Novel coronavirus, monoclonal antibody of mutant of novel coronavirus and application of monoclonal antibody

A monoclonal antibody and antigen technology, applied in the fields of immunology and molecular virology, can solve problems affecting the effect of neutralizing antibodies, and achieve the effect of clinical application value in prevention and treatment

Active Publication Date: 2022-01-14
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Studies targeting the S protein of SARS-CoV-2 and its RBD have been previously reported, and several recent studies have also identified viral mutations that can e

Method used

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  • Novel coronavirus, monoclonal antibody of mutant of novel coronavirus and application of monoclonal antibody
  • Novel coronavirus, monoclonal antibody of mutant of novel coronavirus and application of monoclonal antibody
  • Novel coronavirus, monoclonal antibody of mutant of novel coronavirus and application of monoclonal antibody

Examples

Experimental program
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Effect test

Embodiment 1

[0100] Expression and purification of embodiment 1SARS-CoV-2 virus S protein RBD

[0101] The optimized wild-type nCoV-RBD (residues 319-541, GenBank: YP_009724390.1) coding sequence with 6 His tags at the C-terminus was cloned into the mammalian expression vector pCAGGS. The various mutant RBDs shown in Table 1 (K417N, K417T (present in P.1 (Gamma) RBD), L452R (present in B.1.617.1 (Kappa) RBD and B.1.617.2 (Delta ) RBD), Y453F, N460S, T478K (present in B.1.617.2 (Delta) RBD), E484K, E484A, F486L, N501Y (present in B.1.1.7 (Alpha) RBD, B.1.351 (Beta) ) RBD, P.1 (Gamma) RBD), N501T) coding genes were subcloned into pCAGGS. Then the plasmid (2 μg) and PEI were transiently co-transfected at a mass ratio of 1:3 per milliliter of HEK293F cells. At 310K, 5% CO 2 The cells were cultured with SMM 293-TII medium (Sino Biological) under certain conditions, and then supplemented with SMS M293-SUPI (Sino Biological) at a ratio of 35 mL / L 24 hours after transfection. On the fifth day,...

Embodiment 2

[0102] Example 2 Isolation of memory B cells that specifically recognize RBD protein

[0103] With the informed consent of those infected with the SARS-CoV-2 virus who were cured and discharged, 10 mL of blood was collected and PBMCs were isolated. Separated PBMCs in 10 7 / mL density and final concentration of 400nM RBD protein (prepared by Example 1) were incubated on ice for half an hour; then washed twice with PBS, and then incubated with the following antibodies (both purchased from BD): anti-human CD3 / PE-Cy5, anti-human CD16 / PE-Cy5, anti-human CD235a / PE-Cy5, anti-human CD19 / APC-Cy7, anti-human CD27 / Pacific Blue, anti-human CD38 / APC, anti- human IgG / FITC, and anti-His / PE. After incubation on ice for half an hour, wash PBMCs twice with PBS. Subsequently, sort PBMCs with FACSAria III and collect PE - Cy5 - APCs - APCs - Cy7 + Pacific Blue + FITC + PE + The cells (that is, B cells) were directly collected into a 96-well plate, 1 cell / well.

Embodiment 3

[0104] Example 3 Isolation and identification of 9K antibody and construction of recombinant expression vector

[0105] The B cells obtained in Example 2 were reverse-transcribed using Superscript III reverse transcriptase (Invitrogen) (at 55° C. for 60 minutes), wherein the reverse transcription primers used are shown in Table 2.

[0106] Table 2 Sequence information of reverse transcription primers used

[0107]

[0108]

[0109] Using the reverse transcription product as a template, the first round of PCR (PCRa) was carried out with HotStar Tap Plus enzyme (QIAgen) to amplify the sequence of the variable region of the antibody; wherein, the primers used are shown in Table 3; the reaction conditions used As follows: 95°C, 5min; 35 cycles of (95°C for 30s, 55°C (heavy chain / κ chain) for 30s, 72°C for 90s); 72°C, 7min. Subsequently, the second round of PCR (PCRb) was carried out using the amplified product as a template; wherein, the primers used were as shown in Table ...

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Abstract

The invention relates to the technical field of immunology and molecular virology, and particularly discloses a novel coronavirus, a monoclonal antibody of a mutant of the novel coronavirus and application of the monoclonal antibody. According to the monoclonal antibody or an antigen binding fragment of the monoclonal antibody, CDR1 of a heavy chain variable region has an amino acid sequence shown as SEQ ID NO: 1, CDR2 has an amino acid sequence shown as SEQ ID NO: 2, and CDR3 has an amino acid sequence shown as SEQ ID NO: 3; and/or CDR1 of a light chain variable region has an amino acid sequence shown as SEQ ID NO: 4, CDR2 has an amino acid sequence shown as SEQ ID NO: 5, and CDR3 has an amino acid sequence shown as SEQ ID NO: 6. The monoclonal antibody can be combined with the novel coronavirus and various mutant strain S protein RBD of the novel coronavirus with high affinity, is high in neutralizing activity and has ideal clinical application value for preventing and treating infection of the novel coronavirus and various mutant strains of the novel coronavirus.

Description

technical field [0001] The invention relates to the technical fields of immunology and molecular virology, in particular to a monoclonal antibody to a novel coronavirus and its mutants and applications thereof. Background technique [0002] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel coronavirus, spreads rapidly and widely, with a high fatality rate, posing a major threat to public life and health. [0003] At the same time, various variants represented by Alpha, Beta, Gamma, and Delta of SARS-CoV-2 spread rapidly, and the field is struggling to cope with new virus variants. Studies targeting the S protein of SARS-CoV-2 and its RBD have been previously reported, and several recent studies have also identified viral mutations that can escape certain monoclonal antibodies that occur when neutralizing antibodies bind to the virus The key position, thus affecting the effect of neutralizing antibodies. [0004] SARS-CoV-2 is the causative agent of nove...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N15/13C12N15/85C12N5/10G01N33/577G01N33/569
CPCC07K16/10C12N15/85C12N5/0686G01N33/577G01N33/56983C07K2317/565C07K2317/56G01N2333/165C07K2317/76C07K2317/92C12N2800/107C12N2510/00Y02A50/30
Inventor 高福吴燕李世华张根谭曙光
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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