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Monoclonal antibody against novel coronaviruses and application of monoclonal antibody

A monoclonal antibody and anti-idiotypic antibody technology, which is applied in antiviral agents, antiviral immunoglobulins, applications, etc., can solve the problem that there is no specific drug for the new coronavirus

Active Publication Date: 2021-09-07
THE THIRD PEOPLES HOSPITAL OF SHENZHEN +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is still no specific drug for the treatment of new coronavirus

Method used

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  • Monoclonal antibody against novel coronaviruses and application of monoclonal antibody
  • Monoclonal antibody against novel coronaviruses and application of monoclonal antibody
  • Monoclonal antibody against novel coronaviruses and application of monoclonal antibody

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 connected to 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 tag. The ligation product was transformed into BL21 E. coli competent cells. Then, pick a single clone, inoculate into 40mL LB medium, and cultivate for 6-8 hours; then inoculate into 4L of LB medium, and cultivate 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 culture was continued at 37 degrees Celsius for 4-6 hours. After culturing, the inclusion bodies were harvested and refolded. The refolded protein solution was concentrated and dialyzed into 20mM Tr...

Embodiment 2

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

[0096] With the informed consent of those infected with 2019-nCoV who were cured and discharged, 10 mL of blood was collected to separate PBMCs. Separated PBMCs in 10 7 / mL density and final concentration of 400nM RBD protein (prepared as in Example 1) were incubated on ice for half an hour; then washed 2 times 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 - APC-Cy7 + Pacific Blue + FITC + PE + The cells (i.e. B cells) were directly collected into a 96-well plate, 1 cell / well.

Embodiment 3

[0097] Example 3: Isolation and identification of H4 antibody and construction of recombinant expression vector

[0098] 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.

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

[0100]

[0101] 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 4; the r...

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Abstract

The invention relates to the fields of immunology and molecular virology, in particular to the fields of diagnosis, prevention and treatment of novel coronaviruses. In particular, the invention relates to a monoclonal antibody against novel coronaviruses, and a composition (e.g., diagnostic and therapeutic agents) comprising the antibody. In addition, the invention also relates to an application of the antibody. The antibody can be used for diagnosing, preventing and / or treating infection of the novel coronaviruses and / or diseases caused by the infection (e.g., 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. In particular, the present invention relates to monoclonal antibodies against novel coronaviruses, and compositions (e.g., diagnostic and therapeutic agents) comprising said antibodies. In addition, 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 the infection of novel coronavirus and / or the disease (for example, novel coronavirus pneumonia) caused by said infection. Background technique [0002] As of March 3, 2020, the number of confirmed cases of pneumonia caused by the new coronavirus 2019-nCoV in the country was 80,303, with a total of 2,947 deaths. ) with a total of 9,742 confirmed cases and a total of 171 deaths, posing a major threat to the lives and health of the public. At...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N15/13G01N33/577G01N33/569A61K39/42A61P31/14
CPCC07K16/10G01N33/577G01N33/56983A61P31/14C07K2317/92C07K2317/76A61K2039/505A61K39/42C07K16/00G01N33/569C12N5/16C07K14/165A61P11/00C07K16/1003
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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