Neutralizing epitope peptide in RBD region of SARS-CoV-2 S protein and application of neutralizing epitope peptide

A sars-cov-2, sars-cov-2b technology, applied in the field of protein engineering, can solve the problems of inefficient expression of short peptides, lack of immunogenicity, neutralization of antigenic epitopes, and low immunogenicity

Pending Publication Date: 2022-03-11
MABWELL (SHANGHAI) BIOSCIENCE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far, there is still a lack of detailed information on the immunogenicity and neutralizing epitopes of the S protein of the newly emerged SARS-CoV-2 virus
[0007] At present,

Method used

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  • Neutralizing epitope peptide in RBD region of SARS-CoV-2 S protein and application of neutralizing epitope peptide
  • Neutralizing epitope peptide in RBD region of SARS-CoV-2 S protein and application of neutralizing epitope peptide
  • Neutralizing epitope peptide in RBD region of SARS-CoV-2 S protein and application of neutralizing epitope peptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0136] Embodiment 1 recombinant antigen and antibody preparation

[0137] 1. Preparation of S1RBD

[0138] The SARS-CoV-2 S1RBD (QHD43416.1, 319-533aa) gene was cloned into a eukaryotic expression vector with a His tag at the N-terminal, and the S1RBD eukaryotic expression plasmid with a His tag at the N-terminal was constructed (recombinant RBD- The sequence of His is shown in SEQID NO: 23). The plasmid was transfected into HEK293 cells by transfection reagent 293fectin (Life Technologies, 12347-019, 1977248) for transient expression, and the supernatant was collected after 4 days, and HisTrap HP affinity chromatography column (GE, 17-5248-01 ) to purify the expression supernatant, then ultrafilter through an ultrafiltration concentration tube, replace the buffer with PBS, obtain the pure recombinant protein of SARS-CoV-2 S1RBD antigen, and analyze the purity by SDS-PAGE and HPLC, the results are as follows figure 1 shown.

[0139] 2. Preparation of MW01, MW06 and MW07-Fab...

Embodiment 2

[0143] Embodiment 2 Antigen-antibody complex preparation and analysis

[0144] 1. Preparation of MW01-Fab antigen-antibody complex

[0145] Mix N-terminal His tag SARS-CoV-2 S1RBD and MW01-Fab in 20mM Tris 150mM NaCl solution according to the ratio of SARS-CoV-2 S1RBD: MW01-Fab at 0.9:1, and place at room temperature for 2 hours to make MW01-Fab fully Combined, ultrafiltered through an ultrafiltration concentration tube (Cat: VS2002, Sartorius Stedim), concentrated to 20mg / ml, 0.3ml, and carried out SEC-HPLC and SDS-PAGE detection; according to the results, the antigen-antibody complex retention time Lower than SARS-CoV-2 S1RBD and MW01-Fab, the main peak is a single peak, suggesting that the complex sample meets the requirements of the crystallization experiment ( Figure 6 ).

[0146] 2. Preparation of MW05-Fab antigen-antibody complex

[0147] Mix SARS-CoV-2 S1RBD and MW05-Fab in 20mM Tris 150mM NaCl solution according to the molar ratio SARS-CoV-2 S1RBD: MW05-Fab = 1:2....

Embodiment 3

[0152]Example 3 MW05 Antigen Antibody Crystal Preparation and Diffraction Data Collection and Structure Analysis

[0153] 1. Crystal Index and Diffraction

[0154] Crystals of MW05 antigen-antibody complexes were grown using Crystal Screen1, CrystalScreen2, Index, PEGRx1, PEGRx2 from HAMPTON, and WIZARD I / II kits from Emerald Biosystems. Using the sitting drop method, under the conditions of 0.2M sodium citrate, 21% PEG3350, and 0.02M urea, crystallized for 3 days, and finally obtained crystals with better diffraction, such as Figure 10 shown. The crystal was collected at the Shanghai Synchrotron Radiation Facility Diffraction data of MW317 FAB and covid-19Spike in MW317-PD1 complex structure (PDB ID: 6JJP) using PHASER software RBD -The spike protein RBD in the hACE2 complex structure (PDB ID: 6LZG) is a molecular replacement model, and the complex structure of MW05 is analyzed by the molecular replacement method.

[0155] 2. Analysis of key amino acids involved in S1RB...

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Abstract

On the basis of the neutralizing antibody in the RBD region of the SARS-CoV-2S protein, the Fab segment of the neutralizing antibody is prepared through recombinant expression, the Fab segment reacts with the RBD region of the SARS-CoV-2S protein to form an antigen-antibody compound, and purification, crystallization and X-crystal diffraction analysis are performed. The method comprises the following steps: analyzing an antigen-antibody compound structure by using a homologous compound three-dimensional structure in a PDB database as a model to obtain amino acid residues interacting with a neutralizing antibody on SARS-CoV-2S protein RBD, carrying out site-specific mutagenesis on key amino acid residues, and detecting the affinity of an SARS-CoV-2S protein RBD mutant and the neutralizing antibody Fab. And determining the epitope peptide fragment according to the influence degree of the RBD point mutation on the affinity. The invention also provides application of the epitope peptide fragment in preparation of a fusion antigen for detecting SARS-CoV-2, and application of the epitope peptide fragment in preparation of an immunogen for preparing a vaccine or an antibody.

Description

[0001] This patent application claims priority to the Chinese patent application for invention with application number CN 202010945544.0 filed on September 10, 2020, the entire contents of which are hereby incorporated by reference. technical field [0002] The invention belongs to the field of protein engineering, in particular to a neutralizing epitope peptide in the RBD region of the SARS-CoV-2 S protein and its application, in particular to an anti-SARS-CoV-2 epitope peptide on the RBD region of the SARS-CoV-2 S protein An antigenic epitope interacting with an antibody, its preparation method and application. Background technique [0003] The 2019 novel coronavirus (2019-nCoV), discovered due to cases of viral pneumonia in 2019, was named by the World Health Organization on January 12, 2020. Coronaviruses are a large family of viruses known to cause colds as well as more serious illnesses such as Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndro...

Claims

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Application Information

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IPC IPC(8): C07K14/165C07K16/10C07K19/00C12N15/85C12N15/50C12N15/62C12N5/10
CPCC07K14/005C07K16/10C12N15/85C12N2770/20022C07K2319/40C07K2319/00
Inventor 王荣娟焦莎莎杨莹莹王双
Owner MABWELL (SHANGHAI) BIOSCIENCE CO LTD
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