CN111848750B

CN111848750B - Method and kit for rapidly enriching and detecting 2019-nCoV

Info

Publication number: CN111848750B
Application number: CN202010241930.1A
Authority: CN
Inventors: 杨永芳; 朱庆庆
Original assignee: Zhejiang Jukang Biotech Ltd
Current assignee: Zhejiang Jukang Biotech Ltd
Priority date: 2020-03-31
Filing date: 2020-03-31
Publication date: 2023-09-12
Anticipated expiration: 2040-03-31
Also published as: CN111848750A

Abstract

The application provides a method and a kit for rapidly enriching and detecting 2019-nCoV, which belong to the technical field of biological detection and analysis, can perform immunospecific enrichment on viruses in a larger-volume test sample, then perform cracking on the enriched virus sample to release nucleic acid and protein substances of the viruses, and then detect SPIKE protein with specific amino acid sequences in the 2019-nCoV viruses, so that the detection is rapid and sensitive, the generation of false positive phenomenon is reduced, and the method and the kit have good practical application value.

Description

Method and kit for rapidly enriching and detecting 2019-nCoV

Technical Field

The application belongs to the technical field of biological detection and analysis, and particularly relates to a method and a kit for rapidly enriching and detecting 2019-nCoV.

Background

The information disclosed in the background of the application is only for enhancement of understanding of the general background of the application and is not necessarily to be taken as an admission or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art.

The outbreak of new respiratory viruses is a serious threat to human life safety, and has caused immeasurable losses to social and economic developments. Acute respiratory infections caused by atypical coronavirus (SARS-nCoV) in 2002 quickly became epidemic tides worldwide, accumulated 8422 infections, and 919 deaths. At 17 years apart, a new coronavirus (2019-nCoV) was discovered from 12 months in 2019 to date, and the number of infections has been confirmed to be over 7 ten thousand, and the number of deaths is over 2000.

Coronaviruses belong to the genus Coronaviridae (Coronavirus) of the family Coronaviridae (Nidovirales) of the order Coronavirales in the phylogenetic classification. Coronaviruses are enveloped RNA viruses whose genome is linear single-stranded plus strand, and are a broad class of viruses that are widely found in nature. Coronaviruses infect only vertebrates, such as humans, mice, pigs, cats, dogs, wolves, chickens, cattle, birds. 2019 novel coronavirus (2019-nCoV; SARS-CoV-2) is currently known as the 7 th coronavirus that can infect humans, the remaining 6 are HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV (causing severe acute respiratory syndrome) and MERS-CoV (causing middle east respiratory syndrome), respectively. It is thought that this epidemic is an outbreak of epidemic disease caused by the transmission of Bat-borne coronavirus (Bat-CoV RaTG 13) to humans.

The inventor finds that the virus titer in clinical samples is very low, so that the existing detection method using virus nucleic acid as a target detection substance needs to be amplified and amplified by RT-PCR, and the main disadvantage is that false positives in the amplification and amplification processes or the virus titer is too low and sensitivity is insufficient, so that a large number of suspected cases which cannot be diagnosed exist in the early stage of 2019-nCoV epidemic outbreaks, and the implementation of a treatment scheme is influenced.

Disclosure of Invention

Aiming at the defects in the prior art, the application provides a method and a kit for rapidly enriching and detecting 2019-nCoV. The application can perform immune specificity enrichment aiming at viruses in a large-volume test sample, then perform cracking on the enriched virus sample to release nucleic acid and protein substances of the viruses, and then detect the SPIKE protein with specific amino acid sequences in 2019-nCoV viruses, thereby having rapid and sensitive detection, reducing the generation of false positive phenomena and having good practical application value.

In order to achieve the technical purpose, the application adopts the following technical scheme:

in one aspect of the application, there is provided a polypeptide having any one or more of the following sequences;

a) TPGDSSSGWT (SEQ ID No. 1) and/or multimeric forms thereof;

b) QTNSPRRAR (SEQ ID No. 2) and/or multimeric forms thereof;

wherein the polypeptide multimer is in particular in a form that facilitates host expression and/or purification of the recombinant protein, e.g., in a polypeptide multimerHistidine tag is added to the C-terminal or N-terminal, and thus, in one embodiment of the present application, the polypeptide sequence is (TPGDSSSGWT) ₅ -6His or (QTNSPRRAR) ₅ -6His。

According to the current study, bat-CoV RaTG13 virus carried by bats is closest to the nucleic acid sequence of 2019-nCoV responsible for this disease. Whereas some sequences only appear in the nucleic acid sequences of 2019-nCoV on their nucleic acids encoding the homologous protein SPIKE. Therefore, the application takes the 2-segment specific amino acid TPGDSSSGWT and QTNSPRRAR sequences on the SPIKE protein of the 2019-nCoV virus as detection targets, thereby detecting whether the 2019-nCoV virus exists in a sample.

Further, the polypeptide is obtained by a chemical artificial synthesis method or a biological synthesis method. Wherein the polypeptide is synthesized by a chemical artificial synthesis method such as a solid-phase polypeptide synthesis method or a liquid-phase polypeptide synthesis method or produced by a biosynthesis method such as by gene recombination from a genetically engineered bacterium.

Further, the biosynthesis method specifically includes: synthesizing a nucleotide encoding the above polypeptide; the nucleotide is introduced into an expression vector to construct a recombinant expression vector, and then introduced into a host for culture and collection.

In yet another aspect of the application, there is provided the use of a polypeptide in 2019-nCoV virus detection and/or in the preparation of a 2019-nCoV virus detection product.

In yet another aspect of the application, an antibody is provided that specifically binds to and recognizes the above polypeptide. Wherein the antibody may be a chimeric or humanized or deimmunized antibody.

Further, the antibody is a monoclonal antibody or a polyclonal antibody against the above polypeptide.

Further, the preparation method of the antibody comprises the step of immunizing a non-human animal against the antigen. The non-human animals include, but are not limited to, mice, rats, guinea pigs, rabbits, and monkeys.

Further, the preparation method comprises the following steps: preparing immune antigen, immunizing non-human animal, measuring serum titer, purifying antiserum and detecting antibody specificity.

In yet another aspect of the application, there is provided the use of the above antibody in 2019-nCoV virus detection and/or in the preparation of 2019-nCoV virus detection products.

Further, the 2019-nCoV virus detection method includes, but is not limited to, a method based on ELISA, colloidal gold method, or protein chip technology.

Further, the 2019-nCoV virus detection product includes, but is not limited to, substances required based on ELISA, colloidal gold method, or protein chip technology.

Further, the 2019-nCoV virus detection product includes a kit.

In yet another aspect of the application, there is provided a kit for rapid enrichment and detection of 2019-nCoV, the kit comprising:

immunomagnetic beads for enriching 2019-nCoV virus in a sample; and

the detection reagent strip is specifically a colloidal gold test strip and is used for detecting 2019-nCoV virus in a sample;

wherein the immunomagnetic beads are coupled with the antibodies; the antibody is preferably a monoclonal antibody against the above polypeptide;

the detection test strip is a conventional colloidal gold test strip, in a specific implementation mode, a sample pad 2 is stuck on a PVC bottom plate 1, a colloidal gold pad 3, a nitrocellulose membrane 4 and a water absorption pad 5 are stuck on the colloidal gold pad 3, an antibody marked by colloidal gold is sprayed on the nitrocellulose membrane, the nitrocellulose membrane is coated with the antibody as a test line 6, and an anti-mouse antibody is coated on the nitrocellulose membrane as a control line 7.

In yet another aspect of the present application, a method for rapid enrichment and detection of 2019-nCoV is provided, the method comprising the steps of:

enriching the 2019-nCoV virus in the sample to be detected by using the immunomagnetic beads so as to obtain an enriched sample of the 2019-nCoV virus; and

and detecting the 2019-nCoV virus by using the detection reagent strip.

Further, the samples to be tested include, but are not limited to, blood, saliva, nasal secretions and urine.

The beneficial technical effects of the application are as follows:

the application provides a method and a kit for rapidly enriching and detecting 2019-nCoV, and particularly, the application discovers that two sections of specific polypeptide sequences exist in the SPIKE protein of 2019-nCoV, so that a specific antibody is prepared based on the polypeptide sequences, enrichment and detection of 2019-nCoV are realized, and the application greatly improves the specificity (SPIKE protein unique sequence), sensitivity (virus enrichment >1000 times) and detection speed (time 20-50 minutes) of 2019-nCoV virus, so that the application has good practical application value.

Drawings

The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the application.

FIG. 1 shows the different nucleic acid sequences of the homologous protein SPIKE in different coronaviruses of the application.

FIG. 2 is a schematic diagram of a test strip according to the present application.

FIG. 3 is a graph showing the result judgment of the test strip according to the present application.

In the figure, 1-PVC bottom plate, 2-sample pad, 3-colloidal gold pad, 4-nitrocellulose membrane, 5-absorbent pad, 6-test line (T line), 7-control line (C line).

Detailed Description

It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present application. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.

The application will now be further illustrated with reference to specific examples, which are given for the purpose of illustration only and are not intended to be limiting in any way. If experimental details are not specified in the examples, it is usually the case that the conditions are conventional or recommended by the reagent company; reagents, consumables, etc. used in the examples described below are commercially available unless otherwise specified.

As described above, the virus titer in the clinical sample of 2019-nCoV virus is very low, so that the existing detection method using viral nucleic acid as the target detection substance needs to be amplified and amplified by RT-PCR, and the main disadvantage is that the false positive in the amplification and amplification process or the virus titer is too low and the sensitivity is not enough, so that a large number of suspected cases which cannot be diagnosed exist in the early stage of 2019-nCoV epidemic outbreak, and the implementation of a treatment scheme is affected.

The application provides a polypeptide, wherein the polypeptide sequence is any one or more of the following;

a) TPGDSSSGWT (SEQ ID No. 1) and/or multimeric forms thereof;

b) QTNSPRRAR (SEQ ID No. 2) and/or multimeric forms thereof;

further, the polypeptide multimer form is in particular a form that facilitates host expression and/or purification of the recombinant protein, such as by adding a histidine tag at the C-terminus or N-terminus of the polypeptide multimer, and thus the polypeptide sequence may be (TPGDSSSGWT) 5-6His or (QTNSPRRAR) 5-6His.

According to the current study, bat-CoV RaTG13 virus carried by bats is closest to the nucleic acid sequence of 2019-nCoV responsible for this disease. As shown in FIG. 1, some of the sequences on their nucleic acids encoding the homologous protein SPIKE appear only in the nucleic acid sequence of 2019-nCoV. Therefore, the application takes the sequences of 2-segment specific amino acids TPGDSSSGWT (SEQ ID No. 1) and QTNSPRRAR (SEQ ID No. 2) on the SPIKE protein of the 2019-nCoV virus as detection targets, thereby detecting whether the 2019-nCoV virus exists in a sample.

Thus, in yet another embodiment of the application, there is provided the use of a polypeptide in 2019-nCoV virus detection and/or in the preparation of a 2019-nCoV virus detection product.

Wherein the polypeptide can be synthesized by a chemical artificial synthesis method such as a solid-phase polypeptide synthesis method or a liquid-phase polypeptide synthesis method or produced by a biosynthesis method such as by gene recombination from a genetically engineered bacterium.

Further, the expression vector is any one or more of a viral vector, a plasmid, a phage, a phagemid, a cosmid, an F cosmid, a phage or an artificial chromosome; viral vectors may include adenovirus vectors, retrovirus vectors, or adeno-associated virus vectors, artificial chromosomes including Bacterial Artificial Chromosomes (BAC), phage P1-derived vectors (PAC), yeast Artificial Chromosomes (YAC), or Mammalian Artificial Chromosomes (MAC); further preferred are plasmids; even more preferred is the pET-28a plasmid;

in yet another embodiment of the application, the host includes, but is not limited to, bacteria, fungi and eukaryotic cells, further selected from the group consisting of E.coli, bacillus subtilis, saccharomyces cerevisiae, trichoderma reesei and Penicillium oxalate; more preferably, E.coli BL21 (DE 3) is used.

In yet another embodiment of the application, the host is introduced for cultivation and the specific method of collection comprises inducing the host to express, collecting the polypeptide and purifying.

Wherein, the induction expression adopts an inducer, and the inducer is preferably IPTG; the purification is performed by using Ni2+ affinity chromatography techniques (e.g., ni columns).

An antibody that specifically binds to and recognizes the above polypeptide. The antibody may be a chimeric or humanized or deimmunized antibody. Further, the antibodies are modified and thus chimeric antibodies comprising domains or chains of amino acid residues of different antibodies bound together in a functional antibody, in particular antibodies obtained from different animal species.

In a specific embodiment of the application, humanization may be performed by surface reconstruction according to known techniques or by CDRR (complementarity determining region) grafting. In particular, the reconstruction of the surface is achieved by substitution of the human amino acid residues with rodent residues. Substitution is performed in a manner that preserves the framework structure and CDR presentation of the original antibody, thereby allowing the framework and CDR interactions in the resurfaced antibody to maintain the native conformation of the surface in contact with the antigen such that it retains antigen binding affinity.

In one embodiment of the application, the murine antibody is humanized in order to reduce its immunogenicity in humans, so that deimmunization is also a useful alternative. Deimmunized can be used to reduce the immunogenicity of antibodies in humans by removing epitopes from the variable regions of rodent antibodies that are likely to be immunogenic in humans. Such as removal of potentially immunogenic B-and T-cell epitopes or chemically replacing the constant regions of murine antibodies with human antibody constant regions.

In a specific embodiment of the application, the antibodies are monoclonal antibodies against the antigens described above, and thus the composition of these antibodies is homogeneous or identical in terms of antigen binding specificity and in terms of variable regions. Thus, an antibody may be referred to as a monoclonal antibody even if the antibody is obtained by an alternative technique to the hybridoma technique.

In yet another embodiment of the present application, there is provided a nucleic acid molecule encoding the above antibody.

In yet another embodiment of the application, the method of producing an antibody comprises immunizing a non-human animal against the antigen described above. The non-human animals include, but are not limited to, mice, rats, guinea pigs, rabbits, and monkeys.

In yet another embodiment of the present application, the preparation method includes: preparing immune antigen, immunizing non-human animal, measuring serum titer, purifying antiserum and detecting antibody specificity.

In yet another embodiment of the present application, there is provided the use of the above-described antibodies in 2019-nCoV virus detection and/or in the preparation of 2019-nCoV virus detection products.

In yet another embodiment of the present application, the 2019-nCoV virus detection method includes, but is not limited to, a method based on ELISA, colloidal gold method or protein chip technology.

In yet another embodiment of the present application, the 2019-nCoV virus detection product includes, but is not limited to, substances required based on ELISA, colloidal gold method or protein chip technology.

In yet another embodiment of the present application, the 2019-nCoV virus detection product comprises a kit.

A kit for rapid enrichment and detection of 2019-nCoV, the kit comprising:

immunomagnetic beads for enriching 2019-nCoV virus in a sample; and

in a specific embodiment, as shown in fig. 2, the detection test strip is formed by sticking a sample pad 2, a colloidal gold pad 3, a nitrocellulose membrane 4 and a water absorption pad 5 on a PVC base plate 1, wherein an antibody marked by colloidal gold is sprayed on the colloidal gold pad 3, and the nitrocellulose membrane is coated with an antibody as a test line 6 and an anti-mouse antibody as a control line 7.

A method of rapid enrichment and detection of 2019-nCoV, the method comprising the steps of:

and detecting the 2019-nCoV virus by using the detection reagent strip.

In yet another embodiment of the present application, the sample to be tested includes, but is not limited to, blood, saliva, nasal secretions and urine.

Example 1

Antibody for preparing two sections of specific amino acids of SPIKE protein of 2019-nCoV virus

1. Polypeptide design: specific amino acid sequences on SPIKE proteins of 2 2019-nCoV viruses

Synthesized by company chemistry (simple sequences) or expressed by achieving bacillus (multimeric form)

Sequence 1: TPGDSSSGWT or multimeric forms (TPGDSSSGWT) ₅ -6His

Sequence 2: QTNSPRRAR or multimeric forms (QTNSPRRAR) ₅ -6His

Direct synthesis of amino acid sequence 1 by other companies: TPGDSSSGWT and sequence 2: QTNSPRRAR.

The laboratory expresses a polypeptide polymer form, and comprises the following specific steps:

(1) Two nucleic acid sequences were synthesized by biological companies:

a. sequence 1: polyamino acid sequences (TPGDSSSGWT) ₅ Corresponding nucleic acid sequences

(ACTCCTGGTGATTCTTCTTCAGGATGGACA) ₅ -HHHHHH；

b. Sequence 2: polyamino acid sequences (QTNSPRRAR) ₅ Corresponding nucleic acid sequences

(CAGACTAATTCTCCTCGGCGGGCACGT) ₅ -HHHHHH；

(2) The two nucleic acid sequences were cloned into the PET28a+ plasmid multiple cloning site, respectively.

(3) Transfer of recombinant expression vectors into BL ₂₁ DE ₃ The strain is cultured.

(4) Expression was induced using 0.1mM isopropyl- β -D-thiogalactoside (IPTG).

(5) The pellet was obtained by centrifugation at 3000g for 10 min.

(6) PBS was added to the cells to resuspend the cells, the cells were sonicated in an ice bath, and 10000g of the cells were centrifuged at 4℃for 10 min.

(7) Because both fragments of the protein of interest were in the centrifugation supernatant, the centrifugation supernatant was taken and filtered with a 0.22 μm filter.

(8) Purifying the 2-segment multimeric protein fragment by NTA-His, and preserving at 4 ℃ for later use. The target protein was detected by SDS-gel electrophoresis, and two single specific bands appeared in the vicinity of the 6KD region.

2. Preparation and purification of monoclonal antibodies to two specific protein fragments

(1) 50. Mu.g of protein solution was added to an equal volume (200. Mu.l) of Freund's complete adjuvant and emulsified (as antigen).

(2) The emulsified antigen is injected into the back of the mice subcutaneously and injected at multiple points.

(3) After 3 weeks intervals, an equal volume (200. Mu.l) of Freund's incomplete adjuvant was added with 50. Mu.g of protein solution, mixed and emulsified.

(4) The emulsified antigen is injected into the back of the mice subcutaneously and intraperitoneally.

(5) After an interval of 2 weeks, the tail vein was bled for about 20. Mu.l, the antibody titer was measured using the Elisa method, and 50ug of protein solution was injected intraperitoneally.

(6) The Elisa test results reached 1: on request of 16000, spleen cells were collected and fused with mouse myeloma cell lines within 3-4 days after the third injection to prepare hybridoma cells.

a. Preparing a feeder cell layer: the cell number was adjusted to 1X 10 by suspending macrophages in the abdominal cavity of 6-8 weeks mice and with 20% Foetal Calf Serum (FCS) in culture ⁵ And (3) paving 96-well plates per ml, and placing the plates in a cell incubator for standby.

b. Spleen of immunized mice is taken to prepare spleen lymphocyte suspension for standby.

c. Taking myeloma cells growing in the logarithmic phase, and preparing cell suspension for later use.

d. Myeloma cells and spleen cells were mixed at a ratio of 1:5 and washed 1 time with serum-free medium in a 50ml centrifuge tube.

e, centrifuging at room temperature at 1200rpm for 8min, discarding the supernatant, removing residual liquid by using a suction tube, and lightly flicking the bottom of the centrifugal tube to slightly loosen the cell sediment.

1ml of 45% PEG4000 solution preheated at 37℃was added over 90s with gentle shaking. Standing and water-bathing at 37 ℃ for 90s.

g. The incomplete culture solution was added at 37℃and 1ml, 2ml, 3ml, 4ml, 5ml and 6ml were added every 2 minutes, respectively.

Centrifugation at 800rpm for 5min at room temperature, and discarding the supernatant.

i. Cells were resuspended in medium containing 20% Fetal Bovine Serum (FBS) and HAT.

j. After the cell suspension was homogenized, the suspension was placed in a 96-well plate with feeder cells spread therein, 100 ul/well, and cultured in a cell incubator at 37 ℃.

After k.7 days, the original culture broth in the culture wells was removed and a culture broth containing 20% Fetal Bovine Serum (FBS) and HT was added. Culture was continued for 2 weeks, during which time the culture broth was replaced with a culture broth containing 20% Fetal Bovine Serum (FBS) and HT. Cells were then cultured using normal medium containing 20% Fetal Bovine Serum (FBS).

Hybridoma cloning and screening of specific monoclonal antibodies was then performed. For the antibodies produced by the hybridoma cells in each well, 7 SPIKE proteins of human coronaviruses that were infected with E.coli were screened by the Elisa method, and antibody-expressing fusion cell lines that recognized only the recombinant SPIKE proteins of 2019-nCoV virus were selected. Screening out an antibody corresponding to the sequence 1, namely named as SPIKE-A; the antibody corresponding to sequence 2 was designated as "SPIKE-B".

And m, injecting the selected cell strain into the abdominal cavity of a mice, and generating ascites in the abdominal cavity of the mice after 7 days. The ascites is removed and the antibodies therein are affinity purified by SPG

(7) Antibodies in serum were purified using SPG affinity column.

a. The column was washed with 5 column volumes of pure water to remove the column stock protecting liquid.

b. The column was equilibrated with 5 column volumes of 0.01M Tris base.

c. Antisera to be purified are added.

d. The column was washed with 10 column volumes of 0.01M Tris base to wash off the contaminating proteins.

e. The antibody bound to the column was eluted with 3 column volumes of 0.1M ph=2.5 glycine-HCl buffer and the effluent antibody solution was collected.

f. The antibody titer was detected by the Elisa method to 1:16000 can be used. Through detection, the prepared antibody prepared in the embodiment meets the requirement of the titer.

(1) Antigen coating: the known antigen was diluted to 1. Mu.g/ml with coating buffer, and the ELISA plate was added at 100 ul/well and incubated at 37℃in a constant temperature water bath for 2 hours or overnight at 4 ℃.

(2) Washing the plate: the next day the liquid in the wells was discarded, gently tapped on a paper towel to suck off the residual liquid, 250 ul/well of wash solution was added and rinsed 3 times.

(3) Closing: adding sealing solution at 200 ul/hole, incubating in a constant-temperature water bath at 37 ℃ for 1 hour or sealing overnight at 4 ℃, and discarding the sealing solution in the hole.

(4) Adding a sample: the antibody stock solution is diluted by 1000 times by using a washing solution, then diluted by 10 gradients from 1000 times to 512000 times according to 2-fold gradients, and then added into an ELISA plate according to 100 ul/hole in sequence, and incubated for 1 hour in a constant-temperature water bath at 37 ℃.

(5) Washing the plate: the solution in the wells was discarded, gently tapped on a paper towel to suck off the residual liquid, 250 ul/well of wash solution was added and rinsed 3 times.

(6) Addition of enzyme-labeled secondary antibody (IgG-HRP): 100 ul/well of freshly prepared enzyme-labeled antibody (purchased by Bio Inc.) was added and incubated at 37℃for 30min. (dilution method of enzyme-labeled antibody according to the product Specification)

(7) Washing the plate: the solution in the wells was discarded, gently tapped on a paper towel to suck off the residual liquid, 250 ul/well of wash solution was added and rinsed 3 times.

(8) Adding a color development liquid: the newly prepared TMB color development solution was added at 100 ul/well, and the reaction was conducted at 37℃for 15 minutes in the absence of light.

(9) And (3) terminating: 50ul of stop solution was added to each well.

And (3) judging the result: and (5) measuring the OD value by using an enzyme-labeled instrument to obtain the antibody titer (which is more than or equal to 2.1 times of the OD value of the negative control, namely, positive judgment).

3. Quality control and identification of specific antibodies

3.1. Quality control of antibodies

(1) The purity of the two purified antibodies meets the requirement by using SDS-PAGE protein electrophoresis detection.

(2) The purified antibodies were both found to be satisfactory by the Elisa method, which was the same as "2 (7). F".

3.2. Identification of antibody specificity

(1) The same procedure as in step 4.1 is adopted to prepare homologous protein spike in 7 coronaviruses capable of infecting human

(2) The two antibodies prepared by the Elisa method were identified to bind only to the SPIKE protein of 2019-nCoV virus, and none of them bound, as described in "2. (7). F).

Example 2

Magnetic bead antibody for preparing antibody SPIKE-A and method for establishing immune enrichment

1. Antibody SPIKE-a coupled to magnetic beads

(1) Pretreatment of magnetic beads: the beads were vortexed and separated, 30. Mu.l of the mixture was removed, the storage solution was removed, and the binding solution was added.

(2) Antibody pretreatment: the concentrated antibody was diluted with binding buffer to a final concentration of 50. Mu.g/mL of antibody preparation and placed on ice for use.

(3) Magnetic beads bind to antibodies: removing supernatant of the pretreated magnetic bead suspension, adding 200 μl of pretreated antibody, rapidly and uniformly mixing, turning over at room temperature and mixing for 20min, removing supernatant, and washing the magnetic bead antibody twice with binding buffer solution to obtain magnetic bead antibody.

(4) The magnetic bead antibodies were placed in sample bottles (conjugate bottles) for use at 4 ℃.

2. Sample lysis

(1) Premix reagents in sample lysates 1 and 2: the liquid in tube 2 was shaken up and then all transferred to tube 1 with a syringe, at which time the pellet appears in the bottle as normal.

(2) Processing a sample to be detected: 1-5 ml of saliva/blood/urine was taken and added to the sample lysis tube. If the sample amount is less than 5ml, the sample diluent is used for supplementing to 5ml, and the mixture is uniformly mixed.

(3) Sample lysis: all the treated samples were added to the tube and shaken vigorously by hand until no large lumps remained in the liquid, then the mixed samples were placed in boiling-stopped (about 100 ℃) water for 10min, removed and slightly shaken under 20 at about 5min, placed in a water bath for about 5min, and then slightly shaken under 20min, at which time the tube had normal foam but the liquid was clear and transparent and no insoluble material was present. Standing at room temperature for 5 minutes.

Remarks: the components of the lysate: tube No.1 powder: tris, naCl, guanidine hydrochloride; tube No.2 liquid: SDS, beta-mercaptoethanol, titox-100, 1ml water. Final concentration of each component in the solution after lysis of the sample: 20mM Tris, 2M NaCl, 6M guanidine hydrochloride, 1% SDS, 1% beta-mercaptoethanol, 0.1% Titox-100, pH=7.8 to 8.0.

3. Sample enrichment

(1) 10ml of dilution water was added to the lysed sample and the sample was mixed up and down 10 times.

(2) The sample is injected into a sample bottle with the magnetic bead antibody, and is evenly mixed for 10 times in an upside down way, the reaction is carried out for 30 minutes, the temperature is not lower than 25 ℃ and not higher than 37 ℃, and the reaction is fully carried out at least 10 times in an upside down way every 5 minutes.

(3) After the reaction is finished, the sample bottle is placed on a magnetic rack which is turned upside down for 10 times together, the magnetic rack and the sample bottle cannot be separated, and liquid is poured out.

(4) The vial was removed from the magnet holder and 10 milliliters of wash solution (PBS) was added to the vial.

(5) The reaction sample bottles were placed on a magnetic rack, turned upside down 10 times together, the magnetic rack and the sample bottles were not separated, and then the liquid was poured off.

(6) The above steps "d" and "e" are repeated.

(7) 0.2ml of the eluent was added to the sample bottle, and after shaking by rotation, the sample bottle was placed in hot water stopped boiling for 5 minutes.

(8) After 10 times of rotary shaking of the sample bottles, the sample bottles were placed on a magnetic rack. The supernatant was aspirated with a small pipette for detection.

Example 3

Preparation of colloidal gold test paper and detection of enriched sample

1. Preparation of colloidal gold-labeled antibody by antibody SPIKE-A

(1) Preparation of colloidal gold:

a. a very clean triangular flask was taken, 100ml of ultrapure water or double distilled water was added, and then 1ml of 1% gold chloride solution was added thereto, and heated to boil.

b. 1ml of 1% sodium citrate was added to the boiling solution and mixed by shaking, and the heating was stopped when the solution was orange. To obtain colloidal gold solution, and standing at 4deg.C for use.

(2) Colloidal gold labeled SPIKE-a antibody

a. The SPIKE-A antibody solution was dialyzed overnight at 4℃in 0.005M/L NaCl solution and then centrifuged at 100000g for 1h at 4 ℃.

b. The pH of the colloidal gold solution was adjusted to 0.1M/L K ₂ CO ₃ Or 0.1M/L HCl to 9.0.

c. Determination of the ratio of colloidal gold to the amount of labeled protein

(1) The treated antibodies were dispensed into 10 tubes, 1ml per tube.

(2) Serial dilution of the labeled antibody with 0.005M/L borate buffer solution with pH9.0 to 5 mg/ml-50 mg/ml, adding 1ml of the labeled antibody into the gold colloid solution, and mixing. Only 1ml of the diluent was added to the control tube, and the mixture was allowed to stand for 5 minutes.

(3) To each of the above tubes, 0.1ml of 10% NaCl solution was added, and after mixing, the mixture was allowed to stand for 2 hours, and the result was observed.

(4) And (3) observing results: both the control tube (without antibody) and the tube with insufficient amount of antibody added to stabilize the colloidal gold, exhibited a red-to-blue coagulation; while the tubes to which the amount of antibody added reached or exceeded the minimum stabilizing amount remained red.

(3) Binding of colloidal gold to antibodies: to the electromagnetically-agitated antibody solution, a colloidal gold solution was added, and stirring was continued for 10min, and fetal Bovine Serum (BSA) was added at a final concentration of 1%.

(4) Purification of colloidal gold-labeled proteins

a. Centrifuging at 1000rpm at room temperature for 20min, discarding the coagulated precipitate.

b. Centrifuge at 14000g for 1h at 4 ℃. The supernatant was aspirated and the pellet was resuspended in 1% BSA in PB to 1/10 the volume of the original solution to give sequence 2 colloidal gold-labeled antibody.

2. Preparation of colloidal gold test paper

(1) Sample pad treatment: NC membrane (nitrocellulose membrane) was placed in the sample pad treatment solution for 1 hour, and turned over 1-2 times during this period to thoroughly infiltrate. Drying in a drying box at 37 ℃ for 20 hours, and then drying and preserving for standby.

(2) And (3) treatment of a colloidal gold pad: the PET film (polyester film) is placed in the treatment liquid for 1 hour, and is turned over for 1 to 2 times during the treatment period, so that the PET film is thoroughly soaked. Drying in a drying box at 37 ℃ for 20 hours, and then drying and preserving for standby.

(3) And (5) metal spraying: and uniformly spraying the prepared gold-labeled antibody on the treated gold pad by using a colloidal gold jet pump. Drying in a drying oven at 37 ℃ for 15 hours. Drying and preserving for standby.

(4) The NC film was adhered to a PVC base plate.

(5) Scribing working solution: SPIKE-B antibodies were diluted to 0.8mg/ml as detection line (T line) and rabbit anti-mouse polyclonal antibodies were diluted to 1.0mg/ml as quality control line (C line) with 3% trehalose in PBS, respectively.

(6) Machine scribing: the control line and the detection line were evenly (1. Mu.l/cm) drawn on the NC film. The detection line is 15 mm from the top of the membrane, and the quality control line is 9mm from the top of the membrane.

(7) After membrane cutting, the seeds are placed in a drying oven at 37 ℃ for 20 hours. Drying and preserving for standby.

(8) As shown in fig. 3, the test paper is cut and assembled: gold mark pad: 6mm by 300mm; sample pad: 19mm by 300mm; water absorbing pad: 17mm by 300mm. The gold mark pad was placed on the NC film by 4mm, the sample pad was placed on the gold mark pad by 2mm, and the absorbent paper was placed on the NC film by 2mm.

3. Colloidal gold immunoassay for enriching samples

(1) The enriched sample was applied to the sample pad with a new pipette and the results read after 5-10 minutes.

(2) The result is determined as shown in fig. 3: if T (test) does not appear a band, then the test is negative, namely, no infection is detected; if the T band appears, the positive judgment is that the SARS-CoV-2 infection is caused, and the treatment is isolated. If the quality control line is not provided, the test is invalid. Through actual detection, the detection is positive for the sample containing coronavirus (the detection result is consistent with 2019-nCoV nucleic acid detection reagent), and the detection is negative for the sample not containing coronavirus.

The application is not a matter of the known technology.

The above embodiments are provided to illustrate the technical concept and features of the present application and are intended to enable those skilled in the art to understand the content of the present application and implement the same, and are not intended to limit the scope of the present application. All equivalent changes or modifications made in accordance with the spirit of the present application should be construed to be included in the scope of the present application.

Claims

1. A polypeptide having the sequence: sequence 1: polyamino acid sequences (TPGDSSSGWT) ₅ The method comprises the steps of carrying out a first treatment on the surface of the Corresponding nucleic acid sequence (ACTCCTGGTGATTCTTCTTCAGGATGGACA) ₅ The method comprises the steps of carrying out a first treatment on the surface of the Sequence 2: polyamino acid sequences (QTNSPRRAR) ₅ The method comprises the steps of carrying out a first treatment on the surface of the Corresponding nucleic acid sequence (CAGACTAATTCTCCTCGGCGGGCACGT) ₅ 。

2. A method of producing a polypeptide according to claim 1, wherein: the polypeptide is obtained by a chemical artificial synthesis method or a biosynthesis method, wherein the biosynthesis method specifically comprises the following steps: synthesizing a nucleotide encoding the polypeptide of claim 1; the nucleotide is introduced into an expression vector to construct a recombinant expression vector, and then introduced into a host for culture and collection.

3. The use of a polypeptide according to claim 1, wherein: the application of the polypeptide in preparing 2019-nCoV virus detection products.