WO2022048052A1 - 新冠病毒中和毒性的纳米抗体及其制备方法与应用 - Google Patents

新冠病毒中和毒性的纳米抗体及其制备方法与应用 Download PDF

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WO2022048052A1
WO2022048052A1 PCT/CN2020/132279 CN2020132279W WO2022048052A1 WO 2022048052 A1 WO2022048052 A1 WO 2022048052A1 CN 2020132279 W CN2020132279 W CN 2020132279W WO 2022048052 A1 WO2022048052 A1 WO 2022048052A1
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seq
amino acid
nanobody
acid sequence
neutralizing
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PCT/CN2020/132279
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French (fr)
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庄文超
李福胜
李倩卉
凌志
赵运星
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康维众和(中山)生物药业有限公司
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Priority to US17/261,802 priority Critical patent/US20220372113A1/en
Publication of WO2022048052A1 publication Critical patent/WO2022048052A1/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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • 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]
    • 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
    • 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
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • 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
    • C07K2317/50—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
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • 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
    • 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 present invention relates to the technical field of polypeptides, in particular to a nanobody for neutralizing the toxicity of a novel coronavirus and a preparation method and application thereof.
  • Novel coronavirus pneumonia (Corona Virus Disease 2019, COVID-19) is caused by a new betacoronavirus-novel coronavirus (SARS-CoV-2 virus), with angiotensin converting enzyme 2 (Angiotensin converting enzyme 2, ACE-2) acts as a receptor invading cells to cause lung damage, and the aggravation of the disease is closely related to the secondary systemic inflammatory response. Severe patients will develop acute respiratory distress syndrome (ARDS) and septic shock, and eventually appear Multiple organ failure. At present, most of the therapeutic drugs such as baricitinib, remdesivir and chloroquine are still in the clinical trial stage, and there is no specific drug treatment. COVID-19 is highly contagious and can cause a variety of serious complications, posing a great threat to global public safety.
  • SARS-CoV-2 virus betacoronavirus-novel coronavirus
  • ACE-2 angiotensin converting enzyme 2
  • ARDS acute respiratory distress
  • Neutralizing antibodies are a class of antibodies that the body produces in response to antigenic stimulation with a protective effect, and may be a useful treatment even in relatively advanced stages of the disease.
  • antibody-mediated measures to prevent and treat viral infections have shown good results, and their application prospects have been recognized by experts.
  • polyclonal plasma is not only limited in source, but also its clinical application is limited by conditions such as difficult quality control, mismatch of donor and recipient blood types, and potential infectious factors.
  • Human monoclonal antibodies can effectively overcome the above problems. Therefore, the research and development of new coronavirus neutralizing antibodies can accelerate the clinical evaluation of new coronavirus antibody drugs and enhance the treatment and prevention of new coronavirus pneumonia.
  • the present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes a novel coronavirus-neutralizing nanobody, which can be used for the prevention and treatment of novel coronavirus pneumonia.
  • the present invention also proposes a method for preparing the above-mentioned novel coronavirus-neutralizing nanobody.
  • the present invention also proposes the application of the above-mentioned novel coronavirus-neutralizing nanobody.
  • the present invention also provides a gene encoding a nanobody that neutralizes toxicity of the novel coronavirus.
  • the present invention also provides a recombinant plasmid.
  • the present invention also provides a recombinant cell.
  • the nanobody according to the embodiment of the first aspect of the present invention for neutralizing toxicity of the novel coronavirus comprises a framework region FR and a complementarity determining region CDR; the complementarity determining region CDRs include CDR1, CDR2 and CDR3;
  • the amino acid sequence of the CDR1 is selected from at least one of the amino acid sequences shown in SEQ ID NO.9 and SEQ ID NO.10;
  • the amino acid sequence of the CDR2 is selected from at least one of the amino acid sequences shown in SEQ ID NO.11, SEQ ID NO.12 and SEQ ID NO.13;
  • the amino acid sequence of the CDR3 is selected from at least one of the amino acid sequences shown in SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16 and SEQ ID NO.17.
  • the novel coronavirus-neutralizing nanobody according to the embodiment of the present invention has at least the following beneficial effects: the novel coronavirus-neutralizing nanobody of the present invention has a small molecular weight (only 1/10 the size of a monoclonal antibody) and high binding activity (has high affinity with SARS-CoV-2 virus) and low production cost, it has strong inhibitory ability to the virus at high concentration, and the inhibition rate is as high as 100% at a concentration of 1 ⁇ g/ml, It has strong neutralizing ability to SARS-CoV-2 pseudovirus and has strong antiviral effect; the nanobody of the present invention also has a mature preclinical research basis for effectiveness and safety, a high success rate of industrialization and transformation, and can Fast-track access to clinical research.
  • the amino acid sequence of the CDR of the complementarity determining region is one of the following (1)-(4): (1) CDR1 shown in SEQ ID NO.9, SEQ ID NO.11 CDR2 shown in SEQ ID NO.14, CDR3 shown in SEQ ID NO.14; (2) CDR1 shown in SEQ ID NO.10, CDR2 shown in SEQ ID NO.12, CDR3 shown in SEQ ID NO.15; (3) ) CDR1 shown in SEQ ID NO.10, CDR2 shown in SEQ ID NO.12, CDR3 shown in SEQ ID NO.16; (4) CDR1 shown in SEQ ID NO.10, CDR1 shown in SEQ ID NO.13 CDR2 shown, CDR3 shown in SEQ ID NO.17.
  • the Nanobody further comprises a framework region FR;
  • the framework region FR includes FR1, FR2, FR3 and FR4;
  • the amino acid sequence of the FR1 is selected from at least one of the amino acid sequences shown in SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20 and SEQ ID NO.21;
  • the amino acid sequence of the FR2 is selected from at least one of the amino acid sequences shown in SEQ ID NO.22, SEQ ID NO.23 and SEQ ID NO.24;
  • the amino acid sequence of the FR3 is selected from at least one of the amino acid sequences shown in SEQ ID NO.25, SEQ ID NO.26, SEQ ID NO.27 and SEQ ID NO.28;
  • the amino acid sequence of the FR4 is SEQ ID NO.29.
  • amino acid sequence of the FR in the framework region is one of the following (1)-(4): (1) FR1 shown in SEQ ID NO.18, FR2 shown in SEQ ID NO.22, SEQ ID NO.22 FR3 shown in ID NO.25, FR4 shown in SEQ ID NO.29; (2) FR1 shown in SEQ ID NO.17, FR2 shown in SEQ ID NO.23, FR2 shown in SEQ ID NO.26 FR3, FR4 shown in SEQ ID NO.29; (3) FR1 shown in SEQ ID NO.20, FR2 shown in SEQ ID NO.24, FR3 shown in SEQ ID NO.27, SEQ ID NO.29 (4) FR1 shown in SEQ ID NO.21, FR2 shown in SEQ ID NO.23, FR3 shown in SEQ ID NO.28, and FR4 shown in SEQ ID NO.29.
  • the amino acid sequence of the Nanobody is the amino acid sequence shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4.
  • the nanobody is an amino acid sequence that has high affinity with the novel coronavirus RBD protein, and the high affinity means that the K D value is between 1 ⁇ 10 -12 and 1 ⁇ 10 -6 .
  • the preparation method according to the embodiment of the second aspect of the present invention comprises the following steps:
  • the preparation method according to the embodiment of the present invention has at least the following beneficial effects: the fermentation system used in the preparation method of the present invention is simple and easy to industrialize.
  • the nucleotide sequence screening method of the Nanobody comprises the following steps:
  • the application of the nanobodies for neutralizing toxicity of the novel coronavirus according to the third aspect of the present invention includes the following aspects:
  • the nanobody of the present invention has better SARS-CoV-2 virus neutralization activity and inhibitory activity, and is suitable for preventing and/or treating new coronavirus pneumonia;
  • the nanobody has high affinity with SARS-CoV-2 virus and can be detected by binding to SARS-CoV-2 virus.
  • the nucleotide sequence of the gene is such as SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO.7 or SEQ ID NO.7 ID NO.8.
  • the recombinant plasmid comprises the nucleotide sequence shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO.8 at least one of the gene fragments.
  • the recombinant cell of the sixth aspect of the present invention comprises the nucleotide sequence shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO.8 at least one of the gene fragments.
  • Fig. 2 is llama immune titer detection result in the embodiment of the present invention 1;
  • Fig. 3 is the electrophoresis figure of alpaca PBMC total RNA in the embodiment of the present invention 1;
  • Fig. 4 is the agarose gel electrophoresis obtained by Nanobody nucleic acid fragments in Example 1 of the present invention
  • Fig. 5 is a graph showing the result of groping for the connection conditions of the phage display library in Example 1 of the present invention.
  • Fig. 6 is the ELISA result chart of different Nanobody nucleic acid sequences screened in Example 1 of the present invention.
  • Fig. 7 is the protein electrophoresis diagram of purified Nanobody in Example 1 of the present invention.
  • FIG. 8 is a graph showing the affinity determination between NBS1-2 and RBD-Fc in Example 2 of the present invention.
  • FIG. 9 is a graph showing the affinity determination between NBS1-3 and RBD-Fc in Example 2 of the present invention.
  • Figure 10 is a graph showing the affinity determination between NBS1-10 and RBD-Fc in Example 2 of the present invention.
  • Figure 11 is a graph showing the affinity determination between NBS1-57 and RBD-Fc in Example 2 of the present invention.
  • Figure 12 shows the inhibition rates of samples with different concentrations on SARS-CoV-2 virus in Example 3 of the present invention.
  • FIG. 1 An experimental flow chart of an embodiment of the present invention is shown in Figure 1, including: purchasing S1 protein, immunizing alpaca; collecting blood in large quantities, isolating alpaca peripheral blood mononuclear cells (PBMC); extracting mRNA, and Reverse transcription into cDNA; self-designed primers, two-step PCR amplification of heavy chain antibody variable region; construction of heavy chain antibody variable region phage display library (the size of the immune library is 10 8 ); after 2-3 rounds The library was screened and sequenced; ELISA-positive clones were directly constructed into E. coli expression vectors for small-scale expression; the small-scale expression samples were subjected to virus neutralization test to obtain a set of nanobodies that can neutralize 2019-nCoV.
  • PBMC peripheral blood mononuclear cells
  • S1 protein The full name of S1 protein is spike glycoprotein (spike glycoprotein), which is located in the outermost layer of the new coronavirus, like a protruding "crown". That's how the coronavirus got its name.
  • spike glycoprotein spike glycoprotein
  • the S1 protein used in the examples of the present invention was purchased from Qiaoshenzhou Biotechnology Co., Ltd., item number: 40591-V08H 2019-nCoV Spike Protein S1 (His Tag).
  • Alpaca (llama) immunization program an adult male healthy llama alpaca, number KZL007, is immunized by subcutaneous injection at multiple points. 0.9 mg of S1 protein was injected each time, once every 14 days, for a total of 3 injections. 14 days after the third immunization, 2 ml of blood was collected to measure the titer.
  • the target antigen is the new coronavirus RBD protein
  • the primary antibody is anti-his-HRP
  • TMB color development 450nm detection O.D. value
  • the ELISA result can be above 1.0.
  • the serum was diluted with PBS buffer at pH 7.4, wherein the serum was diluted by 1K times, 2K times, 4K times, 8K times, 16K times, 32K times, 64K times, 128K times, 256K times, 512K times, 1024K times times, 2048K times, 4096K times, 8192K times, 16384K times.
  • Figure 2 shows the results of the llama immune titer test, as can be seen from Figure 2:
  • mRNA acquisition plan Collect blood from alpacas that meet the titer requirements, isolate PBMC, and extract total RNA from 17 ⁇ 10 6 PBMC cells.
  • the method for extracting total RNA is TRZOL portable operation method. The specific operation is as follows: Take the isolated PBMC cells out of the liquid nitrogen tank and thaw them quickly. Centrifuge at 800 g for 5 min, discard the supernatant, add 1 mL of TRIZOL solution to the pellet, and gently blow back and forth with a gun several times to fully lyse the cells.
  • RNA electropherogram is shown in Figure 3: In the figure, M: DNA maker DL2000 (2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp); Lane 1: Alpaca PBMC RNA (28S, 16S) after immunization; from the figure The 28S and 16S bands of the mRNA can be clearly seen.
  • M DNA maker DL2000 (2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp)
  • Lane 1 Alpaca PBMC RNA (28S, 16S) after immunization; from the figure The 28S and 16S bands of the mRNA can be clearly seen.
  • the RNA acquisition in the present invention is successful.
  • cDNA acquisition protocol Reverse transcription of total RNA into cDNA using Thermo's reverse transcription kit, quantitatively aliquot, and store at -80°C for later use.
  • Two-step PCR amplification was performed using the primers in Table 1 below to obtain the target fragment.
  • the PCR reaction program is as follows:
  • the first round of PCR was performed with Kz-001 and Kz-002 as primers, and amplified with TAKARA's high-fidelity PCR polymerase.
  • the amplification procedure was as follows: pre-denaturation at 94°C for 3 minutes; denaturation at 94°C for 30 seconds, and annealing at 53°C for 30 minutes. seconds, 72°C extension for 40 seconds, cycle the program for 18 rounds; 72°C end extension for 10 minutes; 4°C cooling for 1 minute. A 750bp band was recovered.
  • the second round of PCR was carried out with Kz-003, Kz-004, Kz-005 and Kz-006 as primers, and amplified with TAKARA's high-fidelity PCR polymerase.
  • the amplification procedure was as follows: pre-denaturation at 94°C for 3 minutes; Denaturation for 30 seconds, annealing at 53°C for 30 seconds, extension at 72°C for 40 seconds, cycle the program for 20 rounds; end extension at 72°C for 10 minutes; cooling at 4°C for 1 minute. A 450bp band was recovered.
  • the nucleic acid fragment of the above Nanobody was subjected to restriction enzyme digestion, and the two restriction enzyme cleavage sites used in the embodiment of the present invention were sfiI and NotI, which were recovered by a gel recovery kit and quantified; the embodiment of the present invention used a commercial vector pcantab5e, at the same time, this vector was also digested with the above two restriction enzymes, recovered by gel recovery kit and quantified. After digestion, the fragment concentration was 120ng/ ⁇ l, and the vector was 80ng/ ⁇ l.
  • Figure 5 shows the results of groping the connection conditions for constructing a phage display library in the embodiment of the present invention. It can be seen from Figure 5 that the overnight connection efficiency is the highest when the molar ratio of the vector to the nanobody fragment in the 10 ⁇ l connection system is 1:3.
  • digested vectors and the digested nanobody fragments were ligated overnight at a molar ratio of 1:3, and then electroporated into TG1 competent cells, diluted and coated on a plate for colony counting to obtain 3.4 ⁇ 10 8 phage display library.
  • the phage display library screening method in the present invention is as follows: the phage library is screened by a solid-phase screening method.
  • the screening process is to coat the target molecules on the surface of 96 wells, wash off the unadsorbed target molecules, then block them, and then add the background-subtracted phage antibody library to the wells for binding. Unbound phage was washed away and eluted with 0.2M glycine-hydrochloric acid to obtain affinity phage.
  • high-affinity phage is obtained by reducing the concentration of the coated target molecule and increasing the washing intensity (in the embodiment of the present invention, the concentration of the target molecule is reduced and the concentration of Tween 20 in the washing solution is increased).
  • the ELISA identification method was as follows: dilute the target protein with pH9.6 coating solution, let stand at 37°C for 1 hour for coating, and wash 3 times with PBS. Add blocking solution to block and let stand at 37°C for 1 hour, throw off excess blocking solution and wash 3 times with PBS. The amplified product was diluted 10-fold with 1% M-PBS and mixed with 50ul/well for 1 hour at 37°C.
  • Primary antibody Rabbit anti-M13 was diluted 1:1000 with 1% M-PBS, 50 ⁇ l/well, and allowed to stand at 37°C for 1.0 h.
  • the positive sequences in the ELISA were sequenced to obtain different Nanobody nucleic acid sequences.
  • the ELISA results of the sequences are shown in Figure 6 (the abscissa corresponds to each Nanobody nucleic acid sequence, and the ordinate represents the absorption intensity at 490 nm); from the figure It can be seen that the absorption values of all the selected sequences at 490 nm are much greater than that of the blank control group, indicating that the nanobody binds to the RBD protein as a positive result.
  • 4 different nanosequences among the positive sequences were selected for follow-up research.
  • Expression strain construction scheme Amplify the Nanobody sequence by PCR amplification, introduce NdeI and XhoI restriction sites at both ends of the Nanobody fragment, use these two restriction sites to connect to the PET30A vector, and sequence correctly for later use.
  • the successfully constructed clones NBS1-2, NBS1-3, NBS1-10 and NBS1-57 were induced to express in E. coli BL21 (DE3) with IPTG at a concentration of 1 mM at 37°C overnight. After renaturation and purification, the purity was not lower than 95% of the samples.
  • IPTG was added when the OD 600 value was 0.5, the final concentration of IPTG was 1 mM, and the cells were induced overnight at 37°C.
  • dissolved inclusion bodies including but not limited to centrifugation, dialysis to remove oxidants, reducing agents or redox couples, and purification.
  • FIG. 7 is the electrophoresis diagram (SDS-PAGE) of the purified nanobody, wherein, M: marker; 1: NBS1-2; 2: NBS1-3; 3 : NBS1-10; 4: NBS1-57, it can be seen from the figure that the sample purity is greater than 80%.
  • sequence information of the four nanobodies (NBS1-2, NBS1-3, NBS1-10, NBS1-57) that inhibit SARS-CoV-2 virus in the examples of the present invention are as follows:
  • NBS1-2 amino acid sequence SEQ ID NO.1:
  • NBS1-3 amino acid sequence SEQ ID NO.2
  • NBS1-57 amino acid sequence (SEQ ID NO.4):
  • the nucleotide sequence of the Nanobody is as follows:
  • NBS1-2 nucleotide sequence SEQ ID NO.5
  • NBS1-3 nucleotide sequence SEQ ID NO.6
  • NBS1-10 nucleotide sequence SEQ ID NO.7
  • NBS1-57 nucleotide sequence SEQ ID NO.8
  • FR framework regions
  • CDR complementarity determining regions
  • the instrument Gator probe life is used to detect intermolecular interactions by thin film interference method, and the sample to be tested with a concentration of not less than 80% and the antigen RBD-FC protein (new coronavirus RBD protein) of a concentration of not less than 90% are prepared. .
  • Probe species Kinetic index of binding of anti-human Fc antibody and human Fc.
  • Buffer Q buffer (PBS+0.02%Tween-20+0.2%BSA).
  • NBS1-2 sample detection method immobilized antigen RBD-Fc (100nM) to detect nanobody; probe type: anti-human Fc antibody; NBS1-2 was diluted to 60000nM, 30000nM, 15000nM, 7500nM, 3750nM with Q buffer , 1875nM, 937.5nM, OnM.
  • NBS1-3 sample detection method immobilized antigen RBD-Fc (100nM) to detect nanobody; probe type: anti-human Fc antibody; NBS1-3 was diluted with Q buffer to 25nM, 12.5nM, 6.25nM, 3.18 nM, 1.56nM, 0.78nM, OnM.
  • NBS1-10 test sample detection method immobilized antigen RBD-Fc (100nM) to detect nanobody; probe type: anti-human Fc antibody; NBS1-10 was diluted with Q buffer to 25nM, 12.5nM, 6.25nM, 3.18 nM, 1.56nM, 0.78nM, OnM.
  • NBS1-57 test sample detection method immobilized antigen RBD-Fc (100nM) to detect nanobodies; probe type: anti-human Fc antibody; NBS1-57 was diluted with Q buffer to 25nM, 12.5nM, 6.25nM, 3.18 nM, 1.56nM, 0.78nM, OnM.
  • Figure 8 is the NBS1-2-RBD-Fc affinity measurement diagram
  • Figure 9 is the NBS1-3-RBD-Fc affinity measurement diagram
  • Figure 10 is a graph of NBS1-10-RBD-Fc affinity measurement
  • Figure 11 is a graph of NBS1-57-RBD-Fc affinity measurement.
  • Koff refers to the dissociation rate constant
  • kon refers to the association rate constant
  • K D Koff/kon
  • the equilibrium dissociation constant which is used to characterize the affinity of the Nanobody to the RBD-Fc protein.
  • the larger the K D value the more drug concentration required to cause the maximum effect, and the smaller the affinity.
  • the four nanobody samples all have strong affinity with the antigen RBD-Fc protein.
  • the sample NBS1-3 has the strongest affinity, and it can be inferred that its potential effect of neutralizing the new coronavirus is the best.
  • the virus neutralization test is a technology for detecting specific antibodies, that is, the interference ability of the biological activity of the antibody to be tested is determined by using a known virus. In the embodiment of the present invention, this technology is used to detect the activity of the screened nanobody.
  • nanobody samples with a purity of more than 80% were prepared: NBS1-2, NBS1-3, NBS1-10 and NBS1-57; the nanobody used was prepared in Example 1.
  • the diluted protein sample needs to be incubated with an equal volume of pseudovirus, so the final sample concentration is 1/2 of the initial dilution concentration of the sample.
  • the virus concentration used in the present invention is 500 TCID50; where TCID50 refers to half the cell culture infection and is a calculated index.
  • the TCID50 calculation method used in the present invention is the Reed-muench method (there are two TCID50 calculation methods: Reed-muench method or Karber).
  • Figure 12 shows the neutralization ability of protein samples against SARS-CoV-2 pseudovirus at different concentrations.
  • the abscissa is the protein concentration, and the ordinate is the percentage of neutralization inhibition level.
  • Each point represents the mean ⁇ SEM of 3 replicate wells for each experimental condition.
  • the samples of the four nanobodies in the embodiment of the present invention have strong inhibitory ability to the virus at high concentrations, wherein: NBS1-3 and NBS1-57 can reach 1 ⁇ g/ml when the concentration is 1 ⁇ g/ml.
  • the inhibition rate is close to 100%, and it can reach a high inhibition rate of 80% when its concentration is 0.1 ⁇ g/ml; NBS1-10 also has a high inhibition rate close to 90% when the concentration reaches 1 ⁇ g/ml;
  • the inhibition rate is relatively low, but can reach 70% at 100 ⁇ g/ml.
  • the four nanobodies prepared by the present invention have the ability to inhibit virus infection at higher concentrations
  • NBS1-3 and NBS1-57 have strong antiviral effects
  • NBS1-2 showed relatively weak neutralization ability against SARS-CoV-2 pseudovirus.
  • the samples of the four nanobodies in the embodiment of the present invention have strong inhibitory ability to the virus at high concentrations, and the inhibition rate at the concentration of 1 ⁇ g/ml is as high as 100%, and has strong performance against SARS-CoV-2 pseudovirus.
  • Neutralizing ability has strong antiviral effect, and can be used for the treatment of new coronary pneumonia.

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Abstract

本发明公开了新冠病毒中和毒性的纳米抗体及其制备方法与应用，该纳米抗体包括框架区FR和互补决定区CDR；该互补决定区CDR包括CDR1、CDR2和CDR3；该CDR1的氨基酸序列选自如SEQ ID NO.1和SEQ ID NO.2所示的氨基酸序列中的至少一种；该CDR2的氨基酸序列选自如SEQ ID NO.3、SEQ ID NO.4和SEQ ID NO.5所示的氨基酸序列中的至少一种；该CDR3的氨基酸序列选自如SEQ ID NO.6-9任一项所示的氨基酸序列中的至少一种。

Description

新冠病毒中和毒性的纳米抗体及其制备方法与应用 技术领域
本发明涉及多肽技术领域，具体涉及新冠病毒中和毒性的纳米抗体及其制备方法与应用。
背景技术
新型冠状病毒肺炎(Corona Virus Disease 2019，COVID-19)是由一种新的β冠状病毒-新型冠状病毒(SARS-CoV-2病毒)引起，以血管紧张素转化酶2(Angiotensin converting enzyme 2，ACE-2)作为受体侵入细胞导致肺损伤，病情加重与继发引起的全身性炎症反应密切相关,重症患者会出现急性呼吸窘迫综合征(Acuterespiratory distress syndrome，ARDS)和感染性休克，最终出现多器官功能衰竭。目前，大多数治疗药物如巴利替尼、瑞德西韦和氯喹等尚处于临床试验阶段，尚无特效药治疗。COVID-19传染性强，可引起多种严重的并发症，对全球公共安全造成了极大威胁。
新冠病毒疫苗研发是进行病毒防治的一个重要方法，但是，由于疫苗的安全性和疗效实验的验证间期较长，一般至少需要1-2年的时间才能完成评价，目前尚未有有效的疫苗进入市场被广泛使用。即使是重组全人源单克隆抗体也存在免疫原性和生产成本高等缺点。
中和抗体是机体应对抗原刺激而产生的具有保护作用的一类抗体，即使在疾病的相对晚期，中和抗体也可能是一种有用的治疗方法。作为疫苗和化学治疗的补充，由抗体介导的预防和治疗病毒感染的措施已显现良好的效果，其应用前景得到专家的认同。目前已经有将COVID-19患者康复后的血浆，输注给重症病人实现治愈的成功案例。但多抗血浆不仅来源有限，同时其临床应用也受到诸如难以质控、供受体血型不匹配、潜在的传染性因子等条件的限制，而人源单克隆抗体可有效克服上述问题。因此，新冠病毒中和抗体的研发能够加快推动新冠病毒抗体药物临床评价，增强新冠病毒肺炎治疗和预防手段。
发明内容
本发明旨在至少解决现有技术中存在的技术问题之一。为此，本发明提出一种新冠病毒中和毒性的纳米抗体，能够用于新冠病毒肺炎的防治。
本发明还提出上述新冠病毒中和毒性的纳米抗体的制备方法。
本发明还提出上述新冠病毒中和毒性的纳米抗体的应用。
本发明还提出一种编码新冠病毒中和毒性的纳米抗体的基因。
本发明还提出一种重组质粒。
本发明还提出一种重组细胞。
根据本发明的第一方面实施方式的新冠病毒中和毒性的纳米抗体，所述纳米抗体包括框架区FR和互补决定区CDR；所述互补决定区CDR包括CDR1、CDR2和CDR3；
所述CDR1的氨基酸序列选自如SEQ ID NO.9和SEQ ID NO.10所示的氨基酸序列中的至少一种；
所述CDR2的氨基酸序列选自如SEQ ID NO.11、SEQ ID NO.12和SEQ ID NO.13所示的氨基酸序列中的至少一种；
所述CDR3的氨基酸序列选自如SEQ ID NO.14、SEQ ID NO.15、SEQ ID NO.16和SEQ ID NO.17所示的氨基酸序列中的至少一种。
根据本发明实施方式的新冠病毒中和毒性的纳米抗体，至少具有如下有益效果：本发明的新冠病毒中和毒性的纳米抗体具有分子量小(只有单克隆抗体的1/10大小)、结合活性高(具有与SARS-CoV-2病毒的高亲和特性)和生产成本低等优点，其在高浓度下对病毒具有较强的抑制能力，在1μg/ml的浓度下的抑制率高达100％，对SARS-CoV-2假病毒表现较强的中和能力，具有较强的抗病毒效果；本发明的纳米抗体还具有成熟临床前有效性和安全性研究基础、产业化转化成功率高且能快速进入临床研究。
根据本发明的一些实施方式，所述互补决定区CDR的氨基酸序列为下述(1)-(4)中的一种：(1)SEQ ID NO.9所示的CDR1，SEQ ID NO.11所示的CDR2，SEQ ID NO.14所示的CDR3；(2)SEQ ID NO.10所示的CDR1，SEQ ID NO.12所示的CDR2，SEQ ID NO.15所示的CDR3；(3)SEQ ID NO.10所示的CDR1，SEQ ID NO.12所示的CDR2，SEQ ID NO.16所示的CDR3；(4)SEQ ID NO.10所示的CDR1，SEQ ID NO.13所示的CDR2，SEQ ID NO.17所示的CDR3。
根据本发明的一些实施方式，所述纳米抗体还包括框架区FR；所述框架区FR包括FR1、FR2、FR3和FR4；
所述FR1的氨基酸序列选自如SEQ ID NO.18、SEQ ID NO.19、SEQ ID NO.20和SEQ ID NO.21所示的氨基酸序列中的至少一种；
所述FR2的氨基酸序列选自如SEQ ID NO.22、SEQ ID NO.23和SEQ ID NO.24所示的氨基酸序列中的至少一种；
所述FR3的氨基酸序列选自如SEQ ID NO.25、SEQ ID NO.26、SEQ ID NO.27和SEQ ID NO.28所示的氨基酸序列中的至少一种；
所述FR4的氨基酸序列为SEQ ID NO.29。
进一步地，所述框架区FR的氨基酸序列为下述(1)-(4)中的一种：(1)SEQ ID NO.18 所示的FR1，SEQ ID NO.22所示的FR2，SEQ ID NO.25所示的FR3，SEQ ID NO.29所示的FR4；(2)SEQ ID NO.17所示的FR1，SEQ ID NO.23所示的FR2，SEQ ID NO.26所示的FR3，SEQ ID NO.29所示的FR4；(3)SEQ ID NO.20所示的FR1，SEQ ID NO.24所示的FR2，SEQ ID NO.27所示的FR3，SEQ ID NO.29所示的FR4；(4)SEQ ID NO.21所示的FR1，SEQ ID NO.23所示的FR2，SEQ ID NO.28所示的FR3，SEQ ID NO.29所示的FR4。
根据本发明的一些实施方式，所述纳米抗体的氨基酸序列为如SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3或SEQ ID NO.4所示的氨基酸序列。
根据本发明的一些实施方式，所述纳米抗体为与新型冠状病毒RBD蛋白有高亲和力的氨基酸序列，所述高亲和力是指K D值是在1×10 -12～1×10 -6之间。根据本发明的第二方面实施方式的制备方法，包括以下步骤：
(1)将如SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7或SEQ ID NO.8所示的核苷酸序列克隆至表达载体中得到重组质粒，将所述重组质粒转入宿主细胞中诱导表达所述纳米抗体；
(2)从宿主细胞中纯化所述纳米抗体。
根据本发明实施方式的制备方法，至少具有如下有益效果：本发明的制备方法使用的发酵系统简单，很容易产业化。
根据本发明的一些实施方式，所述纳米抗体的核苷酸序列筛选方法包括以下步骤：
(1)使用S1蛋白免疫羊驼，提取外周血单核细胞中的总mRNA，并反转录成cDNA；
(2)通过PCR扩增重链抗体可变区基因，构建重链抗体可变区噬菌体展示库，筛选并测序得到能够表达所述纳米抗体的核酸序列。
根据本发明的第三方面实施方式的新冠病毒中和毒性的纳米抗体的应用，包括以下方面：
所述纳米抗体在制备预防和/或治疗新冠病毒肺炎的药物中的应用；和/或
所述纳米抗体在制备检测新冠病毒的检测试剂中的应用；和/或
所述纳米抗体在制备检测新冠病毒的检测试剂盒中的应用。
根据本发明实施方式的应用，至少具有如下有益效果：本发明的纳米抗体具有较好的SARS-CoV-2病毒中和活性和抑制活性，适用于预防和/或治疗新冠病毒肺炎；同时，所述纳米抗体具有与SARS-CoV-2病毒的高亲和特性，能够通过结合SARS-CoV-2病毒进行检测。
根据本发明的第四方面实施方式的编码新冠病毒中和毒性的纳米抗体的基因，所述基因的核苷酸序列如SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7或SEQ ID NO.8所示。
根据本发明的第五方面实施方式的重组质粒，所述重组质粒中包含如SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7或SEQ ID NO.8所示的核苷酸序列的基因片段中的至少一种。
根据本发明的第六方面实施方式的重组细胞，所述重组细胞中包含如SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7或SEQ ID NO.8所示的核苷酸序列的基因片段中的至少一种。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。
附图说明
图1为本发明实施例的流程图；
图2为本发明实施例1中llama免疫效价检测结果；
图3为本发明实施例1中羊驼PBMC总RNA电泳图；
图4为本发明实施例1中纳米抗体核酸片段获得琼脂糖凝胶电泳；
图5为本发明实施例1中噬菌体展示库连接条件摸索结果图；
图6为本发明实施例1中筛选得到的不同纳米抗体核酸序列ELISA结果图；
图7为本发明实施例1中纯化后纳米抗体的蛋白电泳图；
图8为本发明实施例2中NBS1-2与RBD-Fc亲和力测定图；
图9为本发明实施例2中NBS1-3与RBD-Fc亲和力测定图；
图10为本发明实施例2中NBS1-10与RBD-Fc亲和力测定图；
图11为本发明实施例2中NBS1-57与RBD-Fc亲和力测定图；
图12为本发明实施例3中不同浓度的样品对SARS-CoV-2病毒的抑制率。
具体实施方式
为详细说明本发明的技术内容、所实现目的及效果，以下结合实施方式并配合附图予以说明。
本发明一种实施方式的实验流程图如图1所示，包括：购买S1蛋白，免疫羊驼；大量采血，分离羊驼外周血单核细胞(Peripheral blood mononuclear cell，PBMC)；提取mRNA，并反转录成cDNA；自行设计引物，两步PCR扩增出重链抗体可变区；构建重链抗体可变区噬菌体展示库(该免疫库的库容大小为10 8)；经过2-3轮筛序此文库，并进行测序；将ELISA阳性克隆直接构建到大肠杆菌表达载体里进行小量表达；将小量表达的样品进行病毒中和试验，得到一组可以中和新冠病毒的纳米抗体。
S1蛋白：S 1蛋白全称为spike glycoprotein(刺突糖蛋白)，位于新冠病毒最外层，像一个个突起的“皇冠”。冠状病毒正因此得名。
本发明实施例中使用的S1蛋白购买自翘神州生物技术有限公司，货号：40591-V08H 2019-nCoV Spike Protein S1(His Tag)。
实施例1新冠病毒中和毒性纳米抗体的制备
1.动物免疫方案及结果
羊驼(llama)免疫方案：一只成年雄性健康llama羊驼，编号KZL007，皮下多点注射免疫。每次注射0.9mg的S1蛋白，每隔14天注射一次，共注射3次。第三次免疫后14天采血2毫升测效价。
llama免疫效价检测方法：利用酶联免疫法，靶抗原为新冠病毒RBD蛋白，一抗为抗his-HRP，TMB显色，450nm检测O.D.值)。当血清稀释128K倍(即128000倍，后文中表述含义类似)后酶联免疫结果在1.0以上即可。检测时将血清用pH 7.4的PBS缓冲液稀释，其中血清被稀释倍数为1K倍、2K倍、4K倍、8K倍、16K倍、32K倍、64K倍、128K倍、256K倍、512K倍、1024K倍、2048K倍、4096K倍、8192K倍、16384K倍。
图2为llama免疫效价检测结果，从图2中可以看出：
1)通常免疫效价在128K时OD 450读数大于1时即可认为免疫成功，可停止免疫，本发明免疫效价达到血清稀释倍数1024K，远超行业通常数值。
2)空白对照数值正常，从血清稀释比例1：1K到血清稀释比例1：128K后的OD 450读值变化不大是因为血清中抗体浓度过高造成效价太高，达到仪器最大读值范围；而从稀释比例256K开始，数据呈现梯度正常下降。
2.mRNA、cDNA获得方案及结果
mRNA获得方案：对满足效价要求的羊驼进行大量采血，分离PBMC，用17×10 6个PBMC细胞提取总RNA，提取总RNA的方法为TRZOL手提操作法。具体操作如下：将分离完的PBMC细胞从液氮罐中取出，迅速解冻。800g离心，5min，弃上清，向沉淀中加入1mL的TRIZOL液，用枪轻轻来回吹打几次使细胞充分裂解。加200μl氯仿后，摇剧烈震荡30s，静置5min，13500r/min，离心10min，分层，吸取上层液体相到另一新的无RNA酶1.5mL管中，加入等体积的异丙醇。混匀，-20℃沉淀20min。将沉淀液体离心，13500r/min，离心10min，弃上清。75％冰乙醇洗两遍，超净台吹干，用60μl无RNA酶的水复溶，备用。
得到RNA电泳图如图3所示：图中，M：DNA maker DL2000(2000bp，1000bp，750bp，500bp，250bp，100bp)；泳道1：免疫后羊驼PBMC RNA(28S，16S)；从图中可以清楚看到mRNA的28S条带，16S条带。说明本发明中RNA获取成功。
cDNA获得方案：使用Thermo公司反转录试剂盒将总RNA反转录成cDNA并定量分装，-80℃保存备用。
3.纳米抗体核酸片段获得方案及结果
利用下表1中的引物进行两步PCR扩增，获得目的片段。
表1.构建噬菌体库引物
Figure PCTCN2020132279-appb-000001
PCR反应程序如下：
以Kz-001和Kz-002为引物进行第一轮PCR，用TAKARA公司高保真PCR聚合酶进行扩增，扩增程序如下：94℃预变性3分钟；94℃变性30秒，53℃退火30秒，72℃延伸40秒，循环该程序18轮；72℃末端延伸10分钟；4℃降温1分钟。回收750bp条带。
以Kz-003、Kz-004、Kz-005和Kz-006为引物进行第二轮PCR，用TAKARA公司高保真PCR聚合酶进行扩增，扩增程序如下：94℃预变性3分钟；94℃变性30秒，53℃退火30秒，72℃延伸40秒，循环该程序20轮；72℃末端延伸10分钟；4℃降温1分钟。回收450bp条带。
第二轮PCR结果(纳米抗体核酸片段获得琼脂糖凝胶电泳图)见图4，图中，M：DNA maker DL2000(2000bp，1000bp，750bp，500bp，250bp，100bp)；泳道1-泳道4：第二轮PCR琼脂糖凝胶电泳；从图4中可以清晰地看出一条特异性的目的条带，大小为450bp，此条带即为纳米抗体片段。将这些条带用琼脂糖凝胶回收试剂盒回收，定量。
4.噬菌体展示方案
将上述纳米抗体的核酸片段做限制性酶切，本发明实施例所用的两种限制性酶切位点为sfiI和NotI，利用凝胶回收试剂盒回收后定量；本发明实施例使用商业化载体pcantab5e，同时此载体也采用上述两种限制性酶切，利用凝胶回收试剂盒回收后定量。酶切后片段浓度为120ng/μl，载体80ng/μl。
本发明实施例中构建噬菌体展示库连接条件摸索结果如图5所示，从图5中可以看出10μl连接体系中载体与纳米抗体片段摩尔比为1：3时，过夜连接效率最高。
将所有酶切后的载体与酶切后的纳米抗体片段按照摩尔比1：3过夜连接，之后电击转化进TG1感受态细胞中，稀释涂布平板进行克隆计数，得到3.4×10 8的噬菌体展示库。
5.噬菌体筛选方案及结果
本发明中噬菌体展示库筛选方法如下：采用固相筛选方法筛选噬菌体库。筛选流程是将靶分子包被在96孔表面，洗去未吸附的靶分子，然后封闭，之后用扣除背景的噬菌体抗体库加入到孔中结合。洗去未结合的噬菌体，再用0.2M甘氨酸-盐酸洗脱得到亲和噬菌体。每轮通过降低包被靶分子浓度和加大洗涤力度(本发明实施例是减小靶分子浓度增加洗液中吐温20浓度)得到高亲和力噬菌体。每经一轮淘选扩增试验，就会使能与靶分子结合的纳米抗体在噬菌体库中得到富集。经过3轮筛选后富集达到1000倍以上，进行挑取单克隆验证。3轮筛选，筛选结果如下表2所示：
表2.抗新冠纳米抗体噬菌体库筛选结果
筛选轮数 投入量(pfu) 产出量(pfu) 富集率(投入/产出)
第一轮 3×10 11 9.48×10 6 3.16×10 4
第二轮 3×10 11 4.47×10 7 6.71×10 3
第三轮 3×10 11 6.81×10 8 4.4×10 2
6.ELISA结合检测方案及结果
从第3轮筛选产物中挑取768个单克隆进行ELISA鉴定，ELISA鉴定方法如下：用pH9.6包被液稀释靶蛋白，37℃静置1小时进行包被，PBS洗3次。加入封闭液进行封闭37℃静置1小时，甩出多余的封闭液PBS洗3次。取扩增产物用1％M-PBS10倍稀释混匀50ul/孔37℃静置1小时。一抗：用1％M-PBS按1：1000稀释兔抗M13，50μl/孔37℃静置1.0h。二抗：用1％M-PBS按1：3000稀释HRP-羊抗兔50μl/孔37℃静置1.0h。显色：取0.2M/LNa2HPO4+0.1M/L柠檬酸各4.5ml加入少量OPD(邻苯二胺)，60μl H 2O 2混匀，50μl/孔。2M硫酸终止50μl/孔。490nm检测。Phage ELISA最少要重复一次。阳性克隆保存：取0.5ml Phage+0.3ml 50％甘油，混匀。-80℃保存。
将ELISA中的阳性序列送测序，得到不同纳米抗体核酸序列，序列的ELISA结果如图6所示(横坐标对应每个纳米抗体核酸序列，纵坐标表示在490nm处的吸收强度)；从图中可以看出筛选到的所有序列在490nm处吸收值都远大于空白对照组，显示纳米抗体与RBD蛋白结合呈阳性结果。本实施例中选择阳性序列中4个不同的纳米序列进行后续研究。
7.表达菌株构建方案及结果
表达菌株构建方案：将纳米抗体序列以PCR扩增的方式扩增，在纳米抗体片段两端引入NdeI和XhoI酶切位点，利用这两个酶切位点连入PET30A载体，测序正确备用。
8.小量样品获得方案
将构建成功的克隆NBS1-2、NBS1-3、NBS1-10和NBS1-57在大肠杆菌BL21(DE3)中利用浓度1mM的IPTG，37℃诱导表达过夜，经过复性纯化后得到纯度不低于95％的样品。
具体诱导表达纯化及复性过程如下：
1)接种后OD 600值在0.5时加入IPTG，使IPTG终浓度为1mM，37℃诱导过夜。
2)诱导表达后离心收菌，匀浆破碎。
3)Tris缓冲液进行包涵体清洗，称量包涵体，加入变性液。按1g包涵体：15ml变性液比例进行包涵体溶解。
其中，变性液配方为：4M盐酸胍或者6M尿素或者采用碱变性，氧化还原电对(包括但不限于GSH/GSSG，胱氨酸/半胱氨酸等)pH＝9.0。
4)对溶解后的包涵体进行处理，包括但不限于离心，透析去除氧化剂，还原剂或氧化还原电对，纯化等。
5)对处理后的包涵体溶解液进行复性，包括进行脉冲复性，稀释复性等。低温(4℃)或者室温(25℃)复性24h。复性液配方：2M尿素，氧化还原电对(包括但不限于GSH/GSSG，胱氨酸/半胱氨酸等)，pH＝9.0。
6)对复性后的样品进行处理；包括但不限于离心，调节pH，过滤/超滤，换液浓缩等。
7)对处理后的复性液进行纯化，采用阳离子或者阴离子树脂进行层析。
8)对收集的目的洗脱蛋白进行凝胶层析，收集洗脱的目的蛋白峰。对处理后的复性液进行SDS-PAGE跑胶观察，图7为纯化后纳米抗体的电泳图(SDS-PAGE)，其中，M：marker；1：NBS1-2；2：NBS1-3；3：NBS1-10；4：NBS1-57，图中可以看出，样品纯度大于80％。
本发明实施例中4个抑制SARS-CoV-2病毒的纳米抗体(NBS1-2、NBS1-3、NBS1-10、NBS1-57)的序列信息如下：
i.纳米抗体的氨基酸序列具体如下：
1)NBS1-2氨基酸序列(SEQ ID NO.1)：
Figure PCTCN2020132279-appb-000002
2)NBS1-3氨基酸序列(SEQ ID NO.2)：
Figure PCTCN2020132279-appb-000003
3)NBS1-10氨基酸序列(SEQ ID NO.3)：
Figure PCTCN2020132279-appb-000004
4)NBS1-57氨基酸序列(SEQ ID NO.4)：
Figure PCTCN2020132279-appb-000005
ii.纳米抗体的核苷酸序列具体如下：
1)NBS1-2核苷酸序列(SEQ ID NO.5)：
Figure PCTCN2020132279-appb-000006
2)NBS1-3核苷酸序列(SEQ ID NO.6)：
Figure PCTCN2020132279-appb-000007
3)NBS1-10核苷酸序列(SEQ ID NO.7)：
Figure PCTCN2020132279-appb-000008
4)NBS1-57核苷酸序列(SEQ ID NO.8)：
Figure PCTCN2020132279-appb-000009
使用Vector NTI软件对测序结果进行分析，可确定以下框架区(Framework Regions，FR)和互补决定区(Complementary Determining Resions，CDR)，将这4个序列进行功能区划分如下表3：
表3.功能区划分
Figure PCTCN2020132279-appb-000010
实施例2新冠病毒中和毒性纳米抗体的亲和力检测试验
1.仪器及试剂：
本实施例中使用仪器Gator probe life利用薄膜干涉法检测分子间相互作用，准备浓度不低于80％的待测样品与浓度不低于90％的抗原RBD-FC蛋白(新型冠状病毒RBD蛋白)。
探针种类：抗人Fc抗体和人Fc的结合测定动力学指数。
缓冲液：Q buffer(PBS+0.02％Tween-20+0.2％BSA)。
2.实验方法：
1)NBS1-2待测样品检测方法：固定抗原RBD-Fc(100nM)检测纳米抗体；探针种类：抗人Fc抗体；用Q buffer将NBS1-2稀释到60000nM、30000nM、15000nM、7500nM、3750nM、1875nM、937.5nM、0nM。
2)NBS1-3待测样品检测方法：固定抗原RBD-Fc(100nM)检测纳米抗体；探针种类：抗人Fc抗体；用Q buffer将NBS1-3稀释到25nM、12.5nM、6.25nM、3.18nM、1.56nM、0.78nM、0nM。
3)NBS1-10待测样品检测方法：固定抗原RBD-Fc(100nM)检测纳米抗体；探针种类：抗人Fc抗体；用Q buffer将NBS1-10稀释到25nM、12.5nM、6.25nM、3.18nM、1.56nM、0.78nM、0nM。
4)NBS1-57待测样品检测方法：固定抗原RBD-Fc(100nM)检测纳米抗体；探针种类：抗人Fc抗体；用Q buffer将NBS1-57稀释到25nM、12.5nM、6.25nM、3.18nM、1.56nM、0.78nM、0nM。
3.实验结果及分析：
通过测定抗原-抗体亲和力，亲和力测定图如图8～图11所示(其中，图8为NBS1-2—RBD-Fc亲和力测定图；图9为NBS1-3—RBD-Fc亲和力测定图；图10为NBS1-10—RBD-Fc亲和力测定图；图11为NBS1-57—RBD-Fc亲和力测定图)。可以看出本发明实施例中几个样品亲和力情况如下表4所示：
表4.纳米抗体的亲和力测试情况
样本名称 koff(1/s) kon(1/Ms) K D(M)
NBS1-3 7.16×10 -5 2.38×10 6 3.01×10 -11
NBS1-57 4.91×10 -3 3.80×10 6 1.29×10 -9
NBS1-10 3.11×10 -3 2.37×10 6 1.31×10 -9
NBS1-2 1.27×10 -3 8.95×10 2 1.41×10 -6
其中，koff指解离速率常数，kon指结合速率常数，K D(K D＝koff/kon)指平衡解离常数，用来表征纳米抗体与RBD-Fc蛋白的亲和力。其中，K D值越大，引起最大效应所需药物浓度越多，亲和力越小。从结果可知，四个纳米抗体样本均具有较强的与抗原RBD-Fc蛋白的亲和力，其中，样本NBS1-3的亲和力最强，可推断其潜在的中和新冠病毒的作用效果最好。
实施例3新冠病毒中和毒性纳米抗体的病毒中和试验
病毒中和试验是一种检测特异性抗体的技术，即用已知病毒测定待测抗体生物活性的干涉能力，本发明实施例用此技术检测筛选到的纳米抗体活性。
准备4个纯度为80％以上的纳米抗体样品：NBS1-2，NBS1-3，NBS1-10和NBS1-57； 使用的纳米抗体由实施例1制备得到。
1.实验方法：
1)首先以各蛋白样品的原始浓度作为起始浓度，利用样品稀释液(20mM PB，pH 7.2)对各蛋白样品进行梯度稀释，依次设置1mg/ml、100μg/ml、10μg/ml、1μg/ml、0.1μg/ml、0.01μg/ml等稀释浓度；
2)稀释好的样品与等体积的SARS-CoV-2假病毒共孵育，再用以感染细胞检测蛋白样品抗病毒中和能力。每个样品检测终浓度(实际检测浓度)如下表5所示。
表5.样品检测终浓度
Figure PCTCN2020132279-appb-000011
注：稀释好的蛋白样品需与等体积的假病毒共孵育，因此样品检测终浓度为样品初始稀释浓度的1/2。
本发明中使用的病毒浓度为500TCID50；其中TCID50是指半数细胞培养物感染量并且是一个计算出来的指标。本发明使用的TCID50计算方法为Reed-muench两氏法(TCID50计算方法有2种：Reed-muench两氏法或Karber)。
2.实验结果：
从表5中可以看出，每个样品对SARS-CoV-2假病毒的50％抑制浓度(IC 50)和90％抑制浓度(IC 90)。中和滴度通过SPSS软件经probit回归分析得到。
图12显示蛋白样品在不同浓度下对SARS-CoV-2假病毒的中和能力。横坐标为蛋白浓度，纵坐标为中和抑制水平百分比。每个点表示每个实验条件下3个重复孔的平均值±SEM。
从图12的试验结果可知，本发明实施例的4种纳米抗体的样品在高浓度下对病毒具有较强的抑制能力，其中：NBS1-3和NBS1-57在浓度为1μg/ml时能达到接近100％的抑制率，当其浓度为0.1μg/ml时也能达到80％的高抑制率；NBS1-10在浓度达到1μg/ml时也具有接近90％的高抑制率；NBS1-2的抑制率相对较低，但在100μg/ml也能达到70％。
因此，通过图12分析可得以下结论：
1)本发明制备得到的四种纳米抗体较高浓度下对病毒感染具有抑制能力；
2)NBS1-3和NBS1-57具有较强的抗病毒效果；
3)NBS1-2对SARS-CoV-2假病毒表现出相对较弱的中和能力。
本发明实施例的4种纳米抗体的样品在高浓度下对病毒具有较强的抑制能力，在1μg/ml的浓度下的抑制率高达100％，对SARS-CoV-2假病毒表现较强的中和能力，具有较强的抗病毒效果，可用于新冠肺炎的治疗。
以上所述仅为本发明的实施例，并非因此限制本发明的专利范围，凡是利用本发明说明书及附图内容所作的等同变换，或直接或间接运用在相关的技术领域，均同理包括在本发明的专利保护范围内。

Claims (10)

  1. 一种新冠病毒中和毒性的纳米抗体，其特征在于，所述纳米抗体包括框架区FR和互补决定区CDR；所述互补决定区CDR包括CDR1、CDR2和CDR3；
    所述CDR1的氨基酸序列选自如SEQ ID NO.9和SEQ ID NO.10所示的氨基酸序列中的至少一种；
    所述CDR2的氨基酸序列选自如SEQ ID NO.11、SEQ ID NO.12和SEQ ID NO.13所示的氨基酸序列中的至少一种；
    所述CDR3的氨基酸序列选自如SEQ ID NO.14、SEQ ID NO.15、SEQ ID NO.16和SEQ ID NO.17所示的氨基酸序列中的至少一种。
  2. 根据权利要求1所述的新冠病毒中和毒性的纳米抗体，其特征在于，所述纳米抗体还包括框架区FR；所述框架区FR包括FR1、FR2、FR3和FR4；
    所述FR1的氨基酸序列选自如SEQ ID NO.18、SEQ ID NO.19、SEQ ID NO.20和SEQ ID NO.21所示的氨基酸序列中的至少一种；
    所述FR2的氨基酸序列选自如SEQ ID NO.22、SEQ ID NO.23和SEQ ID NO.24所示的氨基酸序列中的至少一种；
    所述FR3的氨基酸序列选自如SEQ ID NO.25、SEQ ID NO.26、SEQ ID NO.27和SEQ ID NO.28所示的氨基酸序列中的至少一种；
    所述FR4的氨基酸序列为SEQ ID NO.29。
  3. 根据权利要求1所述的新冠病毒中和毒性的纳米抗体，其特征在于，所述纳米抗体的氨基酸序列为如SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3或SEQ ID NO.4所示的氨基酸序列。
  4. 根据权利要求1至3任一项所述的新冠病毒中和毒性的纳米抗体，其特征在于，所述纳米抗体为与新型冠状病毒RBD蛋白有高亲和力的氨基酸序列，所述高亲和力是指K D值是在1×10 -12～1×10 -6之间。
  5. 权利要求1至4任一项所述的新冠病毒中和毒性的纳米抗体的制备方法，其特征在于，包括以下步骤：
    (1)将如SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7或SEQ ID NO.8所示的核苷酸序列克隆至表达载体中得到重组质粒，将所述重组质粒转入宿主细胞中诱导表达所述纳米抗体；
    (2)从宿主细胞中纯化所述纳米抗体。
  6. 权利要求1至4任一项所述的新冠病毒中和毒性的纳米抗体在制备预防和/或治疗新冠病毒肺炎的药物中的应用。
  7. 权利要求1至4任一项所述的新冠病毒中和毒性的纳米抗体在制备检测新冠病毒的检测试剂或试剂盒中的应用。
  8. 一种编码新冠病毒中和毒性的纳米抗体的基因，其特征在于，所述基因的核苷酸序列如SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7或SEQ ID NO.8所示。
  9. 一种重组质粒，其特征在于，所述重组质粒中包含如SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7或SEQ ID NO.8所示的核苷酸序列的基因片段中的至少一种。
  10. 一种重组细胞，其特征在于，所述重组细胞中包含如SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7或SEQ ID NO.8所示的核苷酸序列的基因片段中的至少一种。
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