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A kind of monoclonal antibody against novel coronavirus and application thereof

A technology of monoclonal antibody and antibody, which is applied in the direction of antiviral agents, antiviral immunoglobulins, antibodies, etc., and can solve the problem that there is no specific drug for the new coronavirus

Active Publication Date: 2022-06-07
THE THIRD PEOPLES HOSPITAL OF SHENZHEN +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is still no specific drug for the treatment of the new coronavirus

Method used

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  • A kind of monoclonal antibody against novel coronavirus and application thereof
  • A kind of monoclonal antibody against novel coronavirus and application thereof
  • A kind of monoclonal antibody against novel coronavirus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] Example 1: Expression and purification of 2019-nCoV virus S protein RBD

[0094] Using NdeI and XhoI enzymes, the DNA fragment encoding the 2019-nCoV / 2019 strain spike protein S protein RBD (its amino acid sequence is shown in SEQ ID NO: 17) was ligated into the pET21a vector, and the DNA fragment was in the coding region. A nucleotide sequence encoding a 6*histidine tag (6*His tag) and a stop codon are also connected to the 3' end of . The ligation product was transformed into BL21 E. coli competent cells. Then, pick a single clone, inoculate it into 40 mL of LB medium, and cultivate for 6-8 hours; then transfer it to 4 L of LB medium, and cultivate it to OD600=0.4-0.6 at 37 degrees Celsius. Subsequently, IPTG was added to the culture to a final concentration of 1 mM and the incubation was continued for 4-6 hours at 37°C. After the culture, the inclusion bodies were harvested and renatured. The renatured protein solution was concentrated and dialyzed into 20 mM Tris...

Embodiment 2

[0095] Example 2: Isolation of memory B cells that specifically recognize RBD proteins

[0096] With the informed consent of persons infected with 2019-nCoV virus and recovered and discharged, 10 mL of blood was collected to isolate PBMCs. The isolated PBMCs were divided into 10 7 The density / mL was combined with RBD protein (prepared as in Example 1) at a final concentration of 400 nM and incubated on ice for half an hour; then washed twice with PBS and incubated with the following antibodies (both 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, PBMCs were washed twice with PBS. Subsequently, PBMCs were sorted with FACSAria III and PE was collected - Cy5 - APC - APC - Cy7 + Pacific Blue + FITC + PE + cells (i.e., B cells) were collected directly into a 96-well plate, 1 ce...

Embodiment 3

[0097] Example 3: Isolation and identification of B38 antibody and construction of recombinant expression vector

[0098] The B cells obtained in Example 2 were reverse transcribed (at 55°C, for 60 minutes) using Superscript III reverse transcriptase (Invitrogen), wherein the reverse transcription primers used are shown in Table 2.

[0099] Table 2. Sequence information of reverse transcription primers used

[0100]

[0101] Using the reverse transcription product as a template, the first round of PCR (PCRa) was performed with HotStar Tap Plus enzyme (QIAgen) to amplify the sequence of the variable region of the antibody; the primers used were shown in Table 3; the reaction conditions used were As follows: 95°C, 5 min; 35 cycles (95°C 30s, 55°C (heavy chain / kappa chain) 30s, 72°C 90s); 72°C, 7min. Subsequently, a second round of PCR (PCRb) was performed using the amplified product as a template; the primers used were shown in Table 4; the reaction conditions used were as f...

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Abstract

The present invention relates to the field of immunology and molecular virology, especially the field of diagnosis, prevention and treatment of novel coronavirus. In particular, the present invention relates to monoclonal antibodies against novel coronaviruses, and compositions (eg, diagnostic and therapeutic agents) comprising said antibodies. Furthermore, the present invention also relates to the use of said antibody. The antibody of the present invention can be used for diagnosing, preventing and / or treating novel coronavirus infection and / or diseases caused by said infection (for example, novel coronavirus pneumonia).

Description

technical field [0001] The present invention relates to the field of immunology and molecular virology, especially the field of diagnosis, prevention and treatment of novel coronavirus. Specifically, the present invention relates to monoclonal antibodies against novel coronavirus, and compositions (e.g., diagnostic and therapeutic agents) comprising the antibodies. Furthermore, the present invention also relates to the use of said antibodies. Antibodies of the present invention can be used to diagnose, prevent and / or treat infection with novel coronaviruses and / or diseases caused by such infections (e.g., novel coronavirus pneumonia). Background technique [0002] Pneumonia caused by the novel coronavirus 2019-nCoV has spread widely across the country and around the world. As of March 3, 2020, the number of confirmed cases nationwide was 80,303, and the cumulative number of deaths was 2,947. In countries other than China (Thailand, the United States, Germany, Australia, Ir...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/10C12N15/13C12N15/85C12N5/10A61K39/395A61P31/14G01N33/569G01N33/577
CPCC07K16/10A61P31/14G01N33/56983G01N33/577C12N15/85A61K2039/505C07K2317/56C07K2317/565C07K2317/52C07K2317/92A61K39/395C07K16/00C12N5/10G01N33/569A61K39/42A61P11/00
Inventor 刘映霞吴燕沈晨光刘磊谭曙光李德林王非然高福
Owner THE THIRD PEOPLES HOSPITAL OF SHENZHEN
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