SARS-CoV-2 S protein specific antibody or fragment thereof and application thereof

A sars-cov-2 and sars-cov-2s technology, applied in the field of antibody engineering, can solve the problems of monoclonal antibodies with no sequence structure, vaccine and therapeutic antibody research difficulties, and achieve high clinical application potential and high affinity , The effect of clear amino acid sequence structure

Inactive Publication Date: 2021-10-22
深圳市福田区格物智康病原研究所 +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] So far, monoclonal antibodies that block the binding of 2019-nCoV to the host ACE2 are still at the stage of theoretical research, and there are no prior technical reports on monoclonal antibodies with clear sequence structure and definite function of blocking 2019-nCoV binding
In addition, studies on anti-SARS-CoV-2 S protein antibodies in the prior art have found that there is an antibody-dependent enhancement effect (ADE) in the process of SARS-CoV-2 infection of host cells, which provides a basis for SARS-CoV-2 vaccines and treatments. Difficulties in the study of sexual antibodies

Method used

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  • SARS-CoV-2 S protein specific antibody or fragment thereof and application thereof
  • SARS-CoV-2 S protein specific antibody or fragment thereof and application thereof
  • SARS-CoV-2 S protein specific antibody or fragment thereof and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1: Recombinant expression of SARS-CoV-2 antigen and host receptor

[0061] The fully synthesized gene S1RBD (Accession: QHD43416.1, 319-541aa) was cloned into eukaryotic transient expression vectors with His tag or mFc tag at the C-terminus by enzyme digestion, and the obtained expression plasmid was transformed into Escherichia coli was amplified, the S1RBD-his and S1RBD-mFc expression plasmids were isolated, and according to the instructions of the transfection reagent 293fectin (Cat: 12347019, Gibco), the plasmids were transferred into HEK293 cells for recombinant expression. 5-6 days after cell transfection, the culture supernatant was taken, and S1RBD-mFc was purified by ProA affinity chromatography column to obtain S1RBD-mFc protein. S1RBD-his was purified by HisTrap HP column affinity chromatography to obtain S1RBD-his protein. And the obtained recombinant protein was tested for purity by SDS-PAGE ( Figure 1-2 ). The coding nucleic acid sequence of S1...

Embodiment 2

[0064] Example 2: B cell screening to obtain anti-new coronavirus S1RBD specific antibody

[0065] Using FITC-S1RBD-hFc as an antigen, the specific memory B cells of recovered patients with novel coronavirus infection were sorted. Single-cell PCR technology (method and primer reference New Biotechnology, 2010. Volume 27, Number 2, P110-117 and pages 114 to 117 in the book Human Monoclonal Antibodies) amplified antibody light and heavy chain genes, and performed sequencing analysis. The correctly sequenced antibody light and heavy chain variable region genes were synthesized and cloned into the transient expression vector pKN019 containing the light chain constant region and the transient expression vector pKN041 containing the heavy chain constant region IgG1 gene, and the HEK293 system was used for transient expression and purification to obtain recombinant antibody MW07 .

[0066] The method for single-cell PCR amplification of antibody light and heavy chain genes is as fol...

Embodiment 3

[0102] Example 3. MW07 and S1RBD affinity

[0103] The Fortebio protein interaction system was used to determine the binding affinity of the antibody to the S1-RBD protein antigen. The main steps are as follows:

[0104] Using Fortebio's Octet QKe system, the anti-human antibody Fc fragment capture antibody (AHC) bioprobe was used to capture the binding and dissociation changes of different concentrations of the assay antibody to the monovalent S1RBD. During the measurement, four different concentrations of MW07 (45nM, 30nM, 15nM, 7.5nM) flowed over the surface of the AHC probe (Cat: 18-5060, PALL) for 120s. S1RBD-His diluted in different concentrations was used as mobile phase. The binding time is 300s and the dissociation time is 300s. After the experiment was completed, the response value of the blank control was deducted, and the 1:1 Langmuir binding model was fitted with software to calculate the kinetic constant of antigen-antibody binding. The result is as Figure ...

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Abstract

According to the invention, coronavirus spike protein RBD specific memory B cells are separated from PBMC of a rehabilitation person infected by a new coronavirus, amplification is carried out to obtain antibody light and heavy chain variable region sequences, a carrier is connected and introduced, recombinant expression is carried out to obtain a recombinant human monoclonal antibody, a monoclonal antibody which has high affinity with SARS-CoV-2 spike protein RBD, strong blocking force on the SARS-CoV-2 spike protein RBD and a host receptor ACEII and strong neutralizing activity on virus infected cells is selected from the monoclonal antibody, further screening is performed the ADE effect of the monoclonal antibody, and finally an anti-SARS-CoV-2S protein specific antibody MW07 which does not generate an ADE phenomenon is obtained.

Description

technical field [0001] The invention belongs to the field of antibody engineering, in particular to a monoclonal antibody against coronavirus and its application, in particular to a SARS-CoV-2 S protein specific antibody or its fragment, preparation method and application. Background technique [0002] Although the plasma therapy of recovered patients has achieved certain clinical results, due to the limited source of plasma in patients with antigens, high safety risks of purified antibodies, and unstable titers of specific antibodies. Monoclonal antibodies with high titer, stable performance, and good safety have good application prospects for controlling the new coronavirus epidemic. At present, the existing literature has published or taught reports on the preparation of protective neutralizing monoclonal antibodies against the RBD of the new coronavirus. Use the new coronavirus spike protein RBD to generate protective neutralizing antibodies against the new coronavirus ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13C12N15/85C12N5/10A61K39/395A61P31/14
CPCC07K16/10C12N15/85A61P31/14C07K2317/56C07K2317/565A61K2039/505
Inventor 管轶桂勋郑作宜王双管静王荣娟陈佩雯焦莎莎李利峰张锦超朱华晨刘大涛
Owner 深圳市福田区格物智康病原研究所
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