CN114539394A - Camel source high affinity nano antibody of SARS-CoV-2 alpha mutant strain and beta mutant strain - Google Patents
Camel source high affinity nano antibody of SARS-CoV-2 alpha mutant strain and beta mutant strain Download PDFInfo
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- CN114539394A CN114539394A CN202111663259.0A CN202111663259A CN114539394A CN 114539394 A CN114539394 A CN 114539394A CN 202111663259 A CN202111663259 A CN 202111663259A CN 114539394 A CN114539394 A CN 114539394A
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Images
Classifications
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- C—CHEMISTRY; METALLURGY
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- 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
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- G—PHYSICS
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- 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
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- 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]
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- 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
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- 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
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- 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
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- 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
Abstract
The invention relates to a camel source high-affinity nano antibody of a SARS-CoV-2 alpha mutant strain and a beta mutant strain, in particular to an antibody specifically bound with S protein of coronavirus (such as SARS-CoV-2) and an antigen binding fragment thereof, more particularly to a camel source nano antibody or an antigen binding fragment thereof capable of binding with high affinity to S protein on the surface of coronavirus such as SARS-CoV-2wild type original strain and a mutant strain thereof (especially a B.1.1.7 mutant strain and a B.1.351 mutant strain), and the camel source nano antibody and the antigen binding fragment thereof can be used for preventing, detecting, diagnosing or treating infection caused by coronavirus, especially SARS-CoV-2 virus.
Description
Technical Field
The invention belongs to the fields of biotechnology, immunodetection and biomedicine, and particularly relates to a broad-spectrum high-affinity antibody or antigen-binding fragment and application thereof in detection, diagnosis, prevention and treatment of coronavirus, in particular to application in detection, diagnosis, prevention and treatment of SARS-CoV-2 wild-type original strain, B.1.1.7 mutant strain (namely alpha mutant strain) and/or B.1.351 mutant strain (namely beta mutant strain).
Background
The novel coronavirus SARS-CoV-2 is a beta coronavirus RNA virus. The virus has the characteristics of strong transmission, high lethality rate, high mutation speed and the like. SARS-CoV-2 causes respiratory tract infections, which in some patients results in viral pneumonia and Acute Respiratory Distress Syndrome (ARDS). And simultaneously, cytokine storm can be caused to cause multiple organ injuries. The original strains of the new coronavirus are separated till now, and novel mutant viruses such as a D614G mutant strain, a B.1.1.7 mutant strain, a B.1.351 mutant strain, a B.1.429 mutant strain, a P.1 mutant strain, a B.1.617.2 mutant strain and the like continuously appear in the global transmission process not only greatly enhance the transmission and the fatality rate of the viruses, but also cause the continuous reduction of the vaccine protection capability.
Antiviral treatments using some small molecule drugs and interferons in the treatment of covi-19 patients, however, clinical results have shown to be ineffective or to have limited therapeutic effect only at the early stages of viral infection, with a series of serious drug side effects. Antibody treatment strategies have been demonstrated to be the best solution for treating patients with coronavirus, especially in middle and late stage patients. The use of COVID-19 post-healing patient sera containing large amounts of neutralizing antibodies is an effective therapeutic strategy for treating patients with new coronaviruses. However, the limitations of patient serotherapy are that the plasma is difficult to obtain and less abundant for the convalescent patient, and cannot meet the requirements of a large patient population, so that alternative engineered antibodies are needed for treatment.
The nano antibody (Nanobody) is a single-domain antibody only containing a heavy chain antibody antigen binding domain VHH, and has a plurality of obvious advantages compared with the traditional polyclonal antibody, monoclonal antibody and single-chain antibody, such as small volume and capability of penetrating tissues and organs (such as a tunica vaginalis, a spinal cord, a brain and the like) which cannot be accessed by the conventional antibody; the stability is strong, and cold chain transportation and cold storage are not needed; low immunogenicity and easy humanization modification. The invention takes SARS-CoV-2 virus surface spur protein (Spike protein, namely S protein) as a target, develops a camel source high affinity nano antibody capable of simultaneously identifying various SARS-CoV-2 virus mutant strains by constructing a phage display nano antibody immune library and biological panning, and lays a foundation for mechanism research, clinical diagnosis and treatment of new coronary pneumonia.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a broad-spectrum and high-affinity antibody for coronavirus, which can effectively detect, block and treat coronavirus, especially SARS-CoV-2 virus original strain and mutant strain thereof.
In a particular embodiment of the invention, there are provided nanobodies against coronaviruses such as SARS-CoV-2, which bind to the S1 subunit (also referred to as S1 protein) of the S protein of the original strain (Wild Type), the British mutant strain (B1.1.7), and the south African mutant strain (B.1.351) with an affinity of nanomolar order.
In a specific embodiment of the invention, nanobodies against coronaviruses, such as SARS-CoV-2, are provided that are effective in blocking infection of 293T cells over-expressed by hACE2 by SARS-CoV-2 pseudovirus, at a semi-effective neutralizing concentration on the nanomolar scale.
In the specific embodiment of the invention, the establishment of a plurality of enzyme-linked immunoassay detection methods based on antigen/antibody reaction and the development of detection products can be carried out.
In particular embodiments of the invention, the same or multiple nanobody-based multivalence gene engineering may be performed.
In a specific embodiment of the present invention, there is provided a nanobody against coronavirus such as SARS-CoV-2, said nanobody comprising the following amino acid sequence and functional properties:
i) 1-6 of SEQ ID NO; or the antibody may have the hypervariable region CDR1 amino acid sequence set forth in any of SEQ ID NOs 7-11; 12-16 of the hypervariable region CDR2 amino acid sequence set forth in any of SEQ ID NOs; and the hypervariable region CDR3 amino acid sequence set forth in any of SEQ ID NOS 17-22;
ii) the nanobody has nanomolar level affinity with coronavirus such as SARS-CoV-2 virus Wild Type original strain, B.1.1.7 mutant strain and B.1.351 mutant strain;
iii) the nanobody effectively blocks the infection of 293T cells over-expressed by hACE2 by SARS-CoV-2 pseudovirus.
The invention also provides a biological material containing the nucleic acid molecule for encoding the antibody, wherein the biological material is recombinant DNA, an expression cassette, a transposon, a plasmid vector, a phage vector, a viral vector or an engineering bacterium.
The invention also provides any one of the following uses of the antibody:
1) used for scientific research related to coronavirus such as SARS-CoV-2 virus original strain and mutant strain thereof;
2) the kit is used for detecting the surface S protein of coronavirus such as SARS-CoV-2 virus original strain and mutant strain thereof;
3) it is used for developing coronavirus such as SARS-CoV-2 virus original strain and its mutant strain detection reagent or ELISA detection reagent.
In the invention, during analysis and detection, the nano antibodies with different concentrations are added into each hole of the ELISA plate coated with coronavirus such as SARS-CoV-2 virus Wild Type original strain, B.1.1.7 mutant strain and B.1.351 mutant strain antigen, and because the content of the solid phase antigen in each hole is consistent, when the antibody bound on the solid phase antigen is less, the binding amount of the added enzyme-labeled secondary antibody and the bound nano antibody is less, and finally, substrate solution and developing solution are added, the developing reaction is light, and the OD value detected by an enzyme reader is low; on the contrary, when the nano antibody is combined with the solid phase antigen to a large extent, the measured OD value is high, and the binding curve of the nano antibody and the SARS-CoV-2 is drawn according to the added nano antibody and the OD value of the corresponding hole.
Specifically, the invention provides the following technical scheme:
1. an antibody or antigen-binding fragment thereof, the amino acid sequence of which comprises CDR1 set forth in any one of SEQ ID NOs 7-11, CDR2 set forth in any one of SEQ ID NOs 12-16, CDR3 set forth in any one of SEQ ID NOs 17-22;
preferably, the antigen binding fragment is, for example, Fv, Fab ', scFv, F (ab')2Multivalent or multispecific fragments.
2. The antibody or antigen-binding fragment according to item 1, having an amino acid sequence as set forth in any one of SEQ ID NOs 1 to 6;
or the antibody or the antigen-binding fragment is an antibody containing a sequence obtained by truncating the 1 st to 128 th amino acids from the N terminal of the sequence shown in any one of SEQ ID NO. 1-6, or an antibody or an antigen-binding fragment with the same function obtained by substituting and/or deleting and/or adding one or more amino acid residues of the sequence shown in any one of SEQ ID NO. 1-6.
3. A genetically engineered antibody comprising the antibody or antigen-binding fragment of item 1 or 2; preferably, the genetically engineered antibody is a humanized antibody, a chimeric antibody, a multivalent or multispecific antibody.
4. A fusion protein comprising the antibody or antigen-binding fragment of item 1 or 2 or the genetically engineered antibody of item 3; preferably, the fusion protein further comprises a tag polypeptide, a detection protein or an accessory protein.
5. A conjugate comprising the antibody or antigen-binding fragment of item 1 or 2 or the genetically engineered antibody of item 3 or the fusion protein of item 4; preferably, the conjugate further comprises a detectable label, a contrast agent, a drug, a cytokine, a radionuclide, an enzyme, a gold nanoparticle/nanorod, a nanomagnet, a liposome, a viral coat protein or VLP, or a combination thereof.
6. A nucleic acid molecule encoding the antibody or antigen-binding fragment of items 1-2, the genetically engineered antibody of item 3, the fusion protein of item 4, or the conjugate of item 5, wherein the nucleic acid molecule is RNA, DNA, or cDNA.
7. An expression vector comprising the nucleic acid molecule of item 6;
optionally, the expression vector can be DNA, RNA, viral vectors, plasmids, expression cassettes, transposons, other gene transfer systems, or combinations thereof;
preferably, the expression vector comprises a viral vector, such as a phage vector, lentivirus, adenovirus, AAV virus, retrovirus, other protein expression system, or a combination thereof.
8. A host cell comprising the expression vector of item 7; wherein the host cell is a host cell for expressing a foreign protein, such as a prokaryotic expression cell, a eukaryotic expression cell, a transgenic cell line; preferably, the host cell comprises prokaryotic cells, yeast cells, insect cells, plant cells, animal cells.
9. A tissue sample or culture obtained by culturing the host cell of item 8.
10. A protein or antigen-binding fragment isolated from the tissue sample or culture of item 9.
11. A method of preparing the antibody or antigen-binding fragment of items 1-2, the genetically engineered antibody of item 3, the fusion protein of item 4, or the conjugate of item 5, comprising isolating/recovering the protein or antigen-binding fragment of interest from the tissue sample or culture of item 9.
12. A pharmaceutical composition comprising as an active ingredient an antibody or antigen-binding fragment according to item 1 or 2 or an engineered antibody according to item 3 or a fusion protein according to item 4 or a conjugate according to item 5; for example, the pharmaceutical composition is an inhaled aerosolized drug, a mucosal or epidermal external drug, a subcutaneous injection drug, a vascular infusion drug, or a combination thereof; preferably, the medicament further comprises a pharmaceutically acceptable excipient or carrier.
13. A product comprising the antibody or antigen-binding fragment of item 1 or 2 or the genetically engineered antibody of item 3 or the fusion protein of item 4 or the conjugate of item 5; for example, the product is a mask or air purifier filter element, an environmental, object or human body surface disinfectant, or a combination thereof; preferably, the product is coated in a purifier cartridge or dissolved in a disinfectant for atomized spraying or surface wiping.
14. Use of an antibody or antigen-binding fragment according to item 1 or 2 or a genetically engineered antibody according to item 3 or a fusion protein according to item 4 or a conjugate according to item 5 for the preparation of a product or a medicament for the prevention, treatment and/or diagnosis of a coronavirus infection.
15. Use of an antibody or antigen-binding fragment according to item 1 or 2 or a genetically engineered antibody according to item 3 or a fusion protein according to item 4 or a conjugate according to item 5 for the preparation of a product for:
1) detecting coronavirus antigen, especially SARS-CoV-2 virus original strain and mutant strain thereof;
2) blocking coronavirus infection, especially SARS-CoV-2 virus original strain and mutant strain thereof;
3) killing coronavirus particles, especially SARS-CoV-2 virus original strain and mutant strain thereof;
4) diagnosing related diseases caused by coronavirus, especially SARS-CoV-2 virus original strain and mutant strain thereof;
5) treating related diseases caused by coronavirus, especially SARS-CoV-2 virus original strain and mutant strain thereof;
6) basic scientific research related to coronavirus, especially SARS-CoV-2 virus original strain and its mutant strain are carried out.
In a specific embodiment of the invention, the coronavirus includes HCoV-NL63, SARS-CoV-1, SARS-CoV-2, HCoV-229E, MERS-CoV, HCoV-OC43, HCoV-HKU1 or other coronaviruses having a similar surface S protein structure.
In a specific embodiment of the present invention, the mutant strains of SARS-CoV-2 virus include the D614G mutant strain, the B.1.1.7 mutant strain, the B.1.351 mutant strain, the B.1.429 mutant strain, the P.1 mutant strain, the B.1.617.2 mutant strain, and the like.
In a specific embodiment of the present invention, the tag polypeptide comprises a functional polypeptide such as a purification tag, a detection tag, an identification tag, a coupling tag, a functional verification tag, etc., for example, a His tag, an HA tag, a Flag tag, a c-Myc tag, an Avi tag, etc.
In a specific embodiment of the present invention, the detection protein contained in the fusion protein includes a fluorescent protein, a fluorescein-labeled protein, a peroxidase, and other functional proteins, such as an FPs protein, an HRP protein, an Alexa Fluor-labeled protein, a FITC-labeled protein, and the like.
In a specific embodiment of the present invention, the auxiliary protein contained in the fusion protein is a protein for assisting folding, expression, solubilization, toxic protein shielding, etc., such as GST protein, MBP protein, SUMO protein, NusA protein.
Technical effects
The antibody for resisting coronavirus such as SARS-CoV-2 provided by the invention effectively overcomes the defects of few serum sources, high cost, unstable structure and the like of the prior recovered patients of coronavirus such as SARS-CoV-2, has high affinity, high sensitivity, high neutralization capacity, high yield, high stability and low cost, and can be used for rapid mass production. The antibody provided by the invention can be used for early infection blocking, early infection diagnosis and middle and late infection treatment, and can also be used for scientific research tools and in-vitro rapid detection, such as ELISA detection/diagnosis kit production and colloidal gold detection/diagnosis kit production.
The detection method established by the antibody, such as ELISA detection method, can accurately and sensitively detect whether the sample contains coronavirus, such as SARS-CoV-2 virus. The pretreatment process of the sample is simple, the time consumption is low, a large amount of samples can be detected simultaneously, and the sample detection cost is far lower than that of the traditional nucleic acid detection method. The antibody of the invention can be applied to a colloidal gold detection/diagnosis kit to quickly and accurately detect whether a sample contains coronavirus such as SARS-CoV-2 virus, and has important practical significance for screening and identifying large-scale population infection, environmental pollution and cargo sample pollution.
Drawings
In order to more clearly illustrate the technical solutions of the present invention or the prior art, the drawings needed to be used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 is the binding curve of the nano antibody of the present invention with the Wild Type original strain of SARS-CoV-2 virus, the B.1.1.7 mutant strain and the S1 mutant strain of B.1.351 protein;
FIG. 2 is the affinity curve of the nano antibody of the present invention with the Wild Type original strain, the B.1.1.7 mutant strain and the S1 mutant strain of SARS-CoV-2 virus (taking antibody A1 as an example);
FIG. 3 is the neutralization inhibition curve of the nano antibody of the present invention against SARS-CoV-2 virus Wild Type original strain, B.1.1.7 mutant strain and B.1.351 mutant strain pseudovirus;
FIG. 4 shows the sequences of the antibodies of the invention and the CDR regions thereof;
FIG. 5 shows the binding curves of 6 antibodies according to the present invention and SARS-1 virus S protein;
FIG. 6 is a plasmid map of pComb3Xss used in example 1.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is obvious that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The instruments and the like are conventional products which are purchased by normal distributors and are not indicated by manufacturers. The process is conventional unless otherwise specified, and the starting materials are commercially available from a public source.
Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual,2001), or the conditions as recommended by the manufacturer's instructions.
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention.
The experimental procedures in the following examples are conventional unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The quantitative tests in the following examples, all set up three replicates and the results averaged.
According to some preferred embodiments of the present invention, the nanobody may be prepared as follows: taking an original strain SARS-CoV-2 protein as an immunogen to immunize an experimental animal camel, extracting total RNA of peripheral blood lymphocytes, cloning a gene fragment of a heavy chain (VHH) of a nano antibody through reverse transcription and nested PCR, cloning the gene fragment to a phagemid carrier through restriction enzyme digestion connection, performing high-efficiency electric transformation to escherichia coli, rescuing by an auxiliary phage, constructing a phage nano antibody library, screening out SARS-CoV-2 nano antibody, expressing and purifying the SARS-CoV-2 nano antibody to obtain the SARS-CoV-2 nano antibody with high sensitivity, and having higher cross reaction with a popular mutant strain. The prepared nano antibody has small molecule, strong solubility, high temperature resistance, easy purification and easy expression.
According to some preferred embodiments of the present invention, the SARS-CoV-2 virus wild type original strain S protein and RBD protein as immunogens, SARS-CoV-2 virus wild type original strain S1 protein, and B.1.1.7 mutant B.1.351 mutant S1 protein as coated antigens are purchased from Beijing-Yi-Qiao-Shenzhou organism Limited.
The ELISA plate is a 96-hole ELISA plate, and the coating concentration of the coating antigen is 1 ug/mL.
The enzyme-labeled secondary antibody is an anti-HA label antibody labeled by horseradish peroxidase, and the concentration is 0.1 mu g/mL. Purchased from Abcam corporation under the trade designation ab 1265.
The color developing solution A consists of carbamide peroxide 1g, citric acid 10.3g and Na2HPO4·12H235.8g of O, 20100 mu L of Tween-and 1000mL of distilled water, and the pH value is 5.
The color developing solution B is prepared from 700mg of tetramethylbenzidine, 40mL of DMSO, 10.3g of citric acid and 1000mL of distilled water, and the pH value is 2.4.
The reaction termination solution is 2M sulfuric acid solution.
Example 1 construction of SARS-CoV-2 Nanobody library
200ug of SARS-CoV-2 virus original strain S protein and RBD protein (Beijing Yiqiao Shenzhou biology, Ltd.) are mixed with equal volume of complete Freund 'S adjuvant, fully emulsified and injected into camel, and then boosted once every two weeks, wherein the mixed solution of the complete Freund' S adjuvant and immunogen is used in boosting immunity, and subcutaneous multipoint immunization is carried out on neck and back for 5 times. Starting from the third immunization, blood was taken from jugular vein one week after each immunization and serum titer was measured.
Leukocytes were isolated from 5 th immunized peripheral blood, total RNA was extracted, VHH gene fragments were cloned by reverse transcription PCR and nested PCR (wherein the system and parameters of reverse transcription PCR and nested PCR are described below), cohesive ends were modified with restriction enzyme SfiI, the VHH gene fragments were ligated to phagemid pComb3Xss (as donated by Bruce D Hammock professor laboratories of UC Davis) by T4 ligase, and efficiently transformed into E.coli 2738 (stored in laboratories, commercially available, for example, from NEB, UK) to construct phage nanobody libraries of SARS-CoV-2. The primary reservoir volume is determined to be 109cfu, which was rescued by adding helper phage (multiplicity of infection: 20:1) M13KO7 (purchased from NEB, Inc.: N0315S) to obtain phage nanobody library with a library capacity of 1012pfu/mL, better library diversity.
Reverse transcription PCR:
the reverse transcription kit adopts PrimeScriptTMRT-PCR Kit, purchased from Takara, under trade designation: AK 2701.
The reverse transcription system is as follows:
the reaction was carried out at 65 ℃ for 5 min. Taking out and placing on ice, loading the sample according to the following system, and carrying out first strand cDNA synthesis.
30℃10min；42℃1h；72℃5min。
Nested PCR: (from TAKATA, Cat # 6210A)
First round PCR:
the reaction system is as follows:
the reaction procedure was as follows:
second round PCR:
the reaction system is as follows:
the reaction procedure was as follows:
the nested PCR primer sequences are as follows (5 '-3'):
GSP-RT：CGCCATCAATRTACCAGTTGA(SEQ ID NO:23)
LP-leader：GTGGTCCTGGCTGCTCTW(SEQ ID NO:24)
F：CATGCCATGACTGTGGCCCAGGCGGCCCAGKTGCAGCTCGTG GAGTC(SEQ ID NO:26)
R：CATGCCATGACTCGCGGCCGGCCTGGCCATGGGGGTCTTCGC TGTGGTGCG(SEQ ID NO:25)
wherein R represents a base A/G, W represents a base A/T, and K represents a base G/T.
Example 2 screening of SARS-CoV-2 Nanobody
SARS-CoV-2 original strain virus S protein antigen is coated on the 1 st hole of 96-hole enzyme label plate, the coating concentration is 1ug/mL, and the temperature is kept overnight at 4 ℃; on the next day, pouring out the coating solution, washing with PBST for 3 times, sealing the 1 st hole and the 2 nd hole of the ELISA plate with BSA, and incubating at room temperature for 2 h; pouring out the blocking solution, and washing with PBST for 3 times; adding the phage nanobody library obtained in example 1 into the 1 st well, and reacting for 2 h; pouring out the liquid, patting dry on clean absorbent paper, and washing with PBST for 5 times; adding 100 μ L of SARS-CoV-2 virus original strain S1 protein into the 1 st well, reacting for 1 h; sucking out the liquid in the 1 st hole, adding the liquid into the 2 nd hole, reacting for 1h, and removing the phage bound with the BSA; the eluate was collected, 5. mu.L was used for titer determination, and the remainder was used for amplification.
Adding the phage eluate to fresh E.coli ER2738 bacterial solution (stored in laboratory, or commercially available, such as NEB), standing at 37 deg.C for 15 min; adding carbenicillin and SB culture medium, culturing at 37 deg.C and 220rpm for 2 hr; adding helper phage M13KO7 (MOI 20:1) (from NEB, cat # N0315S) and kanamycin, and culturing overnight; the next day, the supernatant was centrifuged and purified by adding PEG-NaCl solution.
And (3) carrying out next round of screening on the amplified product to ensure that the addition amount of each round of screening is the same, the antigen coating concentration and the S protein competitive elution concentration are decreased progressively by 2 times, calculating the titer of each round, and selecting a monoclonal to carry out amplification and ELISA identification. Positive monoclone is obtained through 3 rounds of panning.
Example 3 expression of SARS-CoV-2 Nanobody
Positive monoclonal plasmids were extracted, transformed into E.coli TOP 10F' competent cells (purchased from Thermo Fishier), recovered and plated on solid media for overnight culture. The next day, selecting a single clone, culturing in a SB-benzyl carboxylate culture medium, and adding IPTG to induce overnight expression; the next day, cells are lysed by a high-pressure homogenizer, the cells are purified by a nickel column after being filtered by a filter membrane, namely, the nano antibody is separated and purified by affinity chromatography of a histidine tag and nickel chloride in the nickel column to obtain the high-purity anti-SARS-CoV-2 nano antibody, namely the antibody A1-A6, and the amino acid sequence of the obtained nano antibody is shown as SEQ ID NO. 1-6 through amino acid sequencing analysis.
Example 4 binding curves of Nanobodies with SARS-CoV-2 Virus S1 protein
Respectively coating SARS-CoV-2 virus Wild Type original strain S1 protein, B.1.1.7 mutant strain S1 protein and B.1.351 mutant strain S1 protein (Beijing Yinqiao Shenzhou biological limited) on 96-hole enzyme label plates, wherein the coating concentration of each hole is 1ug/mL, and reacting at 4 ℃ overnight; the next day, the liquid in the wells was spun off, washed 3 times with PBST containing 0.05% tween, and the microplate was inverted and patted dry on absorbent paper; adding a sealing solution, incubating at 37 ℃ for 30 minutes, throwing the liquid in the hole, washing for 3 times by using 0.05% PBST, and inverting the ELISA plate on water-absorbent paper to dry; respectively adding 100 mu L of the nano antibody liquid obtained in the example 3 with different dilution times, and incubating for 30 minutes at 37 ℃; throwing the liquid in the hole, washing with PBST for 3 times, and inversely arranging the ELISA plate on water-absorbent paper for patting dry; adding enzyme-labeled secondary antibody (horseradish peroxidase-labeled anti-HA-labeled antibody from Roche) and incubating at 37 ℃ for 30 minutes; the liquid in the wells was spun off, washed 3 times with PBST and patted dry; and (3) uniformly mixing the solution A and the solution B in equal volume, adding 100 mu L of solution A into each hole, performing light-shielding color development for 10-15 minutes, adding a stop solution to terminate the reaction, and measuring the OD value of each hole at the wavelength of 450nm on an enzyme-labeling instrument. The binding curves of nanobody and SARS-CoV-2 virus Wild Type original strain S1 protein, B.1.1.7 mutant strain S1 protein and B.1.351 mutant strain S1 protein were plotted according to the antibody concentration and OD value in the corresponding well (see FIG. 1). The experimental result shows that the 6 nano antibodies have stronger affinity with Wild Type original strain S1 protein, B.1.1.7 mutant strain S1 protein and B.1.351 mutant strain S1 protein, which indicates that the nano antibodies have certain broad spectrum.
Example 5 affinity curves of Nanobodies with SARS-CoV-2 Virus S1 protein
The affinity detection method is performed by using an Octec red 96 instrument and adopts an avidin probe, and the affinity detection method is performed by the conventional technology in the field and is specifically performed as follows. Adding 0.02% tween-20 in PBST to 8 wells in the first column of a black, non-binding 96-well plate; to the second 8 wells, biotinylated SARS-CoV-2 Virus Wild Type original strain S1 protein, B.1.1.7 mutant strain S1 protein and B.1.351 mutant strain S1 protein were added at a concentration of 15 ug/ml. PBST is added into the third, fifth, seventh, ninth and eleventh columns, and the nano-antibody of the invention diluted by times is added into the fourth, sixth and eighth columns, wherein PBST is added into the 8 th hole of each column, glycine is added into the twelfth column for 2.0, and the liquids are 200ul per hole. The general procedure is as follows:
1) first 8 avidin probes (streptavidin-sensor, purchased from FORTEBIO, cat #: 18-5019) immersion in the first column of PBST for equilibration for 60 s;
2) then immersing the avidin probe into the SARS-CoV-2S protein diluent to combine for 3 min;
3) returning to the first and third PBST columns for balancing twice;
4) soaking the balanced probe into a fourth column of nano antibody diluent to perform specific binding of the antigen and the antibody for 3 min;
5) and returning to the third column of PBST for dissociation, and dissociating for 10 min.
6) Regenerating the probe in the twelfth column glycine 2.0 for 5s after dissociation, and completely eluting the combined nano antibody;
7) returning to PBST in the eleventh column for neutralization for 5 s;
8) repeating 6)7) two steps;
9) then soaking the probe into a fifth row of PBST for balancing; repeating the steps of 4)5)6)7)8) to sequentially detect the binding capacity of other nano antibodies and SARS-CoV-2S protein;
10) finally, importing the experimental data into an excel table;
results referring to FIG. 2 and Table 1, the results show that six nanobodies have the affinity range for the SARS-CoV-2 virus Wild Type original strain S1 protein: 5.15-25.4nM, with affinity for the B.1.1.7 mutant S1 protein ranging from: 1.27-10.1nM, 10.5-125nM for the B.1.351 mutant S1 protein.
TABLE 1 affinity constants K of Nanobodies with the Wild Type original strain of SARS-CoV-2 virus, the B.1.1.7 mutant strain and the S1 mutant strain protein of B.1.351D(M)
Example 6 detection of neutralizing Capacity of Nanobodies against SARS-CoV-2 pseudoviral infection
Firstly, 10 concentration gradients of the 6 kinds of nano-antibodies are diluted by the DMEM culture medium in a multiple ratio manner, the final volume of each concentration is 50ul, wherein, the 10 th gradient only contains the DMEM culture medium and the nano-antibody concentration is 0, the 10 th gradient is used as a control group, and 3ul can generate about 1x105SARS-CoV-2Wild type original strain pseudovirus (King Haikun researche, Shanghai Pasteur institute of Chinese academy of sciences), which is related to luciferase activity, of RLUs (also available from Beijing Yunzi biotechnology, Inc.) is added into the nano antibody diluent, mixed, incubated at 37 ℃ for 60min, 50ul of HEK293T-hACE2 cells (King Haikun researche, Shanghai Pasteur institute of Chinese academy of sciences, also available from Nanjing Nuo Wei Zan biotechnology, Inc.) are added into the virus-antibody complex, mixed well and added into a 96-well cell culture plate, and 3 repeat wells are provided for each concentration of antibody. The cell culture plate is placed in an incubator at 37 ℃ and cultured for 48h, cell supernatant is discarded, 100ul Bright-glo (Promega) is added into each well, the reaction is carried out for 2min, the cell supernatant is transferred into a white 96-well plate, and a Varioskan Flash multifunctional reader is used for detecting the activity value of firefly luciferase (the neutralization experiment of SARS-CoV-2 virus B.1.1.7 mutant pseudovirus is consistent with the neutralization experiment step of original strain pseudovirus, only the added pseudovirus is different, the pseudovirus and HEK293T-hACE2 cells are both offered by Hekun researchers of Shanghai Baston, Spanish institute of Shanghai, or can be purchased from Beijing Yunnan Cishi Biotechnology GmbH or Nanjing Nozan Biotech GmbH). Taking the infection rate of SARS-CoV-2S pseudovirus to HEK293T-hACE2 cell after adding antibody and the concentration of nano antibody as horizontal and vertical coordinates to draw a neutralization inhibition curve, and finally calculating EC according to the curve50The value is obtained. Infection rate ═ 100% (experimental well RLUs values-background values)/(control well RLUs values-background values) x, background values are the values read with 100ul Bright-Glo addition only. The results are shown in FIG. 3, and the experimental results show that all six kinds of nano antibodies can specifically neutralize SARS-CoV-2S protein pseudovirus. Neutralization of the Wild Type mutant pseudovirus50The range is as follows: 92.7-273.7nM, neutralizing EC against B.1.1.7 mutant pseudovirus50The range is as follows: 71.1-210.8nM, neutralizing EC against B.1.351 mutant pseudovirus50The range is as follows: 0.38-3.35 uM.
Example 7 binding curves of Nanobodies to SARS-CoV-1 Virus S protein
SARS-CoV-1 virus S protein (which is a new gift from New political researcher of institute of biophysics, Zhongkojie, or from Beijing Yiqian Shenzhou biology, Co., Ltd.) was coated on a 96-well ELISA plate, the coating concentration of each well was 1ug/mL, and the reaction was carried out overnight at 4 ℃; the next day, the liquid in the wells was spun off, washed 3 times with PBST containing 0.05% tween, and the microplate was inverted and patted dry on absorbent paper; adding a sealing solution, incubating at 37 ℃ for 30 minutes, throwing the liquid in the hole, washing for 3 times by using 0.05% PBST, and inverting the ELISA plate on water-absorbent paper to dry; respectively adding 100 mu L of the nano antibody liquid obtained in the example 3 with different dilution times, and incubating for 30 minutes at 37 ℃; throwing the liquid in the hole, washing with PBST for 3 times, and inversely arranging the ELISA plate on water-absorbent paper for patting dry; adding enzyme-labeled secondary antibody (horseradish peroxidase-labeled anti-HA-labeled antibody from Roche) and incubating at 37 ℃ for 30 minutes; the liquid in the wells was spun off, washed 3 times with PBST and patted dry; and (3) uniformly mixing the solution A and the solution B in equal volume, adding 100 mu L of solution A into each hole, performing dark color development for 10-15 minutes, adding a stop solution to terminate the reaction, and measuring the OD value of each hole at the wavelength of 450nm on an enzyme-labeling instrument. The binding curves of nanobodies and SARS-CoV-1 virus S protein were plotted according to the antibody concentration and OD value in the corresponding wells (see FIG. 5). The experimental result shows that the 6 nano antibodies can be combined with SARS-CoV-1 virus S protein, which indicates that the nano antibodies can be combined with SARS-CoV-2 and SARS-CoV-1, and indicates that the antibodies have broad spectrum for coronavirus.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (15)
1. An antibody or antigen-binding fragment thereof, the amino acid sequence of which comprises CDR1 set forth in any one of SEQ ID NOs 7-11, CDR2 set forth in any one of SEQ ID NOs 12-16, CDR3 set forth in any one of SEQ ID NOs 17-22;
preferably, the antigen binding fragment is, for example, Fv, Fab ', scFv, F (ab')2Multivalent or multispecific fragments.
2. The antibody or antigen-binding fragment of claim 1, having an amino acid sequence as set forth in any one of SEQ ID NOs 1-6;
or the antibody or the antigen-binding fragment is an antibody containing a sequence obtained by truncating the 1 st to 128 th amino acids from the N terminal of the sequence shown in any one of SEQ ID NO. 1-6, or an antibody or an antigen-binding fragment with the same function obtained by substituting and/or deleting and/or adding one or more amino acid residues of the sequence shown in any one of SEQ ID NO. 1-6.
3. A genetically engineered antibody comprising the antibody or antigen-binding fragment of claim 1 or 2; preferably, the genetically engineered antibody is a humanized antibody, a chimeric antibody, a multivalent or multispecific antibody.
4. A fusion protein comprising the antibody or antigen-binding fragment of claim 1 or 2 or the genetically engineered antibody of claim 3; preferably, the fusion protein further comprises a tag polypeptide, a detection protein or an accessory protein.
5. A conjugate comprising the antibody or antigen-binding fragment of claim 1 or 2 or the genetically engineered antibody of claim 3 or the fusion protein of claim 4; preferably, the conjugate further comprises a detectable label, a contrast agent, a drug, a cytokine, a radionuclide, an enzyme, a gold nanoparticle/nanorod, a nanomagnet, a liposome, a viral coat protein or VLP, or a combination thereof.
6. A nucleic acid molecule encoding the antibody or antigen-binding fragment of claims 1-2, the genetically engineered antibody of claim 3, the fusion protein of claim 4, or the conjugate of claim 5, wherein the nucleic acid molecule is RNA, DNA, or cDNA.
7. An expression vector comprising the nucleic acid molecule of claim 6;
optionally, the expression vector can be DNA, RNA, viral vectors, plasmids, expression cassettes, transposons, other gene transfer systems, or combinations thereof;
preferably, the expression vector comprises a viral vector, such as a phage vector, lentivirus, adenovirus, AAV virus, retrovirus, other protein expression system, or a combination thereof.
8. A host cell comprising the expression vector of claim 7; wherein the host cell is a host cell for expressing a foreign protein, such as a prokaryotic expression cell, a eukaryotic expression cell, a transgenic cell line; preferably, the host cell comprises a prokaryotic cell, a yeast cell, an insect cell, a plant cell, an animal cell.
9. A tissue sample or culture obtained by culturing the host cell of claim 8.
10. A protein or antigen-binding fragment isolated from the tissue sample or culture of claim 9.
11. A method of preparing the antibody or antigen-binding fragment of claims 1-2, the genetically engineered antibody of claim 3, the fusion protein of claim 4, or the conjugate of claim 5, comprising isolating/recovering the protein or antigen-binding fragment of interest from the tissue sample or culture of claim 9.
12. A pharmaceutical composition comprising as an active ingredient an antibody or antigen-binding fragment according to claim 1 or 2 or a genetically engineered antibody according to claim 3 or a fusion protein according to claim 4 or a conjugate according to claim 5; for example, the pharmaceutical composition is an inhaled aerosolized drug, a mucosal or epidermal external drug, a subcutaneous injection drug, a vascular infusion drug, or a combination thereof; preferably, the medicament further comprises a pharmaceutically acceptable excipient or carrier.
13. A product comprising the antibody or antigen-binding fragment of claim 1 or 2 or the genetically engineered antibody of claim 3 or the fusion protein of claim 4 or the conjugate of claim 5; for example, the product is a mask or air purifier filter element, an environmental, object or human body surface disinfectant, or a combination thereof; preferably, the product is coated in a purifier cartridge or dissolved in a disinfectant for atomized spraying or surface wiping.
14. Use of an antibody or antigen-binding fragment according to claim 1 or 2 or a genetically engineered antibody according to claim 3 or a fusion protein according to claim 4 or a conjugate according to claim 5 for the preparation of a product or a medicament for the prevention, treatment and/or diagnosis of a coronavirus infection.
15. Use of the antibody or antigen-binding fragment of claim 1 or 2 or the genetically engineered antibody of claim 3 or the fusion protein of claim 4 or the conjugate of claim 5 in the manufacture of a product for:
1) detecting coronavirus antigen, especially SARS-CoV-2 virus original strain and mutant strain thereof;
2) blocking coronavirus infection, especially SARS-CoV-2 virus original strain and mutant strain thereof;
3) killing coronavirus particles, especially SARS-CoV-2 virus original strain and mutant strain thereof;
4) diagnosing related diseases caused by coronavirus, especially SARS-CoV-2 virus original strain and mutant strain thereof;
5) treating related diseases caused by coronavirus, especially SARS-CoV-2 virus original strain and mutant strain thereof;
6) basic scientific research related to coronavirus, especially SARS-CoV-2 virus original strain and its mutant strain are carried out.
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| CN112094343A (en) * | 2020-09-25 | 2020-12-18 | 中国科学技术大学 | Alpaca source nano antibody combined with SARS-CoV-2RBD |
| CN112094342A (en) * | 2020-09-25 | 2020-12-18 | 中国科学技术大学 | Alpaca source nano antibody combined with SARS-CoV-2RBD |
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| CN112094343A (en) * | 2020-09-25 | 2020-12-18 | 中国科学技术大学 | Alpaca source nano antibody combined with SARS-CoV-2RBD |
| CN112094342A (en) * | 2020-09-25 | 2020-12-18 | 中国科学技术大学 | Alpaca source nano antibody combined with SARS-CoV-2RBD |
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