID No. 9:

[0096] 5'-tgagcctccactgaattcagaagaaacagtaacttgagtacct-3'.

[0097] Obtain nucleic acid molecules encoding anti-RBD Nanobodies. The antibody gene was double digested with NheI and EcoRI, inserted into the antibody expression vector pCDNA4-Fc, and transformed into DH5a E. coli competent cells (purchased from Quanshijin Company) to obtain recombinant bacteria. The recombinant bacteria were inoculated into liquid LB-Amp medium, cultured at 37°C overnight, and plasmids were extracted with a plasmid extraction kit. 16 Nanobody recombinant plasmids were obtained. pCDNA4-Fc was transformed from pCDNA4 / myc-HisA (purchased from Thermo Fis...

Embodiment 3

[0100] Example 3: Assay of SARS-CoV-2 pseudotyped virus neutralizing activity of Nanobodies

[0101] Step 1: SARS-CoV-2 Pseudotyped Virus Packaging

[0102] 6×10 6 HEK293T cells (from the Cell Resource Center, Institute of Basic Medicine, Chinese Academy of Medical Sciences) were seeded in a 10cm petri dish, and the medium was DMEM high-glucose medium (purchased from Thermo Fisher) containing 10% fetal bovine serum (purchased from Thermo Fisher). from Thermo Fisher Scientific), 12 hours later, transfected with 10 μg of S gene expression plasmid (Golden Wisdom) and 10 μg of pNL4.3-Luc-R-E-plasmid (BioVectorNTCC), and replaced with 2% fetal bovine serum 12 hours after transfection DMEM high glucose medium, continue to culture for 48 hours and harvest the culture supernatant containing SARS-CoV-2 pseudotyped virus, aliquot and store at -80°C.

[0103] Step 2: SARS-CoV-2 pseudotyped virus invasion inhibition experiment

[0104] 10 4 Calu-3 cells (from the Cell Resource Center,...

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Abstract

The invention discloses a neutralizing nano antibody for resisting novel coronavirus SARS-CoV-2 and application thereof, and belongs to the technical field of biological medicine. Variable regions of the neutralizing nano antibody are provided with three complementary determining regions CDR1, CDR2 and CDR3; and the neutralizing nano antibody is any one of (a1)-(a6). The neutralizing nano antibody for resisting novel coronavirus SARS-CoV-2 provided by the invention can be effectively combined with an RBD antigen, can effectively compete with human ACE2 protein to be combined with the RBD antigen after being fused with a human IgGFc tag, has high neutralizing activity on SARS-CoV-2 pseudotype virus, and has important scientific significance and application prospects for prevention and clinical treatment of diseases caused by the novel coronavirus SARS-CoV-2 and research and development of diagnostic reagents.

Description

technical field [0001] The invention relates to a neutralizing nanobody against novel coronavirus SARS-CoV-2 and an application thereof, belonging to the technical field of biomedicine. Background technique [0002] The novel coronavirus, known as severe acute respiratory syndrome (SARS)-coronavirus-2 (SARS-CoV-2), is the causative agent of novel coronavirus pneumonia (COVID-19). SARS-CoV-2 uses angiotensin converting enzyme 2 (Angiotensin converting enzyme 2, ACE-2) as a receptor to invade cells and cause lung damage. Respiratory distress syndrome (Acuterespiratory distress syndrome, ARDS) and septic shock, eventually multiple organ failure. Since the outbreak of the epidemic, scientists from all over the world have begun to work on the development of vaccines, antiviral drugs, and antibody drugs. [0003] Antibodies are glycoproteins produced by B cells that proliferate and differentiate into plasma cells after being stimulated by antigens. They can specifically bind to ...

Claims

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Application Information

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IPC IPC(8): C07K16/10C07K19/00C12N15/85C12N5/10A61K39/42A61P31/14G01N33/569
CPCC07K16/10C12N15/85C12N5/0686A61P31/14G01N33/56983C07K2317/569C07K2317/76C07K2317/565C07K2317/567C07K2319/30C12N2800/107C12N2510/02G01N2333/165G01N2469/10
Inventor 赵振东黄鹤朱悦王蓓王健伟
Owner INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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