阅读说明：本技术 检测新型冠状病毒的多肽或其组合 (Polypeptide or combination thereof for detecting novel coronavirus ) 是由 向志光 刘晓立 秦川 刘云波 鲍琳琳 高虹 佟巍 孔琪 郭智 于 2020-06-15 设计创作，主要内容包括：本发明涉及生物医药领域,特别涉及检测新型冠状病毒的多肽或其组合。经序列抗原预测分析合成12条多肽,12条多肽的混合物PM与新型冠状病毒重组蛋白RN和RS进行抗原性比较分析,PM与RN、RS的抗原性相当。无病原接触者亦存在阳性反应；将12条肽分别与无病原接触者样品进行测试,其中多肽P5和P8具有良好的抗原特异性；利用P5与P8的混合物P58进行多样本测试,在ELISA反应体系中其敏感性与重组抗原无明显差异,特异性达96%。本发明提供的新型冠状病毒的特异性多肽抗原具有较好的血清学诊断应用价值。(The invention relates to the field of biological medicine, in particular to polypeptide or a combination thereof for detecting novel coronavirus, wherein 12 polypeptides are synthesized through sequence antigen prediction analysis, the antigenicity of a mixture PM of the 12 polypeptides is equivalent to that of RN and RS through antigenicity comparison analysis, a positive reaction also exists for a patient without pathogen contact, the 12 peptides are respectively tested with a sample of the patient without pathogen contact, wherein the polypeptides P5 and P8 have good antigenic specificity, and the mixture P58 of P5 and P8 is used for multi-sample test, so that the sensitivity and the recombinant antigen have no obvious difference in an E L ISA reaction system, and the specificity reaches 96%.)

1. Polypeptide or a combination thereof, wherein the amino acid sequence of the polypeptide is shown as SEQ ID No.1 or 2.

2. The polypeptide or combination of claim 1, wherein in the combination the molar ratio of the polypeptide having the amino acid sequence shown as SEQ ID No.1 to the polypeptide having the amino acid sequence shown as SEQ ID No.2 is 1: 10-10: 1.

3. use of the polypeptides of claim 1 or 2 or a combination thereof for the preparation of a novel coronavirus detection reagent or detection means.

4. A reagent for detecting a novel coronavirus comprising the polypeptide of claim 1 or 2 or a combination thereof and an auxiliary agent acceptable for detection.

5. A kit for detecting a novel coronavirus comprising the polypeptide of claim 1 or 2 or a combination thereof and a detection-acceptable adjuvant or carrier.

Technical Field

The invention relates to the field of biomedicine, in particular to a polypeptide for detecting novel coronavirus or a combination thereof.

Background

The coronavirus is a common human pathogen, such as β coronavirus, such as HKU1 and OS43, can also cause respiratory tract infection, and has high infection rate in people, the novel coronavirus belongs to β coronavirus, and the protein sequences of the novel coronavirus have certain similarity.

The specific polypeptide antigen of the novel coronavirus can be found, so that the cross with other common coronavirus antigens can be avoided, and the reduction of the interference of other virus infections on the recognition of the virus infection antibody has important practical significance.

Disclosure of Invention

In view of the above, the present invention provides polypeptides or combinations thereof for detecting novel coronaviruses. The novel coronavirus specific polypeptide is used for diagnosing a patient virus antibody.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a polypeptide or a combination thereof, wherein the amino acid sequence of the polypeptide is shown as SEQ ID No.1 or 2.

In some embodiments of the invention, in the combination of polypeptides, the molar ratio of the polypeptide having an amino acid sequence as shown in SEQ ID No.1 to the polypeptide having an amino acid sequence as shown in SEQ ID No.2 is 1: 10-10: 1; preferably 1: 1.

More importantly, the invention also provides the application of the polypeptide or the combination thereof in preparing a detection reagent or a detection tool for the novel coronavirus.

The invention also provides a reagent for detecting the novel coronavirus, which comprises the polypeptide or the combination thereof and an acceptable auxiliary agent for detection.

The invention also provides a kit for detecting the novel coronavirus, which comprises the polypeptide or the combination thereof and an acceptable auxiliary agent or carrier for detection.

The invention analyzes the protein sequence of the novel coronavirus, searches for a site which is different from other coronaviruses and has strong antigenicity, synthesizes a polypeptide antigen substance, and utilizes an enzyme-linked immunosorbent assay to determine the antigenicity and the specificity of the polypeptide antigen substance, wherein the antigen substance is a serum sample of a patient, a patient without the pathogen and an infected experimental animal.

The method comprises the steps of screening and synthesizing 12 polypeptides through virus antigen protein amino acid sequence prediction analysis, performing antigenicity comparison analysis on a mixture of 12 Polypeptides (PM), novel coronavirus Recombinant protein NP (Recombinant NP, RN) and novel coronavirus Recombinant protein S (Recombinant S, RS), wherein the PM is equivalent to the antigenicity of RN and RS, immunological reactivity exists between a pathogen-free contactor and PM, RN and RS, testing 12 Peptides and a plurality of serum samples of the pathogen-free contactor respectively, screening 2 polypeptides P5 and P8, having no immunoreactivity with the serum samples of the novel coronavirus non-contactor, and having good antigen specificity with polypeptides P5 and P8, performing multi-sample test by utilizing a mixture P58 of P5 and P8, wherein the sensibility of the polypeptide P368652 and the Recombinant antigen RN and RS is not obviously different in an E L reaction system, and an E L ISA detection method taking P58 as the antigen has good diagnostic value in the specificity of the novel antigen ISA P8 and the antigen 58 when the specificity of the ISA 96 and the novel coronavirus ISA P8 is high.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows the comparison result of NP protein sequences of β genus 5 coronavirus, NP protein sequences of novel coronavirus (SARS-CoV-2), HKU1, OS43, SARS coronavirus and mouse MHV coronavirus were analyzed by ClustalW (Slow/Accurate, Gonnet) method using L aseegene magicAlign software;

FIG. 2 shows the reactivity of SARS-CoV-2 infected mice with different antigenic substances;

FIG. 3 shows data analysis of the reactivity of SARS-CoV-2 infected mice with different antigenic substances;

FIG. 4 shows the reactivity of SARS-CoV-2 infected monkey sera with different antigenic substances;

FIG. 5 shows data analysis of the reactivity of SARS-CoV-2 infected monkey sera with different antigenic substances;

FIG. 6 shows the reactivity of SARS-CoV-2 patient serum with different antigenic substances;

FIG. 7 shows data analysis of the reactivity of SARS-CoV-2 patient serum with different antigenic substances;

FIG. 8 shows the reactivity of human serum with different polypeptide antigens without SARS-CoV-2 exposure to healthy human;

FIG. 9 shows the reactivity of polypeptide P58 mixed antigen with human samples;

FIG. 10 shows data analysis of the reactivity of polypeptide P58 mixed antigen with human samples;

FIG. 11 shows the reaction of early stage (5-7 days) of SARS-CoV-2 infection in a IgM antibody detection system using P58 as an antigen species;

FIG. 12 shows data analysis of the reaction of early monkey infection with SARS-CoV-2 (5 to 7 days) in the IgM antibody detection system using P58 as an antigen substance;

FIG. 13 shows the results of the test conducted in effect example 5 in which 10 portions of serum free from SARS-CoV-2 infection were randomly selected and exposed to healthy human;

FIG. 14 shows the results of the measurement in effect example 5 with the negative sample ratio expanded.

Detailed Description

The invention discloses a polypeptide for detecting novel coronavirus or a combination thereof, and the method can be realized by appropriately modifying process parameters by the technical personnel in the field by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The raw materials and reagents used in the polypeptide or the combination thereof for detecting the novel coronavirus provided by the invention are all available in the market.

The 12 polypeptides are synthesized by Beijing Yamei polypeptide biotechnology limited, and the purity of the polypeptides is more than 95%. The coating of the polypeptide was continued using a carbonate coating system, with an antigen amount of 1. mu.g per well, and blocked with a phosphate buffer containing 1% bovine serum albumin. The ELISA plate is a Costar product. The use of a Hrp-labeled secondary antibody and TMB single-component color developing solution was from Solebao. Control antigen recombinant protein NP (RN) and recombinant protein S (RS) were from Beijing Yinqiao Shenzhou.

The invention is further illustrated by the following examples: