WO2022257237A1 - 一种新冠病毒SARS-CoV-2广谱多肽抗原及其特异性中和抗体和应用 - Google Patents

一种新冠病毒SARS-CoV-2广谱多肽抗原及其特异性中和抗体和应用 Download PDF

Info

Publication number
WO2022257237A1
WO2022257237A1 PCT/CN2021/107615 CN2021107615W WO2022257237A1 WO 2022257237 A1 WO2022257237 A1 WO 2022257237A1 CN 2021107615 W CN2021107615 W CN 2021107615W WO 2022257237 A1 WO2022257237 A1 WO 2022257237A1
Authority
WO
WIPO (PCT)
Prior art keywords
cov
sars
broad
spectrum
seq
Prior art date
Application number
PCT/CN2021/107615
Other languages
English (en)
French (fr)
Inventor
叶建强
李拓凡
阚秋琪
秦爱建
万志敏
邵红霞
谢泉
Original Assignee
扬州大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 扬州大学 filed Critical 扬州大学
Priority to US17/620,528 priority Critical patent/US20240002451A1/en
Publication of WO2022257237A1 publication Critical patent/WO2022257237A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • G—PHYSICS
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • 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 object of the present invention is to provide a SARS-CoV-2 broad-spectrum polypeptide antigen, which has no mutations in the SARS-CoV-2 prototype strain and many popular mutant strains, and can specifically recognize the new coronavirus SARS -CoV-2 antibody with good antigenicity.
  • the present invention provides a new coronavirus SARS-CoV-2 broad-spectrum polypeptide antigen, the amino acid sequence of the broad-spectrum polypeptide antigen is shown in SEQ ID NO:1.
  • amino acid sequence of the fusion protein is shown in SEQ ID NO:8.
  • step 3 fast recombination and cloning of the linearized pCold I expression vector described in step 1), the first fragment, the second fragment and the third fragment described in step 2) using recombinase to obtain a recombinant plasmid;
  • the reaction program of PCR amplification described in step 2) is denaturation at 94°C for 5 minutes; denaturation at 94°C for 30 s, annealing at 57°C for 30 s, extension at 72°C for 3 min, 30 cycles; and finally extension at 72°C for 10 min;
  • the PCR amplification reaction system is 33 ⁇ l water, 10 ⁇ l 5 times buffer, 1 ⁇ l 10mM dNTP, 2 ⁇ l 10 ⁇ M upstream primer, 2 ⁇ l 10 ⁇ M downstream primer, 1 ⁇ l 10ng/ ⁇ l pCold I plasmid or pcDNA3.1-SARS-CoV-2 -S plasmid, 1 ⁇ l commercial Phanta Super-Fidelity DNA polymerase.
  • the immunoassay includes colloidal gold immunoassay, immunoprecipitation assay, fluorescence immunoassay and/or enzyme-linked immunoassay.
  • the novel coronavirus SARS-CoV-2 broad-spectrum polypeptide antigen provided by the present invention has an amino acid sequence as shown in SEQ ID NO:1.
  • the polypeptide antigen provided by the present invention is synthesized based on the polypeptide sequence of the S2 segment of the SARS-CoV-2 S gene with good antigenicity.
  • the antigen has no mutations in the prototype strain of SARS-CoV-2 and many popular mutant strains, and has the same amino acid sequence in other coronaviruses SARS-CoV and BatCoV RaTG13, so it has excellent broad-spectrum antigen performance.
  • the present invention determines that the polypeptide has strong specificity by measuring the reaction of the antigen with the serum of a SARS-CoV-2 patient. The antigen can be applied.
  • the SARS-CoV-2 triple broad-spectrum polypeptide tandem fusion protein prepared by the present invention can be used to immunize animals, which can produce specific anti-SARS-CoV-2 broad-spectrum neutralizing antibodies, laying an important foundation for the development of SARS-CoV-2 broad-spectrum vaccines. Base.
  • the present invention also provides a novel coronavirus SARS-CoV-2 broad-spectrum neutralizing antibody, which is produced by immunizing animals with the triple novel coronavirus SARS-CoV-2 broad-spectrum polypeptide tandem fusion protein.
  • the broad-spectrum neutralizing antibody can block the occurrence and development of the new coronavirus by specifically binding to the antigenic site of the new coronavirus SARS-CoV-2, which provides a new idea for the prevention and treatment of the new coronavirus.
  • Fig. 1 is that the ELISA that coats polypeptide antigen detects the positive serum of SARS-CoV-2 patient;
  • Figure 2 is the electropherogram of 3 gene fragments corresponding to SEQ ID NO: 1 amplified by PCR method, wherein swimming lane 1, the first fragment; swimming lane 2, the second fragment; swimming lane 3, the third fragment; swimming lane M, DNA Marker ;
  • Figure 3 is the electrophoresis diagram of linearized vector pCold I amplified by PCR method, wherein lane 1 is the PCR product of linearized vector pCold I; lane M is DNA Marker;
  • Figure 4 shows the expression results of the SARS-CoV-2 triple broad-spectrum polypeptide tandem fusion protein analyzed by SDS-PAGE, in which lane 1, pCold I ultrasonic lysis precipitation; lane 2, His-3 ⁇ P4 ultrasonic lysis precipitation; lane 3, pCold I Ultrasonic lysed supernatant; lane 4, His-3 ⁇ P4 supernatant ultrasonically lysed; lane 5, purified His-3 ⁇ P4 protein; lane M, protein marker;
  • Figure 5 is the result of Western blot identification of the expression of SARS-CoV-2 triple broad-spectrum polypeptide tandem fusion protein, in which lane 1, pCold I ultrasonic lysed precipitate; lane 2, pCold I ultrasonic lysed supernatant; lane 3, His-3 ⁇ P4 ultrasonic lysed supernatant; lane 4, His-3 ⁇ P4 ultrasonic lysed precipitate; lane M, protein marker;
  • Figure 6 is the ELISA detection of anti-triple broad-spectrum polypeptide tandem fusion protein mouse serum coated with polypeptide antigen
  • Figure 8 is a SARS-CoV-2 pseudovirus neutralization test identifying the neutralizing activity of anti-triple broad-spectrum polypeptide tandem fusion protein mouse serum.
  • the present invention provides a new coronavirus SARS-CoV-2 broad-spectrum polypeptide antigen, the amino acid sequence of the broad-spectrum polypeptide antigen is shown in SEQ ID NO: 1 (DPLQPELDSFKEELDKYFKNHTSPDVDLGDIS).
  • the polypeptide antigen has no mutations in the SARS-CoV2 prototype strain and each popular mutant strain, and has the same sequence in other coronaviruses (SARS-CoV and BatCoV RaTG13). And the specificity of the polypeptide antigen was determined by reacting with SARS-CoV-2 human positive sera. Therefore, a simple and quick ELISA method for specifically detecting SARS-CoV-2 antibody (S2 antibody) can be established based on the polypeptide antigen.
  • S2 antibody simple and quick ELISA method for specifically detecting SARS-CoV-2 antibody
  • the invention provides a triple novel coronavirus SARS-CoV-2 broad-spectrum polypeptide tandem fusion protein, the fusion protein comprising three sequences of the novel coronavirus SARS-CoV-2 broad-spectrum polypeptide antigens arranged in tandem.
  • the fusion protein preferably has a connecting peptide between the novel coronavirus SARS-CoV-2 broad-spectrum polypeptide antigens.
  • the present invention has no special limitation on the amino acid sequence of the connecting peptide, and the amino acid sequence of the connecting peptide well known in the art can be used.
  • the connecting peptide is GGGGS (SEQ ID NO: 9).
  • the amino acid sequence of the fusion protein is preferably as shown in SEQ ID NO:8.
  • the 5' end of the fusion protein contains a histidine tag, which facilitates subsequent separation and purification.
  • the fusion protein can mimic the trimer pattern of the SARS-CoV-2 S protein in its natural state, and the fusion protein can be used as an antigen to immunize mice to produce anti-SARS-CoV-2 specific neutralizing antibodies.
  • the invention provides a method for constructing the triple novel coronavirus SARS-CoV-2 broad-spectrum polypeptide tandem fusion protein, comprising the following steps:
  • the pCold I plasmid is used as a template, and the primer pair shown in SEQ ID NO: 6 (AAGCTTGTCGACCTGCAGTCTAGAT) and SEQ ID NO: 7 (GGTACCGAGCTCCATATGCCTACC) is used for PCR amplification to obtain a linearized pCold I expression vector.
  • the present invention does not specifically limit the source of the pCold I plasmid, and the source of the pCold I plasmid well known in the art can be used.
  • the pCold I plasmid was purchased from a commercially available plasmid from Takara (Dalian Bao Biology Co., Ltd.).
  • the reaction program of the PCR amplification is preferably denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 57°C for 30 s, extension at 72°C for 3 min, 30 cycles; and finally extension at 72°C for 10 min.
  • the reaction system of the PCR amplification is preferably 33 ⁇ l of water, 10 ⁇ l of 5-fold buffer, 1 ⁇ l of 10 mM dNTP, 2 ⁇ l of 10 ⁇ M upstream primer, 2 ⁇ l of 10 ⁇ M downstream primer, 1 ⁇ l of 10 ng/ ⁇ l pCold I plasmid, and 1 ⁇ l of commercialized Phanta Super-Fidelity DNA polymerase.
  • the present invention uses the pcDNA3.1-SARS-CoV-2-S plasmid as a template, uses the primer pair shown in SEQ ID NO: 2 and SEQ ID NO: 3 to carry out PCR amplification of the first fragment, and uses SEQ ID NO: 4 and The primer pair shown in SEQ ID NO: 3 is used for PCR amplification of the second fragment, and the primer pair shown in SEQ ID NO: 4 and SEQ ID NO: 5 is used for PCR amplification of the third fragment.
  • the pcDNA3.1-SARS-CoV-2-S plasmid was purchased from Shanghai Jihaoge Biotechnology Co., Ltd.
  • the primer pair for amplifying the first fragment includes the primer pair shown in SEQ ID NO: 2 (ATGGAGCTCGGTACCGACCCATTGCAACCCGAACTTG) and SEQ ID NO: 3 (ACTTCCACCTCCACCGCTAATGTCGCCCAGGTCCAC).
  • the primer pair for amplifying the second fragment preferably includes primers shown in SEQ ID NO: 4 (GGTGGAGGTGGAAGTGACCCATTGCAACCCGAACTTG) and SEQ ID NO: 3.
  • the primer pair for amplifying the third fragment includes primers shown in SEQ ID NO: 4 and SEQ ID NO: 5 (CAGGTCGACAAGCTTTCAGCTAATGTCGCCCAGGTCCAC).
  • the present invention uses recombinase to carry out the linearized pCold I expression vector, the first fragment, the second fragment and the third fragment Rapid recombination and cloning to obtain recombinant plasmids.
  • the recombinase is preferably MultiS One Step Cloning Kit.
  • the reaction system of the recombinant clone is 2 ⁇ l of linearized vector (containing 100 ng), 1 ⁇ l of each of the first fragment, the second fragment and the third fragment (each containing 10 ng), 4 ⁇ l of CE MultiS Buffer, 2 ⁇ l of Exnase MultiS, and 9 ⁇ l of water.
  • the reaction program of the recombinant clone is 37° C. for 30 min and then lowered to 4° C.
  • the present invention preferably carries out the identification of the positive recombinant plasmid.
  • the identification method preferably includes transforming the recombinant plasmid into a conventional DH5 ⁇ competent bacterium, coating an LB plate, picking bacterial clones the next day, and extracting the plasmid to obtain a positive recombinant plasmid after positive bacteria are obtained from positive clones detected by PCR.
  • the present invention transforms the cells with the recombinant plasmid, cultures and induces with IPTG to obtain the fusion protein.
  • the transformation method there is no special limitation on the transformation method, and the transformation method well known in the art can be used.
  • the transformation method it is preferred to identify the positive clones of the transformed cells, and to obtain 3 polypeptide antigen fragments through PCR amplification, indicating that the transformation is successful.
  • the identified positive clones were cultured and induced by IPTG to obtain cells expressing the fusion protein.
  • the type of the cells is preferably BL21 competent bacteria.
  • the fusion protein is preferably isolated and purified.
  • the present invention has no special limitation on the separation and purification method, and the separation and purification method known in the art can be used.
  • the present invention provides the new coronavirus SARS-CoV-2 broad-spectrum polypeptide antigen, the triple new coronavirus Application of viral SARS-CoV-2 broad-spectrum polypeptide tandem fusion protein in preparation of SARS-CoV-2 broad-spectrum subunit vaccine and/or immunological detection kit for SARS-CoV-2 S2 antibody of new coronavirus.
  • the immunoassay preferably includes colloidal gold immunoassay, immunoprecipitation assay, fluorescence immunoassay and/or enzyme-linked immunoassay.
  • the invention provides an ELISA detection kit for detecting antibodies against the new coronavirus SARS-CoV-2, comprising an antigen-coated microtiter plate; the antigen includes the new coronavirus SARS-CoV-2 broad-spectrum polypeptide antigen or The triple new coronavirus SARS-CoV-2 broad-spectrum polypeptide tandem fusion protein.
  • enzyme-labeled antibody In the present invention, enzyme-labeled antibody, substrate solution, stop solution, washing solution, negative control and positive control.
  • the enzyme-labeled antibody is horseradish peroxidase-labeled mouse anti-human IgG.
  • the substrate solution is a solution obtained by mixing citric acid solution and Na 2 HPO 4 ⁇ 12H 2 O, and adding tetramethylbenzidine.
  • the compounding method of described substrate liquid is preferably as follows: 100mmol/L citric acid solution (21g citric acid is dissolved in deionized water, constant volume to 1L) 24.3mL, 200mmol/LNa 2 HPO 4 12H 2 O (71.6g Na 2 HPO 4 ⁇ 12H 2 O was dissolved in deionized water, and the volume was adjusted to 1 L) (25.7 mL) and mixed evenly, 50 mg of tetramethylbenzidine (TMB) was added, and 50 ⁇ L of 30% H 2 O 2 was added just before use.
  • the stop solution was 2M H 2 SO 4 aqueous solution.
  • the washing solution is preferably 10 mmol/L PBS solution with pH 7.4 containing 5% Tween-20. Negative control was negative human serum.
  • the positive control was mouse anti-SARS-CoV-2 serum.
  • the ELISA detection kit is prepared based on the capture method.
  • the present invention imposes special restrictions on the use method of the kit, and the use method of the capture method ELISA detection kit well known in the art can be used.
  • the invention provides a novel coronavirus SARS-CoV-2 broad-spectrum neutralizing antibody, a triple novel coronavirus SARS-CoV constructed by the triple novel coronavirus SARS-CoV-2 broad-spectrum polypeptide tandem fusion protein or the construction method -2 broad-spectrum polypeptide tandem fusion protein produced by immunizing animals.
  • the animals preferably include mice.
  • the immunization method preferably carries out two immunizations. The interval between two immunizations is preferably 15 days.
  • the antibody prepared by the present invention can not only effectively recognize the SARS-CoV-2 S protein, but also effectively neutralize the SARS-CoV-2 S protein pseudovirus, and inhibit the virus from invading 293T cells stably expressing human ACE2, which is SARS -CoV-2 virus broad-spectrum neutralizing antibody.
  • the present invention Based on the neutralizing antibody suppressing virus invasion into eukaryotic cells, the present invention provides a drug for preventing and/or treating the new coronavirus SARS-CoV-2, the new coronavirus SARS-CoV-2 broadly Spectrum neutralizing antibodies.
  • the present invention has no special limitation on the dosage form and preparation method of the drug, and the dosage form and preparation method of antibody drugs well known in the art can be used.
  • the amino acid sequence of the S2 protein of SARS-CoV-2 is DPLQPELDSFKEELDKYFKNHTSPDVDLGDIS (SEQ ID NO: 1)
  • SEQ ID NO: 1 The polypeptide.
  • SEQ ID NO: 1 has no change in the S protein of SARS, SARS-CoV-2 of the ⁇ -coronavirus genus, and all variants of SARS-CoV-2 found so far.
  • the polypeptide antigen preparation kit described in Example 1 is composed as follows:
  • Enzyme-labeled antibody horseradish peroxidase-labeled mouse anti-human IgG;
  • Substrate solution preparation 100mmol/L citric acid solution (21g citric acid dissolved in deionized water, dilute to 1L) 24.3mL, 200mmol/L Na 2 HPO 4 ⁇ 12H 2 O (71.6g Na 2 HPO 4 ⁇ 12H 2 O was dissolved in deionized water, and the volume was adjusted to 1 L) (25.7 mL) and mixed evenly, 50 mg of tetramethylbenzidine (TMB) was added, and 50 ⁇ L of 30% H 2 O 2 was added just before use.
  • TMB tetramethylbenzidine
  • Stop solution 2M H 2 SO 4 Mix 89 mL of distilled water and 11 mL of concentrated H 2 SO 4 .
  • Washing solution add 0.5mL Tween-20 to 1000mL 10mmo1/L PBS with pH 7.4;
  • Negative control human negative serum
  • positive control human SARS-CoV-2 inactivated positive serum
  • horseradish peroxidase-labeled mouse anti-human secondary antibody contains 5% skim milk powder and 1% newborn cow Serum PBST was diluted at a ratio of 1:40000;
  • the serum to be tested is the positive serum and negative human serum of inactivated SARS-CoV-2 patients, and all the results in the examples are the average value of OD 450nm , as shown in Figure 1.
  • the upstream primer for amplifying the pCold I linearization vector is located at position 337-361 of the pCold I plasmid; amplifying the pCold I linearization vector
  • the downstream primers of the vector are located at positions 295-318 of the pCold I plasmid.
  • the amplified fragment gene containing SEQ ID NO: 1 is located at the 3415-3510 position of the SARS-CoV-2 S gene; the upstream primer for amplifying the first fragment has 15 reverse primers at the 5' end that are opposite to the downstream primers of the pCold I linearized vector.
  • the reverse complementary sequence to the base corresponding to the expression of Linker GGGGS is added downstream; the upstream and downstream primers for the amplification of the second fragment are respectively added to the base sequence corresponding to the expression of Linker GGGGS and its reverse complementary sequence;
  • the downstream primers of the three fragments have 15 reverse complementary bases at the 5' end to the upstream primers of the pCold I linearized vector, and the upstream sequence is added with the corresponding bases of the expression Linker GGGGS. See attached table 1 for specific primer sequences, which were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.
  • Table 1 The sequence information of the primers used in the construction of the fusion protein
  • the PCR amplification reaction system is: 33 ⁇ l water, 10 ⁇ l 5 times buffer, 1 ⁇ l 10mM dNTP, 2 ⁇ l 10 ⁇ mol upstream primer, 2 ⁇ l 10 ⁇ mol downstream primer, 1 ⁇ l 10ng/ ⁇ l pCold I plasmid or pcDNA3.1-SARS-CoV-2-S plasmid (purchased from Shanghai Jihaoge Biotechnology Co., Ltd.), 1 ⁇ l of commercialized Phanta Super-Fidelity DNA polymerase.
  • the cycle parameters of the PCR amplification reaction were: denaturation at 94°C for 5 minutes, followed by 30 cycles (denaturation at 94°C for 30 seconds, annealing at 57°C for 30 seconds, extension at 72°C for 3 minutes); and finally extension at 72°C for 10 minutes.
  • the PCR products were electrophoresed on 1% agarose gel.
  • lane M is DNA Marker
  • lane 1 is the PCR product of the first fragment
  • lane 2 is the PCR product of the second fragment
  • lane 3 is the PCR product of the third fragment.
  • lane M is the DNA Marker
  • lane 1 is the PCR product of the linearized vector pCold I.
  • the recombinant product was transformed into conventional DH5 ⁇ competent bacteria and coated with LB plates.
  • the reaction conditions and reaction procedures are the same as step 2) recorded.
  • the triple broad-spectrum polypeptide tandem fusion protein His-3 ⁇ P4 can exist in soluble form in the supernatant of sonicated samples. Based on the determination of the soluble expression of His-3 ⁇ P4, the supernatant of the sonicated sample was purified by His purification column. Lane 5 in Fig. 4 is the result of SDS-PAGE analysis after purification of His-3 ⁇ P4 protein.
  • lane 4 is the result of Western blot analysis after His-3 ⁇ P4 protein purification.
  • the above results show that the present invention can efficiently express the triple broad-spectrum polypeptide tandem fusion protein of SARS-CoV-2, and the fusion protein will have a good application prospect in the development of SARS-CoV-2 serological diagnosis and broad-spectrum epitope vaccine .
  • mice serum was collected to obtain a broad-spectrum neutralizing antibody to SARS-CoV-2.
  • mice serum is diluted and added to a microtiter plate coated with polypeptide antigen, and incubated;
  • the above three experimental results show that the SARS-CoV-2 broad-spectrum antibody prepared by the present invention can not only effectively recognize the SARS-CoV-2 S protein, but also effectively neutralize the SARS-CoV-2 S protein pseudovirus and suppress virus invasion.
  • 293T cells stably expressing human ACE2.
  • the above results show that the SARS-CoV-2 broad-spectrum neutralizing antibody that can be prepared by the present invention has a good effect, and will have a good application prospect in SARS-CoV-2 anti-infection treatment, vaccine research and development, and detection kit development.
  • a temperature-sensitive gel damage repair preparation provided by the present invention and its application will be described in detail below in conjunction with the examples, but they should not be construed as limiting the protection scope of the present invention.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Virology (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Physics & Mathematics (AREA)
  • Urology & Nephrology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Plant Pathology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Communicable Diseases (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Oncology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)

Abstract

提供了一种新冠病毒SARS-CoV-2广谱多肽抗原及其特异性中和抗体和应用，属于病毒免疫检测技术领域。一种新冠病毒SARS-CoV-2广谱多肽抗原，氨基酸序列如SEQ ID NO:1所示，与SARS-CoV-2人阳性血清的反应，能够特异性与新冠病毒抗体结合。基于的多肽序列，利用PCR、原核表达及蛋白纯化技术，制备了三重SARS-CoV-2广谱多肽串联融合蛋白，模拟SARS-CoV-2 S蛋白自然状态下三聚体模式，以该融合蛋白作为抗原免疫小鼠，可以产生抗SARS-CoV-2特异性中和抗体，所述中和抗体在SARS-CoV-2抗感染治疗、疫苗研发和检测试剂盒研制中将具有良好的应用前景。

Description

一种新冠病毒SARS-CoV-2广谱多肽抗原及其特异性中和抗体和应用
本申请要求于2021年06月07日提交中国专利局的申请号为202110631147.0、发明名称为“一种新冠病毒SARS-CoV-2广谱多肽抗原及其特异性中和抗体和应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于病毒免疫检测技术领域，具体涉及一种新冠病毒SARS-CoV-2广谱多肽抗原及其特异性中和抗体和应用。
背景技术
新型冠状病毒SARS-CoV-2是一种传染性极强的病毒。SARS-CoV-2的S蛋白包含两个亚基：S1和S2。S1亚基通过其RBD与细胞受体血管紧张素转换酶2(ACE2)结合，而S2亚基通过融合肽(FP)介导病毒与细胞膜的融合。由于S2在感染过程中的融合过程中的重要作用，可以作为广泛保护的靶点。目前，SARS-CoV-2逃逸突变体的频繁出现，给当下针对具有免疫优势的受体结合域(RBD)新冠疫苗策略带来了挑战。
然而，目前尚无针对SARS-CoV-2 S2的广谱疫苗及检测抗S2抗体的快速检测方法。
发明内容
有鉴于此，本发明的目的在于提供一种SARS-CoV-2广谱多肽抗原，所述抗原在SARS-CoV-2原型株以及众多流行突变株中均无突变，可特异性识别新冠病毒SARS-CoV-2的抗体，具有较好的抗原性。
本发明提供了一种新冠病毒SARS-CoV-2广谱多肽抗原，所述广谱多肽抗原的氨基酸序列如SEQ ID NO:1所示。
本发明提供了一种三重新冠病毒SARS-CoV-2广谱多肽串联融合蛋白，所述融合蛋白包括3条所述新冠病毒SARS-CoV-2广谱多肽抗原的序列串联排列。
优选的，所述融合蛋白的氨基酸序列如SEQ ID NO:8所示。
本发明提供了所述三重新冠病毒SARS-CoV-2广谱多肽串联融合蛋白的构建方法，包括以下步骤：
1)以pCold I质粒为模板，用核苷酸序列如SEQ ID NO:6和SEQ ID NO:7所示的引物对进行PCR扩增，得到线性化pCold I表达载体；
2)以pcDNA3.1-SARS-CoV-2-S质粒为模板，用SEQ ID NO:2和SEQ ID NO:3所示的引物对进行PCR扩增第一片段，用SEQ ID NO:4和SEQ ID NO:3所示的引物对进行PCR扩增第二片段，用SEQ ID NO:4和SEQ ID NO:5所示的引物对进行PCR扩增第三片段；
3)利用重组酶对步骤1)中所述线性化pCold Ⅰ表达载体、步骤2)中所述第一片段、第二片段和第三片段进行快速重组克隆，得到重组质粒；
4)将步骤3)中所述重组质粒转化细胞，经培养、IPTG诱导得到融合蛋白。
优选的，步骤2)中所述PCR扩增的反应程序为94℃变性5min；94℃变性30s，57℃退火30s，72℃延伸3min，30个循环；最后72℃延伸10min；
所述PCR扩增的反应体系为33μl水，10μl 5倍缓冲液，1μl 10mM dNTP，2μl 10μM上游引物，2μl 10μM下游引物，1μl 10ng/μl的pCold Ⅰ质粒或pcDNA3.1-SARS-CoV-2-S质粒，1μl商品化的Phanta Super-Fidelity DNA聚合酶。
本发明提供了所述新冠病毒SARS-CoV-2广谱多肽抗原、所述三重新冠病毒SARS-CoV-2广谱多肽串联融合蛋白在制备SARS-CoV-2广谱亚单位疫苗和/或免疫检测新冠病毒SARS-CoV-2 S2抗体的试剂盒中的应用。
优选的，所述免疫检测包括胶体金免疫检测、免疫沉淀检测、荧光免疫检测和/或酶联免疫检测。
本发明提供了一种用于检测新冠病毒SARS-CoV-2抗体的ELISA检测试剂盒，包括抗原包被的酶标板；所述抗原包括所述新冠病毒SARS-CoV-2广谱多肽抗原或所述三重新冠病毒SARS-CoV-2广谱多肽串联融合蛋白。
本发明提供了一种新冠病毒SARS-CoV-2广谱中和抗体，由所述三重新冠病毒SARS-CoV-2广谱多肽串联融合蛋白或所述构建方法构建得到的三重新冠病毒SARS-CoV-2广谱多肽串联融合蛋白免疫动物产生的。
本发明提供了一种用于预防和/或治疗新冠病毒SARS-CoV-2的药物，所述新冠病毒SARS-CoV-2广谱中和抗体。
本发明提供的新冠病毒SARS-CoV-2广谱多肽抗原，氨基酸序列如SEQ ID NO:1所示。本发明所提供的多肽抗原是基于抗原性较好的SARS-CoV-2 S基因S2段多肽序列合成。所述抗原在SARS-CoV-2原型株以及众多流行突变株中均无突变，且在其他冠状病毒SARS-CoV和BatCoV RaTG13中均具有相同的氨基酸序列，因此具有优异的广谱抗原性能。本发明通过将所述抗原与SARS-CoV-2病人血清反应测定，确定了多肽具有较强的特异性。所述抗原可应用所述多肽抗原可以应用于SARS-CoV-2的S2抗体检测，与其他方法比较，此方法具有快速安全可靠，可操作性高的优点。利用多肽抗原作为抗原检测样本中的特异性抗体，使得试剂盒的制备和样品的检测更加快速简便，在免疫人群或SARS-CoV-2病人广谱抗体水平的评估指标。
本发明还提供了三重新冠病毒SARS-CoV-2广谱多肽串联融合蛋白，所述融合蛋白包括3条所述新冠病毒SARS-CoV-2广谱多肽抗原的序列串联排列。所述融合蛋白既可以提高抗原表达量，为实际大规模生产打下基础，又可以模拟SARS-CoV-2 S蛋白在感染时状态，具有三聚体特性。同时应用本发明制备的SARS-CoV-2三重广谱多肽串联融合蛋白免疫动物，可以产生特异性抗SARS-CoV-2广谱中和抗体，为SARS-CoV-2广谱疫苗的研制打下重要基础。
本发明还提供了一种新冠病毒SARS-CoV-2广谱中和抗体，由所述三重新冠病毒SARS-CoV-2广谱多肽串联融合蛋白免疫动物产生的。所述广谱中和抗体能够通过特异性结合新冠病毒SARS-CoV-2抗原位点，从而阻断新冠病毒的发生和发展，为预防和治疗新冠病毒提供了新的思路。
附图说明
图1为包被多肽抗原的ELISA检测SARS-CoV-2病人的阳性血清；
图2为采用PCR法扩增3个含SEQ ID NO：1对应基因片段的电泳图，其中泳道1，第一片段；泳道2，第二片段；泳道3，第三片段；泳道M，DNA Marker；
图3为采用PCR法扩增线性化载体pCold Ⅰ的电泳图，其中泳道1，线性化载体pCold Ⅰ的PCR产物；泳道M，DNA Marker；
图4为用SDS-PAGE分析SARS-CoV-2三重广谱多肽串联融合蛋白 的表达结果，其中泳道1，pCold Ⅰ超声裂解沉淀；泳道2，His-3×P4超声裂解沉淀；泳道3，pCold Ⅰ超声裂解上清；泳道4，His-3×P4超声裂解上清；泳道5，纯化的His-3×P4蛋白；泳道M，蛋白Marker；
图5为用Western blot鉴定SARS-CoV-2三重广谱多肽串联融合蛋白的表达结果，其中泳道1，pCold Ⅰ超声裂解沉淀；泳道2，pCold Ⅰ超声裂解上清；泳道3，His-3×P4超声裂解上清；泳道4，His-3×P4超声裂解沉淀；泳道M，蛋白Marker；
图6为包被多肽抗原的ELISA检测抗三重广谱多肽串联融合蛋白小鼠血清；
图7为间接免疫荧光鉴定抗三重广谱多肽串联融合蛋白小鼠血清与SARS-CoV-2 S蛋白反应性；
图8为SARS-CoV-2假病毒中和试验鉴定抗三重广谱多肽串联融合蛋白小鼠血清的中和活性。
具体实施方式
本发明提供了一种新冠病毒SARS-CoV-2广谱多肽抗原，所述广谱多肽抗原的氨基酸序列如SEQ ID NO:1(DPLQPELDSFKEELDKYFKNHTSPDVDLGDIS)所示。所述多肽抗原在SARS-CoV2原型株以及各流行突变株中均无突变，且在其他冠状病毒(SARS-CoV和BatCoV RaTG13)中均具有相同序列。并且通过与SARS-CoV-2人阳性血清的反应，确定了该多肽抗原的特异性。因此，基于所述多肽抗原可建立了简单快捷的ELISA方法特异性检测SARS-CoV-2抗体(S2抗体)的方法。
本发明提供了一种三重新冠病毒SARS-CoV-2广谱多肽串联融合蛋白，所述融合蛋白包括3条所述新冠病毒SARS-CoV-2广谱多肽抗原的序列串联排列。所述融合蛋白优选在所述新冠病毒SARS-CoV-2广谱多肽抗原之间还有连接肽。本发明对所述连接肽的氨基酸序列没有特殊限制，采用本领域所熟知的连接肽的氨基酸序列即可。在本发明实施例中，所述连接肽为GGGGS(SEQ ID NO:9)。所述融合蛋白的氨基酸序列优选如SEQ ID NO:8所示。所述融合蛋白的5′端包含组氨酸标签，便于后续分离纯化。所述融合蛋白可模拟SARS-CoV-2 S蛋白自然状态下三聚体模式，以该融合蛋白作为抗原免疫小鼠，可以产生抗SARS-CoV-2特异性中和抗体。
本发明提供了所述三重新冠病毒SARS-CoV-2广谱多肽串联融合蛋白的构建方法，包括以下步骤：
1)以pCold I质粒为模板，用核苷酸序列如SEQ ID NO:6和SEQ ID NO:7所示的引物对进行PCR扩增，得到线性化pCold I表达载体；
2)以pcDNA3.1-SARS-CoV-2-S质粒为模板，用核苷酸序列如SEQ ID NO:2和SEQ ID NO:3所示的引物对进行PCR扩增第一片段，用核苷酸序列如SEQ ID NO:4和SEQ ID NO:3所示的引物对进行PCR扩增第二片段，用核苷酸序列如SEQ ID NO:4和SEQ ID NO:5所示的引物对进行PCR扩增第三片段；
3)利用重组酶对步骤1)中所述线性化pCold Ⅰ表达载体、步骤2)中所述第一片段、第二片段和第三片段进行快速重组克隆，得到重组质粒；
4)将步骤3)中所述重组质粒转化细胞，经培养、IPTG诱导得到融合蛋白。
本发明以pCold I质粒为模板，用核苷酸序列如SEQ ID NO:6(AAGCTTGTCGACCTGCAGTCTAGAT)和SEQ ID NO:7(GGTACCGAGCTCCATATGCCTACC)所示的引物对进行PCR扩增，得到线性化pCold I表达载体。
本发明对所述pCold I质粒的来源不做特殊限制，采用本领域所熟知的pCold I质粒的来源即可。在本发明实施例中，所述pCold I质粒购自Takara商品化的质粒(大连宝生物公司)。所述PCR扩增的反应程序优选为94℃变性5min；94℃变性30s，57℃退火30s，72℃延伸3min，30个循环；最后72℃延伸10min。所述PCR扩增的反应体系优选为33μl水，10μl 5倍缓冲液，1μl 10mM dNTP，2μl 10μM上游引物，2μl 10μM下游引物，1μl 10ng/μl的pCold Ⅰ质粒，1μl商品化的Phanta Super-Fidelity DNA聚合酶。
本发明以pcDNA3.1-SARS-CoV-2-S质粒为模板，用SEQ ID NO:2和SEQ ID NO:3所示的引物对进行PCR扩增第一片段，用SEQ ID NO:4和SEQ ID NO:3所示的引物对进行PCR扩增第二片段，用SEQ ID NO:4和SEQ ID NO:5所示的引物对进行PCR扩增第三片段。
在本发明中，所述pcDNA3.1-SARS-CoV-2-S质粒购自上海吉浩格生 物科技有限公司。扩增第一片段的引物对包括SEQ ID NO:2(ATGGAGCTCGGTACCGACCCATTGCAACCCGAACTTG)和SEQ ID NO:3(ACTTCCACCTCCACCGCTAATGTCGCCCAGGTCCAC)所示的引物对。扩增第二片段的引物对优选包括SEQ ID NO:4(GGTGGAGGTGGAAGTGACCCATTGCAACCCGAACTTG)和SEQ ID NO:3所示引物。扩增第三片段的引物对包括SEQ ID NO:4和SEQ ID NO:5(CAGGTCGACAAGCTTTCAGCTAATGTCGCCCAGGTCCAC)所示引物。
在本发明中，所述第一片段、第二片段以及第三片段PCR扩增的反应程序优选为94℃变性5min；94℃变性30s，57℃退火30s，72℃延伸3min，30个循环；最后72℃延伸10min；所述PCR扩增的反应体系优选为33μl水，10μl 5倍缓冲液，1μl 10mM dNTP，2μl 10μM上游引物，2μl 10μM下游引物，1μl 10ng/μl的pCold Ⅰ质粒或pcDNA3.1-SARS-CoV-2-S质粒，1μl商品化的Phanta Super-Fidelity DNA聚合酶。
得到线性化pCold Ⅰ表达载体、第一片段、第二片段和第三片段后，本发明利用重组酶对所述线性化pCold Ⅰ表达载体、所述第一片段、第二片段和第三片段进行快速重组克隆，得到重组质粒。
在本发明中，所述重组酶优选为
Figure PCTCN2021107615-appb-000001
MultiS One Step Cloning Kit。所述重组克隆的反应体系为线性化载体2μl(含100ng)，第一片段、第二片段和第三片段各1μl(各含10ng)，CE MultiS Buffer 4μl，Exnase MultiS 2μl，水9μl。所述重组克隆的反应程序为37℃30min，降至4℃。
得到重组质粒后，本发明优选进行阳性重组质粒的鉴定。所述鉴定的方法优选包括将所述重组质粒转化到常规DH5α感受态细菌，涂LB板，次日挑取细菌克隆，PCR检测阳性克隆得到阳性菌后，提取质粒，得到阳性重组质粒。
鉴定后，本发明将所述重组质粒转化细胞，经培养、IPTG诱导得到融合蛋白。
在本发明对所述转化方法没有特殊限制，采用本领域所熟知的转化方法即可。所述转化后，优选对转化后的细胞进行阳性克隆鉴定，通过PCR扩增，得到3个的多肽抗原片段说明转化成功。将鉴定的阳性克隆进行培 养、IPTG诱导得到表达有融合蛋白的细胞。所述细胞的种类优选为BL21感受态细菌。
得到表达有融合蛋白的细胞后，优选对融合蛋白进行分离纯化。本发明对所述分离纯化的方法没有特殊限制，采用本领域所熟知的分离纯化方法即可。
基于所述多肽抗原在包括新冠病毒在内的多种冠状病毒中高度保守，且具有优异的抗原性，因此本发明提供了所述新冠病毒SARS-CoV-2广谱多肽抗原、所述三重新冠病毒SARS-CoV-2广谱多肽串联融合蛋白在制备SARS-CoV-2广谱亚单位疫苗和/或免疫检测新冠病毒SARS-CoV-2 S2抗体的试剂盒中的应用。所述免疫检测优选包括胶体金免疫检测、免疫沉淀检测、荧光免疫检测和/或酶联免疫检测。
本发明提供了一种用于检测新冠病毒SARS-CoV-2抗体的ELISA检测试剂盒，包括抗原包被的酶标板；所述抗原包括所述新冠病毒SARS-CoV-2广谱多肽抗原或所述三重新冠病毒SARS-CoV-2广谱多肽串联融合蛋白。
在本发明中，酶标抗体、底物溶液、终止液、洗涤液、阴性对照和阳性对照。所述酶标抗体为辣根过氧化物酶标记鼠抗人IgG。所述底物溶液为柠檬酸溶液和Na 2HPO 4·12H 2O混匀，加入四甲基联苯胺得到的溶液。所述底物液的配方法优选如下：100mmol/L的柠檬酸溶液(21g柠檬酸溶于去离子水，定容至1L)24.3mL，200mmol/LNa 2HPO 4·12H 2O(71.6g Na 2HPO 4·12H 2O溶于去离子水，定容至1L)25.7mL混匀，加入50mg的四甲基联苯胺(TMB)，临用前加入50μL的30％H 2O 2。终止液为2M H 2SO 4水溶液。所述洗涤液优选为含5％Tween-20的10mmo1/L pH 7.4的PBS溶液。阴性对照为阴性人血清。阳性对照为鼠抗SARS-CoV-2血清。
在本发明中，所述ELISA检测试剂盒是基于捕获法制备得到。本发明对所述试剂盒的使用方法做特殊限制，采用本领域所熟知的捕获法ELISA检测试剂盒的使用方法即可。
本发明提供了一种新冠病毒SARS-CoV-2广谱中和抗体，由所述三重新冠病毒SARS-CoV-2广谱多肽串联融合蛋白或所述构建方法构建得到 的三重新冠病毒SARS-CoV-2广谱多肽串联融合蛋白免疫动物产生的。所述动物优选包括小鼠。所述免疫方法优选进行两次免疫。两次免疫间隔优选为15d。试验表明，本发明制备的抗体不仅能有效识别SARS-CoV-2 S蛋白，还能有效中和SARS-CoV-2 S蛋白假病毒，阻抑病毒入侵稳定表达人ACE2的293T细胞，即为SARS-CoV-2病毒广谱中和抗体。
基于所述中和抗体阻抑病毒入侵真核细胞，因此本发明提供了一种用于预防和/或治疗新冠病毒SARS-CoV-2的药物，所述所述新冠病毒SARS-CoV-2广谱中和抗体。本发明对所述药物的剂型和制备方法均没有特殊限制，采用本领域所熟知的抗体药物的剂型和制备方法即可。
下面结合实施例对本发明提供的一种新冠病毒SARS-CoV-2广谱多肽抗原及其特异性中和抗体和应用进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
实施例1
一种新冠病毒SARS-CoV-2广谱多肽抗原的确定以及抗原特异性检测方法
利用生物软件分析SARS-CoV-2的S2蛋白的氨基酸序列，从亲水性、抗原指数、表面位点指数以及S2蛋白的保守性可能的抗原性片段，得到氨基酸序列为DPLQPELDSFKEELDKYFKNHTSPDVDLGDIS(SEQ ID NO:1)的多肽。经比对，SEQ ID NO:1所示肽段在β-冠状病毒属的SARS、SARS-CoV-2以及截止目前发现的所有SARS-CoV-2的变体的S蛋白中均无任何变化。
实施例2
一.采用多肽抗原制备ELISA检测试剂盒
将实施例1所述多肽抗原制备试剂盒，组成如下：
1、包被多肽抗原的酶标板；
2、酶标抗体：辣根过氧化物酶标记鼠抗人IgG；
3、底物液配制：100mmol/L的柠檬酸溶液(21g柠檬酸溶于去离子水，定容至1L)24.3mL，200mmol/L Na 2HPO 4·12H 2O(71.6g Na 2HPO 4·12H 2O溶于去离子水，定容至1L)25.7mL混匀，加入50mg的四甲基联苯胺(TMB)，临用前加入50μL的30％H 2O 2。
4、终止液2M H 2SO 4：分别取蒸馏水89mL和浓H 2SO 411mL混合即可。
5、洗涤液：1000mL 10mmo1/L pH 7.4的PBS中加入0.5mL Tween-20；
6、阴性对照(人阴性血清)和阳性对照(人SARS-CoV-2灭活阳性血清)。
二.所述试剂盒中包被多肽抗原的酶标板的制备和检测方法
将得到的多肽委托上海德翅生物科技有限公司进行多肽抗原合成，采用SARS-CoV-2特异性抗体ELISA检测方法的步骤包被上述多肽抗原，具体包括以下步骤：
1.将所述多肽抗原包被96孔酶标板，包被量为0.5μg/孔，包被浓度为5μg/mL，每孔总体积100μL包被后用含1％新生牛血清和5％脱脂奶粉的PBST封闭过夜；
2.待检血清用含5％脱脂奶粉和1％新生牛血清的PBST按1:100的比例稀释后加入包被的多肽抗原的酶标板，孵育；
3.洗涤多次后，加入辣根过氧化物酶标记的鼠抗人二抗再次孵育；所述辣根过氧化物酶标记的鼠抗人二抗用含5％脱脂奶粉和1％新生牛血清的PBST按1:40000的比例稀释；
4.再次洗涤多次，加TMB显色液反应；
5.最后用硫酸终止反应，以450nm波长下读取OD值，检测出个体中SARS-CoV-2抗体水平。
所述待检血清为灭活的SARS-CoV-2病人的阳性血清及阴性人血清，实施例中的所有结果均为OD 450nm的平均值，结果如图1所示。
通过该ELISA检测OD值发现，多肽抗原可以和11份阳性血清反应，但是与其余8份阳性血清不反应，说明多肽抗原能有效识别人体内产生针对新冠病毒SARS-CoV-2的抗体。
实施例3
一种SARS-CoV-2三重广谱多肽串联融合蛋白的制备方法
1)设计扩增含pCold Ⅰ线性化载体以及3个含SEQ ID NO：1对应基因片段的引物：扩增pCold Ⅰ线性化载体上游引物位于pCold Ⅰ质粒 337-361位；扩增pCold Ⅰ线性化载体下游引物位于pCold Ⅰ质粒295-318位。扩增含SEQ ID NO：1的片段基因位于SARS-CoV-2 S基因的3415-3510位；扩增第一片段的上游引物在5′端带有15个与pCold Ⅰ线性化载体下游引物反向互补碱基，下游则添加与表达Linker GGGGS对应碱基的反向互补序列；扩增第二片段的上游和下游引物分别添加表达Linker GGGGS对应碱基序列和其反向互补序列；扩增第三片段的下游引物在5’端带有15个与pCold Ⅰ线性化载体上游引物反向互补碱基，上游则添加与表达Linker GGGGS对应碱基的序列。具体引物序列见附表1，由苏州金唯智生物科技有限公司合成。
表1融合蛋白构建过程中所用引物序列信息
Figure PCTCN2021107615-appb-000002
2)pCold Ⅰ线性化载体以及3个含SEQ ID NO：1对应基因片段PCR扩增：以pCold Ⅰ质粒以及pcDNA3.1-SARS-CoV-2-S质粒分别为模板，表1所述引物为引物进行PCR扩增。PCR扩增反应体系为：33μl水，10μl5倍缓冲液，1μl 10mM dNTP，2μl 10μmol上游引物，2μl 10μmol下游引物，1μl 10ng/μl的pCold Ⅰ质粒或pcDNA3.1-SARS-CoV-2-S质粒(购自上海吉浩格生物科技有限公司)，1μl商品化的Phanta Super-Fidelity DNA聚合酶。PCR扩增反应循环参数为：94℃变性5分钟，随后进行30个循环(94℃变性30秒，57℃退火30秒，72℃延伸3分钟)；最后72℃延伸10分钟。PCR结束后，PCR产物在1％的琼脂糖凝胶中进行电泳。如图2所示，其中泳道M为DNA Marker，其中泳道1为第一片段PCR产物，泳道2为第二片段PCR产物，泳道3为第三片段PCR产物。 如图3所示，其中泳道M为DNA Marker，泳道1为线性化载体pCold Ⅰ的PCR产物。
3)将上述三个片段快速克隆进pCold Ⅰ载体：将以上纯化的表达线性化载体pCold Ⅰ以及3个含SEQ ID NO：1对应基因片段PCR产物在商品化重组酶(
Figure PCTCN2021107615-appb-000003
MultiS One Step Cloning Kit)的作用下进行重组克隆；反应体系：线性化载体2μl(含100ng)，第一片段、第二片段和第三片段各1μl(各含10ng)，CE MultiS Buffer4μl，Exnase MultiS 2μl，水9μl。反应条件：37℃30min，降至4℃。随后将重组产物转化到常规DH5α感受态细菌，涂LB板。次日挑取细菌克隆进行质粒制备，阳性克隆鉴定，具体采用SEQ ID NO：2和SEQ ID NO：5作为上下游引物对所挑取的克隆进行PCR鉴定，反应条件和反应程序同步骤2)记载。
4)三重广谱多肽串联融合蛋白诱导表达及其纯化:将获得的含3个含SEQ ID NO：1对应基因片段的阳性克隆(命名为pCold Ⅰ-3×P4)转化BL21感受态细菌，经IPTG诱导(0.1mmol/ml)后收集细菌，进行超声(40赫兹)破碎。将超声破碎样品离心后分上清与沉淀进行SDS-PAGE(5％的浓缩胶，10％的分离胶)以及Western blot分析(以抗P4单抗G5为一抗，羊抗鼠HRP标记的IgG为二抗)鉴定表达。
在图4中，三重广谱多肽串联融合蛋白His-3×P4可在超声破碎样品上清中以可溶性形式存在。在确定His-3×P4的可溶性表达基础上，将超声破碎样品上清通过His纯化柱进行了His-3×P4蛋白的纯化。图4中泳道5是His-3×P4蛋白纯化后SDS-PAGE分析结果。
在图5中，泳道4是His-3×P4蛋白纯化后Western blot分析结果。以上结果表明本发明可以高效表达SARS-CoV-2的三重广谱多肽串联融合蛋白，在该融合蛋白在SARS-CoV-2血清学诊断和广谱表位疫苗的研制中将具有良好的应用前景。
实施例4
一种SARS-CoV-2广谱中和抗体的制备方法
用上述SARS-CoV-2的三重广谱多肽串联融合蛋白His-3×P4为免疫原免疫小鼠制备抗SARS-CoV-2广谱中和抗体，具体方法如下：
1)将His-3×P4与弗氏完全佐剂混合(佐剂和抗原的体积比为1:1)后 对BALB/c小鼠进行首次免疫，每次免疫剂量为50μg/只；
2)免疫15天后，将His-3×P4与弗氏不完全佐剂混合(佐剂和抗原的体积比为1:1)后对BALB/c小鼠进行二次免疫，每次小鼠免疫剂量为50μg/只；
3)二次免疫20天后，采集小鼠血清，得到SARS-CoV-2广谱中和抗体。
实施例5
SARS-CoV-2广谱中和抗体的验证
1.SARS-CoV-2广谱中和抗体的抗性检测方法
1)用实施例2制备的多肽抗原包被酶标板对采集的小鼠血清进行检测；
2)采集的小鼠血清稀释后加入包被有多肽抗原的酶标板，孵育；
3)洗涤多次后，加入辣根过氧化物酶标记的羊抗鼠二抗再次孵育；
4)再次洗涤多次，加TMB显色液反应；
5)最后用硫酸终止反应，以450nm波长下读取OD值，检测出小鼠体内抗SARS-CoV-2抗体水平，结果如图6所示。
2.抗原抗体特异性结合能力检测方法
1)将表达SARS-CoV-2 S蛋白的真核表达质粒pcDNA3.1-SARS-CoV-2-S转染293T细胞；
2)转染48小时后，将细胞用丙酮乙醇固定液固定；
3)采集的小鼠血清稀释后固定的细胞孵育；
4)洗涤多次后，加入FITC标记的羊抗鼠二抗再次孵育；
5)在倒置荧光显微镜中观察，结果如图7所示。
3.抗体的中和特异性检测方法
1)将采集的小鼠血清倍比稀释后与等量的SARS-CoV-2 S蛋白假病毒(含GFP-Luciferase报告基因)共孵育；
2)将孵育后的抗体-假病毒混合物接种稳定表达人ACE2的293T细胞；
3)8小时后弃去细胞培养上清，换新鲜培养液继续培养；
4)病毒感染60小时后，用报告基因试剂盒检测细胞的Luciferase荧 光强度，并按照公式I计算病毒的感染率，结果如图8所示；
病毒感染率(％)＝(血清组的Luciferase荧光强度/PBS组的Luciferase荧光强度)×100％公式I。
上述三个实验结果显示，本发明制备的SARS-CoV-2广谱抗体不仅能有效识别SARS-CoV-2 S蛋白，还能有效中和SARS-CoV-2 S蛋白假病毒，阻抑病毒入侵稳定表达人ACE2的293T细胞。以上结果表明本发明可以制备出的SARS-CoV-2广谱中和抗体效果好，在SARS-CoV-2抗感染治疗、疫苗研发和检测试剂盒研制中将具有良好的应用前景。
下面结合实施例对本发明提供的一种温度敏感型凝胶损伤修复制剂及其应用进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。对这些实施例的多种修改对本领域的专业技术人员来说是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。

Claims (10)

  1. 一种新冠病毒SARS-CoV-2广谱多肽抗原，其特征在于，所述广谱多肽抗原的氨基酸序列如SEQ ID NO:1所示。
  2. 一种三重新冠病毒SARS-CoV-2广谱多肽串联融合蛋白，其特征在于，所述融合蛋白包括3条权利要求1所述新冠病毒SARS-CoV-2广谱多肽抗原的序列串联排列。
  3. 根据权利要求2所述三重新冠病毒SARS-CoV-2广谱多肽串联融合蛋白，其特征在于，所述融合蛋白的氨基酸序列如SEQ ID NO:8所示。
  4. 权利要求2或3所述三重新冠病毒SARS-CoV-2广谱多肽串联融合蛋白的构建方法，其特征在于，包括以下步骤：
    1)以pCold I质粒为模板，用核苷酸序列如SEQ ID NO:6和SEQ ID NO:7所示的引物对进行PCR扩增，得到线性化pCold I表达载体；
    2)以pcDNA3.1-SARS-CoV-2-S质粒为模板，用SEQ ID NO:2和SEQ ID NO:3所示的引物对进行PCR扩增第一片段，用SEQ ID NO:4和SEQ ID NO:3所示的引物对进行PCR扩增第二片段，用SEQ ID NO:4和SEQ ID NO:5所示的引物对进行PCR扩增第三片段；
    3)利用重组酶对步骤1)中所述线性化pCold Ⅰ表达载体、步骤2)中所述第一片段、第二片段和第三片段进行快速重组克隆，得到重组质粒；
    4)将步骤3)中所述重组质粒转化细胞，经培养、IPTG诱导得到融合蛋白。
  5. 根据权利要求4所述构建方法，其特征在于，步骤2)中所述PCR扩增的反应程序为94℃变性5min；94℃变性30s，57℃退火30s，72℃延伸3min，30个循环；最后72℃延伸10min；
    所述PCR扩增的反应体系为33μl水，10μl 5倍缓冲液，1μl 10mM dNTP，2μl 10μM上游引物，2μl 10μM下游引物，1μl 10ng/μl的pCold Ⅰ质粒或pcDNA3.1-SARS-CoV-2-S质粒，1μl商品化的Phanta Super-Fidelity DNA聚合酶。
  6. 权利要求1所述新冠病毒SARS-CoV-2广谱多肽抗原、权利要求2或3所述三重新冠病毒SARS-CoV-2广谱多肽串联融合蛋白在制备 SARS-CoV-2广谱亚单位疫苗和/或免疫检测新冠病毒SARS-CoV-2 S2抗体的试剂盒中的应用。
  7. 根据权利要求6所述应用，其特征在于，所述免疫检测包括以下检测方法中的一种或几种：胶体金免疫检测、免疫沉淀检测、荧光免疫检测和酶联免疫检测。
  8. 一种用于检测新冠病毒SARS-CoV-2抗体的ELISA检测试剂盒，其特征在于，包括抗原包被的酶标板；所述抗原包括权利要求1所述新冠病毒SARS-CoV-2广谱多肽抗原或权利要求2或3所述三重新冠病毒SARS-CoV-2广谱多肽串联融合蛋白。
  9. 一种新冠病毒SARS-CoV-2广谱中和抗体，其特征在于，由权利要求2或3所述三重新冠病毒SARS-CoV-2广谱多肽串联融合蛋白或权利要求4或5所述构建方法构建得到的三重新冠病毒SARS-CoV-2广谱多肽串联融合蛋白免疫动物产生的。
  10. 一种用于预防和/或治疗新冠病毒SARS-CoV-2的药物，其特征在于，所述权利要求9所述新冠病毒SARS-CoV-2广谱中和抗体。
PCT/CN2021/107615 2021-06-07 2021-07-21 一种新冠病毒SARS-CoV-2广谱多肽抗原及其特异性中和抗体和应用 WO2022257237A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US17/620,528 US20240002451A1 (en) 2021-06-07 2021-07-21 Broad-spectrum peptide antigen of the novel coronavirus sars-cov-2, specific neutralizing antibody and use thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202110631147.0 2021-06-07
CN202110631147.0A CN113354717B (zh) 2021-06-07 2021-06-07 一种新冠病毒SARS-CoV-2广谱多肽抗原及其特异性中和抗体和应用

Publications (1)

Publication Number Publication Date
WO2022257237A1 true WO2022257237A1 (zh) 2022-12-15

Family

ID=77532745

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/107615 WO2022257237A1 (zh) 2021-06-07 2021-07-21 一种新冠病毒SARS-CoV-2广谱多肽抗原及其特异性中和抗体和应用

Country Status (3)

Country Link
US (1) US20240002451A1 (zh)
CN (1) CN113354717B (zh)
WO (1) WO2022257237A1 (zh)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113917139A (zh) * 2021-10-18 2022-01-11 扬州大学 基于重组荧光病毒的血清4型禽腺病毒中和抗体的检测方法
CN116751305A (zh) * 2022-02-08 2023-09-15 苏州方舟生物科技有限公司 β属冠状病毒融合重组蛋白及其制备方法和应用
CN114805560B (zh) * 2022-03-21 2022-11-15 中国科学院微生物研究所 纳米抗体r14的构建体及其应用
CN114703147A (zh) * 2022-04-18 2022-07-05 扬州大学 一种抗SARS-CoV-2广谱中和性单克隆抗体及其杂交瘤细胞株、检测试剂盒和应用
CN115976078B (zh) * 2022-12-30 2023-08-15 广州恩宝生物医药科技有限公司 一种广谱的腺病毒载体新冠疫苗

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111217919A (zh) * 2020-03-04 2020-06-02 中山大学 一种基于火球菌铁蛋白的新型冠状病毒s蛋白双区域亚单位纳米疫苗
CN111848753A (zh) * 2020-07-20 2020-10-30 中国科学院过程工程研究所 新型冠状病毒抗原表位及其应用
US20200407402A1 (en) * 2020-06-29 2020-12-31 The Scripps Research Institute Stabilized Coronavirus Spike (S) Protein Immunogens and Related Vaccines
CN112409496A (zh) * 2020-11-26 2021-02-26 焦顺昌 一种跨膜表达新型冠状病毒抗原s2的融合蛋白、重组载体、重组树突状细胞及其应用
CN112535730A (zh) * 2020-11-27 2021-03-23 常州文松生物技术有限公司 新型冠状病毒多肽疫苗及其应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111607002B (zh) * 2020-02-24 2021-07-09 中山大学 一种基于幽门螺旋杆菌铁蛋白的新型冠状病毒s蛋白双区域亚单位纳米疫苗

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111217919A (zh) * 2020-03-04 2020-06-02 中山大学 一种基于火球菌铁蛋白的新型冠状病毒s蛋白双区域亚单位纳米疫苗
US20200407402A1 (en) * 2020-06-29 2020-12-31 The Scripps Research Institute Stabilized Coronavirus Spike (S) Protein Immunogens and Related Vaccines
CN111848753A (zh) * 2020-07-20 2020-10-30 中国科学院过程工程研究所 新型冠状病毒抗原表位及其应用
CN112409496A (zh) * 2020-11-26 2021-02-26 焦顺昌 一种跨膜表达新型冠状病毒抗原s2的融合蛋白、重组载体、重组树突状细胞及其应用
CN112535730A (zh) * 2020-11-27 2021-03-23 常州文松生物技术有限公司 新型冠状病毒多肽疫苗及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE PROTEIN 18 March 2020 (2020-03-18), ANONYMOUS : "surface glycoprotein [Severe acute respiratory syndrome coronavirus 2] ", XP055925288, retrieved from NCBI Database accession no. QHD43416 *
ZHENG, ZHIQIANG ET AL.: "Monoclonal Antibodies for the S2 Subunit of Spike of SARS-CoV-1 Cross-React with the Newly-Emerged SARS-CoV-2", EURO SURVEILL, vol. 25, no. 28, 16 July 2020 (2020-07-16), pages 19 - 28, XP055850940, DOI: 10.2807/1560-7917.ES.2020.25.28.2000291 *

Also Published As

Publication number Publication date
CN113354717A (zh) 2021-09-07
US20240002451A1 (en) 2024-01-04
CN113354717B (zh) 2022-04-08

Similar Documents

Publication Publication Date Title
WO2022257237A1 (zh) 一种新冠病毒SARS-CoV-2广谱多肽抗原及其特异性中和抗体和应用
WO2021254327A1 (zh) 一种包膜替换型病毒载体疫苗及其构建方法
WO2022262142A1 (zh) 一种产生广谱交叉中和活性的重组新型冠状病毒rbd三聚体蛋白疫苗、其制备方法和应用
WO2021243974A1 (zh) 一种SARS-CoV-2的融合蛋白及其疫苗组合物
Embers et al. Protective immunity to rabbit oral and cutaneous papillomaviruses by immunization with short peptides of L2, the minor capsid protein
JP2003529319A (ja) ＨＩＶ−１ｇｐ４１を標的化する広範に中和する抗体を誘発する方法
CN112574318B (zh) 非洲猪瘟病毒p22蛋白纳米颗粒及其制备方法与应用
CN111778264A (zh) 基于新型腺病毒载体Sad23L和/或Ad49L的新型冠状病毒肺炎疫苗
CN113018427B (zh) 基于新冠病毒中和抗原表位的多价融合蛋白疫苗
US9884894B2 (en) Fine epitope peptide capable of inducing cross-reactive antibodies among homologous proteins in human papilloma virus E6 protein
CN107586322B (zh) 牛传染性鼻气管炎病毒gD蛋白抗原表位多肽及其抑制剂和单抗及其应用
CN112574319B (zh) 非洲猪瘟病毒p12蛋白纳米颗粒及其制备方法与应用
KR20210123234A (ko) 코로나바이러스 감염증­１９의 진단 및 백신을 위한 재조합 뉴클레오캡시드 단백질 및 이의 용도
WO2021206638A1 (en) Vaccine and/or antibody for viral infection
WO2023138333A1 (zh) 一种重组新型冠状病毒蛋白疫苗、其制备方法和应用
CN105085672B (zh) 3d蛋白特异性单克隆免疫球蛋白a抗体及其组合物
CN110257405A (zh) 牛支原体乙醇脱氢酶基因及其编码蛋白与应用
US20240148850A1 (en) N protein epitope mutation marker for preparing epitope deletion-marked vaccine strain of type ii porcine reproductive and respiratory syndrome virus (prrsv) and use thereof
WO2021170090A1 (zh) SARS-CoV-2病毒的检测方法及检测试剂盒
CN113151331B (zh) SARS-ConV-2病毒S蛋白膜外BD端结构域高靶向性重组蛋白及其亚单位疫苗
TWI490229B (zh) 豬瘟病毒封套醣蛋白Ｅｒｎｓ之特異性單株抗體ＣＷ８１３及其於間接三明治ＥＬＩＳＡ抗體檢測之應用
CN113234654A (zh) 一种重组枯草芽孢杆菌及其应用
CN111514287A (zh) 甲型流感通用型dna疫苗及其制备方法和应用
KR20100105974A (ko) 재조합 ｎ 단백질에 대한 단클론 항체를 이용한 리프트계곡열 경합적 효소결합면역측정법
CN116217717A (zh) 一种鸭坦布苏病毒多克隆抗体及其制备方法

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 17620528

Country of ref document: US

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21944740

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE