Reagent for detecting novel coronavirus SARS-CoV-2 antibody and application thereof

Abstract

The invention discloses a reagent for detecting a novel coronavirus SARS-CoV-2 antibody and application of the reagent, and particularly relates to a polypeptide. The sequence of the polypeptide comprises one or more of partial amino acid sites corresponding to a S protein sequence and / or N protein sequence of a novel coronavirus SARS-CoV-2 shown as SEQ ID NO.1, and the polypeptide has S protein antigenicity and / or N protein antigenicity; the invention further relates to a primer composition for synthesizing the polypeptide, a preparation method of the polypeptide and application of the polypeptide in preparation of the reagent for detecting or diagnosing the novel coronavirus SARS-CoV-2, and particularly can be used for preparing a colloidal gold reagent strip and a test kit thereof. Thespecific primer designed by the invention can successfully synthesize the S protein / N protein antigen peptide with excellent binding activity, and the colloidal gold chromatography reagent strip prepared from the specific primer can rapidly and effectively detect the S protein antibody / N protein antibody of the novel coronavirus SARS-CoV-2, the generation of false negative results is avoided, thedetection efficiency is high, and epidemic diffusion can be prevented as soon as possible.

Description

Technical field

[0001] The present invention relates to the technical field of virus antibody detection, in particular to a reagent for detecting a novel coronavirus SARS-CoV-2 antibody and its application, specifically for detecting the spike protein Spike and the novel coronavirus (SARS-CoV-2) / Or reagents for IgM and IgG antibodies of N protein, more specifically, preparation of novel coronavirus SARS-CoV-2 antigen peptides for antibody detection by colloidal gold lateral chromatography. Background technique

[0002] The new coronavirus pneumonia (COVID-19) that appeared at the end of 2019 spread rapidly in a short period of time. According to the World Health Organization (WHO) report, as of May 25, 2020, the number of confirmed cases worldwide has exceeded 2.8 million, with hundreds of thousands. Death, the disease seriously threatens human survival and health. The situation for the prevention and control of the novel coronavirus pneumonia epidemic is very severe. China has...

Examples

Embodiment 1

[0088] Example 1-Construction of S protein antigen peptide display library and preparation and screening of antigen peptide display library Phage S protein (Spike protein) is a membrane protein, which constitutes the envelope particles of the virus and is the main component of the virus infecting host cells. To infect cells, the new coronavirus must first introduce its genetic material RNA into the host cell. This process is mainly done through the S protein. The protrusion of S protein is the key part of its interaction with receptor and membrane fusion. The experimental steps for synthesizing S protein antigen peptide include:

[0089] 1. Construction of S protein antigen peptide display library;

[0090] (1) According to the above-mentioned Spike protein full-length sequence shown in SEQ ID NO.1 and the peptide sequence to be synthesized, the corresponding primer pair is designed. The peptide numbers to be synthesized are S1~S126, which correspond to the sequence of Spike prote...

Embodiment 2

[0110] Example 2-Preparation of colloidal gold detection reagent strips by using S protein antigen peptide and its effect verification

[0111] In this example, the predominant epitope peptide of the SARS-CoV-2 spike protein Spike screened in Example 1 is used as the solid phase, and the indirect method is used to detect whether the serum contains antibodies against the new coronavirus. The colloidal gold detection reagent strip The preparation steps include:

[0112] (1) Synthesize the dominant epitope peptides S22, S25 and S29 according to the steps in Example 1;

[0113] (2) Preparation of coating film: 0.01mol / L PBS diluted antibody, mouse anti-human IgG antibody concentration 1.0mg / mL sprayed on the NC membrane, marked as G line; mouse anti-human IgM antibody concentration 1.0mg / mL spray On the NC membrane, mark it as the M line; the goat anti-mouse IgG antibody concentration is 1.0mg / mL, spray on the NC membrane, and mark it as the C line

[0114] (3) Preparation of gold-labele...

Embodiment 3-N

[0136] Example 3-Construction of N protein antigen peptide display library and preparation and screening of antigen peptide display library Phage

[0137] The N protein is an important structural protein in the coronavirus. The N protein is in the core part of the virus particle and exists in the form of binding to genomic RNA. The experimental steps of synthesizing N protein antigen peptide include:

[0138] 1. Construction of N protein antigen peptide display library;

[0139] (1) According to the above-mentioned N protein full-length sequence as shown in SEQ ID NO. 2 and the peptide sequence to be synthesized, the corresponding primer pair is designed. The peptides to be synthesized are numbered N1 to N42, which correspond to the N protein For partial amino acid sites, the corresponding primer numbers are N1F / N1R~N42F / N42R (SEQ ID NO.255~338), and each primer has sticky ends. The polypeptide sequence, its corresponding amino acid positions and the designed specific primers are s...

Technical field

[0001] The present invention relates to the technical field of virus antibody detection, in particular to a reagent for detecting a novel coronavirus SARS-CoV-2 antibody and its application, specifically for detecting the spike protein Spike and the novel coronavirus (SARS-CoV-2) / Or reagents for IgM and IgG antibodies of N protein, more specifically, preparation of novel coronavirus SARS-CoV-2 antigen peptides for antibody detection by colloidal gold lateral chromatography. Background technique

[0002] The new coronavirus pneumonia (COVID-19) that appeared at the end of 2019 spread rapidly in a short period of time. According to the World Health Organization (WHO) report, as of May 25, 2020, the number of confirmed cases worldwide has exceeded 2.8 million, with hundreds of thousands. Death, the disease seriously threatens human survival and health. The situation for the prevention and control of the novel coronavirus pneumonia epidemic is very severe. China has...

Examples

Embodiment 1

[0088] Example 1-Construction of S protein antigen peptide display library and preparation and screening of antigen peptide display library Phage S protein (Spike protein) is a membrane protein, which constitutes the envelope particles of the virus and is the main component of the virus infecting host cells. To infect cells, the new coronavirus must first introduce its genetic material RNA into the host cell. This process is mainly done through the S protein. The protrusion of S protein is the key part of its interaction with receptor and membrane fusion. The experimental steps for synthesizing S protein antigen peptide include:

[0089] 1. Construction of S protein antigen peptide display library;

[0090] (1) According to the above-mentioned Spike protein full-length sequence shown in SEQ ID NO.1 and the peptide sequence to be synthesized, the corresponding primer pair is designed. The peptide numbers to be synthesized are S1~S126, which correspond to the sequence of Spike prote...

Embodiment 2

[0110] Example 2-Preparation of colloidal gold detection reagent strips by using S protein antigen peptide and its effect verification

[0111] In this example, the predominant epitope peptide of the SARS-CoV-2 spike protein Spike screened in Example 1 is used as the solid phase, and the indirect method is used to detect whether the serum contains antibodies against the new coronavirus. The colloidal gold detection reagent strip The preparation steps include:

[0112] (1) Synthesize the dominant epitope peptides S22, S25 and S29 according to the steps in Example 1;

[0113] (2) Preparation of coating film: 0.01mol / L PBS diluted antibody, mouse anti-human IgG antibody concentration 1.0mg / mL sprayed on the NC membrane, marked as G line; mouse anti-human IgM antibody concentration 1.0mg / mL spray On the NC membrane, mark it as the M line; the goat anti-mouse IgG antibody concentration is 1.0mg / mL, spray on the NC membrane, and mark it as the C line

[0114] (3) Preparation of gold-labele...

Embodiment 3-N

[0136] Example 3-Construction of N protein antigen peptide display library and preparation and screening of antigen peptide display library Phage

[0137] The N protein is an important structural protein in the coronavirus. The N protein is in the core part of the virus particle and exists in the form of binding to genomic RNA. The experimental steps of synthesizing N protein antigen peptide include:

[0138] 1. Construction of N protein antigen peptide display library;

[0139] (1) According to the above-mentioned N protein full-length sequence as shown in SEQ ID NO. 2 and the peptide sequence to be synthesized, the corresponding primer pair is designed. The peptides to be synthesized are numbered N1 to N42, which correspond to the N protein For partial amino acid sites, the corresponding primer numbers are N1F / N1R~N42F / N42R (SEQ ID NO.255~338), and each primer has sticky ends. The polypeptide sequence, its corresponding amino acid positions and the designed specific primers are s...

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