WO2021168968A1 - 一种基于s蛋白配体与ace2受体竞争法层析的冠状病毒快速检测的试剂盒 - Google Patents

一种基于s蛋白配体与ace2受体竞争法层析的冠状病毒快速检测的试剂盒 Download PDF

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WO2021168968A1
WO2021168968A1 PCT/CN2020/081636 CN2020081636W WO2021168968A1 WO 2021168968 A1 WO2021168968 A1 WO 2021168968A1 CN 2020081636 W CN2020081636 W CN 2020081636W WO 2021168968 A1 WO2021168968 A1 WO 2021168968A1
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ace2
protein
kit
chromatography
detection
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French (fr)
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范春雷
朱晓进
程向荣
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范春雷
朱晓进
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Priority to US16/942,666 priority Critical patent/US11549114B2/en
Publication of WO2021168968A1 publication Critical patent/WO2021168968A1/zh
Priority to US18/063,578 priority patent/US20230212589A1/en

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    • C12N15/86—Viral vectors
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/43—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a FLAG-tag
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    • C07—ORGANIC CHEMISTRY
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus

Definitions

  • the invention belongs to the field of biotechnology, and specifically relates to a rapid detection kit for coronavirus based on S protein ligand and ACE2 receptor competition chromatography.
  • Coronaviruses belong to the genus Coronavirus in the family Coronaviridae (Coronaviridae).
  • the virus of the genus Coronavirus is a positive-stranded single-stranded RNA virus with an envelope, with a diameter of about 80-120nm. Its genetic material is the largest among all RNA viruses.
  • the host of infection includes humans, mice, bats, pigs, cats, Pangolins, dogs, wolves, chickens, cattle, snakes, poultry and other vertebrates.
  • the 2019 new type of coronavirus (SARS-CoV-2, formerly known as 2019-nCoV) is currently the seventh known coronavirus that can infect humans.
  • HCoV-229E The remaining six are HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-CoV.
  • SARS-CoV-19, SARS-CoV and HCoV-NL63 infect humans through the viral envelope spike glycoprotein (S-protein) mediated interaction between the virus and the ACE2 receptor on the host cell membrane.
  • S-protein viral envelope spike glycoprotein
  • COVID-19 2019 novel coronavirus pneumonia
  • the early detection of new viruses generally uses nucleic acid detection methods, because the virus genome can be quickly deciphered, and specific nucleic acid fluorescent probes can be synthesized Prepared the fluorescent quantitative PCR detection kit and put it into use.
  • This method has the advantages of mature technology, short R&D cycle, good specificity, and accurate detection; but at the same time, it also has disadvantages such as sample pre-processing, long detection time, operation by professional instrument professionals, and expensive reagents. It also has certain disadvantages. False negatives.
  • Another quick test method is colloidal gold immunochromatographic test paper, including virus antigen test and serological antiviral protein antibody (IgM/IgG) test.
  • the advantages of this kind of reagents are that they are fast and simple, do not require equipment and professional operation, and have low reagent costs.
  • the disadvantage is that for new viruses, protein antigens and specific antibodies of new viruses must be prepared first, resulting in a long research and development cycle that cannot be used in time for epidemics. Detection, and the detection accuracy of the kit is closely related to the quality of protein antigens and specific antibodies.
  • the spike protein of Coronavirus CoV has two subunits, S1 and S2, among which the S1 subunit acts as a ligand to interact with the ACE2 receptor on the human cell membrane to form a specific binding.
  • S1 and S2 the spike protein of Coronavirus CoV
  • the principle of competitive immunochromatography we replaced one of the anti-S-protein monoclonal antibodies with recombinant human ACE2 protein to label quantum dot fluorescence and sprayed it on the release pad of the test strip.
  • the detection can be passed on the coronavirus spike protein S1
  • the RBD combined with the coronavirus in the sample makes the virus particles fluorescent with quantum dots;
  • the detection line (T line) of the test strip is coated with recombinant coronavirus spike protein S1, which is used to capture the ACE2 labeled with quantum dots in the chromatography (Principle of ligand-receptor binding). If there is no coronavirus in the test sample, the T line has a strong fluorescent signal of quantum dots. If there is a coronavirus in the test sample, it neutralizes the ACE2 labeled by the quantum dot and the T line quantum The spot fluorescence signal is weakened, similar to the principle of competitive immunochromatography.
  • the advantage of the present invention is that it retains the advantages of colloidal gold immunochromatography such as fast, simple, cheap, stable, and realizable at-home detection, and adopts the principle of ligand-receptor interaction, avoiding long antibody development cycles and antibody development.
  • the cross-reverse problem can not only improve the specificity of detection but also quickly provide an effective kit.
  • it is universal in the detection of coronaviruses mediated by the ACE2 receptor. Even if the virus is mutated and suddenly new, the test kit can be put into use immediately.
  • quantum dots have many advantages such as wide excitation spectrum, narrow emission peak, wide Stokes shift, good biocompatibility, stable signal, long fluorescence lifetime, and strong photobleaching resistance.
  • the use of quantum dots instead of colloidal gold labels not only The detection sensitivity can be improved by 1-2 orders of magnitude, and quantitative analysis can be performed.
  • kit for rapid detection of coronavirus based on S protein ligand and ACE2 receptor competition chromatography is characterized in that the kit method includes the ACE2 protein labeled with quantum dots, and the preparation of the ACE2 protein labeled with quantum dots The method includes the following steps:
  • step 2) Transfect the plasmid expressing the ACE2 fusion protein constructed in step 1) into eukaryotic immortalized cells, and establish a stable transgenic cell line expressing the ACE2 fusion protein gene, and carry out culture amplification to obtain the culture supernatant containing the ACE2 fusion protein liquid;
  • step 3 Use a protein purification column to obtain the ACE2 fusion protein from the culture supernatant containing the ACE2 fusion protein prepared in step 2), and then use a specific protease to cut off the purification tag of the ACE2 fusion protein to obtain the ACE2 protein;
  • the ACE2 protein obtained in step 3) is biotinylated at the C-terminus with biotin protein ligase to obtain the protein ACE2-Biotin;
  • the human ACE2 extracellular domain gene is the extracellular domain of human ACE2 Part, that is, the coding gene sequence of the 1-739aa part of GenBank: AB046569.1; the protein site-directed biotinylation sequence is biotin protein ligase BirA, and its recognition site sequence is GSGGSGGSAGGGLNDIFEAQKIEW; the specific protease is EK enzyme
  • the cleavage site tag is the Flag tag;
  • the purification tag is mouse IgG Fc segment (mFc);
  • the codon-optimized host is human;
  • the vector containing EF1 to initiate the expression of fluorescent protein is the lentiviral expression vector pCDH- CMV-MCS-EF1-copGFP, the fluorescent protein expressed by EF1 is the green fluorescent protein copGFP, and the plasmid of the constructed ACE2 fusion protein is named
  • the human-derived ACE2 extracellular domain gene is sequentially connected to the C-terminal of the protein site-directed biotinylation sequence, the specific protease cleavage site tag, and the artificially designed sequence of the purification tag.
  • the nucleic acid sequence after the host cell codon optimization is as SEQ ID NO :1
  • the amino acid sequence of the ACE2 fusion protein expressed by the constructed pCDH-ACE2mFc.copGFP plasmid is shown in SEQ ID NO: 2.
  • the said kit for rapid detection of coronavirus based on S protein ligand and ACE2 receptor competition chromatography is characterized in that in step 2), the eukaryotic immortalized cell is HEK293, and it is established to express ACE2 fusion protein The stable gene transfer cell line of ACE2mFc.copGFP/293.
  • ACE2mFc.copGFP/293 stable transfection cell line The establishment process of ACE2mFc.copGFP/293 stable transfection cell line is as follows: pCDH-ACE2mFc.copGFP plasmid, pH1 plasmid, pH2 plasmid are co-transfected into lentiviral packaging line cell 293V to prepare ACE2mFc.copGFP lentivirus and transfect HEK293 Cells, clones were picked under a fluorescence microscope to establish a stable ACE2mFc.copGFP/293 cell line.
  • the said kit for rapid detection of coronavirus based on S protein ligand and ACE2 receptor competition chromatography is characterized in that in step 3), the protein purification column is a Protein A/G column; The amino acid sequence of the ACE2 protein without purification tag is shown in SEQ ID NO: 3.
  • the kit for rapid detection of coronavirus based on S protein ligand and ACE2 receptor competition chromatography is characterized in that in step 4), the site of the site-directed biotinylation is the amino acid sequence SEQ of the ACE2 protein ID NO: At the end of 3, the lysine (K) on the biotin protein ligase recognition site GSGGSGGSAGGGLNDIFEAQKIEW.
  • the kit for rapid detection of coronavirus based on S protein ligand and ACE2 receptor competition chromatography is characterized in that in step 5), the carboxyl quantum dot microspheres are quantum dots with carboxyl functional groups on the surface.
  • Dot fluorescent microspheres, activated with EDC/NHS cross-linking agent couple streptavidin SA to quantum dot fluorescent microspheres through peptide bonds to obtain QM-SA; biotinylated ACE2-Biotin protein is passed through streptavidin Harmonin-biotin reaction is connected to obtain the fluorescently labeled ACE2 protein with quantum dots.
  • the said kit for rapid detection of coronavirus based on S protein ligand and ACE2 receptor competition chromatography is characterized in that the kit also includes an immunochromatographic test strip, and the immunochromatographic test strip includes Bottom liner, nitrocellulose membrane, sample pad, release pad and absorbent paper; the sample pad, release pad, nitrocellulose membrane and absorbent paper are assembled on the backing by lap, and the release pad and absorbent paper are laminated on the nitrocellulose.
  • the two ends of the membrane form a detection area on the surface of the nitrocellulose membrane, and the sample pad is laminated on the release pad; the nitrocellulose membrane in the detection area is provided with a detection line area T line close to the release pad and a control line close to the absorbent paper Area C line;
  • the cellulose membrane is coated with the ligand of ACE2 protein; the nitrocellulose membrane in the C-line area is coated with GAR; the result is to irradiate the immunochromatographic test strip with a UV flashlight, and visually observe for qualitative judgment of Yin and Yang, or use a dry fluorescent layer Analytical analyzer for quantitative analysis.
  • the said kit for rapid detection of coronavirus based on S protein ligand and ACE2 receptor competition chromatography is characterized in that the ligand of the ACE2 protein is a recombinant new coronavirus spike protein S1; S protein ligand and ACE2 receptor competition chromatography coronavirus rapid detection kit is suitable for the detection of oral mucosal fluid, respiratory tract, whole blood, plasma, serum or excrement, and can be applied to coronaviruses with ACE2 as the receptor Rapid detection and diagnosis of infectious diseases.
  • the above-prepared kit can be used for rapid detection of coronaviruses.
  • the method is as follows:
  • the immunochromatographic test strips of the kit are used to improve the biological safety, that is, to add RNase to the sample pad pretreatment solution and photochemically kill Activator to inhibit or inactivate the virus in the test sample;
  • the kit is equipped with an EP tube containing virus inactivation agent. After sampling, put it into the EP tube containing virus inactivation agent to inactivate the virus in the test sample, and then add the sample buffer containing RNase to dilute Post-spotting detection; wherein the photochemical inactivation agent is at least one of riboflavin, psoralen, and methylene blue; the concentration of RNase in the sample pad pretreatment solution is 0.1-1 U/ml.
  • the virus inactivating agent is at least one of alcohol, hydrogen peroxide, ether, formaldehyde, glutaraldehyde, sodium hypochlorite, chloroform, caprylic acid, and peracetic acid;
  • the sample pad pretreatment solution before the spot detection contains 0.2 U/ml RNAse PBS buffer (pH 7.4), the dilution factor of the test sample can be 2-50 times.
  • the present invention uses quantum dot fluorescent labeling and multi-stage coupling to amplify the signal to improve detection sensitivity, utilizes the principle of ligand and receptor binding to improve detection specificity and avoids the antibody development cycle Quickly provide an effective detection kit and establish a virus inactivation system to ensure biological safety in the detection process.
  • the kit is suitable for the detection of various biological samples and environmental samples such as oral mucosal fluid, respiratory tract, whole blood, plasma, serum, excrement, etc. It can be applied to SARS-CoV-2, SARS-CoV, and HCoV-NL63 with ACE2 Rapid detection of coronavirus as recipient.
  • Figure 1 is a plasmid map of pCDH-ACE2omFc.copGFP
  • FIG 2 is a schematic diagram of a rapid detection kit for coronavirus based on S protein ligand and ACE2 receptor competition chromatography.
  • the principle of rapid detection of oral mucosal fluid samples SARS-CoV-2 test strip based on S1 protein and ACE2 receptor Competitive Chromatography;
  • Figure 3 is a schematic diagram of the principle of specific binding of S protein ligand to ACE2 receptor
  • Figure 4 is the standard curve of the kit for the determination of recombinant novel coronavirus spike protein S1;
  • FIG. 5 shows the specific sequence result of SEQ ID NO:1
  • FIG. 6 shows the specific sequence result of SEQ ID NO: 2;
  • Figure 7 shows the specific sequence result of SEQ ID NO: 3.
  • the lentiviral packaging cell 293V was purchased from Beijing Yingmaoshengye Biotechnology Co., Ltd.; the recombinant coronavirus spike protein S1 was provided by Hangzhou Mile Biotechnology Co., Ltd.
  • the C-terminus of the human ACE2 extracellular domain gene is sequentially connected with a protein site-directed biotinylation sequence, a specific protease cleavage site tag and a purification tag to form an artificially designed sequence, wherein the human ACE2 extracellular domain gene is the cell membrane of human ACE2
  • the outer part that is, the coding gene sequence of the 1-739aa part of GenBank: AB046569.1;
  • the protein site-directed biotinylation sequence is biotin protein ligase BirA, and the recognition site sequence is GSGGSGGSAGGGLNDIFEAQKIEW;
  • the specific protease is EK
  • its cutting site tag is a Flag tag;
  • the purification tag is a mouse IgG Fc segment (mFc).
  • nucleic acid sequence of the genetically designed ACE2-Flag-mFc fusion protein is obtained.
  • the sequence is shown in SEQ ID NO:1 after the host (human) codon optimization.
  • the two ends are designed with EcoR I and Not I restriction enzymes and expressed
  • the latter amino acid sequence is shown in SEQ ID NO: 2; after the gene synthesis sequence, it is cloned under the CMV promoter of the lentiviral expression vector pCDH-CMV-MCS-EF1-copGFP by ligating the restriction sites EcoR I and Not I, Construct the eukaryotic expression plasmid pCDH-ACE2mFc.copGFP (shown in Figure 1).
  • SEQ ID NO: 1 is an artificial sequence of the DNA gene encoding the ACE2mFc fusion protein. Restriction enzymes EcoRI (GAATTC)/NotI (GCGGCCGC) at both ends are used for gene subcloning; 7-2223bp is the gene sequence encoding the extracellular domain (1-739aa) of human ACE2 protein; 2224-2295bp is encoding BirA enzyme.
  • the gene sequence of the site-specific biochemical recognition site (the first underlined part in Figure 5); 2302-2325bp is the gene sequence of the Flag tag (the second underlined part in Figure 5); 2326-2979bp is the gene of the purification tag mFc sequence.
  • SEQ ID NO: 2 The specific sequence of SEQ ID NO: 2 is shown in Figure 6.
  • the amino acid sequence of the ACE2 fusion protein expressed by the constructed pCDH-ACE2mFc.copGFP plasmid is shown in SEQ ID NO: 2.
  • 1-739aa is the human ACE2 protein extracellular domain gene part
  • the amino acid sequence of (1-17aa is the signal peptide)
  • 740-763 is the amino acid sequence of the recognition site of BirA enzyme site-directed biochemical action (the first underlined part in Figure 6)
  • 766-773aa is the amino acid sequence of the Flag tag ( The second underlined part in Figure 6)
  • 774-990aa is the amino acid sequence of the purification tag mFc.
  • SEQ ID NO: 3 is the amino acid sequence of the ACE2 protein artificial sequence (with the signal peptide MSSSSWLLLSLVAVTAA removed, and the mFc tag), and 723-746aa is the site-directed biochemical effect of BirA enzyme The amino acid sequence of the recognition site (underlined in Figure 7).
  • the pCDH-ACE2mFc.copGFP plasmid, pH1 plasmid, and pH2 plasmid were co-transfected into lentiviral packaging line cells 293V to prepare ACE2mFc.copGFP lentivirus and transfected into HEK293 cells.
  • the clones were picked under a fluorescent microscope to establish ACE2mFc.copGFP/293 stable Transform cell lines. Specific steps are as follows:
  • the infected HEK293 cells were cultured for 2 days and then passaged into two 6-well plates with a total of 12 wells; the single cells to be dispersed formed a clonal cell cluster (about 1 week), and the single cells with high expression of green fluorescent protein were picked under a fluorescence microscope.
  • the cloned cell group was expanded and cultured to establish a stable ACE2mFc.copGFP/293 cell line.
  • the precipitated protein is reconstituted with 1/10 ⁇ 1/100 stock solution volume of PBS (pH 7.4), it is desalted, concentrated, and replaced with binding/washing buffer (0.5M NaCl) with a 30KD protein cut-off ultrafiltration tube , 20mM Na 2 HPO 4 , pH8.0);
  • the desalted protein solution is applied to the Protein A/G column to adsorb the target protein. After washing with the washing buffer, add EK enzyme buffer (250mM Tris-HCl, 50mM NaCl, pH7.4, 2.5mM CaCl 2 ), according to the product The instructions use EK enzyme to cut the ACE2 part from the column;
  • ACE2 protein Concentrate the excised ACE2 protein with a 10KD protein cut-off ultrafiltration tube, and replace it with a biotin protein ligase (BirA enzyme) ligation buffer (10mM ATP, 10mM MgOAc, 50 ⁇ M D-Biotin), and use it according to the product instructions
  • the BirA enzyme makes the ACE2 protein site-specific biotinylation, that is, linking biotin to the lysine (K) in the GSGGSGGSAGGGLNDIFEAQKIEW sequence to obtain the biotinylated protein ACE2-Biotin.
  • ACE2-Biotin By coupling streptavidin (SA) to carboxyl quantum dot microspheres, ACE2-Biotin can be firmly labeled with quantum dot fluorescence through the SA-Biotin system. This method not only avoids the problem of ACE2 protein function inactivation caused by the direct labeling of ACE2 through the condensation reaction of -NH 4 and -COOH, but also the detection signal forms multi-level amplification. Specific steps are as follows:
  • rabbit IgG is also coupled to carboxyl quantum dot microspheres to obtain QM-rabbit IgG, which is used in the test strip quality control system;
  • the quantum dot fluorescent ligand-receptor competition chromatography test paper was prepared, and the virus inactivation biological safety system was established.
  • sample pad pretreatment solution 1) Add photochemical inactivation agent psoralen and RNase to the sample pad pretreatment solution.
  • psoralen/UV ultraviolet rays
  • coronavirus belongs to RNA virus, so RNase inhibits or inactivates virus by degrading its nucleic acid.
  • the sample pad pretreatment solution is a Tris buffer solution containing 0.5% Tween-20, pH 7.4, and the concentration of psoralen in the sample pad pretreatment solution is 1 ⁇ g/ml and the RNase concentration is 0.2 U/ml.
  • the sample pad Lap the sample pad, release pad, nitrocellulose membrane and absorbent paper on the bottom liner.
  • the release pad and absorbent paper are laminated on both ends of the nitrocellulose membrane and formed on the surface of the nitrocellulose membrane.
  • the sample pad is laminated on the release pad and assembled to form a test paperboard, which is cut into strips with a width of 3.78mm to make the S protein-based ligand Coronavirus rapid detection test paper for chromatography with ACE2 receptor competition method.
  • coronavirus rapid detection kit based on S protein ligand and ACE2 receptor competition chromatography developed based on the technical scheme of the present invention can be applied to all coronaviruses with ACE2 as the receptor (including but not limited to SARS-CoV-2, SARS) -CoV, and HCoV-NL63) rapid detection and the diagnosis of infectious diseases; suitable for the detection of various biological samples and environmental samples including oral mucosal fluid, respiratory tract, whole blood, plasma, serum, excrement, etc.
  • Standard curve the recombinantly expressed SARS-CoV-2 spike protein S1 is used as the standard, and the concentration of 50, 100, 200, 400, 800 ng/ml is prepared with pH 7.4 PBS, and the blank is pH 7.4 PBS.

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Abstract

一种基于S蛋白配体与ACE2受体竞争法层析的冠状病毒快速检测试剂盒，包括量子点标记的ACE2蛋白、量子点标记的兔IgG、重组冠状病毒棘突蛋白S1、羊抗兔IgG的多抗、以及免疫层析试纸条等材料。通过量子点荧光标记和多级偶联放大信号提高检测灵敏度、利用配体与受体结合原理提高检测特异性并避开抗体研发周期，提供一种能够快速检测冠状病毒的试剂盒，通过建立病毒灭活系统保障检测过程中的生物安全性；试剂盒适用于口腔黏膜液、呼吸道、全血、血浆、血清、排泄物等多种生物样本及环境样本的检测，可应用于SARS-CoV-2、SARS-CoV和HCoV-NL63等以ACE2为受体的冠状病毒的快速检测。

Description

一种基于S蛋白配体与ACE2受体竞争法层析的冠状病毒快速检测的试剂盒 技术领域
本发明属于生物技术领域，具体涉及一种基于S蛋白配体与ACE2受体竞争法层析的冠状病毒快速检测的试剂盒。
背景技术
冠状病毒在系统分类上属冠状病毒科(Coronaviridae)冠状病毒属(Coronavirus)。冠状病毒属的病毒是具外套膜(envelope)的正链单股RNA病毒，直径约80～120nm，其遗传物质是所有RNA病毒中最大的，感染宿主包括人、鼠、蝙蝠、猪、猫、穿山甲、犬、狼、鸡、牛、蛇类、禽类等脊椎动物。2019新型冠状病毒(SARS-CoV-2,曾用名2019-nCoV)是目前已知的第7种可以感染人的冠状病毒，其余6种分别是HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV和MERS-CoV。研究表明，其中SARS-CoV-19、SARS-CoV和HCoV-NL63是通过病毒包膜spike glycoprotein(S-protein)介导病毒与宿主细胞膜上的ACE2受体相互作用来感染人的。在新疫情突发时，如2019新型冠状病毒肺炎(COVID-19)，新病毒的早期检测一般采用核酸检测的方法，因为病毒的基因组可以很快被破译，即可合成特异性核酸荧光探针制备荧光定量PCR检测试剂盒投入使用。该方法具有技术成熟、研发周期短、特异性好、检测准确等优点；但同时也存在样本需前处理、检测时间长、需专业仪器专业人员操作、以及试剂贵等缺点，而且也存在一定的假阴性。另外一种快检方法就是胶体金免疫层析试纸，包括病毒抗原检测和血清学抗病毒蛋白抗体(IgM/IgG)检测。这类试剂的优点在于快速、简单，无需仪器和专业人员操作、试剂成本低等；但缺点在于对于新病毒首先要制备新病毒的蛋白抗原和特异性抗体，造成研发周期长不能及时用于疫情检测，而试剂盒的检测准确性与蛋白抗原和特异性抗体的质量密切相关。
发明内容
针对现有技术存在的上述技术问题，我们根据配体与受体特异性结合的原理，发明了一种基于S蛋白配体与ACE2受体竞争法层析的冠状病毒快速检测的试剂盒。如图3所示，冠状病毒CoV的棘突蛋白有2个亚基，S1和S2，其中S1亚基作为配体与人细胞膜上的ACE2受体相互作用形成特异性结合。依据竞争法免疫层析原理，我们用重组的人ACE2蛋白替代其中一个抗S-蛋白单抗来标记量子点荧光并喷涂于试纸条的释放垫上，检测时可通过冠状病毒棘突蛋白S1上的RBD与样本中的冠状病毒结合使病毒颗粒带上量子点荧光；在试纸条的检测线(T线)包被重组冠状病毒棘突蛋白S1，用来捕获层析中量子点标记的ACE2(配体-受体结合原理)，如果检测样本中没有冠状病毒，则T线有较强的量子点荧光信号，如果检测样本中有冠状病 毒，则中和量子点标记的ACE2，T线量子点荧光信号减弱，类似于竞争法免疫层析原理。
本发明的优势在于在保留了胶体金免疫层析快速、简便、廉价、稳定好、可实现居家检测等优点外，采用配体与受体相互作用的原理，避免了抗体研发周期长、以及抗体交叉反的问题，既提高检测特异性又可快速提供一种有效的试剂盒。同时，在以ACE2受体介导感染的冠状病毒检测方面又具有通用性，既使病毒突变变异、突发新疫性，该检测试剂盒可即时投入应用。另外，量子点具有激发光谱宽、发射峰窄、斯托克斯位移宽大、生物相容性好、信号稳定、荧光寿命长、抗光漂白能力强等诸多优点，用量子点替代胶体金标记不仅可提高检灵敏度1-2个数量级，而且可进行定量分析。
所述的一种基于S蛋白配体与ACE2受体竞争法层析的冠状病毒快速检测的试剂盒，其特征在于所述试剂盒方法包含标记量子点的ACE2蛋白,标记量子点的ACE2蛋白制备方法包括以下步骤：
1)将人源ACE2胞外域基因的C端顺序接上蛋白定点生物素化序列、特异性蛋白酶切位点标签和纯化标签形成人工设计序列，该人工设计序列经宿主细胞密码子优化后，亚克隆到一个含有EF1启动表达荧光蛋白的载体的CMV启动子下，构建表达ACE2-定点生物素化序列-蛋白酶切位点标签-纯化标签融合蛋白(简称ACE2融合蛋白，下同)的质粒；
2)将步骤1)构建好的表达ACE2融合蛋白的质粒转染到真核永生化细胞，并建立表达ACE2融合蛋白的基因稳转细胞系，进行培养扩增得到含ACE2融合蛋白的培养上清液；
3)用蛋白纯化柱从步骤2)制备的含ACE2融合蛋白的培养上清液中获得ACE2融合蛋白，随后用特异性蛋白酶切去ACE2融合蛋白的-纯化标签获得ACE2蛋白；
4)将步骤3)获得的ACE2蛋白用生物素蛋白连接酶使其C-末端定点生物素化，获得蛋白ACE2-Biotin；
5)将链霉亲和素SA偶联于羧基量子点微球，获得QM-SA；将步骤4)获得的ACE2-Biotin与QM-SA共孵育，获得牢固标记量子点的ACE2蛋白，将其标记为QM-SB-ACE2。
所述的一种基于S蛋白配体与ACE2受体竞争法层析的冠状病毒快速检测的试剂盒，其特征在于步骤1)中，所述人源ACE2胞外域基因为人源ACE2的细胞膜外部分，即GenBank:AB046569.1的1-739aa部分的编码基因序列；所述蛋白定点生物素化序列为生物素蛋白连接酶BirA，其识别位点序列为GSGGSGGSAGGGLNDIFEAQKIEW；所述特异性蛋白酶为EK酶，其切位点标签为Flag标签；所述纯化标签为小鼠IgG Fc段(mFc)；所述密码子优化的宿主为人；所述含有EF1启动表达荧光蛋白的载体为慢病毒表达载体pCDH-CMV-MCS-EF1-copGFP，EF1启动表达的荧光蛋白为绿色荧光蛋白copGFP，构建好的ACE2融合蛋白的质粒命名为pCDH-ACE2mFc.copGFP。
其中，在人源ACE2胞外域基因的C端顺序接上蛋白定点生物素化序列、特异性蛋白酶切位点标签和纯化标签的人工设计序列经宿主细胞密码子优化后的核酸序列如SEQ ID NO：1所示，构建好的pCDH-ACE2mFc.copGFP质粒所表达的ACE2融合蛋白的氨基酸序列如SEQ ID NO:2所示。
所述的一种基于S蛋白配体与ACE2受体竞争法层析的冠状病毒快速检测的试剂盒，其特征在于步骤2)中，所述真核永生化细胞为HEK293，建立表达ACE2融合蛋白的基因稳转细胞系为ACE2mFc.copGFP/293。
其中ACE2mFc.copGFP/293稳转细胞系的建立过程为：将pCDH-ACE2mFc.copGFP质粒、pH1质粒、pH2质粒共转染到慢病毒包装系细胞293V，制备ACE2mFc.copGFP慢病毒，并转染HEK293细胞，荧光显微镜下挑取克隆建立ACE2mFc.copGFP/293稳转细胞系。
所述的一种基于S蛋白配体与ACE2受体竞争法层析的冠状病毒快速检测的试剂盒，其特征在于步骤3)中，所述蛋白纯化柱为Protein A/G柱；所述切去纯化标签的ACE2蛋白的氨基酸序列如SEQ ID NO:3所示。
所述的一种基于S蛋白配体与ACE2受体竞争法层析的冠状病毒快速检测的试剂盒，其特征在于步骤4)中，所述定点生物素化的位置是ACE2蛋白的氨基酸序列SEQ ID NO:3末端，生物素蛋白连接酶识别位点GSGGSGGSAGGGLNDIFEAQKIEW上的赖氨酸(K)。
所述的一种基于S蛋白配体与ACE2受体竞争法层析的冠状病毒快速检测的试剂盒，其特征在于步骤5)中，所述羧基量子点微球为表面具有羧基功能团的量子点荧光微球，用EDC/NHS交联剂活化，将链霉亲和素SA通过肽键偶联于量子点荧光微球，获得QM-SA；生物素化的ACE2-Biotin蛋白通过链霉亲和素-生物素反应连接，获得量子点荧光标记的ACE2蛋白。
所述的一种基于S蛋白配体与ACE2受体竞争法层析的冠状病毒快速检测的试剂盒，其特征在于所述试剂盒还包括免疫层析试纸条，免疫层析试纸条包括底衬、硝酸纤维素膜、样品垫、释放垫和吸水纸；样品垫、释放垫、硝酸纤维素膜和吸水纸搭接组装在底衬上，释放垫和吸水纸分别叠压在硝酸纤维素膜的两端，并在硝酸纤维素膜的表面形成检测区，样品垫叠压在释放垫上；检测区的硝酸纤维素膜上设置靠近释放垫的检测线区T线和靠近吸水纸的控制线区C线；
将兔IgG偶联于羧基量子点微球，获得QM-兔IgG；将标记量子点的ACE2蛋白和QM-兔IgG混合后喷涂在免疫层析试纸条释放垫上，并在T线区的硝酸纤维素膜上包被ACE2蛋白的配体；C线区的硝酸纤维素膜上包被GAR；结果用紫外手电筒照射免疫层析试纸条，肉眼观察做阴阳定性判断，或采用干式荧光层析分析仪定量分析。
所述的一种基于S蛋白配体与ACE2受体竞争法层析的冠状病毒快速检测的试剂盒，其特 征在于所述ACE2蛋白的配体为重组新型冠状病毒棘突蛋白S1；所述基于S蛋白配体与ACE2受体竞争法层析的冠状病毒快速检测试剂盒适用于口腔黏膜液、呼吸道、全血、血浆、血清或排泄物的检测，能应用于以ACE2为受体的冠状病毒的快速检测和及其感染疾病的诊断。
采用上述制备的试剂盒，能够对冠状病毒进行快速检测，方法如下：所述试剂盒的免疫层析试纸条进行生物安全性改良，即在样本垫预处理液中添加RNA酶以及光化学法灭活剂，以抑制或灭活检测样本中的病毒；
试剂盒中配有装病毒灭活剂的EP管，取样后先放入装有病毒灭活剂的EP管中对检测样本中的病毒进行灭活处理，然后加入含有RNA酶的样本缓冲液稀释后点样检测；其中，所述光化学法灭活剂为核黄素、补骨脂素、亚甲蓝中的至少一种；RNA酶在样本垫预处理液中浓度为0.1～1U/ml。
所述的病毒灭活剂为酒精、双氧水、乙醚、甲醛、戊二醛、次氯酸钠、氯仿、辛酸、过氧乙酸中的至少一种；所述点样检测前的样本垫预处理液为含有0.2U/ml RNA酶的PBS缓冲液(pH7.4)，其对检测样本的稀释倍数可以是2～50倍。
相对于现有技术，本发明取得的有益效果是：本发明通过量子点荧光标记和多级偶联放大信号提高检测灵敏度、利用配体与受体结合原理提高检测特异性并避开抗体研发周期快速提供一种有效的检测试剂盒、通过建立病毒灭活系统保障检测过程中的生物安全性。该试剂盒适合口腔黏膜液、呼吸道、全血、血浆、血清、排泄物等多种生物样本及环境样本的检测，可应用于SARS-CoV-2、SARS-CoV、和HCoV-NL63等以ACE2为受体的冠状病毒的快速检测。
附图说明
图1为pCDH-ACE2omFc.copGFP质粒图谱；
图2为基于S蛋白配体与ACE2受体竞争法层析的冠状病毒快速检测的试剂盒，对口腔黏膜液样本进行快速检测的原理示意图(SARS-CoV-2 test strip based on S1 protein and ACE2 receptor competitive chromatography)；
图3为S蛋白配体与ACE2受体特异性结合的原理示意图；
图4为试剂盒测定重组新型冠状病毒棘突蛋白S1的标准曲线；
图5为SEQ ID NO：1的具体序列结果；
图6为SEQ ID NO：2的具体序列结果；
图7为SEQ ID NO：3的具体序列结果。
具体实施方式
下面结合具体实施例对本发明作进一步说明，但本发明的保护范围并不限于此。
以下实施例中，慢病毒包装细胞293V购自于北京英茂盛业生物科技有限公司；重组冠状 病毒棘突蛋白S1由杭州迈尔德生物科技有限公司提供。
实施例1 pCDH-ACE2mFc.copGFP质粒构建
将人源ACE2胞外域基因的C端顺序接上蛋白定点生物素化序列、特异性蛋白酶切位点标签和纯化标签形成人工设计序列，其中所述人源ACE2胞外域基因为人源ACE2的细胞膜外部分，即GenBank:AB046569.1的1-739aa部分的编码基因序列；所述蛋白定点生物素化序列为生物素蛋白连接酶BirA，其识别位点序列为GSGGSGGSAGGGLNDIFEAQKIEW；所述特异性蛋白酶为EK酶，其切位点标签为Flag标签；所述纯化标签为小鼠IgG Fc段(mFc)。最终得到基因设计ACE2-Flag-mFc融合蛋白的核酸序列，经宿主(人)密码子优化后序列如SEQ ID NO：1所示，两端分别设计EcoR I和Not I限制性内切酶，表达后氨基酸序列如SEQ ID NO:2所示；基因合成序列后，通过连接酶切位点EcoR I和Not I，克隆到慢病毒表达载体pCDH-CMV-MCS-EF1-copGFP的CMV启动子下，构建真核表达质粒pCDH-ACE2mFc.copGFP(如图1所示)。
其中SEQ ID NO：1的具体序列如图5所示，SEQ ID NO:1为编码ACE2mFc融合蛋白的DNA基因人工序列。位于两端的限制性内切酶EcoRI(GAATTC)/NotI(GCGGCCGC)用于基因亚克隆；7-2223bp为编码人ACE2蛋白胞外域(1-739aa)部分的基因序列；2224-2295bp为编码BirA酶定点生物化作用识别位点的基因序列(图5中第1个下划线部分)；2302-2325bp为Flag标签的基因序列(图5中第2个下划线部分)；2326-2979bp为纯化标签mFc的基因序列。
其中SEQ ID NO:2的具体序列如图6所示，构建好的pCDH-ACE2mFc.copGFP质粒所表达的ACE2融合蛋白的氨基酸序列如SEQ ID NO:2。1-739aa为人ACE2蛋白胞外域基因部分的氨基酸序列(其中1-17aa为信号肽)；740-763为BirA酶定点生物化作用识别位点的氨基酸序列(图6中第1个下划线部分)；766-773aa为Flag标签的氨基酸序列(图6中第2个下划线部分)；774-990aa为纯化标签mFc的氨基酸序列。
其中SEQ ID NO:3的具体序列如图7所示，SEQ ID NO:3为ACE2蛋白人工序列(去掉了信号肽MSSSSWLLLSLVAVTAA，以及mFc标签)的氨基酸序列，723-746aa为BirA酶定点生物化作用识别位点的氨基酸序列(图7中下划线部分)。
实施例2 建立ACE2mFc.copGFP/293稳转细胞系
将pCDH-ACE2mFc.copGFP质粒、pH1质粒、pH2质粒共转染到慢病毒包装系细胞293V，制备ACE2mFc.copGFP慢病毒，并转染HEK293细胞，荧光显微镜下挑取克隆建立ACE2mFc.copGFP/293稳转细胞系。具体步骤如下：
1)实验前一天对5个15cm dish进行细胞铺板，保证在转染之前，细胞均达70％-80％汇合度。
2)转染前1～2h，更换dish内培养基为无血清无抗生素的DMEM培养基。
3)准备一支15mL离心管，加入5mL 1×HBS，再依次加入100μg的pCDH-ACE2mFc.copGFP质粒、100μg PH1/PH2混合质粒(PH1:PH2＝3:1)，轻轻混匀。
4)加入4mL PEI工作液(10μM)，轻轻混匀，置37℃孵育20min。
5)将转染复合液等分5份，均匀加入5个待转染的15cm dish内，轻轻晃动使复合物分布均匀。
6)转染5.5h后，再准备一支15mL离心管，加入5mL 1×HBS，再依次加入100μg的pCDH-ACE2mFc.copGFP质粒、100μg PH1/PH2混合质粒(PH1:PH2＝3:1)，轻轻混匀。
7)加入4mL PEI工作液(10μM)，轻轻混匀，置37℃孵育20min。
8)更换dish内培养基为新鲜无血清无抗生素的DMEM培养基。
9)将转染复合液等分5份，均匀加入5个待转染的15cm dish内，轻轻晃动使复合物分布均匀。
10)转染6h后换液，更换为DMEM完全培养基(+10％FBS+1％青霉素/链霉素双抗)。
11)转染48h收集上清，于8000g离心15min后，冻于-80℃冰箱。
12)转染72h收集上清，于8000g离心15min后，合并转染48h所收集上清，过0.45μm滤膜，再以85000g离心2h。
13)弃上清，沉淀以1mL完全培养基(+10％FBS+1％双抗)重悬，感染HEK293细胞(6孔板1个孔，细胞60％-70％汇合度)。
14)感染的HEK293细胞培养2天后传代分至2个6孔板共12个孔；待分散的单个细胞形成克隆细胞团(约1周)，在荧光显微镜下挑取高表达绿色荧光蛋白的单克隆细胞团进行扩增培养建立ACE2mFc.copGFP/293稳转细胞系。
实施例3 ACE2-Biotin蛋白制备
用无蛋白293细胞培养基扩增培养ACE2mFc.copGFP/293稳转细胞，收集上清培养液，并用protein A/G纯化ACE2mFc融合蛋白，用EK酶将ACE2从柱上切下，通过生物素蛋白连接酶将生物素偶联于ACE2的(GSGGSGGSAGGGLNDIFEAQKIEW)位点，获得ACE2-Biotin蛋白。具体步骤如下：
1)将ACE2omFc.copGFP/293稳转细胞扩增至五层细胞工厂，用不含蛋白和多肽的HektorHEK293细胞培养基培养；
2)收集培养液，1200g、4℃离心20min后，取上清液，用饱和硫酸铵法沉淀蛋白；
3)沉淀蛋白用1/10～1/100原液体积的PBS(pH7.4)复溶后，用30KD蛋白截留分子量的超滤管脱盐、浓缩、并置换为结合/洗涤缓冲液(0.5M NaCl，20mM Na 2HPO 4，pH8.0)；
4)脱盐后的蛋白液上Protein A/G柱，吸附目标蛋白，用洗涤缓冲液洗涤后加入EK酶缓冲液(250mM Tris-HCl，50mM NaCl，pH7.4，2.5mM CaCl 2)，按产品说明书用EK酶将ACE2部分从柱上切下；
5)切下的ACE2蛋白用10KD蛋白截留分子量的超滤管浓缩，并置换为生物素蛋白连接酶(BirA酶)连接缓冲液(10mM ATP,10mM MgOAc,50μM D-Biotin)，按产品说明书用BirA酶使ACE2蛋白定点生物素化，即将生物素连在GSGGSGGSAGGGLNDIFEAQKIEW序列中赖氨酸(K)上，获得生物素化蛋白ACE2-Biotin。
实施例4 量子点荧光标记蛋白QM-SB-ACE2和QM-兔IgG的制备
将链霉亲和素(SA)偶联于羧基量子点微球，ACE2-Biotin即可通过SA-Biotin系统牢固地标记上量子点荧光。该方法不仅避免了ACE2直接通过-NH 4和-COOH缩合反应标记造成的ACE2蛋白功能失活的问题，而且检测信号形成多级放大。具体步骤如下：
1)按产品说明书的方法将链霉亲和素(SA)偶联于羧基量子点微球(所述羧基量子点微球为表面具有羧基功能团的量子点荧光微球)，获得QM-SA；
2)同样将兔IgG也偶联于羧基量子点微球，获得QM-兔IgG，用于试纸条质控系统；
3)在pH7.4的PBS缓冲液中将QM-SA和ACE2-Biotin以1:4的摩尔比混合(QM-SA以标记的SA计)，37℃、150rpm振荡孵育1小时，获得QM-SB-ACE2；
4)8000g、4℃离心30分钟，去上清，沉淀用PB缓冲液(pH7.0、1％BSA、8％蔗糖、0.05％NaN 3)重悬。
实施例5 基于S蛋白配体与ACE2受体竞争法层析的冠状病毒快速检测试剂盒的制备
在参考竞争法免疫层析试纸制备工艺的基础上进行量子点荧光配体-受体竞争法层析试纸的制备，并建立病毒灭活生物安全系统。
1)样品垫预处理液中添加光化学法灭活剂补骨脂素和RNA酶。在后期用紫外线手电筒观察或仪器分析时，补骨脂素/UV(紫外线)形成病毒光化学法灭活系统；冠状病毒属于RNA病毒，因而RNA酶通过降解其核酸抑制或灭活病毒。样品垫预处理液为含0.5％吐温-20的Tris缓冲液，pH7.4，在样品垫预处理液中的补骨脂素的浓度为1μg/ml和RNA酶浓度为0.2U/ml。
2)将上述实施例4制备的QM-SB-ACE2和QM-兔IgG混合后喷涂在试剂盒的释放垫上，37℃烘干12h备用；
3)用包被稀释液(150mM PB，pH 7.4)分别将重组新型冠状病毒棘突蛋白S1和羊抗兔IgG多克隆抗体稀释成0.2mg/mL，以膜液量30μL/30cm分别均匀喷涂划线于硝酸纤维素膜检测线区(标记为T线区)和控制线区(标记为C线区)上，37℃烘干处理12h，封袋备用；
4)将样品垫、释放垫、硝酸纤维素膜和吸水纸搭接组装在底衬上，释放垫和吸水纸分别 叠压在硝酸纤维素膜的两端，并在硝酸纤维素膜的表面形成检测区(T线区靠近释放垫，C线区靠近吸水纸)，样品垫叠压在释放垫上，组装后形成试纸板，切割成宽度3.78mm条状，制成所述的基于S蛋白配体与ACE2受体竞争法层析的冠状病毒快速检测试纸。
实施例6 基于S蛋白配体与ACE2受体竞争法层析的冠状病毒快速检测试剂盒的应用
基于本发明技术方案研发的基于S蛋白配体与ACE2受体竞争法层析的冠状病毒快速检测试剂盒可应用于所有以ACE2为受体的冠状病毒(包括不仅限于SARS-CoV-2、SARS-CoV、和HCoV-NL63)的快速检测和及其感染疾病的诊断；适合包括口腔黏膜液、呼吸道、全血、血浆、血清、排泄物等多种生物样本及环境样本的检测。
1)标准曲线：以重组表达的SARS-CoV-2棘突蛋白S1为标准品，用pH7.4的PBS分别配制成50、100、200、400、800ng/ml的浓度，空白为pH7.4的PBS。分别点样本发明的试纸条，每孔100μl，层析10-15分钟后用干式荧光层析分析仪测定T线荧光值；每个浓度设5个重复，以平均荧光值为纵坐标，浓度为横坐标绘制标准曲线，结果见图4；曲线方程为y＝-4.7382x+3645，R 2＝0.967。
2)目前没有针对新型冠状病毒(SARS-CoV-2)抗原检测的产品，不能作同类产品的对比。我们取5个健康人的血清和3个临床确诊COVID-19病人的血清，用本发明技术方案研发的试剂盒进行检测，结果3个确诊病人的血清均为阳性，5个健康人的血清均为阴性，结果准确。

Claims (10)

  1. 一种基于S蛋白配体与ACE2受体竞争法层析的冠状病毒快速检测的试剂盒，其特征在于所述试剂盒方法包含标记量子点的ACE2蛋白,标记量子点的ACE2蛋白制备方法包括以下步骤：
    1)将人源ACE2胞外域基因的C端顺序接上蛋白定点生物素化序列、特异性蛋白酶切位点标签和纯化标签形成人工设计序列，该人工设计序列经宿主细胞密码子优化后，亚克隆到一个含有EF1启动表达荧光蛋白的载体的CMV启动子下，构建表达ACE2-定点生物素化序列-蛋白酶切位点标签-纯化标签融合蛋白的质粒，简称为ACE2融合蛋白的质粒；
    2)将步骤1)构建好的表达ACE2融合蛋白的质粒转染到真核永生化细胞，并建立表达ACE2融合蛋白的基因稳转细胞系，进行培养扩增得到含ACE2融合蛋白的培养上清液；
    3)用蛋白纯化柱从步骤2)制备的含ACE2融合蛋白的培养上清液中获得ACE2融合蛋白，随后用特异性蛋白酶切去ACE2融合蛋白的纯化标签获得ACE2蛋白；
    4)将步骤3)获得的ACE2蛋白用生物素蛋白连接酶使其C-末端定点生物素化，获得蛋白ACE2-Biotin；
    5)将链霉亲和素SA偶联于羧基量子点微球，获得QM-SA；将步骤4)获得的ACE2-Biotin与QM-SA共孵育，获得牢固标记量子点的ACE2蛋白，将其标记为QM-SB-ACE2。
  2. 如权利要求1所述的一种基于S蛋白配体与ACE2受体竞争法层析的冠状病毒快速检测的试剂盒，其特征在于步骤1)中，所述人源ACE2胞外域基因为人源ACE2的细胞膜外部分，即GenBank:AB046569.1的1-739aa部分的编码基因序列；所述蛋白定点生物素化序列为生物素蛋白连接酶BirA，其识别位点序列为GSGGSGGSAGGGLNDIFEAQKIEW；所述特异性蛋白酶为EK酶，其切位点标签为Flag标签；所述纯化标签为小鼠IgG Fc段；所述密码子优化的宿主为人；所述含有EF1启动表达荧光蛋白的载体为慢病毒表达载体pCDH-CMV-MCS-EF1-copGFP，EF1启动表达的荧光蛋白为绿色荧光蛋白copGFP，构建好的ACE2融合蛋白的质粒命名为pCDH-ACE2mFc.copGFP；
    其中，在人源ACE2胞外域基因的C端顺序接上蛋白定点生物素化序列、特异性蛋白酶切位点标签和纯化标签的人工设计序列经宿主细胞密码子优化后的核酸序列如SEQ ID NO：1所示，构建好的pCDH-ACE2mFc.copGFP质粒所表达的ACE2融合蛋白的氨基酸序列如SEQ ID NO:2所示。
  3. 如权利要求1所述的一种基于S蛋白配体与ACE2受体竞争法层析的冠状病毒快速检测的试剂盒，其特征在于步骤2)中，所述真核永生化细胞为HEK293，建立表达ACE2融合蛋白的基因稳转细胞系为ACE2mFc.copGFP/293；
    其中ACE2mFc.copGFP/293稳转细胞系的建立过程为：将pCDH-ACE2mFc.copGFP质粒、pH1质粒、pH2质粒共转染到慢病毒包装系细胞293V，制备ACE2mFc.copGFP慢病毒，并转染HEK293细胞，荧光显微镜下挑取克隆建立ACE2mFc.copGFP/293稳转细胞系。
  4. 如权利要求1所述的一种基于S蛋白配体与ACE2受体竞争法层析的冠状病毒快速检测的试剂盒，其特征在于步骤3)中，所述蛋白纯化柱为Protein A/G柱；所述切去纯化标签后的ACE2蛋白的氨基酸序列如SEQ ID NO:3所示。
  5. 如权利要求1所述的一种基于S蛋白配体与ACE2受体竞争法层析的冠状病毒快速检测的试剂盒，其特征在于步骤4)中，所述定点生物素化的位置是ACE2蛋白的氨基酸序列SEQ ID NO:3末端，生物素蛋白连接酶识别位点GSGGSGGSAGGGLNDIFEAQKIEW上的赖氨酸K。
  6. 如权利要求1所述的一种基于S蛋白配体与ACE2受体竞争法层析的冠状病毒快速检测的试剂盒，其特征在于步骤5)中，所述羧基量子点微球为表面具有羧基功能团的量子点荧光微球，用EDC/NHS交联剂活化，将链霉亲和素SA通过肽键偶联于量子点荧光微球，获得QM-SA；生物素化的ACE2-Biotin蛋白通过链霉亲和素-生物素反应连接，获得量子点荧光标记的ACE2蛋白。
  7. 如权利要求1所述的一种基于S蛋白配体与ACE2受体竞争法层析的冠状病毒快速检测的试剂盒，其特征在于所述试剂盒还包括免疫层析试纸条，免疫层析试纸条包括底衬、硝酸纤维素膜、样品垫、释放垫和吸水纸；样品垫、释放垫、硝酸纤维素膜和吸水纸搭接组装在底衬上，释放垫和吸水纸分别叠压在硝酸纤维素膜的两端，并在硝酸纤维素膜的表面形成检测区，样品垫叠压在释放垫上；检测区的硝酸纤维素膜上设置靠近释放垫的检测线区T线和靠近吸水纸的控制线区C线；
    将兔IgG偶联于羧基量子点微球，获得QM-兔IgG；将标记量子点的ACE2蛋白和QM-兔IgG混合后喷涂在免疫层析试纸条释放垫上，并在T线区的硝酸纤维素膜上包被ACE2蛋白的配体；C线区的硝酸纤维素膜上包被GAR。
  8. 如权利要求7所述的一种基于S蛋白配体与ACE2受体竞争法层析的冠状病毒快速检测的试剂盒，其特征在于所述ACE2蛋白的配体为重组新型冠状病毒棘突蛋白S1；所述试剂盒适用于口腔黏膜液、呼吸道、全血、血浆、血清或排泄物的检测，能应用于以ACE2为受体的冠状病毒的快速检测。
  9. 如权利要求1所述的一种基于S蛋白配体与ACE2受体竞争法层析的冠状病毒快速检测的试剂盒，其特征在于所述试剂盒的免疫层析试纸条进行生物安全性改良，即在样本垫预处理液中添加RNA酶以及光化学法灭活剂，以抑制或灭活检测样本中的病毒；其中，所述光化学法灭活剂为核黄素、补骨脂素、亚甲蓝中的至少一种；
    试剂盒中配有装病毒灭活剂的EP管，取样后先放入装有病毒灭活剂的EP管中对检测样本中的病毒进行灭活处理，然后加入含有RNA酶的样本缓冲液稀释后点样检测，结果用紫外手电筒照射免疫层析试纸条，肉眼观察做阴阳定性判断，或采用干式荧光层析分析仪定量分析；RNA酶在样本垫预处理液中浓度为0.1～1U/ml。
  10. 如权利要求9所述的一种基于S蛋白配体与ACE2受体竞争法层析的冠状病毒快速检测的试剂盒，其特征在于所述的病毒灭活剂为酒精、双氧水、乙醚、甲醛、戊二醛、次氯酸钠、氯仿、辛酸、过氧乙酸中的至少一种；所述点样检测前的样本垫预处理液为含有0.2U/ml RNA酶的PBS缓冲液pH7.4，其对检测样本的稀释倍数是2～50倍。
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114252601A (zh) * 2021-11-08 2022-03-29 润和生物医药科技(汕头)有限公司 一种新冠总抗体和中和抗体乳胶快速检测试纸及制备方法
CN116400070A (zh) * 2023-06-07 2023-07-07 济南德亨医学科技有限公司 高通量定量检测过敏原特异性IgE抗体试剂的制备方法

Families Citing this family (43)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111273006A (zh) * 2020-03-10 2020-06-12 四川沃文特生物技术有限公司 一种新型冠状病毒SARS-CoV-2 S蛋白检测方法
US20230176054A1 (en) * 2020-03-20 2023-06-08 Lumiradx Uk Ltd. Coronavirus assay
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WO2021254476A1 (zh) * 2020-06-19 2021-12-23 南京金斯瑞生物科技有限公司 检测SARS-CoV-2病毒中和抗体的磁微粒化学发光试剂盒及其应用
DE102020209412B4 (de) * 2020-07-02 2024-02-08 Friedrich-Schiller-Universität Jena Mittel und Verfahren zur An- oder Abreicherung und zum Nachweis von Coronaviren
CN112043825B (zh) * 2020-07-13 2023-12-05 中国医学科学院医学生物学研究所 一种基于新型冠状病毒突刺蛋白s1区域预防新型冠状病毒感染的亚单位疫苗
CN112079906B (zh) * 2020-07-15 2022-11-04 张影 一种新型冠状病毒刺突蛋白、相关生物材料及其应用、检测试纸和检测试剂盒
CN113945714B (zh) * 2020-07-16 2023-01-31 南京蓬勃生物科技有限公司 新型冠状病毒中和抗体类药物中和能力的检测方法
WO2022012688A1 (en) * 2020-07-17 2022-01-20 Shenzhen Bay Laboratory Ace2-ig fusion variants
CN112098643B (zh) * 2020-08-20 2022-02-15 广东省农业科学院农业生物基因研究中心 一种评估冠状病毒跨物种传染风险的方法和试纸条及其应用
CN112213497B (zh) * 2020-09-24 2023-10-20 杭州医学院 检测新型冠状病毒s蛋白独特性抗体的多肽-elisa试剂盒
CN112409488B (zh) * 2020-10-23 2022-07-01 中国科学院上海药物研究所 针对多种冠状病毒的单克隆抗体及应用
CN112098660B (zh) * 2020-11-03 2021-02-09 北京百普赛斯生物科技股份有限公司 新型冠状病毒中和性抗体检测试剂盒
EP4241085A1 (en) * 2020-11-04 2023-09-13 Sorrento Therapeutics, Inc. Lateral flow devices for high sensitivity detection of coronavirus infection, and methods of making and using the same
CN112707968B (zh) * 2020-12-17 2023-10-20 苏州科锐迈德生物医药科技有限公司 一种用于检测新冠病毒中和抗体的重组的受体结合蛋白、重组的受体蛋白
CN112626089B (zh) * 2020-12-08 2022-03-11 杭州百凌生物科技有限公司 一种SARS-CoV-2病毒S蛋白受体结合区域编码基因、抗体及应用
CN112229994B (zh) * 2020-12-10 2021-03-16 丹娜(天津)生物科技股份有限公司 一种基于磁微粒化学发光的新型冠状病毒抗体检测试剂盒
CN112798795A (zh) * 2020-12-11 2021-05-14 贵州美鑫达医疗科技有限公司 用于检测阻断分析物的测定
US11105804B1 (en) * 2020-12-14 2021-08-31 Raybiotech Life, Inc. COVID-19 spike-ACE2 binding assay for drug and antibody screening
CN112748243A (zh) * 2020-12-23 2021-05-04 北京美康基因科学股份有限公司 一种新型冠状病毒中和抗体检测试剂盒及其制备方法
CN113156129B (zh) * 2021-01-13 2022-04-05 广东菲鹏生物有限公司 中和抗体高敏检测方法及产品
CN112625125B (zh) * 2021-01-18 2021-12-14 中国人民解放军军事科学院军事医学研究院 一株中和新型冠状病毒感染的单抗
CN112782401A (zh) * 2021-02-08 2021-05-11 聊城大学 一种体外快速检测新型冠状病毒的方法及应用
CN113009132B (zh) * 2021-02-10 2024-02-23 天津中新科炬生物制药股份有限公司 高灵敏的新型冠状病毒中和抗体检测试剂盒及检测方法
CN112986580B (zh) * 2021-02-10 2024-03-01 天津中新科炬生物制药股份有限公司 一种新型冠状病毒中和抗体检测方法及试剂盒
KR102434714B1 (ko) * 2021-02-16 2022-08-23 바디텍메드(주) 중화 항체 및 인터페론-감마 생성 여부를 이용한 능동 면역 진단을 위한 정보 제공 방법
CN114672474B (zh) * 2021-02-24 2023-07-04 中国科学院生物物理研究所 一种ace2突变体及其突变方法和应用
CN113514636A (zh) * 2021-03-03 2021-10-19 广州中医药大学顺德医院(佛山市顺德区中医院) 一种新冠中和抗体荧光免疫层析检测试纸条及其制备方法
CN113024641B (zh) * 2021-03-11 2022-05-17 广东省农业科学院动物卫生研究所 新型冠状病毒的重组s蛋白及其制备方法和应用
CN113049831B (zh) * 2021-03-12 2022-11-11 北京博晖创新生物技术股份有限公司 检测新型冠状病毒中和抗体的试剂盒及其制备方法和应用
CN113358867A (zh) * 2021-04-26 2021-09-07 北京润博福得生物科技发展有限公司 新型冠状病毒中和抗体的检测组合物、检测试剂、检测试剂盒
CN113189333A (zh) * 2021-04-28 2021-07-30 杭州宝临生物科技有限公司 量子点免疫荧光检测试剂条包含其的试剂盒及其应用
CN113484522B (zh) * 2021-06-03 2022-05-10 上海捷诺生物科技有限公司 SARS-CoV-2中和抗体检测试剂盒及其制备方法
CN113588945A (zh) * 2021-06-30 2021-11-02 杭州迈尔德生物科技有限公司 一种基于化学发光的新型冠状病毒中和抗体检测试剂及试剂盒
CN113671171A (zh) * 2021-07-06 2021-11-19 安徽惠邦生物工程有限公司 信号放大量子点荧光免疫探针及其制备方法和应用
CN113563477B (zh) * 2021-07-06 2023-08-04 上海健康医学院 新冠病毒重组蛋白和人血管紧张素转换酶-2重组蛋白及其制备方法和应用
CN113588941A (zh) * 2021-07-18 2021-11-02 杭州迈尔德生物科技有限公司 一种基于间接胶体金法的新型冠状病毒中和抗体的检测方法及试剂盒
CN113832188A (zh) * 2021-07-18 2021-12-24 杭州迈尔德生物科技有限公司 一种基于荧光蛋白的新型冠状病毒中和抗体快速检测试剂盒及应用
CN113740534A (zh) * 2021-11-04 2021-12-03 北京乐普诊断科技股份有限公司 新型冠状病毒中和抗体检测卡及制备方法
CN114134121A (zh) * 2021-11-30 2022-03-04 南通大学 一种基于荧光素酶的新冠病毒棘突蛋白和人血管紧张素酶互作检测细胞模型及应用
CN114740199A (zh) * 2022-03-18 2022-07-12 北京安奇生物医药科技有限公司 一种SARS-CoV-2中和抗体试剂盒及其应用
CN114384245B (zh) * 2022-03-24 2022-06-24 江苏美克医学技术有限公司 新型冠状病毒s-rbd蛋白和n蛋白双表位识别的联检试剂盒及其应用
CN114606218B (zh) * 2022-04-01 2023-10-27 北京大学 冠状病毒中和效应蛋白及其应用

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050113298A1 (en) * 2003-09-15 2005-05-26 The Brigham And Women's Hospital, Inc. Receptor binding peptides derived from the SARS S protein
CN1815234A (zh) * 2004-11-22 2006-08-09 香港大学 靶向引起病毒感染的sars相关冠状病毒刺突蛋白上的关键位点的合成肽以及其使用方法
CN1826356A (zh) * 2003-07-22 2006-08-30 克鲁塞尔荷兰公司 针对sars冠状病毒的结合分子及其应用
CN1884303A (zh) * 2005-06-20 2006-12-27 中国医学科学院基础医学研究所 SARS－CoV病毒结构蛋白的融合蛋白及其高量表达与纯化和用途
CN1914226A (zh) * 2003-11-25 2007-02-14 达纳-法伯癌症研究院有限公司 SARS－CoV抗体及其使用方法
CN102015767A (zh) * 2008-01-17 2011-04-13 胡马斯有限公司 针对SARS-CoV的交叉中和人单克隆抗体及其使用方法
CN101228267B (zh) * 2005-07-22 2013-01-23 克鲁塞尔荷兰公司 生产冠状病毒的细胞系
CN107690582A (zh) * 2015-04-03 2018-02-13 雅培制药有限公司 用于样品分析的装置和方法

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101915848B (zh) * 2010-07-06 2013-09-25 中国医学科学院病原生物学研究所 Rbd蛋白在制备sars病人辅助诊断试剂中的应用
CN102520165B (zh) * 2011-12-29 2014-07-30 北京康美天鸿生物科技有限公司 一种量子点荧光免疫层析高灵敏定量检测的方法

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1826356A (zh) * 2003-07-22 2006-08-30 克鲁塞尔荷兰公司 针对sars冠状病毒的结合分子及其应用
US20050113298A1 (en) * 2003-09-15 2005-05-26 The Brigham And Women's Hospital, Inc. Receptor binding peptides derived from the SARS S protein
CN1914226A (zh) * 2003-11-25 2007-02-14 达纳-法伯癌症研究院有限公司 SARS－CoV抗体及其使用方法
CN1815234A (zh) * 2004-11-22 2006-08-09 香港大学 靶向引起病毒感染的sars相关冠状病毒刺突蛋白上的关键位点的合成肽以及其使用方法
CN1884303A (zh) * 2005-06-20 2006-12-27 中国医学科学院基础医学研究所 SARS－CoV病毒结构蛋白的融合蛋白及其高量表达与纯化和用途
CN101228267B (zh) * 2005-07-22 2013-01-23 克鲁塞尔荷兰公司 生产冠状病毒的细胞系
CN102015767A (zh) * 2008-01-17 2011-04-13 胡马斯有限公司 针对SARS-CoV的交叉中和人单克隆抗体及其使用方法
CN107690582A (zh) * 2015-04-03 2018-02-13 雅培制药有限公司 用于样品分析的装置和方法

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114252601A (zh) * 2021-11-08 2022-03-29 润和生物医药科技(汕头)有限公司 一种新冠总抗体和中和抗体乳胶快速检测试纸及制备方法
CN116400070A (zh) * 2023-06-07 2023-07-07 济南德亨医学科技有限公司 高通量定量检测过敏原特异性IgE抗体试剂的制备方法
CN116400070B (zh) * 2023-06-07 2023-08-18 济南德亨医学科技有限公司 高通量定量检测过敏原特异性IgE抗体试剂的制备方法

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