US-12617823-B2 - Broad-spectrum peptide antigen of the novel coronavirus SARS-COV-2, specific neutralizing antibody and use thereof

US12617823B2US 12617823 B2US12617823 B2US 12617823B2US-12617823-B2

Abstract

The present disclosure provides a broad-spectrum peptide antigen of SARS-COV-2, a specific neutralizing antibody and use thereof. A broad-spectrum peptide antigen of SARS-COV-2, with an amino acid sequence comprising SEQ ID NO: 1, reacts with human SARS-COV-2 positive serum, and can specifically bind to a novel coronavirus antibody. Based on the peptide sequence of the present disclosure, a fusion protein with broad-spectrum triple tandem peptides of SARS-COV-2 is prepared using PCR, prokaryotic expression, and protein purification technology which simulates the trimeric mode of SARS-COV-2 S protein in its natural state. The fusion protein is used as an antigen to immunize mice, and can produce a specific anti-SARS-CoV-2 neutralizing antibody. The neutralizing antibody may be promising in anti-infective treatment, vaccine development and detection kit development for SARS-COV-2.

Inventors

Jianqiang Ye
Tuofan LI
Qiuqi KAN
Aijian Qin
Zhimin Wan
Hongxia Shao
Quan Xie

Assignees

YANGZHOU UNIVERSITY

Dates

Publication Date: 20260505
Application Date: 20210721
Priority Date: 20210607

Claims (1)

1 . A fusion protein having broad-spectrum triple tandem peptides for SARS-COV-2, wherein the fusion protein comprises three tandemly arranged sequences of a broad-spectrum peptide antigen of SARS-COV-2, and the broad-spectrum peptide antigen comprises the amino acid sequence of SEQ ID NO: 1, and wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 8.

Description

CROSS REFERENCE TO RELATED APPLICATION This patent application is a National Stage application that claims the benefit and priority to International Application No. PCT/CN2021/107615, filed on Jul. 21, 2021, entitled “BROAD-SPECTRUM PEPTIDE ANTIGEN OF THE NOVEL CORONAVIRUS SARS-COV-2, SPECIFIC NEUTRALIZING ANTIBODY AND USE THEREOF”, which claims the benefit of and priority to Chinese Patent Application No. 202110631147.0 filed on Jun. 7, 2021, the disclosures of which is are incorporated by reference herein in their its entirety as part of the present application. TECHNICAL FIELD The present disclosure belongs to the technical filed of immunoassay of virus, and particularly relates to a broad-spectrum peptide antigen of the novel coronavirus SARS-CoV-2, a specific neutralizing antibody and use thereof. BACKGROUND ART The novel coronavirus SARS-CoV-2 is a highly infectious virus. The S protein of SARS-CoV-2 includes two subunits, i.e. S1 and S2. The S1 subunit binds to the cell receptor angiotensin-converting enzyme 2 (ACE2) via its receptor binding domain (RBD), and the S2 subunit mediates the fusion between the virus and the cell membrane through a fusion peptide (FP). Due to the important role in the fusion process during infection, S2 can be used as a target for extensive protection. At present, the frequent emergence of SARS-CoV-2 escape mutants has brought challenges to current immunologically advantageous strategy of receptor-binding-domain (RBD)-based vaccine against SARS-CoV-2. However, there are currently no broad-spectrum vaccine against SARS-CoV-2 and rapid detection method for detecting anti-S2 antibodies. SUMMARY In view of this, the objective of the present disclosure is to provide a broad-spectrum peptide antigen of SARS-CoV-2. The antigen shows no mutation in the SARS-CoV-2 prototype strain and various epidemic mutant strains, is capable of specifically identifying the antibody to SARS-CoV-2 and has good antigenicity. The present disclosure provides a broad-spectrum peptide antigen of SARS-CoV-2, in which the amino acid sequence of the broad-spectrum peptide antigen is set forth in SEQ ID NO: 1. The present disclosure provides a fusion protein having broad-spectrum triple tandem peptides for SARS-CoV-2, in which the fusion protein includes three tandemly arranged sequences of the broad-spectrum peptide antigen of SARS-CoV-2. Preferably, the amino acid sequence of the fusion protein is set forth in SEQ ID NO: 8. The present disclosure provides a generation method of the fusion protein having broad-spectrum triple tandem peptides for SARS-CoV-2, including the following steps: 1) performing PCR amplification with a primer pair having nucleotide sequences set forth in SEQ ID NO: 6 and SEQ ID NO: 7 using plasmid pCold I as template to obtain a linearized pCold I expression vector;2) amplifying a first fragment by PCR with a primer pair as set forth in SEQ ID NO: 2 and SEQ ID NO: 3, amplifying a second fragment by PCR with a primer pair as set forth in SEQ ID NO: 4 and SEQ ID NO: 3, and amplifying a third fragment by PCR with a primer pair as set forth in SEQ ID NO: 4 and SEQ ID NO: 5, using plasmid pcDNA3.1-SARS-CoV-2-S as template;3) performing rapid recombinant cloning of linearized pCold I expression vector in step 1) and the first fragment, the second fragment and the third fragment in step 2) with a recombinase to obtain a recombinant plasmid; and4) transforming cells with the recombinant plasmid in step 3), generating a fusion protein through cell culture and IPTG induction. Preferably, an amplification procedure of the PCR in step 2) includes denaturation at 94° C. for 5 min, 30 cycles of denaturation at 94° C. for 30 s, annealing at 57° C. for 30 s and extension at 72° C. for 3 min and a final extension at 72° C. for 10 min. An amplification reaction system for the PCR includes 33 μl water, 10 μl of 5× buffer, 1 μl of 10 mM dNTP, 2 μl of 10 μM upstream primer, 2 μl of 10 μM downstream primer, 1 μl of 10 ng/μl pCold I plasmid or pcDNA3.1-SARS-CoV-2-S plasmid, and 1 μl of commercial Phanta Super-Fidelity DNA polymerase. The present disclosure provides the use of the broad-spectrum peptide antigen of SARS-CoV-2 and a fusion protein having broad-spectrum triple tandem peptides for novel coronavirus SARS-CoV-2 in preparing a broad-spectrum subunit vaccine against SARS-CoV-2 and/or a kit for immunoassay of S2 antibody to novel coronavirus SARS-CoV-2. Preferably, the immunoassay includes colloidal gold immunoassay, immunoprecipitation, fluorescence immunoassay and/or enzyme-linked immunoassay. The present disclosure provides an ELISA kit for detecting an antibody to SARS-CoV-2, including an ELISA plate coated with an antigen; the antigen includes the broad-spectrum peptide antigen of SARS-CoV-2 or the fusion protein having broad-spectrum triple tandem peptides of SARS-CoV-2. The present disclosure provides a broad-spectrum neutralizing antibody to novel coronavirus SARS-CoV-2, in which the antib