WO2022089044A1 - 抗新型冠状病毒的抗体、检测新型冠状病毒试剂和试剂盒 - Google Patents

抗新型冠状病毒的抗体、检测新型冠状病毒试剂和试剂盒 Download PDF

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WO2022089044A1
WO2022089044A1 PCT/CN2021/117802 CN2021117802W WO2022089044A1 WO 2022089044 A1 WO2022089044 A1 WO 2022089044A1 CN 2021117802 W CN2021117802 W CN 2021117802W WO 2022089044 A1 WO2022089044 A1 WO 2022089044A1
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cdr
antibody
functional fragment
mutation
novel coronavirus
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PCT/CN2021/117802
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English (en)
French (fr)
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崔鹏
何志强
孟媛
钟冬梅
娄文娟
范凌云
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东莞市朋志生物科技有限公司
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Priority to CA3200148A priority Critical patent/CA3200148A1/en
Priority to KR1020237017479A priority patent/KR20230096021A/ko
Priority to JP2023526469A priority patent/JP2023551103A/ja
Priority to US18/034,394 priority patent/US20230391853A1/en
Publication of WO2022089044A1 publication Critical patent/WO2022089044A1/zh

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    • 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
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • 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
    • 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
    • C07K16/1002—Coronaviridae
    • 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
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host

Definitions

  • the present disclosure relates to the technical field of antibodies, and in particular, to an antibody against novel coronavirus, a novel coronavirus detection reagent, and a kit.
  • the structural proteins of the new coronavirus 2019-nCoV are divided into: spike glycoprotein (S protein), envelope glycoprotein (E protein), membrane glycoprotein (M protein) and nucleocapsid protein (N protein). These proteins include multiple antigenic epitopes.
  • S protein spike glycoprotein
  • E protein envelope glycoprotein
  • M protein membrane glycoprotein
  • N protein nucleocapsid protein
  • the N protein and the viral genome RNA are intertwined to form the viral nucleocapsid, which plays an important role in the synthesis of viral RNA.
  • the N protein is relatively conservative and occupies the largest proportion in the structural proteins of the virus.
  • the body can produce high-level antibodies against the N protein in the early stage of infection.
  • the N protein is an important marker protein of the new coronavirus. Using the principle of specific binding of antigens and antibodies, the presence of the antigen can be detected by the N protein monoclonal antibody, thereby directly proving that the sample contains the new coronavirus and realizing the detection
  • the detected antibodies are mainly divided into two categories: IgM and IgG.
  • IgM antibodies are produced early, and once infected, they are rapidly produced, last for a short time, and disappear quickly.
  • a positive test in the blood can reflect that the body is in an acute infection state and can be used as an indicator of early infection.
  • antibody detection samples are serum or plasma, which are less affected by sample sampling, which is conducive to early diagnosis and exclusion of suspicious cases. At the same time, the detection is fast, convenient, and suitable for large-scale screening.
  • 2019-nCoV Since the outbreak of the novel coronavirus 2019-nCoV pneumonia, it has spread around the world, posing a serious threat to human life and health. Respiratory droplets and close contact transmission are the main transmission routes of novel coronavirus pneumonia, and there is the possibility of aerosol transmission in the case of prolonged exposure to high concentrations of aerosols in a relatively closed environment. 2019-nCoV is highly contagious, and most patients develop clinical symptoms after infection, but some patients are asymptomatic. Common signs of people infected with coronavirus include respiratory symptoms, fever, cough, shortness of breath, and difficulty breathing. In more severe cases, the infection can lead to pneumonia, severe acute respiratory syndrome, kidney failure, and even death. Although there is currently no specific treatment for the disease caused by the new coronavirus, treatment for mild or asymptomatic patients can greatly improve the cure rate and shorten the treatment time. Therefore, the detection or identification of patients becomes particularly important.
  • nucleic acid detection and viral gene sequencing are mainly used as etiological confirmation evidence. Due to various factors such as sampling, operation, and reagents, nucleic acid detection will produce false negative results.
  • the positive detection rate of viral nucleic acid in patients with highly suspected 2019 novel coronavirus (2019-nCoV) infection is only 30% to 50%.
  • nucleic acid testing has high requirements on equipment, testing sites and environmental conditions, and has shortcomings such as long testing time and low throughput, which is not convenient for large-scale testing of people under the current epidemic situation. Therefore, there is an urgent need to develop a rapid and convenient detection kit for clinical detection, so as to isolate the infected population as soon as possible to block the spread of the virus. Therefore, antibody detection kits have become more important.
  • the present disclosure provides an antibody against a novel coronavirus or its N protein or a functional fragment thereof, the antibody or functional fragment thereof comprising the following complementarity determining regions:
  • CDR-VH1 G-X1-T-F-S-X2-F-X3-M-H; where: X1 is V or F; X2 is S or T; X3 is G or A;
  • CDR-VH2 Y-X1-N-S-X2-S-N-X3-I-Y-Y-A-D-T-X4-K; where: X1 is L or I; X2 is G or A; X2 is I, V or L; X3 is I, V or L;
  • CDR-VH3 X1-R-H-X2-M; wherein: X1 is A or T; X2 is A or V;
  • CDR-VL1 S-Q-S-X1-D-Y-X2-G-D-S-X3-M; where: X1 is I, V or L; X2 is D or N; X3 is F or Y;
  • CDR-VL2 X1-A-S-N-X2-E-S; where: X1 is A or D; X2 is I, V or L;
  • CDR-VL3 Q-X1-S-N-E-X2-P-Y; wherein: X1 is N, H or Q; X2 is D or E.
  • X1 is F
  • X1 is I
  • X1 is A
  • X3 is Y.
  • X2 is S.
  • X2 is T.
  • X3 is G.
  • X3 is A.
  • X2 is G.
  • X2 is A in the CDR-VH2.
  • X3 is 1.
  • X3 is V.
  • X3 is L.
  • X4 is 1.
  • X4 is V.
  • X4 is L.
  • X2 is A.
  • X2 is V.
  • X1 is 1.
  • X1 is V.
  • X1 is L.
  • X2 is D in CDR-VL1.
  • X2 is N.
  • X1 is A in CDR-VL2.
  • X1 is D.
  • X2 is 1.
  • X2 is V.
  • X2 is L.
  • X1 is N.
  • X1 is H.
  • X1 is Q.
  • X2 is D.
  • X2 is E.
  • each complementarity determining region of the antibody or functional fragment thereof is selected from any one of the following mutation combinations 1-68:
  • the antibody or its functional fragment binds to the N protein of the novel coronavirus with an affinity of K D ⁇ 8 ⁇ 10 -9 mol/L. In some embodiments, K D ⁇ 7 ⁇ 10 ⁇ 10 mol/L.
  • X1 is V
  • X1 is L
  • X1 is T
  • X3 is F.
  • each complementarity determining region of the antibody or functional fragment thereof is selected from any one of the following mutation combinations 69-76:
  • the antibody comprises light chain framework regions FR1-L, FR2-L, FR3-L and FR1-L, FR2-L, FR3-L, and FR4-L, and/or, heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H, FR2-H, FR3-H, and FR4-H having at least 80% homology with sequences of SEQ ID NOs: 5, 6, 7, 8, respectively H.
  • amino acid sequence of FR1-H is set forth in SEQ ID NO:15.
  • the antibody further comprises a constant region.
  • the constant region is selected from the constant region of any one of IgGl, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.
  • the species source of the constant region is bovine, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck , goose, turkey, cockfight or man.
  • the constant region is derived from a mouse.
  • the light chain constant region sequence of the constant region is set forth in SEQ ID NO:9 or has at least 80% homology
  • the heavy chain constant region sequence of the constant region is set forth in SEQ ID NO:10 shown or have at least 80% homology.
  • the heavy chain constant region sequence is set forth in SEQ ID NO:16.
  • the functional fragment is selected from any one of VHH, F(ab')2, Fab', Fab, Fv and scFv of the antibody.
  • the present disclosure provides a reagent or kit for detecting a novel coronavirus or its N protein, which includes the antibody or functional fragment thereof described in any one of the above.
  • the antibody or functional fragment thereof is labeled with a detectable label.
  • the detectable label is selected from the group consisting of fluorescent dyes, enzymes that catalyze color development of substrates, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.
  • the fluorescent dye is selected from fluorescein dyes and derivatives thereof, rhodamine dyes and derivatives thereof, Cy series dyes and derivatives thereof, Alexa series dyes and derivatives thereof, and protein dyes and its derivatives.
  • the enzyme that catalyzes coloration of the substrate is selected from the group consisting of horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6- Phosphoglucose deoxygenase.
  • the radioisotope is selected from212Bi , 131I , 111In , 90Y , 186Re , 211At , 125I , 188Re , 153Sm , 213Bi , 32P , 94mTc ,99mTc , 203 Pb, 67 Ga, 68 Ga, 43 Sc, 47 Sc, 110 mIn, 97 Ru, 62 Cu, 64 Cu, 67 Cu, 68 Cu, 86 Y, 88 Y, 121 Sn, 161 Tb, 166 Ho, 105 Rh, 177 Lu, 172 Lu, and 18 F.
  • the chemiluminescent reagent is selected from the group consisting of luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine esters and its derivatives, Oxetane and its derivatives, Lofenine and its derivatives, and peroxyoxalate and its derivatives.
  • the nanoparticle-based label is selected from the group consisting of nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.
  • the colloid is selected from the group consisting of colloidal metals, disperse dyes, dye-labeled microspheres, and latex.
  • the colloidal metal is selected from colloidal gold, colloidal silver, and colloidal selenium.
  • the present disclosure provides a nucleic acid molecule encoding the antibody or functional fragment thereof of any one of the above.
  • the present disclosure provides a vector containing a nucleic acid fragment encoding the antibody or functional fragment thereof according to any one of the above.
  • the present disclosure provides a recombinant cell containing the vector.
  • the present disclosure provides the use of the antibody or its functional fragment, the reagent or the kit in detecting novel coronavirus.
  • the present disclosure provides the use of the antibody or its functional fragment, the reagent or the kit for detecting novel coronavirus.
  • the present disclosure provides a method for detecting a novel coronavirus, comprising:
  • the present disclosure provides a method for diagnosing a subject infected with a novel coronavirus or a disease associated with the novel coronavirus infection, comprising:
  • the disease associated with novel coronavirus infection includes respiratory symptoms, fever, cough, shortness of breath, dyspnea, pneumonia, severe acute respiratory syndrome, renal failure.
  • the subjects infected with the novel coronavirus include asymptomatic infected persons, asymptomatic infected persons, and symptomatic infected persons.
  • the present disclosure provides a method for preparing the antibody or functional fragment thereof according to any one of the above, comprising: culturing the recombinant cell, and separating and purifying the antibody or the functional fragment thereof from the culture product.
  • FIG. 1 shows the results of reducing SDS-PAGE of the anti-novel coronavirus antibody of Example 1.
  • the term "complementarity determining regions” means: an intact or complete antibody comprises two heavy chains and two light chains; each heavy chain comprises a variable region (VH) and a constant region (CH); each The light chain contains a variable region (VL) and a constant region (CL); the antibody has a "Y" shape with the stem of the Y consisting of the second and third of the two heavy chains held together by disulfide bonds
  • the constant region consists of; each arm of Y includes the variable region and the first constant region of a single heavy chain combined with the variable region and constant region of a single light chain; the variable region and heavy chain of the light chain
  • the variable regions of the are responsible for antigen binding; the variable regions in both chains typically contain three hypervariable regions, called complementarity determining regions.
  • the term "functional fragment” is intended to refer to a portion of an antibody comprising a heavy or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment was derived.
  • These functional fragments may include, for example, Fd, Fv, Fab, F(ab'), F(ab)2, F(ab')2, single chain Fv (scFv), diabodies, triple chain Antibodies (triabodies), tetrabodies (tetrabodies) and minibodies (minibodies).
  • Other functional fragments may include, for example, heavy or light chain polypeptides, variable region polypeptides or CDR polypeptides or portions thereof, so long as these functional fragments retain binding activity.
  • constant region refers to a relatively stable region of the light and heavy chains of an antibody molecule near the C-terminal amino acid sequence.
  • variable region refers to the region of the light and heavy chains of an antibody molecule that varies greatly in amino acid sequence near the N-terminus.
  • naked anti-stable refers to an antibody or functional fragment thereof that is not labeled, eg, an antibody or functional fragment thereof that is not labeled with a detectable label.
  • Some embodiments of the present disclosure provide antibodies against novel coronaviruses, reagents and kits for detecting novel coronaviruses, the antibodies can specifically bind to the N protein of novel coronaviruses and have a high affinity for them, and the antibodies are used to detect novel coronaviruses Coronaviruses have better sensitivity and specificity.
  • the present disclosure provides a richer selection of antibodies for the detection of novel coronaviruses.
  • One embodiment of the present disclosure provides an antibody against a novel coronavirus or its N protein or a functional fragment thereof, wherein the antibody or functional fragment thereof has the following complementarity determining regions:
  • CDR-VH1 G-X1-T-F-S-X2-F-X3-M-H; where: X1 is V or F; X2 is S or T; X3 is G or A;
  • CDR-VH2 Y-X1-N-S-X2-S-N-X3-I-Y-Y-A-D-T-X4-K; where: X1 is L or I; X2 is G or A; X2 is I, V or L; X3 is I, V or L;
  • CDR-VH3 X1-R-H-X2-M; wherein: X1 is A or T; X2 is A or V;
  • CDR-VL1 S-Q-S-X1-D-Y-X2-G-D-S-X3-M; where: X1 is I, V or L; X2 is D or N; X3 is F or Y;
  • CDR-VL2 X1-A-S-N-X2-E-S; where: X1 is A or D; X2 is I, V or L;
  • CDR-VL3 Q-X1-S-N-E-X2-P-Y; wherein: X1 is N, H or Q; X2 is D or E.
  • the anti-novel coronavirus antibody or its functional fragment provided by the present disclosure has the above-mentioned complementarity determining region structure, and the above-mentioned complementarity determining region structure enables the antibody or its functional fragment to specifically bind to the anti-novel coronavirus N protein, and its It has good affinity, and the antibody or its functional fragment is used to detect novel coronavirus with good specificity and sensitivity.
  • the present disclosure provides a richer selection of antibodies for the detection of novel coronaviruses.
  • X1 in CDR-VH1, X1 is F; in CDR-VH2, X1 is I; in CDR-VH3, X1 is A; and in CDR-VL1, X3 is Y.
  • X2 is S.
  • X2 is T.
  • X3 is G.
  • X3 is A in CDR-VH1.
  • X2 is G.
  • X2 is A in CDR-VH2.
  • X3 is 1.
  • X3 is V.
  • X3 is L.
  • X4 is 1.
  • X4 is V.
  • X4 is L.
  • X2 is A in the CDR-VH3.
  • X2 is V.
  • X1 is I.
  • X1 is V.
  • X1 is L.
  • X2 is D in CDR-VL1.
  • X2 is N.
  • X1 is A.
  • X1 is D.
  • X2 is 1.
  • X2 is V.
  • X2 is L.
  • X1 is N.
  • X1 is H.
  • X1 is Q.
  • X2 is D.
  • X2 is E.
  • each complementarity determining region of the antibody or functional fragment thereof is selected from any one of the following mutation combinations 1-68:
  • the antibody or its functional fragment binds to the anti-novel coronavirus N protein with an affinity of K D ⁇ 8 ⁇ 10 -9 mol/L.
  • K D ⁇ 7 ⁇ 10 ⁇ 10 mol/L.
  • K D The detection of K D is performed with reference to the methods in the embodiments of the present disclosure.
  • X1 in CDR-VH1, X1 is V; in CDR-VH2, X1 is L; in CDR-VH3, X1 is T; in CDR-VL1, X3 is F.
  • each complementarity determining region of the antibody or functional fragment thereof is selected from any one of the following mutation combinations 69-76:
  • the antibody comprises the light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L, and/or the sequences of the light chain framework regions shown in SEQ ID NOs: 1-4 in sequence.
  • variable region (VH) of the heavy chain and the variable region (VL) of the light chain can be obtained by linking the following numbered CDRs and FRs in the following combinations: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the antibody comprises light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L, FR2-L, FR3-L and FR4- L, and/or, heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H having at least 80% homology with sequences of SEQ ID NOs: 5, 6, 7, 8, respectively.
  • the antibody comprises light chain framework regions FR1-L, FR2-L, FR3-L, and FR4-L that are at least 80% identical to the sequences of SEQ ID NOs: 1, 2, 3, 4, respectively, in order , and/or, heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H having at least 80% identity to the sequences of SEQ ID NOs: 5, 6, 7, 8, respectively.
  • each framework region of the antibody or its functional fragment provided by the present disclosure may be the same as the above-mentioned corresponding framework region (SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 or 8) may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology.
  • the amino acid sequence of FR1-H can also be SEQ ID NO:15.
  • the antibody further comprises a constant region.
  • the constant region is selected from the constant region of any one of IgGl, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD.
  • the species source of the constant region is mammalian or poultry.
  • mammals include cows, horses, dairy cows, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, minks, or humans.
  • poultry animals include chickens, ducks, geese, turkeys or fighting cocks.
  • the species source of the constant region is bovine, equine, dairy cattle, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken , duck, goose, turkey, cockfight or man.
  • the constant region is derived from a mouse.
  • the light chain constant region (CL) sequence of the constant region is shown in SEQ ID NO:9
  • the heavy chain constant region (CH) sequence of the constant region is shown in SEQ ID NO:10 Show.
  • the constant region sequence of the antibody provided by the present disclosure may have at least 80%, 81%, 82%, 83%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology sex.
  • the heavy chain constant region can also be SEQ ID NO:16.
  • the functional fragment is selected from any one of VHH, F(ab')2, Fab', Fab, Fv and scFv of the antibody.
  • Functional fragments of the above antibodies generally have the same binding specificity as the antibody from which they were derived.
  • the functional fragments of the above-mentioned antibodies can be cleaved by methods including but not limited to enzymatic digestion (including but not limited to pepsin or papain) and/or by chemical reduction Sulfur bond method.
  • enzymatic digestion including but not limited to pepsin or papain
  • chemical reduction Sulfur bond method On the basis of the structure of the complete antibody provided in the present disclosure, those skilled in the art can easily obtain the above-mentioned functional fragments.
  • Functional fragments of the above-described antibodies can also be obtained by recombinant genetic techniques, also known to those skilled in the art, or by, for example, automated peptide synthesizers such as those sold by including, but not limited to, Applied BioSystems and the like.
  • One embodiment of the present disclosure provides a reagent or kit for detecting a novel coronavirus or its N protein, which includes the antibody or its functional fragment as described in any of the above.
  • the antibody or functional fragment thereof in the above reagent or kit is labeled with a detectable label.
  • detectable label refers to a class of substances having properties that can be directly observed by the naked eye or detected or detected by instruments, such as luminescence, coloration, radioactivity, etc., by which the corresponding Qualitative or quantitative detection of a target.
  • the detectable labels include, but are not limited to, fluorescent dyes, enzymes that catalyze color development of substrates, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.
  • the fluorescent dyes include, but are not limited to, fluorescein dyes and derivatives thereof (for example, including but not limited to fluorescein isothiocyanate (FITC) hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or its analogs), rhodamine dyes and their derivatives (such as, but not limited to, red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or the like compounds), Cy series dyes and their derivatives (such as but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy3, etc.
  • fluorescein dyes and derivatives thereof for example, including but not limited to fluorescein isothiocyanate (FITC) hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or its analogs
  • Alexa series dyes and their derivatives such as including But not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs
  • protein dyes and their derivatives for example, including but Not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydinoxanthin-chlorophyll protein (preCP), etc.
  • the enzymes that catalyze the coloration of the substrate include but are not limited to horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase enzyme and glucose 6-phosphate deoxygenase.
  • the radioisotopes include but are not limited to 212Bi , 131I , 111In , 90Y , 186Re , 211At , 125I , 188Re , 153Sm , 213Bi , 32P , 94 mTc, 99 mTc, 203 Pb, 67 Ga, 68 Ga, 43 Sc, 47 Sc, 110 mIn, 97 Ru, 62 Cu, 64 Cu, 67 Cu, 68 Cu, 86 Y, 88 Y, 121 Sn, 161 Tb, 166 Ho, 105 Rh, 177 Lu, 172 Lu and 18 F.
  • the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine esters and its derivatives Derivatives, Dioxetane and its Derivatives, Lopine and its Derivatives and Peroxyoxalate and its Derivatives.
  • the nanoparticle-based labels include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.
  • the colloids include, but are not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.
  • the colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.
  • An embodiment of the present disclosure provides a nucleic acid molecule encoding the above-mentioned antibody or a functional fragment thereof.
  • One embodiment of the present disclosure provides a vector containing the above-mentioned nucleic acid molecule.
  • One embodiment of the present disclosure provides a recombinant cell containing the above-mentioned vector.
  • One embodiment of the present disclosure provides a method for preparing an antibody or a functional fragment thereof, comprising: culturing the above-mentioned recombinant cells, and separating and purifying the antibody or its functional fragment from the cultured product.
  • restriction endonuclease and Prime Star DNA polymerase were purchased from Takara Company.
  • MagExtractor-RNA extraction kit was purchased from TOYOBO Company.
  • BD SMART TM RACE cDNA Amplification Kit was purchased from Takara Company.
  • the pMD-18T vector was purchased from Takara Company.
  • Plasmid extraction kit was purchased from Tiangen Company. Primer synthesis and gene sequencing were performed by Invitrogen.
  • the mRNA was extracted from the hybridoma cell line that secreted the antibody against the new coronavirus N protein, and the DNA product was obtained by RT-PCR method.
  • the heavy chain (Heavy Chain) and the light chain (Light Chain) gene clones were taken after the colonies grew, and 4 clones were sent to a gene sequencing company for sequencing.
  • the gene sequences obtained by the above sequencing were placed in the IMGT antibody database (IMGT antibody database from: http://www.imgt.org) for analysis, and the VNTI11.5 software was used for analysis to determine the amplification of heavy chain and light chain primer pairs.
  • the added genes are all correct.
  • the VL gene sequence is 342 bp, belonging to the VkII gene family, and there is a 57 bp leader peptide sequence in front of it;
  • the gene fragment amplified by the heavy chain primer pair Among them, the VH gene sequence is 336 bp, belonging to the VH1 gene family, and there is a 57 bp leader peptide sequence in front of it.
  • pcDNATM 3.4 vector is the constructed recombinant antibody eukaryotic expression vector.
  • the expression vector has introduced polyclonal restriction sites such as HindIII, BamHI and EcoRI, and is named pcDNA3.4A expression vector, hereinafter referred to as 3.4A expression vector; according to the above pMD-18T
  • 3.4A expression vector pcDNA3.4A expression vector
  • the VL and VH gene-specific primers of the antibody were designed, with HindIII and EcoRI restriction sites and protective bases at both ends, respectively, and a 0.72kb light chain was amplified by PCR amplification method. Gene fragment and 1.41kb heavy chain gene fragment.
  • the heavy chain and light chain gene fragments were digested with HindIII/EcoRI double enzymes respectively, and the 3.4A vector was digested with HindIII/EcoRI double enzymes. After the fragments and vectors were purified and recovered, the heavy chain genes and light chain genes were connected to the 3.4A expression vector to obtain the heavy chain. Chain and light chain recombinant expression plasmids.
  • the recombinant antibody expression plasmid was transiently transfected into CHO cells to determine the activity of the expression plasmid
  • plasmid obtained in step 1-(3) Dilute the plasmid obtained in step 1-(3) to 400ng/ml with ultrapure water, adjust CHO cells to 1.43 ⁇ 10 7 cells/ml in a centrifuge tube, mix 100 ⁇ L of plasmid with 700 ⁇ L of cells, transfer into an electroporation cup, and electroporate, The samples were counted on the 3rd, 5th, and 7th days, and the samples were collected and tested on the 7th day.
  • the coating solution (the main component NaHCO 3 ) diluted the 2019-nCoV N protein antigen to 1 ⁇ g/ml, 100 ⁇ L per well, overnight at 4°C; the next day, the washing solution was washed twice and patted dry; the blocking solution (20% BSA+80 %PBS), 120 ⁇ L per well, 37°C, 1h, pat dry; add diluted cell supernatant, 100 ⁇ L/well, 37°C, 30min (partial supernatant for 1h); wash 5 times with washing solution, pat dry; add sheep Anti-mouse IgG-HRP (where HPR is labeled with horseradish peroxidase), 100 ⁇ L per well, 37°C, 30 min; wash 5 times with washing solution, pat dry; add chromogenic solution A (50 ⁇ L/well, containing citric acid) + sodium acetate + acetanilide + carbamide peroxide), add color developer solution B (50 ⁇ L/well, containing citric acid + EDTA
  • reaction OD was still greater than 1.0 after the cell supernatant was diluted 1000 times, and the reaction OD of the unadded cell supernatant was less than 0.1, indicating that the antibody generated after transient transfection of the plasmid was active against the 2019-nCoV N protein antigen.
  • step 2-(2) Dilute the plasmid obtained in step 2-(2) with ultrapure water to 400ng/ml, adjust CHO cells to 1.43 ⁇ 10 7 cells/ml in a centrifuge tube, mix 100 ⁇ L of plasmid with 700 ⁇ L of cells, transfer into an electroporation cup, and electroporate, Count the next day; 25umol/L MSX 96-well pressurized culture for about 25 days.
  • the cells obtained in step 2-(3) were recovered by passage, they were first cultured in a 125ml shake flask, the inoculation volume was 30ml, and the medium was 100% Dynamis medium, placed at a rotating speed of 120r/min and a temperature of 37 °C in a shaker with 8% carbon dioxide. After culturing for 72 hours, inoculate and expand the culture at an inoculation density of 500,000 cells/ml, and the expansion volume is calculated according to the production demand, and the medium is 100% Dynamis medium. After that, the culture was expanded every 72h. When the cell volume meets the production requirements, the seeding density is strictly controlled to be about 500,000 cells/ml for production.
  • Shaking flask parameters rotating speed 120r/min, temperature 37°C, carbon dioxide 8%.
  • Feed feeding start feeding every day after culturing in the shake flask for 72 hours.
  • HyClone Cell Boost Feed 7a is fed 3% of the initial culture volume every day, and Feed 7b is fed daily at 1/1000 of the initial culture volume. Supplement until the 12th day (feeding on the 12th day).
  • Glucose was supplemented with 3 g/L on the sixth day. Samples were collected on the 13th day.
  • Affinity purification was performed using a protein A affinity chromatography column. Take 4 ⁇ g of purified antibody for reducing SDS-PAGE, and 4 ⁇ g foreign control antibody as control.
  • the electropherogram is shown in Figure 1 below. After reducing SDS-PAGE, two bands are displayed, and one Mr is 50KD (heavy chain, SEQ ID NO: 14), the other Mr is 28KD (light chain, SEQ ID NO: 13).
  • the antibody (WT) sequence of Example 1 was analyzed, and its heavy chain variable region was shown in SEQ ID NO: 12, wherein the amino acid sequence of each complementarity determining region on the heavy chain variable region was as follows:
  • CDR-VH1 G-V(X1)-T-F-S-T(X2)-F-A(X3)-M-H;
  • CDR-VH2 Y-L(X1)-N-S-A(X2)-S-N-L(X3)-I-Y-Y-A-D-T-L(X4)-K;
  • CDR1-VL S-Q-S-I(X1)-D-Y-D(X2)-G-D-S-F(X3)-M;
  • CDR-VL2 D(X1)-A-S-N-V(X2)-E-S;
  • CDR-VL3 Q-H(X1)-S-N-E-D(X2)-P-Y.
  • the coating solution (the main component NaHCO 3 ) diluted the 2019-nCoV N protein antigen to 1 ⁇ g/ml for microplate coating, 100 ⁇ l per well, overnight at 4°C; the next day, the washing solution was washed twice and patted dry; the blocking solution was added (20%BSA+80%PBS), 120 ⁇ l per well, 37°C, 1h, pat dry; add the diluted monoclonal antibody in Table 1, 100 ⁇ l/well, 37°C, 30min-60min; wash 5 times with washing solution , pat dry; add goat anti-mouse IgG-HRP, 100 ⁇ l per well, 37°C, 30 min; wash 5 times with washing solution (PBS), pat dry; add chromogenic solution A (50 ⁇ l/well, containing 2.1 g/L lemon acid, 12.25g/L citric acid, 0.07g/L acetanilide and 0.5g/L carbamide peroxide), add color developing solution B (50 ⁇ l/well, containing 1.05g/L
  • the purified antibody was diluted to 10 ⁇ g/mL with PBST (phosphate Tween buffer, the main component Na 2 HPO 4 +NaCl+TW-20), and the 2019-nCoV N protein antigen was serially diluted with PBST: 1.41 ⁇ g/mL, 0.70 ⁇ g/mL, 0.35 ⁇ g/mL, 0.18 ⁇ g/mL, 0.09 ⁇ g/mL, 0.04 ⁇ g/mL.
  • PBST phosphate Tween buffer, the main component Na 2 HPO 4 +NaCl+TW-20
  • Running process equilibrate in buffer 1 (PBST) for 60s, immobilize antibody in antibody solution for 300s, incubate in buffer 2 (PBST) for 180s, bind in antigen solution for 420s, dissociate in buffer 2 for 1200s, use 10mM pH 1.69 GLY solution and buffer 3 to regenerate the sensor and output data.
  • K D is the equilibrium dissociation constant or affinity; kon is the association rate; kdis is the dissociation rate.
  • the above antibodies were placed at 4°C (refrigerator), -80°C (refrigerator), and 37°C (incubator) for 21 days, and the 7-day, 14-day, and 21-day samples were taken for state observation, and the 21-day sample was tested for activity. , the results showed that there was no obvious change in the protein state of the antibodies under the three test conditions for 21 days, and the activity did not show a downward trend with the increase of the test temperature, indicating that the above antibodies were stable.
  • the following table 7 mutation 1 antibody is the OD results of the enzyme immunoassay activity detection for 21 days.
  • the present disclosure provides an antibody against the new coronavirus, a reagent and a kit for detecting the new coronavirus, the antibody can specifically bind to the N protein of the new coronavirus, and has a high affinity for it, and the antibody can be used to detect the new coronavirus. Good sensitivity and specificity.
  • the present disclosure provides a richer selection of antibodies for the detection of novel coronaviruses.
  • the detection kit provided by the present disclosure also has the same technical effect as the antibody, and has broad application prospects and high market value.

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Abstract

一种抗新型冠状病毒的抗体、检测新型冠状病毒试剂和试剂盒，涉及抗体技术领域。抗新型冠状病毒的抗体包括重链互补决定区和轻链互补决定区。该抗体对新型冠状病毒的N蛋白的亲和力较好，使用该抗体检测新型冠状病毒具有较好的灵敏度和特异性。为新型冠状病毒的检测提供了抗体选择。

Description

抗新型冠状病毒的抗体、检测新型冠状病毒试剂和试剂盒
相关申请的交叉引用
本公开要求于2020年10月29日提交中国专利局的申请号为“202011182628.X”名称为“抗新型冠状病毒的抗体、检测新型冠状病毒试剂和试剂盒”的中国专利申请的优先权，其全部内容通过引用结合在本公开中。
技术领域
本公开涉及抗体技术领域，具体而言，涉及一种抗新型冠状病毒的抗体、检测新型冠状病毒试剂和试剂盒。
背景技术
新型冠状病毒2019-nCoV的结构蛋白分为：刺突糖蛋白(S蛋白)、包膜糖蛋白(E蛋白)、膜糖蛋白(M蛋白)和核衣壳蛋白(N蛋白)，这些蛋白包括多个抗原表位。N蛋白与病毒基因组RNA相互缠绕形成病毒核衣壳，在病毒RNA的合成过程中发挥着重要的作用。同时，N蛋白相对保守，在病毒的结构蛋白中所占比例最大，感染早期机体就能产生抗N蛋白的高水平抗体。最后，N蛋白是新型冠状病毒重要的标志蛋白，利用抗原与抗体特异性结合的原理，可通过N蛋白单克隆抗体检测抗原的存在，从而直接证明样本中含有新型冠状病毒，实现新型冠状病毒的检测。
检测的抗体主要分为IgM和IgG两类。目前对新型冠状病毒的这两类抗体的产生和持续时间尚缺乏系统性研究。通常情况下，IgM抗体产生早，一经感染，快速产生，维持时间短，消失快，血液中检测阳性可反应机体处于急性感染状态，可作为早期感染的指标。与核酸检测方法相比，抗体检测样本为血清或血浆，受样本采样的影响较小，有利于早期诊断和排除可疑病例，同时检测快速、方便、适合大规模筛查。
新型冠状病毒2019-nCoV肺炎疫情发生以来，在全球蔓延，严重威胁着人类的生命安全和身体健康。呼吸道飞沫和密切接触传播是新型冠状病毒肺炎主要的传播途径，在相对封闭的环境中长时间暴露于高浓度气溶胶情况中存在经气溶胶传播的可能。2019-nCoV具有很强的传染性，多数患者感染后出现临床症状，但也有部分患者为无症状感染者。人感染了冠状病毒后常见体征有呼吸道症状、发热、咳嗽、气促和呼吸困难等。在较严重病例中，感染可导致肺炎、严重急性呼吸综合征、肾衰竭，甚至死亡。虽然目前对于新型冠状病毒所致疾病没有特异治疗方法，但是轻症、或者无症状患者通过治疗可以大大的提高治愈率，缩短治疗时间。因此，对患者的检测或者鉴定变的尤为重要。
目前主要以核酸检测和病毒基因测序为病原学确诊证据，由于采样、操作以及试剂等多方面因素影响，核酸检测会出现假阴性结果。高度疑似2019新型冠状病毒(2019-nCoV)感染患者的病毒核酸阳性检出率仅为30％～50％。另外，核酸检测对仪器设备、检测场地及环境条件要求高，存在检测时间长、通量低等缺点，不便于目前疫情下的人群大规模检测。因此，迫切需要研发出一种快速便捷检测试剂盒用于临床检测，从而尽快隔离感染人群以阻断病毒传播。因此，抗体检测试剂盒变得更为重要。
目前2019-nCoV N蛋白单克隆抗体产品较少，性能存在差异。
发明内容
本公开提供一种抗新型冠状病毒或其N蛋白的抗体或其功能性片段，所述抗体或其功能性片段包括如下互补决定区：
CDR-VH1：G-X1-T-F-S-X2-F-X3-M-H；其中：X1是V或F；X2是S或T；X3是G或A；
CDR-VH2：Y-X1-N-S-X2-S-N-X3-I-Y-Y-A-D-T-X4-K；其中：X1是L或I；X2是G或A；X2是I、V或L；X3是I、V或L；
CDR-VH3：X1-R-H-X2-M；其中：X1是A或T；X2是A或V；
CDR-VL1：S-Q-S-X1-D-Y-X2-G-D-S-X3-M；其中：X1是I、V或L；X2是D或N；X3是F或Y；
CDR-VL2：X1-A-S-N-X2-E-S；其中：X1是A或D；X2是I、V或L；
CDR-VL3：Q-X1-S-N-E-X2-P-Y；其中：X1是N、H或Q；X2是D或E。
在一些实施方式中，
CDR-VH1中，X1是F；
CDR-VH2中，X1是I；
CDR-VH3中，X1是A；
CDR-VL1中，X3是Y。
在一些实施方式中，CDR-VH1中，X2是S。
在一些实施方式中，CDR-VH1中，X2是T。
在一些实施方式中，CDR-VH1中，X3是G。
在一些实施方式中，CDR-VH1中，X3是A。
在一些实施方式中，CDR-VH2中，X2是G。
在一些实施方式中，CDR-VH2中，X2是A。
在一些实施方式中，CDR-VH2中，X3是I。
在一些实施方式中，CDR-VH2中，X3是V。
在一些实施方式中，CDR-VH2中，X3是L。
在一些实施方式中，CDR-VH2中，X4是I。
在一些实施方式中，CDR-VH2中，X4是V。
在一些实施方式中，CDR-VH2中，X4是L。
在一些实施方式中，CDR-VH3中，X2是A。
在一些实施方式中，CDR-VH3中，X2是V。
在一些实施方式中，CDR-VL1中，X1是I。
在一些实施方式中，CDR-VL1中，X1是V。
在一些实施方式中，CDR-VL1中，X1是L。
在一些实施方式中，CDR-VL1中，X2是D。
在一些实施方式中，CDR-VL1中，X2是N。
在一些实施方式中，CDR-VL2中，X1是A。
在一些实施方式中，CDR-VL2中，X1是D。
在一些实施方式中，CDR-VL2中，X2是I。
在一些实施方式中，CDR-VL2中，X2是V。
在一些实施方式中，CDR-VL2中，X2是L。
在一些实施方式中，CDR-VL1中，X1是N。
在一些实施方式中，CDR-VL1中，X1是H。
在一些实施方式中，CDR-VL1中，X1是Q。
在一些实施方式中，CDR-VL2中，X2是D。
在一些实施方式中，CDR-VL2中，X2是E。
在一些实施方式中，所述抗体或其功能性片段的各互补决定区选自如下突变组合1-68中的任意一种：
Figure PCTCN2021117802-appb-000001
Figure PCTCN2021117802-appb-000002
Figure PCTCN2021117802-appb-000003
在一些实施方式中，所述抗体或其功能性片段与新型冠状病毒的N蛋白以K D≤8×10 -9mol/L的亲和力结合。在一些实施方式中，K D≤7×10 -10mol/L。
在一些实施方式中，
CDR-VH1中，X1是V；
CDR-VH2中，X1是L；
CDR-VH3中，X1是T；
CDR-VL1中，X3是F。
在一些实施方式中，所述抗体或其功能性片段的各互补决定区选自如下突变组合69-76中的任意一种：
Figure PCTCN2021117802-appb-000004
在一些实施方式中，所述抗体包括分别依次与序列SEQ ID NO:1、2、3、4具有至少80％的同源性的轻链骨架区FR1-L、FR2-L、FR3-L及FR4-L，和/或，分别依次与序列SEQ ID NO:5、6、7、8具有至少80％的同源性的重链骨架区FR1-H、FR2-H、FR3-H及FR4-H。
在一些实施方式中，所述FR1-H的氨基酸序列如SEQ ID NO:15所示。
在一些实施方式中，所述抗体还包含恒定区。
在一些实施方式中，所述恒定区选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE和IgD中的任意一者的恒定区。
在一些实施方式中，所述恒定区的种属来源为牛、马、乳牛、猪、绵羊、山羊、大鼠、小鼠、狗、猫、兔、骆驼、驴、鹿、貂、鸡、鸭、鹅、火鸡、斗鸡或人。
在一些实施方式中，所述恒定区来源于小鼠。
在一些实施方式中，所述恒定区的轻链恒定区序列如SEQ ID NO:9所示或其具有至少80％同源性，所述恒定区的重链恒定区序列如SEQ ID NO:10所示或其具有至少80％同源性。
在一些实施方式中，所述重链恒定区序列如SEQ ID NO:16所示。
在一些实施方式中，所述功能性片段选自所述抗体的VHH、F(ab’)2、Fab’、Fab、Fv和scFv中的任意一种。
本公开提供一种检测新型冠状病毒或其N蛋白的试剂或试剂盒，其包括上述任一项所述的抗体或其功能性片段。
在一些实施方式中，所述抗体或其功能性片段标记有可被检测的标记物。
在一些实施方式中，所述可被检测的标记物选自荧光染料、催化底物显色的酶、放射性同位素、化学发光试剂和纳米颗粒类标记物。
在一些实施方式中，所述荧光染料选自荧光素类染料及其衍生物、罗丹明类染料及其衍生物、Cy系列染料及其衍生物、Alexa系列染料及其衍生物和蛋白类染料及其衍生物。
在一些实施方式中，所述催化底物显色的酶选自辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、葡萄糖氧化酶、碳酸酐酶、乙酰胆碱酯酶以及6-磷酸葡萄糖脱氧酶。
在一些实施方式中，所述放射性同位素选自 212Bi、 131I、 111In、 90Y、 186Re、 211At、 125I、 188Re、 153Sm、 213Bi、 32P、 94mTc、 99mTc、 203Pb、 67Ga、 68Ga、 43Sc、 47Sc、 110mIn、 97Ru、 62Cu、 64Cu、 67Cu、 68Cu、 86Y、 88Y、 121Sn、 161Tb、 166Ho、 105Rh、 177Lu、 172Lu和 18F。
在一些实施方式中，所述化学发光试剂选自鲁米诺及其衍生物、光泽精、甲壳动物荧光素及其衍生物、联吡啶钌及其衍生物、吖啶酯及其衍生物、二氧环乙烷及其衍生物、洛粉碱及其衍生物和过氧草酸盐及其衍生物。
在一些实施方式中，所述纳米颗粒类标记物选自纳米颗粒、胶体、有机纳米颗粒、磁性纳米颗粒、量子点纳米颗粒和稀土络合物纳米颗粒。
在一些实施方式中，所述胶体选自胶体金属、分散型染料、染料标记的微球和乳胶。
在一些实施方式中，所述胶体金属选自胶体金、胶体银和胶体硒。
本公开提供一种核酸分子，所述核酸分子编码上述任一项所述的抗体或其功能性片段。
本公开提供一种载体，其含有编码如上述任一项所述的抗体或其功能性片段的核酸片段。
本公开提供一种重组细胞，其含有所述载体。
本公开提供所述抗体或其功能性片段、所述试剂或试剂盒在检测新型冠状病毒中的用途。
本公开提供所述抗体或其功能性片段、所述试剂或试剂盒，用于检测新型冠状病毒的用途。
本公开提供一种检测新型冠状病毒中的方法，包括：
A)在足以发生结合反应的条件下，使上述任一项所述的抗体或其功能性片段与样品接触以进行结合反应；以及
B)检测结合反应产生的免疫复合物。
本公开提供一种诊断受试者在感染新型冠状病毒或与新型冠状病毒感染相关疾病中的方法，包括：
A)在足以发生结合反应的条件下，使上述任一项所述的抗体或其功能性片段与来自所述受试者的样品接触以进行结合反应；以及
B)检测结合反应产生的免疫复合物。
在一些实施方式中，所述与新型冠状病毒感染相关的疾病包括呼吸道症状、发热、咳嗽、气促、呼吸困难、肺炎、严重急性呼吸综合征、肾衰竭。
在一些实施方式中，所述感染新型冠状病毒的受试者包括无症状感染者、无明显症状感染者、有症状感染者。
本公开提供一种制备上述任一项所述的抗体或其功能性片段的方法，其包括：培养所述重组细胞，从培养产物中分离纯化得到所述抗体或其功能性片段。
附图说明
为了更清楚地说明本公开实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本公开的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1为实施例1的抗新型冠状病毒的抗体的还原性SDS-PAGE的结果。
具体实施方式
为使本公开实施例的目的、技术方案和优点更加清楚，下面将对本公开实施例中的技术方案进行清楚、完整地描述。以下实施方式和实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
除非另有定义，否则本文使用的所有技术和科学术语具有与本公开内容所属领域的普通技术人员通常理解的含义相同的含义。尽管与本文描述的那些方法和材料类似或等同的任何方法和材料都可用于本文的制剂或单位剂量的实践或测试，但现在描述一些方法和材料。除非另有说明，否则本文采用或考虑的技术是标准方法。材料、方法和实例仅是说明性而非限制性的。
除非另外指明，否则实践本公开将采用细胞生物学、分子生物学(包含重组技术)、微生物学、生物化学和免疫学的常规技术，所述常规技术在本领域技术人员的能力范围内。文献中充分解释了这种技术，如《分子克隆：实验室手册(Molecular Cloning:A Laboratory Manual)》，第二版(Sambrook等人，1989)；《寡核苷酸合成(Oligonucleotide Synthesis)》(M.J.Gait编，1984)；《动物细胞培养(Animal Cell Culture)》(R.I.Freshney编，1987)；《酶学方法(Methods in Enzymology)》(学术出版社有限公司(Academic Press，Inc.)；《实验免疫学手册(Handbook of Experimental Immunology)》(D.M.Weir和C.C.Blackwell编)；《哺乳动物细胞用基因转移载体(Gene Transfer Vectors for Mammalian Cells)》(J.M.Miller和M.P.Calos编，1987)；《当代分子生物学方法(Current Protocols in Molecular Biology)》(F.M.Ausubel等人编，1987)；《PCR:聚合酶链反应(PCR:The Polymerase Chain Reaction)》(Mullis等人编，1994)；以及《当代免疫学方法(Current Protocols in Immunology)》(J.E.Coligan等人编，1991)，所述文献中的每个文献均通过引用明确并入本文中。
术语定义
如本文所使用，术语“互补决定区”是指：一个完整或完全的抗体包含两条重链及两条轻链；每条重链含有可变区(VH)及恒定区(CH)；每条轻链含有一可变区(VL)及一恒定区(CL)；抗体具有“Y”形状，Y的茎部由透过双硫键结合 在一起的两条重链的第二及第三恒定区所组成；Y的每个臂部包括与一单个轻链的可变区及恒定区结合的一单个重链的可变区及第一恒定区；该轻链的可变区及重链的可变区负责抗原结合；两条链中的可变区通常包含三个高度可变区，称为互补决定区。
如本文所使用，当用于表示抗体时，术语“功能性片段”是指在表示包含重链或轻链多肽的抗体的一部分，所述多肽保留了片段来源的抗体的一些或所有结合活性。这些功能性片段可以包括(例如)Fd、Fv、Fab、F(ab′)、F(ab)2、F(ab′)2、单链Fv(scFv)、双链抗体(diabody)、三链抗体(triabody)、四链抗体(tetrabody)和微抗体(minibody)。其他功能性片段可以包括(例如)重链或轻链多肽、可变区多肽或CDR多肽或其部分，只要这些功能性片段保留了结合活性即可。
如本文所使用，术语“恒定区”是指抗体分子的轻链和重链中靠近C端氨基酸序列相对稳定的区域。
如本文所使用，术语“可变区”是指抗体分子的轻链和重链中靠近N端氨基酸序列变化较大的区域。
如本文所用，术语“裸抗稳定性”是指未进行标记的抗体或其功能性片段，例如未标记有可被检测的标记物的抗体或其功能性片段。
本公开的一些实施方式提供了抗新型冠状病毒的抗体、检测新型冠状病毒试剂和试剂盒，该抗体可以特异性结合新型冠状病毒的N蛋白，对其具有较高的亲和力，用该抗体检测新型冠状病毒具有较好的灵敏度和特异性。本公开为新型冠状病毒的检测提供了更为丰富的抗体选择。
本公开一实施方式提供一种抗新型冠状病毒或其N蛋白的抗体或其功能性片段，所述抗体或其功能性片段具有如下互补决定区：
CDR-VH1：G-X1-T-F-S-X2-F-X3-M-H；其中：X1是V或F；X2是S或T；X3是G或A；
CDR-VH2：Y-X1-N-S-X2-S-N-X3-I-Y-Y-A-D-T-X4-K；其中：X1是L或I；X2是G或A；X2是I、V或L；X3是I、V或L；
CDR-VH3：X1-R-H-X2-M；其中：X1是A或T；X2是A或V；
CDR-VL1：S-Q-S-X1-D-Y-X2-G-D-S-X3-M；其中：X1是I、V或L；X2是D或N；X3是F或Y；
CDR-VL2：X1-A-S-N-X2-E-S；其中：X1是A或D；X2是I、V或L；
CDR-VL3：Q-X1-S-N-E-X2-P-Y；其中：X1是N、H或Q；X2是D或E。
本公开提供的抗新型冠状病毒的抗体或其功能性片段，具有上述互补决定区结构，上述互补决定区结构可使抗体或其功能性片段能够特异性结合抗新型冠状病毒的N蛋白，对其具有较好的亲和力，用该抗体或其功能性片段检测新型冠状病毒，具有较好的特异性和灵敏度。本公开为新型冠状病毒的检测提供了更为丰富的抗体选择。
在可选的实施方式中，CDR-VH1中，X1是F；CDR-VH2中，X1是I；CDR-VH3中，X1是A；CDR-VL1中，X3是Y。
通过本实施例的实验结果显示，在各互补决定区中的上述突变位点为上述氨基酸残基时，该抗体对抗新型冠状病毒的N蛋白表现出更高的亲和力。
在可选的实施方式中，CDR-VH1中，X2是S。
在可选的实施方式中，CDR-VH1中，X2是T。
在可选的实施方式中，CDR-VH1中，X3是G。
在可选的实施方式中，CDR-VH1中，X3是A。
在可选的实施方式中，CDR-VH2中，X2是G。
在可选的实施方式中，CDR-VH2中，X2是A。
在可选的实施方式中，CDR-VH2中，X3是I。
在可选的实施方式中，CDR-VH2中，X3是V。
在可选的实施方式中，CDR-VH2中，X3是L。
在可选的实施方式中，CDR-VH2中，X4是I。
在可选的实施方式中，CDR-VH2中，X4是V。
在可选的实施方式中，CDR-VH2中，X4是L。
在可选的实施方式中，CDR-VH3中，X2是A。
在可选的实施方式中，CDR-VH3中，X2是V。
在可选的实施方式中，CDR-VL1中，X1是I。
在可选的实施方式中，CDR-VL1中，X1是V。
在可选的实施方式中，CDR-VL1中，X1是L。
在可选的实施方式中，CDR-VL1中，X2是D。
在可选的实施方式中，CDR-VL1中，X2是N。
在可选的实施方式中，CDR-VL2中，X1是A。
在可选的实施方式中，CDR-VL2中，X1是D。
在可选的实施方式中，CDR-VL2中，X2是I。
在可选的实施方式中，CDR-VL2中，X2是V。
在可选的实施方式中，CDR-VL2中，X2是L。
在可选的实施方式中，CDR-VL1中，X1是N。
在可选的实施方式中，CDR-VL1中，X1是H。
在可选的实施方式中，CDR-VL1中，X1是Q。
在可选的实施方式中，CDR-VL2中，X2是D。
在可选的实施方式中，CDR-VL2中，X2是E。
在可选的实施方式中，所述抗体或其功能性片段的各互补决定区选自如下突变组合1-68中的任意一种：
Figure PCTCN2021117802-appb-000005
Figure PCTCN2021117802-appb-000006
在可选的实施方式中，所述抗体或其功能性片段与抗新型冠状病毒的N蛋白以K D≤8×10 -9mol/L的亲和力结合。
在可选的实施方式中，K D≤7×10 -10mol/L。
在可选的实施方式中，K D≤8×10 -9mol/L、K D≤7×10 -9mol/L、K D≤6×10 -9mol/L、K D≤5×10 -9mol/L、K D≤4×10 -9mol/L、K D≤3×10 -9mol/L、K D≤2×10 -9mol/L、K D≤1×10 -9mol/L、K D≤9×10 -10mol/L、K D≤8×10 -10mol/L、K D≤7×10 -10mol/L、K D≤6×10 -10mol/L、K D≤5×10 -10mol/L、K D≤4×10 -10mol/L、K D≤3×10 -10mol/L、K D≤2×10 -10mol/L、K D≤1×10 -10mol/L、K D≤9×10 -11mol/L或K D≤8×10 -11mol/L。
在可选的实施方式中，8.75×10 -11mol/L≤K D≤7.08×10 -10mol/L。
K D的检测参考本公开实施例中的方法进行。
在可选的实施方式中，CDR-VH1中，X1是V；CDR-VH2中，X1是L；CDR-VH3中，X1是T；CDR-VL1中，X3是F。
在可选的实施方式中，所述抗体或其功能性片段的各互补决定区选自如下突变组合69-76中的任意一种：
Figure PCTCN2021117802-appb-000007
Figure PCTCN2021117802-appb-000008
在可选的实施方式中，所述抗体包括序列依次如SEQ ID NO:1-4所示的轻链骨架区FR1-L、FR2-L、FR3-L及FR4-L，和/或，序列依次如SEQ ID NO:5-8所示的重链骨架区FR1-H、FR2-H、FR3-H及FR4-H。
通常情况下，重链可变区(VH)和轻链的可变区(VL)可由以下编号的CDR与FR按如下组合排列连接获得：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。
在一些实施方式中，抗体包括分别依次与序列SEQ ID NO:1、2、3、4具有至少80％的同源性的轻链骨架区FR1-L、FR2-L、FR3-L及FR4-L，和/或，分别依次与序列SEQ ID NO:5、6、7、8具有至少80％的同源性的重链骨架区FR1-H、FR2-H、FR3-H及FR4-H。
在一些实施方式中，抗体包括分别依次与序列SEQ ID NO:1、2、3、4具有至少80％的同一性的轻链骨架区FR1-L、FR2-L、FR3-L及FR4-L，和/或，分别依次与序列SEQ ID NO:5、6、7、8具有至少80％的同一性的重链骨架区FR1-H、FR2-H、FR3-H及FR4-H。
需要说明的是，在其他的实施方式和实施例中，本公开提供的抗体或其功能性片段的各骨架区氨基酸序列可以与上述对应骨架区(SEQ ID NO:1、2、3、4、5、6、7或8)可以具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同源性。例如，FR1-H的氨基酸序列还可以是SEQ ID NO:15。
在可选的实施方式中，所述抗体还包含恒定区。
在可选的实施方式中，所述恒定区选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE和IgD中的任意一者的恒定区。
在可选的实施方式中，所述恒定区的种属来源为哺乳动物或家禽类动物。在可选的实施方式中，哺乳动物包括牛、马、乳牛、猪、绵羊、山羊、大鼠、小鼠、狗、猫、兔、骆驼、驴、鹿、貂或人。在可选的实施方式中，家禽类动物包括鸡、鸭、鹅、火鸡或斗鸡。
在可选的实施方式中，所述恒定区的种属来源为牛、马、乳牛、猪、绵羊、山羊、大鼠、小鼠、狗、猫、兔、骆驼、驴、鹿、貂、鸡、鸭、鹅、火鸡、斗鸡或人。
在可选的实施方式中，所述恒定区来源于小鼠。
在可选的实施方式中，所述恒定区的轻链恒定区(CL)序列如SEQ ID NO:9所示，所述恒定区的重链恒定区(CH)序列如SEQ ID NO:10所示。
需要说明的是，在其他的实施例中，本公开提供的抗体的恒定区序列可以与上述恒定区(SEQ ID NO:9或10)可以具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同源性。例如，重链恒定区还可以是SEQ ID NO:16。
在可选的实施方式中，所述功能性片段选自所述抗体的VHH、F(ab’)2、Fab’、Fab、Fv和scFv中的任意一种。
上述抗体的功能性片段通常具有与其来源抗体相同的结合特异性。本领域技术人员根据本公开记载的内容容易理解到，上述抗体的功能性片段可以通过比如包括但不限于酶消化的方法(包括但不限于胃蛋白酶或木瓜蛋白酶)和/或通过化学还原分裂二硫键的方法获得。在本公开提供了完整抗体的结构基础上，本领域技术人员容易获得上述的功能性片段。
上述抗体的功能性片段还可以通过也是本领域技术人员所知的重组遗传学技术或通过例如自动肽合成仪，比如包括但不限于Applied BioSystems等销售的自动肽合成仪合成获得。本公开一实施方式提供一种检测新型冠状病毒或其N蛋白的试剂或试剂盒，其包括如上任一项所述的抗体或其功能性片段。
在可选的实施方式中，上述试剂或试剂盒中所述抗体或其功能性片段标记有可被检测的标记物。
如本文所用，“可被检测的标记物”是指具有能够被肉眼直接观察或被仪器检测或探测到的特性例如发光、显色、放射性等特性的一类物质，通过该特性可以实现对相应目标物的定性或定量检测。
在可选的实施方式中，所述可被检测的标记物包括但不限于荧光染料、催化底物显色的酶、放射性同位素、化 学发光试剂和纳米颗粒类标记物。
在实际的使用过程中，本领域技术人员可以根据检测条件或实际需要选择合适的标记物，无论使用何种标记物，其均属于本公开的保护范围。
在可选的实施方式中，所述荧光染料包括但不限于荧光素类染料及其衍生物(例如包括但不限于异硫氰酸荧光素(FITC)羟基光素(FAM)、四氯光素(TET)等或其类似物)、罗丹明类染料及其衍生物(例如包括但不限于红色罗丹明(RBITC)、四甲基罗丹明(TAMRA)、罗丹明B(TRITC)等或其类似物)、Cy系列染料及其衍生物(例如包括但不限于Cy2、Cy3、Cy3B、Cy3.5、Cy5、Cy5.5、Cy3等或其类似物)、Alexa系列染料及其衍生物(例如包括但不限于AlexaFluor350、405、430、488、532、546、555、568、594、610、33、647、680、700、750等或其类似物)和蛋白类染料及其衍生物(例如包括但不限于藻红蛋白(PE)、藻蓝蛋白(PC)、别藻蓝蛋白(APC)、多甲藻黄素-叶绿素蛋白(preCP)等)。
在可选的实施方式中，所述催化底物显色的酶包括但不限于辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、葡萄糖氧化酶、碳酸酐酶、乙酰胆碱酯酶以及6-磷酸葡萄糖脱氧酶。
在可选的实施方式中，所述放射性同位素包括但不限于 212Bi、 131I、 111In、 90Y、 186Re、 211At、 125I、 188Re、 153Sm、 213Bi、 32P、 94mTc、 99mTc、 203Pb、 67Ga、 68Ga、 43Sc、 47Sc、 110mIn、 97Ru、 62Cu、 64Cu、 67Cu、 68Cu、 86Y、 88Y、 121Sn、 161Tb、 166Ho、 105Rh、 177Lu、 172Lu和 18F。
在可选的实施方式中，所述化学发光试剂包括但不限于鲁米诺及其衍生物、光泽精、甲壳动物荧光素及其衍生物、联吡啶钌及其衍生物、吖啶酯及其衍生物、二氧环乙烷及其衍生物、洛粉碱及其衍生物和过氧草酸盐及其衍生物。
在可选的实施方式中，所述纳米颗粒类标记物包括但不限于纳米颗粒、胶体、有机纳米颗粒、磁性纳米颗粒、量子点纳米颗粒和稀土络合物纳米颗粒。
在可选的实施方式中，所述胶体包括但不限于胶体金属、分散型染料、染料标记的微球和乳胶。
在可选的实施方式中，所述胶体金属包括但不限于胶体金、胶体银和胶体硒。本公开一实施方式提供一种编码上述抗体或其功能性片段的核酸分子。本公开一实施方式提供含有上述核酸分子的载体。本公开一实施方式提供含有上述载体的重组细胞。本公开一实施方式提供一种制备抗体或其功能性片段的方法，其包括：培养如上所述的重组细胞，从培养产物中分离纯化得到所述抗体或其功能性片段。
在本公开提供了抗体或其功能性片段的氨基酸序列的基础上，本领域技术人员容易想到采用基因工程技术或其他技术(化学合成、杂交瘤细胞)制备得到该抗体或其功能性片段，例如从能够重组表达如上任一项所述的抗体或其功能性片段的重组细胞的培养产物中分离纯化得到该抗体或其功能性片段，这对本领域技术人员来说是容易实现的，基于此，无论采用何种技术制备本公开的抗体或其功能性片段，其均属于本公开的保护范围。
实施例
以下结合实施例对本公开的特征和性能作进一步的详细描述。
实施例1
本实施例中限制性内切酶、Prime Star DNA聚合酶购自Takara公司。MagExtractor-RNA提取试剂盒购自TOYOBO公司。BD SMART TM RACE cDNA Amplification Kit试剂盒购自Takara公司。pMD-18T载体购自Takara公司。质粒提取试剂盒购自天根公司。引物合成和基因测序由Invitrogen公司完成。
1重组质粒的构建
(1)抗体基因制备
从分泌抗新型冠状病毒N蛋白的抗体的杂交瘤细胞株中提取mRNA，通过RT-PCR方法获得DNA产物，该产物用rTaq DNA聚合酶进行加A反应后插入到pMD-18T载体中，转化到DH5α感受态细胞中，长出菌落后分别取重链(Heavy Chain)及轻链(Light Chain)基因克隆，各4个克隆送基因测序公司进行测序。
(2)抗体可变区基因的序列分析
将上述测序得到的基因序列放在IMGT抗体数据库(IMGT抗体数据库来源于：http://www.imgt.org)中进行分析，并利用VNTI11.5软件进行分析确定重链和轻链引物对扩增出的基因都是正确的，其中轻链扩增出的基因片段中，VL基因序列为342bp，属于VkII基因家族，其前方有57bp的前导肽序列；重链引物对扩增出的基因片段中，VH基因序列为336bp，属于VH1基因家族，其前方有57bp的前导肽序列。
(3)重组抗体表达质粒的构建
pcDNA TM 3.4
Figure PCTCN2021117802-appb-000009
vector为构建的重组抗体真核表达载体，该表达载体已经引入HindIII、BamHI、EcoRI等多克隆酶切位点，并命名为pcDNA3.4A表达载体，后续简称3.4A表达载体；根据上述pMD-18T中抗体可变区基因测序结果，设计该抗体的VL和VH基因特异性引物，两端分别带有HindIII、EcoRI酶切位点和保护碱基，通过PCR扩增方法扩出0.72kb的轻链基因片段和1.41kb的重链基因片段。
重链和轻链基因片段分别采用HindIII/EcoRI双酶切，3.4A载体采用HindIII/EcoRI双酶切，将片段和载体纯化回收后重链基因和轻链基因连接3.4A表达载体中，得到重链和轻链的重组表达质粒。
2稳定细胞株筛选
(1)重组抗体表达质粒瞬时转染CHO细胞，确定表达质粒活性
将步骤1-(3)得到的质粒用超纯水稀释至400ng/ml，调节CHO细胞1.43×10 7个细胞/ml于离心管中，100μL质粒与700μL细胞混合，转入电转杯，电转，第3、5、7天取样计数，第7天收样检测。
包被液(主要成分NaHCO 3)稀释2019-nCoV N蛋白抗原到1μg/ml，每孔100μL，4℃过夜；次日，洗涤液清洗2次，拍干；加入封闭液(20％BSA+80％PBS)，每孔120μL，37℃，1h，拍干；加入稀释后的细胞上清，100μL/孔，37℃，30min(部分上清1h)；洗涤液清洗5次，拍干；加入羊抗鼠IgG-HRP(其中HPR为辣根过氧化物酶标记)，每孔100μL，37℃，30min；洗涤液清洗5次，拍干；加入显色液A液(50μL/孔，含柠檬酸+醋酸钠+乙酰苯胺+过氧化脲)，加入显色液B液(50μL/孔，含柠檬酸+EDTA·2Na+TMB+浓HCL)，10min；加入终止液(50μL/孔，含EDTA·2Na+浓H 2SO 4)；酶标仪上450nm(参考630nm)处读OD值。结果显示细胞上清稀释1000倍后反应OD仍大于1.0，未加细胞上清孔反应OD小于0.1，表明质粒瞬转后产生的抗体对2019-nCoV N蛋白抗原有活性。
(2)重组抗体表达质粒线性化
准备下述试剂：Buffer 50μL、DNA 100μg/管、PuvⅠ酶10μL、无菌水补至500μL，37℃水浴酶切过夜；先用等体积酚/氯仿/异戊醇(下层)25:24:1，再用氯仿(水相)依次进行抽提；0.1倍体积(水相)3M醋酸钠和2倍体积乙醇冰上沉淀，70％乙醇漂洗沉淀，去除有机溶剂，待乙醇挥发完全用适量的灭菌水进行复融，最后进行浓度的测定。
(3)重组抗体表达质粒稳定转染，加压筛选稳定细胞株
将步骤2-(2)得到的质粒用超纯水稀释至400ng/ml，调节CHO细胞1.43×10 7个细胞/ml于离心管中，100μL质粒与700μL细胞混合，转入电转杯，电转，次日计数；25umol/L MSX 96孔加压培养约25天。
显微镜下观察标记长有细胞的克隆孔,并记录汇合度；取培养上清，送样检测；挑选抗体浓度、相对浓度高的细胞株转24孔，3天左右转6孔；3天后保种批培，调整细胞密度0.5×10 6个细胞/ml，2.2ml进行批培养，细胞密度0.3×10 6个细胞/ml，2ml进行保种；7天6孔批培上清送样检测，挑选抗体浓度及细胞直径较小的细胞株转TPP保种传代。
3重组抗体生产
(1)细胞扩培
将步骤2-(3)获得的经传代获得的细胞复苏之后先在125ml规格的摇瓶中培养，接种体积为30ml，培养基为100％Dynamis培养基，放置于转速120r/min，温度为37℃，二氧化碳为8％的摇床中。培养72h，以50万个细胞/ml接种密度接种扩培，扩培体积根据生产需求进行计算，培养基为100％Dynamis培养基。之后每72h扩培一次。当细胞量满足生产需求时，严格控制接种密度为50万个细胞/ml左右进行生产。
(2)摇瓶生产及纯化
摇瓶参数：转速120r/min，温度为37℃，二氧化碳为8％。流加补料：在摇瓶中培养至72h时开始每天补料，HyClone Cell Boost Feed 7a每天流加初始培养体积的3％，Feed 7b每天流加量为初始培养体积的千分之一，一直补到第12天(第12天补料)。葡萄糖在第六天补加3g/L。第13天收样。用蛋白A(proteinA)亲和层析柱进行亲和纯化。取4μg纯化的抗体进行还原性SDS-PAGE，4μg外来对照抗体作为对照，电泳图如下图1所示，在还原性SDS-PAGE后显示两条带，1条Mr为50KD(重链，SEQ ID NO:14)，另一条Mr为28KD(轻链，SEQ ID NO:13)。
实施例2
抗体的性能检测
(1)实施例1抗体及其突变体的活性检测
分析实施例1的抗体(WT)序列，其重链可变区如SEQ ID NO:12所示，其中，重链可变区上的各互补决定区的氨基酸序列如下：
CDR-VH1：G-V(X1)-T-F-S-T(X2)-F-A(X3)-M-H；
CDR-VH2：Y-L(X1)-N-S-A(X2)-S-N-L(X3)-I-Y-Y-A-D-T-L(X4)-K；
CDR-VH3：T(X1)-R-H-A(X2)-M；
其轻链可变区如SEQ ID NO:11所示，其中，轻链可变区上的各互补决定区的氨基酸序列如下：
CDR1-VL：S-Q-S-I(X1)-D-Y-D(X2)-G-D-S-F(X3)-M；
CDR-VL2：D(X1)-A-S-N-V(X2)-E-S；
CDR-VL3：Q-H(X1)-S-N-E-D(X2)-P-Y。
在实施例1的抗新型冠状病毒抗体(WT)基础上，在互补决定区中对于抗体活性有关的位点进行突变，其中，X1、X2、X3、X4均为突变位点。见下表1。
表1 与抗体活性有关的突变位点
Figure PCTCN2021117802-appb-000010
Figure PCTCN2021117802-appb-000011
对表1中的抗体结合活性检测：
包被液(主要成分NaHCO 3)稀释2019-nCoV N蛋白抗原到1μg/ml进行微孔板包被，每孔100μl，4℃过夜；次日，洗涤液清洗2次，拍干；加入封闭液(20％BSA+80％PBS)，每孔120μl，37℃，1h，拍干；加入稀释后的表1中的单克隆抗体，100μl/孔，37℃，30min-60min；洗涤液清洗5次，拍干；加入羊抗鼠IgG-HRP，每孔100μl，37℃，30min；洗涤液(PBS)清洗5次，拍干；加入显色液A液(50μl/孔，含2.1g/L柠檬酸、12.25g/L柠檬酸、0.07g/L乙酰苯胺和0.5g/L过氧化脲)，加入显色液B液(50μl/孔，含1.05g/L柠檬酸、0.186g/LEDTA·2Na、0.45g/L TMB和0.2ml/L浓HCl)，10min；加入终止液(50μl/孔，含0.75g/EDTA·2Na和10.2ml/L浓H 2SO 4)；酶标仪上450nm(参考630nm)处读OD值。结果见下表2。
表2 WT抗体及其突变体的活性数据
抗体浓度(ng/ml) 5000 1000 500 250 125 0.00
WT 1.165 1.061 0.985 0.650 0.284 0.029
突变1 1.382 1.251 1.148 0.858 0.493 0.028
突变2 1.3014 1.2518 1.145 0.854 0.507 0.023
突变3 1.2201 1.296 1.195 0.802 0.501 0.025
突变4 1.284 1.291 1.156 0.864 0.484 0.022
突变5 1.204 1.130 1.052 0.743 0.358 0.090
从表2结果可以看出，WT和突变体都有较好的结合活性，其中，突变1的活性最佳。
(2)抗体及其突变体的亲和力检测
(a)在突变1的基础上，对各CDR区的其他位点进行突变，各突变的序列见下表3。
表3 与抗体亲和力有关的突变位点
Figure PCTCN2021117802-appb-000012
Figure PCTCN2021117802-appb-000013
Figure PCTCN2021117802-appb-000014
亲和力分析
利用AMC传感器，将纯化出来的抗体用PBST(磷酸盐吐温缓冲液，主要成分Na 2HPO 4+NaCl+TW-20)稀释到10μg/mL，2019-nCoV N蛋白抗原用PBST进行梯度稀释：1.41μg/mL、0.70μg/mL、0.35μg/mL、0.18μg/mL、0.09μg/mL、0.04μg/mL。
运行流程：缓冲液1(PBST)中平衡60s，抗体溶液中固化抗体300s，缓冲液2(PBST)中孵育180s，抗原溶液中结合420s，缓冲液2中解离1200s，用10mM pH 1.69GLY溶液及缓冲液3进行传感器再生，输出数据。K D表示平衡解离常数即亲和力；kon表示结合速率；kdis表示解离速率。结果见下表4。
表4 亲和力检测数据
  K D(M) kon(1/Ms) kdis(1/s)
突变1 1.20E-10 1.82E+06 2.18E-04
突变1-1 1.28E-10 1.63E+06 2.08E-04
突变1-2 1.44E-10 2.15E+06 3.09E-04
突变1-3 1.09E-10 2.55E+06 2.77E-04
突变1-4 1.35E-10 1.90E+06 2.57E-04
突变1-5 1.38E-10 2.97E+06 4.09E-04
突变1-6 3.27E-10 1.97E+06 6.44E-04
突变1-7 2.65E-10 2.85E+06 7.55E-04
突变1-8 3.57E-10 2.05E+06 7.31E-04
突变1-9 1.84E-10 2.66E+06 4.90E-04
突变1-10 5.72E-10 1.16E+06 6.63E-04
突变1-11 8.75E-11 2.97E+06 2.60E-04
突变1-12 2.20E-10 2.92E+06 6.41E-04
突变1-13 1.51E-10 2.73E+06 4.13E-04
突变1-14 3.83E-10 1.25E+06 4.79E-04
突变1-15 4.24E-10 1.73E+06 7.33E-04
突变1-16 1.18E-10 2.48E+06 2.93E-04
突变1-17 1.78E-10 2.95E+06 5.26E-04
突变1-18 4.74E-10 1.16E+06 5.50E-04
突变1-19 1.61E-10 2.43E+06 3.92E-04
突变1-20 1.70E-10 1.56E+06 2.65E-04
突变1-21 7.08E-10 1.11E+06 7.86E-04
突变1-22 9.77E-11 2.22E+06 2.17E-04
突变1-23 1.29E-10 1.85E+06 2.38E-04
突变1-24 1.45E-10 2.98E+06 4.33E-04
突变1-25 9.19E-11 2.71E+06 2.49E-04
突变1-26 2.36E-10 2.41E+06 5.68E-04
突变1-27 3.25E-10 1.73E+06 5.63E-04
突变1-28 5.11E-10 1.47E+06 7.51E-04
突变1-29 4.37E-10 1.22E+06 5.33E-04
突变1-30 1.91E-10 2.97E+06 5.66E-04
突变1-31 1.96E-10 2.53E+06 4.97E-04
突变1-32 4.48E-10 1.66E+06 7.43E-04
突变1-33 2.17E-10 1.15E+06 2.49E-04
突变1-34 1.32E-10 2.03E+06 2.67E-04
突变1-35 1.55E-10 2.79E+06 4.33E-04
突变1-36 3.23E-10 1.49E+06 4.82E-04
突变1-37 2.49E-10 2.17E+06 5.40E-04
突变1-38 3.37E-10 2.17E+06 7.32E-04
突变1-39 4.74E-10 1.21E+06 5.74E-04
突变1-40 5.56E-10 1.28E+06 7.12E-04
突变1-41 2.63E-10 2.92E+06 7.67E-04
突变1-42 1.98E-10 2.46E+06 4.87E-04
突变1-43 2.05E-10 2.12E+06 4.34E-04
突变1-44 1.53E-10 2.36E+06 3.60E-04
突变1-45 1.74E-10 2.56E+06 4.45E-04
突变1-46 2.56E-10 2.26E+06 5.79E-04
突变1-47 2.19E-10 1.13E+06 2.47E-04
突变1-48 1.34E-10 2.88E+06 3.86E-04
突变1-49 5.12E-10 1.41E+06 7.22E-04
突变1-50 5.12E-10 1.44E+06 7.37E-04
突变1-51 2.08E-10 1.93E+06 4.01E-04
突变1-52 1.45E-10 2.10E+06 3.04E-04
突变1-53 1.99E-10 2.20E+06 4.37E-04
突变1-54 2.26E-10 2.99E+06 6.76E-04
突变1-55 1.83E-10 1.16E+06 2.12E-04
突变1-56 2.13E-10 1.33E+06 2.83E-04
突变1-57 3.09E-10 2.22E+06 6.85E-04
突变1-58 4.63E-10 1.72E+06 7.97E-04
突变1-59 2.90E-10 2.48E+06 7.18E-04
突变1-60 5.62E-10 1.25E+06 7.02E-04
突变1-61 1.66E-10 2.80E+06 4.64E-04
突变1-62 4.47E-10 1.60E+06 7.15E-04
突变1-63 2.86E-10 1.91E+06 5.46E-04
突变1-64 1.74E-10 2.86E+06 4.99E-04
突变1-65 1.25E-10 1.80E+06 2.25E-04
突变1-66 1.55E-10 2.53E+06 3.92E-04
突变1-67 6.65E-10 1.04E+06 6.92E-04
表4数据显示，突变1及其系列突变体都具有较好的亲和力，说明在突变1的基础上按表3的突变方式突变得到的抗体都具有较好的亲和力。
(b)在WT的基础上，对其他位点进行突变，并利用上述2(a)的亲和力测定方法检测各突变体的亲和力，各 突变的序列见下表5，对应的亲和力数据见表6。
表5 以WT为骨架进行的突变
  CDR-VH1 CDR-VH2 CDR-VH3 CDR-VL1 CDR-VL2 CDR-VL3
WT T/A A/L/L A I/D D/V H/D
WT1 T/G G/L/I V I/N D/V Q/D
WT2 S/A G/V/L A L/N D/V N/D
WT3 S/G G/L/L A V/N A/V Q/D
WT4 S/G A/L/L V L/D D/L Q/E
WT5 S/G G/I/L V L/D D/L H/E
WT6 T/A A/L/I A L/D A/L H/D
WT7 T/G G/I/L V V/D A/I N/E
表6 WT抗体及其突变体的亲和力检测结果
  K D(M) kon(1/Ms) kdis(1/s)
WT 8.03E-09 1.75E+05 1.41E-03
WT1 5.33E-09 1.35E+05 7.19E-04
WT2 2.12E-09 3.71E+05 7.87E-04
WT3 2.60E-09 2.78E+05 7.22E-04
WT4 4.01E-09 2.29E+05 9.18E-04
WT5 2.90E-09 3.28E+05 9.50E-04
WT6 4.92E-09 1.58E+05 7.78E-04
WT7 3.55E-09 2.37E+05 8.41E-04
表6数据显示，WT及其系列突变体对抗原也具有不错的亲和力，说明在WT的基础上，按表5的突变方式突变得到的抗体都具有不错的亲和力。
(3)裸抗稳定性考核
将上述抗体置于4℃(冰箱)、-80℃(冰箱)、37℃(恒温箱)放置21天，取7天、14天、21天样品进行状态观察，并对21天样品进行活性检测，结果显示三种考核条件下抗体放置21天均未见明显蛋白状态变化，活性也未随考核温度的升高呈下降趋势，说明上述抗体稳定。下表7突变1抗体为考核21天的酶免活性检测OD结果。
表7
样品浓度(ng/ml) 500 250 0
4℃，21天样品 1.189 0.951 0.030
-80℃，21天样品 1.250 0.998 0.033
37℃，21天样品 1.165 0.905 0.047
以上所述仅为本公开的优选实施例而已，并不用于限制本公开，对于本领域的技术人员来说，本公开可以有各种更改和变化。凡在本公开的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本公开的保护范围之内。
工业实用性
本公开提供了抗新型冠状病毒的抗体、检测新型冠状病毒试剂和试剂盒，该抗体可以特异性结合新型冠状病毒的N蛋白，对其具有较高的亲和力，用该抗体检测新型冠状病毒具有较好的灵敏度和特异性。本公开为新型冠状病毒的检测提供了更为丰富的抗体选择。同时本公开提供的检测试剂盒同样具有与该抗体相同的技术效果，具有广泛 的应用前景和较高的市场价值。

Claims (18)

  1. 一种抗新型冠状病毒或其N蛋白的抗体或其功能性片段，其特征在于，所述抗体或其功能性片段包括如下互补决定区：
    CDR-VH1：G-X1-T-F-S-X2-F-X3-M-H；其中：X1是V或F；X2是S或T；X3是G或A；
    CDR-VH2：Y-X1-N-S-X2-S-N-X3-I-Y-Y-A-D-T-X4-K；其中：X1是L或I；X2是G或A；X2是I、V或L；X3是I、V或L；
    CDR-VH3：X1-R-H-X2-M；其中：X1是A或T；X2是A或V；
    CDR-VL1：S-Q-S-X1-D-Y-X2-G-D-S-X3-M；其中：X1是I、V或L；X2是D或N；X3是F或Y；
    CDR-VL2：X1-A-S-N-X2-E-S；其中：X1是A或D；X2是I、V或L；
    CDR-VL3：Q-X1-S-N-E-X2-P-Y；其中：X1是N、H或Q；X2是D或E。
  2. 根据权利要求1所述的抗体或其功能性片段，其特征在于，
    CDR-VH1中，X1是F；
    CDR-VH2中，X1是I；
    CDR-VH3中，X1是A；
    CDR-VL1中，X3是Y；
    优选的，CDR-VH1中，X2是S；
    优选的，CDR-VH1中，X2是T；
    优选的，CDR-VH1中，X3是G；
    优选的，CDR-VH1中，X3是A；
    优选的，CDR-VH2中，X2是G；
    优选的，CDR-VH2中，X2是A；
    优选的，CDR-VH2中，X3是I；
    优选的，CDR-VH2中，X3是V；
    优选的，CDR-VH2中，X3是L；
    优选的，CDR-VH2中，X4是I；
    优选的，CDR-VH2中，X4是V；
    优选的，CDR-VH2中，X4是L；
    优选的，CDR-VH3中，X2是A；
    优选的，CDR-VH3中，X2是V；
    优选的，CDR-VL1中，X1是I；
    优选的，CDR-VL1中，X1是V；
    优选的，CDR-VL1中，X1是L；
    优选的，CDR-VL1中，X2是D；
    优选的，CDR-VL1中，X2是N；
    优选的，CDR-VL2中，X1是A；
    优选的，CDR-VL2中，X1是D；
    优选的，CDR-VL2中，X2是I；
    优选的，CDR-VL2中，X2是V；
    优选的，CDR-VL2中，X2是L；
    优选的，CDR-VL1中，X1是N；
    优选的，CDR-VL1中，X1是H；
    优选的，CDR-VL1中，X1是Q；
    优选的，CDR-VL2中，X2是D；
    优选的，CDR-VL2中，X2是E；
    优选的，所述抗体或其功能性片段的各互补决定区选自如下突变组合1-68中的任意一种：
    Figure PCTCN2021117802-appb-100001
    Figure PCTCN2021117802-appb-100002
    Figure PCTCN2021117802-appb-100003
  3. 根据权利要求2所述的抗体或其功能性片段，其特征在于，所述抗体或其功能性片段与新型冠状病毒的N蛋白以K D≤8×10 -9mol/L的亲和力结合；优选的，K D≤7×10 -10mol/L。
  4. 根据权利要求1所述的抗体或其功能性片段，其特征在于，
    CDR-VH1中，X1是V；
    CDR-VH2中，X1是L；
    CDR-VH3中，X1是T；
    CDR-VL1中，X3是F；
    优选的，所述抗体或其功能性片段的各互补决定区选自如下突变组合69-76中的任意一种：
      CDR-VH1 CDR-VH2 CDR-VH3 CDR-VL1 CDR-VL2 CDR-VL3 突变组合69 T/A A/L/L A I/D D/V H/D 突变组合70 T/G G/L/I V I/N D/V Q/D 突变组合71 S/A G/V/L A L/N D/V N/D 突变组合72 S/G G/L/L A V/N A/V Q/D 突变组合73 S/G A/L/L V L/D D/L Q/E 突变组合74 S/G G/I/L V L/D D/L H/E 突变组合75 T/A A/L/I A L/D A/L H/D 突变组合76 T/G G/I/L V V/D A/I N/E
    。
  5. 根据权利要求1-4任一项所述的抗体或其功能性片段，其特征在于，所述抗体包括序列依次如SEQ ID NO:1-4所示或其具有至少80％同源性的轻链骨架区FR1-L、FR2-L、FR3-L及FR4-L，和/或，序列依次如SEQ ID NO:5-8或其具有至少80％同源性所示的重链骨架区FR1-H、FR2-H、FR3-H及FR4-H；
    优选的，所述FR1-H的氨基酸序列如SEQ ID NO:15所示；
    优选的，所述抗体还包含恒定区；
    优选的，所述恒定区选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE和IgD中的任意一者的恒定区；
    优选的，所述恒定区的种属来源为牛、马、乳牛、猪、绵羊、山羊、大鼠、小鼠、狗、猫、兔、骆驼、驴、鹿、 貂、鸡、鸭、鹅、火鸡、斗鸡或人；
    优选的，所述恒定区来源于小鼠；
    优选的，所述恒定区的轻链恒定区序列如SEQ ID NO:9所示或其具有至少80％同源性，所述恒定区的重链恒定区序列如SEQ ID NO:10所示或其具有至少80％同源性；
    优选的，所述重链恒定区序列如SEQ ID NO:16所示；
    优选的，所述功能性片段选自所述抗体的VHH、F(ab’)2、Fab’、Fab、Fv和scFv中的任意一种。
  6. 根据权利要求1-4中任一项所述的抗新型冠状病毒的抗体或其功能性片段，其特征在于，所述抗体包括分别依次与序列SEQ ID NO:1、2、3、4具有至少80％的同源性的轻链骨架区FR1-L、FR2-L、FR3-L及FR4-L，和/或，分别依次与序列SEQ ID NO:5、6、7、8具有至少80％的同源性的重链骨架区FR1-H、FR2-H、FR3-H及FR4-H。
  7. 一种检测新型冠状病毒或其N蛋白的试剂或试剂盒，其特征在于，其包括如权利要求1-6任一项所述的抗体或其功能性片段。
  8. 根据权利要求7所述的试剂或试剂盒，其特征在于，所述抗体或其功能性片段标记有可被检测的标记物；
    优选的，所述可被检测的标记物选自荧光染料、催化底物显色的酶、放射性同位素、化学发光试剂和纳米颗粒类标记物；
    优选的，所述荧光染料选自荧光素类染料及其衍生物、罗丹明类染料及其衍生物、Cy系列染料及其衍生物、Alexa系列染料及其衍生物和蛋白类染料及其衍生物；
    优选的，所述催化底物显色的酶选自辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、葡萄糖氧化酶、碳酸酐酶、乙酰胆碱酯酶以及6-磷酸葡萄糖脱氧酶；
    优选的，所述放射性同位素选自 212Bi、 131I、 111In、 90Y、 186Re、 211At、 125I、 188Re、 153Sm、 213Bi、 32P、 94mTc、 99mTc、 203Pb、 67Ga、 68Ga、 43Sc、 47Sc、 110mIn、 97Ru、 62Cu、 64Cu、 67Cu、 68Cu、 86Y、 88Y、 121Sn、 161Tb、 166Ho、 105Rh、 177Lu、 172Lu和 18F；
    优选的，所述化学发光试剂选自鲁米诺及其衍生物、光泽精、甲壳动物荧光素及其衍生物、联吡啶钌及其衍生物、吖啶酯及其衍生物、二氧环乙烷及其衍生物、洛粉碱及其衍生物和过氧草酸盐及其衍生物；
    优选的，所述纳米颗粒类标记物选自纳米颗粒、胶体、有机纳米颗粒、磁性纳米颗粒、量子点纳米颗粒和稀土络合物纳米颗粒；
    优选的，所述胶体选自胶体金属、分散型染料、染料标记的微球和乳胶；
    优选的，所述胶体金属选自胶体金、胶体银和胶体硒。
  9. 一种核酸分子，所述核酸分子编码根据权利要求1-6任一项所述的抗体或其功能性片段。
  10. 一种载体，其特征在于，其含有编码如权利要求1-6任一项所述的抗体或其功能性片段的核酸片段。
  11. 一种重组细胞，其特征在于，其含有权利要求10所述的载体。
  12. 如权利要求1-6任一项所述的抗体或其功能性片段或者如权利要求7或8所述的试剂或试剂盒在检测新型冠状病毒中的用途。
  13. 如权利要求1-6任一项所述的抗体或其功能性片段或者如权利要求7或8所述的试剂或试剂盒，用于检测新型冠状病毒的用途。
  14. 一种检测新型冠状病毒的方法，包括：
    A)在足以发生结合反应的条件下，使权利要求1-6任一项所述的抗体或其功能性片段与样品接触以进行结合反应；以及
    B)检测结合反应产生的免疫复合物。
  15. 一种诊断受试者在感染新型冠状病毒或与新型冠状病毒感染相关疾病中的方法，包括：
    A)在足以发生结合反应的条件下，使权利要求1-6任一项所述的抗体或其功能性片段与来自所述受试者的样品接触以进行结合反应；以及
    B)检测结合反应产生的免疫复合物。
  16. 根据权利要求15所述的方法，其中，所述与新型冠状病毒感染相关的疾病包括呼吸道症状、发热、咳嗽、气促、呼吸困难、肺炎、严重急性呼吸综合征、肾衰竭。
  17. 根据权利要求15所述的方法，其中，所述感染新型冠状病毒的受试者包括无症状感染者、无明显症状感染者、有症状感染者。
  18. 一种制备如权利要求1-6任一项所述的抗体或其功能性片段的方法，其特征在于，其包括：培养权利要求11所述的重组细胞，从培养产物中分离纯化得到所述抗体或其功能性片段。
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