Nano antibody for novel coronavirus and application of nano antibody

A coronavirus, nanobody technology, applied in the field of biomedicine or biopharmaceuticals

Active Publication Date: 2021-03-16
SHANGHAI NOVAMAB BIOPHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In view of the current situation that there is no specific drug for the treatment of new coronary pneumonia, the development of a nanobody that has high blocking activity against ACE2 / S-RBD and can bind to a variety of virus mutants will have extremely high market value and clinical application value

Method used

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  • Nano antibody for novel coronavirus and application of nano antibody
  • Nano antibody for novel coronavirus and application of nano antibody
  • Nano antibody for novel coronavirus and application of nano antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0222] Example 1: Screening of SARS-COV2 s protein-specific nanocodies

[0223] In order to obtain nanocodies specific for human SARS-COV2 s proteins, first-use mammalian cell HEK293F transient expression of human SARS-COV2 s protein, after the lucrants were used for camel immunity. Briefly, the purified SARS-COV2 s protein was immunized from two Xinjiang double peak camel. After 7 immunization, Total RNA was separated from camel peripheral blood, and then the vHH gene was amplified by reverse transcription and PCR, and then clone the VHH gene. To the phage carrier PMECs, transformed into TG1 in a structural complexed phage display library. The build library storage capacity is 4.7 × 10, respectively. 9 CFU, 3.3 × 10 9 CFU, 1.3 × 10 9 CFU, 1.3 × 10 8 CFU, insertion rate is 100%, 91.7%, 95.8%, 95.8%, respectively. The library screening is then performed, and the four documents are selected by 3 rounds of phage enrichment containing antibody genes. 400 clones were selected from each...

Embodiment 2

[0224] Example 2: SARS-COV2 S protein nanoparticles to block activity detection of ACE2 / S-RBD

[0225] The cultured 293F / ACE2 steady cells were dispensed into a 96-well plate, 3E5 cells per well, 3000 rpm centrifuge for 3 min to remove the supernatant, add diluted antibody and S-RBD-Biotin protein to incubate for 20 min, antibody concentration Three times from 160 ug / ml, 12 gradients were diluted. EtOAc. Also dishearted again, and 200 ul PBS resuscitation is added to each well, and the flow cytometry detects the sample PE signal.

[0226] Such as Figures 1A-1D As shown, the results show that 32 sets of candidate antibodies have good ACE2 / SARS-COV2 S-RBD blocking activity.

Embodiment 3

[0227] Example 3: Combined with reference to different types of viral s proteins with different types of viral s proteins

[0228] 1 ug / ml Sars-Cov1 S-RBD protein, MERS S-RBD protein, SARS WIV1 S-RBD protein package was laid out at night. PBST washed 5 times, and 300 ul 1% BSA was added to 2 hours per well. PBST washed 5 times, and then gradient diluted SARS-COV2 S-RBD nanoparticles were added to room temperature for 1 hour. PBST washed 5 times, and 100 ul diluted Mouseanti-HA Antibody was incubated for 1 hour. PBST washed 5 times, 100 ul of alkaline phosphatase-labeled anti-mouse antibody, 37 ° C for 30 minutes. The color coloration was added, and the enzyme label was measured at 405 nm.

[0229] The result of the combination of nanoparticles and SARS-COV1 S-RBD is figure 2 As shown, all candidate nanocodies can identify SARS-COV-1S-RBD.

[0230] The result of the combination of nanoparticles and SARS-WIV1 S-RBD image 3 As shown, NB16-68 and NB11-59 can identify SARS WIV1 S-R...

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Abstract

The invention discloses a nano antibody for a novel coronavirus and application of the nano antibody. Specifically, the invention provides the nano antibody for the novel coronavirus and a sequence ofthe nano antibody. The invention also provides polynucleotide coding the nano antibody, a corresponding expression vector, a host cell capable of expressing the nano antibody and a production methodof the nano antibody. The nano antibody disclosed by the invention can be specifically combined with human SARS-CoV2; the nano antibody disclosed by the invention has good ACE2 / SARS-CoV2S-RBD blockingactivity; the nano antibody disclosed by the invention can be combined with various mutants of new coronavirus; the nano antibody disclosed by the invention has relatively high neutralizing activityon SARS-CoV2 euvirus; and the antibody disclosed by the invention is stable in quality before and after being atomized.

Description

Technical field [0001] The present invention relates to the field of biomedical or biopharmaceutical techniques, and more particularly to nanopartys and applications for new coronaviruses. Background technique [0002] Coronavirus (COV) has single-stranded, non-segmented positive electrode RNA genome, which contains four major structural proteins: nucleus (N) protein, transmembrane (M) protein, envelope (E) protein and thorn Sudden (S) protein. Among them, the probular protein S plays a key role in the virus, fused, enters, and propagates, including the N-terminal responsible for the S1 subunit and C-terminal responsible for viral-cell membrane fusion, S1 can be subjected to N-N-N- Terminal domain (NTD) and receptor binding domain (RBD). [0003] The new crown virus (SARS-COV2) is a pathogen of new coronavirus pneumonia (COVID-19). Neutralizing antibodies are considered to be against COVID-19 candidate therapies. In terms of principles, the new crown virus recognizes and binds th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13C12N15/85C12N5/10
CPCC07K16/10C12N15/85C07K2317/569C07K2317/565C07K2317/567
Inventor 万亚坤朱敏盖军伟李光辉沈晓宁
Owner SHANGHAI NOVAMAB BIOPHARM CO LTD
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