CN114805563A - Camel source high affinity nano antibody of SARS-CoV-2alpha, gamma, delta and omicron mutant strain - Google Patents
Camel source high affinity nano antibody of SARS-CoV-2alpha, gamma, delta and omicron mutant strain Download PDFInfo
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Abstract
The present invention relates to a camelid-derived high affinity nanobody of SARS-CoV-2. Alpha.,. Gamma., (. Delta.) and.o mutant strains, specifically to an antibody specifically binding to the S protein of a novel coronavirus (SARS-CoV-2) and an antigen-binding fragment thereof, and more specifically to an antibody or an antigen-binding fragment thereof capable of binding with high affinity to the S protein on the surface of a mutant strain of a coronavirus such as SARS-CoV-2Alpha (B.1.1.7), Gamma (P.1), Delta (B.1.617.2) and Omicron (B.1.1.529), the amino acid sequence of which comprises the amino acid sequence represented by SEQ ID NO: 9-15, CDR1 set forth in any one of SEQ ID NOs: 16-21, CDR2 set forth in any one of SEQ ID NOs: 22-29, or a CDR 3; it can be used for preventing, detecting, diagnosing or treating infections caused by coronaviruses, especially SARS-CoV-2 virus.
Description
Technical Field
The invention belongs to the fields of biotechnology, immunoassay and biomedicine, and particularly relates to a specific nano antibody or antigen binding fragment, an antigen recognition epitope and application thereof in detection, diagnosis, prevention and treatment of coronavirus such as SARS-CoV-2, in particular to application in detection, diagnosis, prevention and treatment of mutant strains of SARS-CoV-2Alpha (B.1.1.7), Gamma (P.1), Delta (B.1.617.2) and Omicron (B.1.1.529).
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, a B.1.1.529 mutant strain and the like which 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 the quantity is small for the convalescent patient, and the requirement of huge patient population can not be met, so that alternative engineering 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 Spike protein (Spike protein, namely S protein) as a target, develops a camel source high affinity nano antibody which can simultaneously identify a plurality of novel coronaviruses such as Alpha (B.1.1.7), Gamma (P.1), Delta (B.1.617.2) and Omicron (B.1.1.529) mutant strains by constructing a phage display nano antibody immune library and biopanning, and lays a foundation for the 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 specific embodiment of the invention, there is provided an anti-SARS-CoV-2 nanobody, which can bind to the S1 subunit (also called S1 protein) of the S protein of the Alpha (B.1.1.7), Gamma (P.1), Delta (B.1.617.2) and Omicron (B.1.1.529) mutants, with an affinity on the nanomolar scale.
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) SEQ ID NO: 1-8; alternatively, the antibody may have the amino acid sequence of SEQ ID NO: 9-15 of any one of the hypervariable region CDR1 amino acid sequences; SEQ ID NO: 16-21, the hypervariable region CDR2 amino acid sequence set forth in any one of seq id nos; and SEQ ID NO: 22-29, or a hypervariable region CDR3 amino acid sequence set forth in any one of seq id nos;
ii) the nanobody has nanomolar affinity for coronavirus such as SARS-CoV-2 virus Alpha (B.1.1.7), Gamma (P.1), Delta (B.1.617.2) and Omicron (B.1.1.529) mutants;
iii) the nanobody effectively blocks the infection of 293T cells over-expressed by hACE2 by SARS-CoV-2 pseudovirus.
iiii) the nanobody specifically recognizes an epitope of the SARS-CoV-2 virus surface S protein, which comprises the TR amino acid sequence located at position 345-356, the NLDSKVGGNYN amino acid sequence located at position 440-450 and the PT amino acid sequence located at position 499 500 of the conserved region of the RBD domain, particularly comprising the amino acid residues K444, N450, N448, R346, T345, L441, V445, P499, N440, V445 and T500;
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 present invention, in the assay, the nanobodies are added at different concentrations to each well of an elisa plate coated with coronavirus, such as the SARS-CoV-2 virus Alpha (B.1.1.7), Gamma (P.1), Delta (B.1.617.2) and Omicron (B.1.1.529) mutant antigen, because the content of the solid phase antigen in each well is consistent, when the antibody bound to the solid phase antigen is small, the amount of the bound nanobody bound to the enzyme-labeled secondary antibody is small, and finally, a substrate solution and a 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 a sequence consisting of SEQ ID NO: 9-15, CDR1 set forth in any one of SEQ ID NOs: 16-21, and a CDR2 set forth by SEQ ID NO: 22-29, or a CDR 3;
preferably, the antigen binding fragment is, for example, Fv, Fab ', scFv, F (ab') 2 Multivalent or multispecific fragments.
2. The antibody or antigen-binding fragment according to item 1, having an amino acid sequence as set forth in SEQ ID NO: 1-8;
or the antibody or antigen-binding fragment is a monoclonal antibody comprising the amino acid sequence of SEQ ID NO: 1-8, or an antibody or antigen-binding fragment thereof, which is a sequence obtained by truncating the sequence from amino acid 1 to amino acid 121 from the N-terminus of any one of SEQ ID NOs: 1-8 through substitution and/or deletion and/or addition of one or more amino acid residues to obtain the antibody or antigen binding fragment with the same function.
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 an antibody or antigen-binding fragment according to items 1-2, a genetically engineered antibody according to item 3, a fusion protein according to item 4, or a conjugate according to item 5, comprising isolating/recovering a protein or polypeptide of interest from a tissue sample or culture according to 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 pharmaceutical composition 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.
16. Epitope located in the conserved region of the surface Spike protein RBD domain of SARS-CoV-2 virus and the combination thereof, wherein the epitope has the following amino acid sequence:
1) the TR amino acid sequence is positioned at 345-346 th site of the surface Spike protein RBD structural domain of the SARS-CoV-2 virus;
2) NLDSKVGGNYN amino acid sequence located at 440-450 site of the surface Spike protein RBD structural domain of SARS-CoV-2 virus; and
3) PT amino acid sequence located at 499 nd 500 th site of surface Spike protein RBD structure domain of SARS-CoV-2 virus.
17. Use of an epitope according to item 16 in the preparation of a product for:
1) developing broad-spectrum coronavirus antigen or vaccine, especially antigen vaccine aiming at SARS-CoV-2 virus original strain and mutant strain thereof;
2) preparing antibodies, antigen-binding fragments, medicaments or products for preventing, treating and/or diagnosing infection of the broad-spectrum coronavirus, particularly for the SARs-CoV-2 virus original strain and mutant strains thereof;
3) basic scientific research related to coronavirus, especially basic scientific research related to SARS-CoV-2 virus original strain and mutant strain thereof is 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 invention, the mutant strains of SARS-CoV-2 virus include the B.1.1.7 mutant strain, the P.1 mutant strain, the B.1.617.2 mutant strain, the B.1.1.529 mutant strain, and the like.
In a specific embodiment of the 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., e.g., 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, or a FITC-labeled protein.
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, or 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 epitope provided by the invention can be used for researching and developing coronavirus broad-spectrum antigens and vaccines, and can also be used for scientific research tools and in-vitro rapid detection, such as production of ELISA detection/diagnosis kits and colloidal gold detection/diagnosis kits.
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 nanobody of the present invention to SARS-CoV-2 virus Alpha (B.1.1.7), Gamma (P.1), Delta (B.1.617.2) and Omicron (B.1.1.529) mutant S1 proteins;
FIG. 2 is the affinity curve of the nano-antibody of the present invention with SARS-CoV-2 virus Alpha (B.1.1.7), Gamma (P.1), Delta (B.1.617.2) and Omicron (B.1.1.529) mutant strain S1 protein (taking A1 as an example);
FIG. 3 is the neutralization and inhibition curve of the nano antibody of the present invention against SARS-CoV-2 virus Alpha (B.1.1.7), Gamma (P.1), Delta (B.1.617.2) and Omicron (B.1.1.529) mutant pseudoviruses.
FIG. 4 shows the sequences of the antibodies of the invention and the CDR regions thereof;
FIG. 5 is a plasmid map of pComb3Xss used in example 1;
FIG. 6 is the complex of the nano antibody and SARS-CoV-2 virus S protein (trimer) (taking antibody A1 as an example);
FIG. 7 is the binding pattern diagram of the nano antibody of the present invention and SARS-CoV-2 virus RBD subunit (taking antibody A1 as an example).
FIG. 8 shows the binding epitope of the nano antibody CDR and SARS-CoV-2 virus RBD subunit (taking antibody A1 as an example).
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 the open literature.
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, SARS-CoV-2 virus wild type original strain S protein and RBD protein as immunogens and SARS-CoV-2 virus Alpha (B.1.1.7), Gamma (P.1), Delta (B.1.617.2) and Omicron (B.1.1.529) mutant strain S1 protein as coating antigens are purchased from Beijing-Yi Qiao Shenzhou organism Co.
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, trade number: ab 1265.
The color developing solution A consists of carbamide peroxide 1g, citric acid 10.3g and Na 2 HPO 4 ·12H 2 35.8g 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 Wild Type original strain S protein and RBD protein (Beijing Yiqian Shenzhou biology, Ltd.) are mixed with equal volume of Freund 'S adjuvant, fully emulsified and injected into camel, and then boosted once every two weeks, wherein the mixed solution of Freund' S adjuvant and immunogen is used in boosting immunization, 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 (the Huffi Provisions of Bruce D Hammock professor laboratories of DAVIs, see FIG. 5) by T4 ligase, and efficiently electrotransformed into E.coli ER2738 (stored in laboratories, also commercially available, for example, from NEB, UK) to construct phage nanobody libraries for SARS-CoV-2. The primary reservoir volume is determined to be 10 9 cfu, supplemented with helper phage (multiplicity of infection: 20: 1) M13KO7 (from NEB, cat # N0315S) for rescue to obtain phage nanobody library with a library capacity of 10 12 pfu/mL, better library diversity.
Reverse transcription PCR:
the reverse transcription kit adopts PrimeScript TM RT-PCR Kit, purchased from TaKaRa, under the 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：30)
LP-leader：GTGGTCCTGGCTGCTCTW(SEQ ID NO：31)
R：CATGCCATGACTCGCGGCCGGCCTGGCCATGGGGGTCTTCGC TGTGGTGCG(SEQ ID NO：32)
F：CATGCCATGACTGTGGCCCAGGCGGCCCAGKTGCAGCTCGTG GAGTC(SEQ ID NO：33)
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 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 ℃; the next day, pouring out the coating solution, washing with PBST for 3 times, blocking the 1 st and 2 nd holes of the ELISA plate with BSA, and incubating at room temperature for 2 h; pouring out the sealing liquid, and washing for 3 times by using PBST; adding the phage nanobody library obtained in the example 1 into the 1 st hole, 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 Wild type 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 into fresh Escherichia coli ER2738 bacterial liquid (stored in laboratory, or obtained commercially, such as from 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 amplification products 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 according to 2 times, calculating the titer of each round, and selecting a monoclonal for 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 a high-purity anti-SARS-CoV-2 nano antibody, namely, the antibody A1-A8, and the amino acid sequence of the obtained nano antibody is shown as SEQ ID NO: 1-8.
Example 4 binding curves of Nanobodies with SARS-CoV-2 Virus S1 protein
Respectively coating SARS-CoV-2 virus Alpha (B.1.1.7), Gamma (P.1), Delta (B.1.617.2) and Omicron (B.1.1.529) mutant strain S1 protein (Beijing Yizhushangzhou biological limited) on a 96-well enzyme label plate, wherein the coating concentration of each well 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. Binding curves for nanobodies and SARS-CoV-2 virus Alpha (B.1.1.7), Gamma (P.1), Delta (B.1.617.2) and Omicron (B.1.1.529) mutant S1 protein were plotted based on antibody concentration and OD values in the corresponding wells (see FIG. 1). The experimental result shows that the 8 nano-antibodies have stronger affinity with Alpha (B.1.1.7), Gamma (P.1), Delta (B.1.617.2) and Omicron (B.1.1.529) mutant S1 proteins, 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 avidin probe and utilizing an Octet red 96 instrument, and is performed by the conventional technology in the field. 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 Alpha (B.1.1.7), Gamma (P.1), Delta (B.1.617.2) and Omicron (B.1.1.529) mutant S1 proteins 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) was immersed in the first column of PBSTs 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;
referring to FIG. 2 and Table 1, the results show that the affinity of eight nanobodies for the SARS-CoV-2 virus Alpha mutant S1 protein is in the range: 0.18-0.9nM, and the range of affinity for Gamma mutant S1 protein: 0.52-9.3nM, and an affinity for the Delta mutant S1 protein ranging from: 0.53-7.79nM, with an affinity range for the Omicron mutant S1 protein: 0.88-9.9 nM.
TABLE 1 affinity constants K of Nanobodies with the protein of the Alpha mutant S1, the protein of the Gamma mutant S1, the protein of the Delta mutant S1 and the protein of the Omicron mutant S1 of SARS-CoV-2 virus D (M)
Example 6 detection of neutralizing Capacity of Nanobodies against SARS-CoV-2 pseudoviral infection
Firstly, eight kinds of nano-antibodies of the invention are diluted 10 concentration gradients in a DMEM culture medium, the final volume of each concentration is 50ul, wherein, the 10 th gradient only contains the DMEM culture medium and the concentration of the nano-antibody is 0, and the 10 th gradient is used as a control group, then 3ul SARS-CoV-2Alpha mutant strain pseudovirus (Hosta of Shanghai Pasteur institute of China, or Hokkaia sp. Biotechnology Co., Ltd.) capable of generating about 1x105 RLUs (related luciferase activity) is added into the nano-antibody dilution solution, after mixing uniformly, the mixture is incubated for 60min at 37 degrees, and 50ul SARS-HEK 293 hA 2 cells (Hokkaiun research institute of Shanghai Pasteur of China, or Hokkaian bioscience Co., Ltd.) are added into the virus-antibody compound, mix well and add to 96 well cell culture plate, each concentration of antibody set 3 repeat hole. Placing the cell culture plate in an incubator at 37 ℃, culturing for 48h, discarding cell supernatant, adding 100ul Bright-glo (Promega) into each well, reacting for 2min, transferring to a white 96-well plate, detecting the activity value of firefly luciferase (the neutralization experiment of the SARS-CoV-2 virus Gamma mutant strain, Delta mutant strain and Omicron mutant strain pseudovirus is consistent with the neutralization experiment step of the Alpha mutant strain pseudovirus by using a Varioskan Flash multifunctional reading instrument, and only addingThe pseudoviruses of (2) are different; both pseudoviruses and HEK293T-hACE2 cells were either offered by Shanghai Pasteur King Haikun researcher, or purchased from Nanjing NouWei Zan Biotech, Inc.). 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 curve 50 The 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 eight kinds of nano antibodies can specifically neutralize SARS-CoV-2S protein pseudovirus. Neutralization of Alpha mutant pseudoviruses EC 50 The range is as follows: 0.56-9.91nM, neutralizing EC against Gamma mutant pseudovirus 50 The range is as follows: 0.56-7.73nM, neutralizing EC against Delta mutant pseudovirus 50 The range is as follows: 0.67-6.26nM, EC for neutralization of Omicron mutant pseudovirus 50 The range is as follows: 3.07-31.38 nM.
Example 7 structural analysis of the complex between Nanobody and SARS-CoV-2 Virus S glycoprotein
In this example, the antibody A1 is taken as an example, the structural analysis of the complex between the nano antibody and SARS-CoV-2 virus S glycoprotein is studied, and the data results in this example are also applicable to the antibody A2-A8.
The S glycoprotein extracellular region of SARS-CoV-2 virus is secreted and expressed by using an insect-baculovirus expression system (stored in the laboratory, or commercially available, for example, from Invitrogen, USA, catalog nos. A11100), S protein is enriched from the culture supernatant by using a nickel affinity chromatography column, target protein is eluted by using imidazole gradient, the fraction containing the target protein is collected and concentrated, and then gel filtration chromatography (superdex 200 incubation chromatography column) is performed to identify the protein by SDS-PAGE, thus obtaining the S glycoprotein trimer with high purity and high uniformity. Glutaraldehyde (final concentration 0.25%) was added to the S protein solution and cross-linked for 30 minutes on ice. A frozen sample-preparing grid (GIG 411) on which a continuous carbon film with holes was laid was treated with oxygen, argon, 50W glow discharge for 1 minute for future use. Adding the protein S into the fixed protein S in a ratio of 1: adding the nano antibody in a proportion of 3 mol, quickly blowing and uniformly mixing in an EP tube, and immediately preparing a sample for a cryoelectron microscope by using an EMGP (Leica) sample preparation instrument. The final concentration of the protein solution is 0.7 mg/mL, the sample loading volume is 3 microliters, the freezing setting is that the temperature of an ethane cup is-183 ℃, the temperature of a chamber is 10 ℃, the humidity is 85%, prebot is 0 second, and the blot is 4 seconds. And screening the prepared sample for the cryoelectron microscope by using a Talos 200KV cryoelectron microscope, and reserving the sample with good particle contrast, integrity and uniformity for later use.
And collecting the screened cryoelectron microscope samples by using a high-end cryoelectron microscope Titan Krios 300KV equipped with a K2 camera. Data collection conditions were pixel size 1.04, 32 frames, total electron dose
And (3) carrying out data processing on the cryoelectron microscope data by using Relion 3.0 software to obtain a high-resolution cryoelectron microscope electron density image. The protein S takes PDB 7DK3 as an initial model, the model obtained by predicting the nano antibody by using manicure software is taken as the initial model, the initial model is put into an electron density map by using Chimera software Fit, and then manual adjustment and model building are carried out by using Coot software. And (3) carrying out model correction through PHENIX software to finally obtain a structural model of the S protein and nano antibody compound with good parameters.
As can be seen from FIGS. 6-8, the structural model of the complex of SARS-CoV-2 glycoprotein and Nanobody P2-1 by cryoelectron microscopy showed that the complex captured in this example consists of one S glycoprotein trimer and two Nanobody P2-1. The S glycoprotein trimer is in a partially open conformation: one RBD domain is in the "half-open" state; the two RBD domains are in the "closed" state. Two nanobodies P2-1 each independently bind to a "semi-open" RBD domain and an adjacent "closed" RBD domain. Nanobody P2-1 interacts with RBDs primarily through its CDR3 region, and portions of its CDR1, FR2 and FR3 regions are also involved in complex binding. The amino acid residues D99, S101, A103, D104, W105, R106, A107 and W109 in the CDR3 region of P2-1 may interact with the TR amino acid sequence at position 345 and 346, the NLDSKVGGNYN amino acid sequence at position 440 and 450 and the amino acid residues K444, N450, N448, R346, T345, L441, V445 and P499 in the PT amino acid sequence at position 499 and 500 of the RBD domain of SARS-CoV-2 glycoprotein; the amino acid residue D33 in the CDR1 region probably binds with the amino acid residues K444 and V445 in the NLDSKVGGNYN amino acid sequence at the 440-450 position of the RBD domain of SARS-CoV-2 glycoprotein; amino acid residues Y47 and T50 in the FR2 region probably interacted with amino acid residues P499, N440 and V445 in NLDSKVGGNYN amino acid sequence at position 440-450 and PT amino acid sequence at position 499-500 of the RBD domain of SARS-CoV-2 glycoprotein; the amino acid residues Y60 and S61 in the FR3 region probably interacted with the amino acid residue T500 in the PT amino acid sequence at position 499 and 500 in the RBD domain of SARS-CoV-2 glycoprotein. The antibody binding sites are located in the conserved region of the RBD structural domain of the surface Spike protein of SARS-CoV-2 virus, which proves that the antibody of the invention has broad spectrum to SARS-CoV-2 original strain and each mutant strain.
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.
Sequence listing
Nano antibody amino acid sequence
A1(SEQ ID NO：1)
A2(SEQ ID NO：2)
A3(SEQ ID NO：3)
A4(SEQ ID NO：4)
A5(SEQ ID NO：5)
A6(SEQ ID NO：6)
A7(SEQ ID NO：7)
A8(SEQ ID NO：8)
CDR1
A9(SEQ ID NO：9)：GFAFDSHD(VHH A1：CDR1)
A10(SEQ ID NO: 10): GFTLDSHD (VHH A2 and A3: CDR1)
A11(SEQ ID NO：11)：GFTFDSHD(VHH A4：CDRI)
A12(SEQ ID NO：12)：GFTFWSHE(VHH A5：CDR1)
A13(SEQ ID NO：13)：GFTFWSHD(VHH A6：CDR1)
A14(SEQ ID NO：14)：AFTFDSHE(VHH A7：CDR1)
A15(SEQ ID NO：15)：AATFDSHD(VHH A8：CDR1)
CDR2
A16(SEQ ID NO: 16): SVGYNT (VHH A1 and A7: CDR2)
A17(SEQ ID NO：17)：AVGSNT(VHH A2：CDR2)
A18(SEQ ID NO: 18): SVGSNT (VHHA3 and A4: CDR2)
A19(SEQ ID NO：19)：SVWYNT(VHH A5：CDR2)
A20(SEQ ID NO：20)：AYLSNT(VHH A6：CDR2)
A21(SEQ ID NO：21)：SAGSNT(VHH A8：CDR2)
CDR3
A22(SEQ ID NO：22)：SATLSTADSRAYWA(VHH A1：CDR3)
A23(SEQ ID NO：23)：AADLSTYDWRANWF(VHH A2：CDR3)
A24(SEQ ID NO：24)：AATLSTWDWRANRA(VHH A3：CDR3)
A25(SEQ ID NO：25)：AADLSTADWRANWA(VHH 4：CDR3)
A26(SEQ ID NO：26)：AATLSTSD-RYAW/A(VHH A5：CDR3)
A27(SEQ ID NO：27)：SADGSTYDWKNWAF(VHH A6：CDR3)
A28(SEQ ID NO：28)：AADLSTAD-RAYW(VHH A7：CDR3)
A29(SEQ ID NO：29)：SADGSTYDWKNWAF(VHH A8：CDR3)
Primer GSP-RT: CGCCATCAATRTACCAGTTGA (SEQ ID NO: 30)
Primer LP-leader: GTGGTCCTGGCTGCTCTW (SEQ ID NO: 31)
Primer R (SEQ ID NO: 32):
primer F (SEQ ID NO: 33):
Claims (17)
1. an antibody or antigen-binding fragment thereof, the amino acid sequence of which comprises a sequence consisting of SEQ ID NO: 9-15, CDR1 set forth in any one of SEQ ID NOs: 16-21, and a CDR2 set forth by SEQ ID NO: 22-29, or a CDR 3;
preferably, the antigen binding fragment is, for example, Fv, Fab ', scFv, F (ab') 2 Multivalent or multispecific fragments.
2. The antibody or antigen-binding fragment of claim 1, having an amino acid sequence as set forth in SEQ ID NO: 1-8;
or the antibody or antigen-binding fragment is a monoclonal antibody comprising the amino acid sequence of SEQ ID NO: 1-8, or an antibody or antigen-binding fragment thereof, which is a sequence obtained by truncating the sequence from amino acid 1 to amino acid 121 from the N-terminus of any one of SEQ ID NOs: 1-8 through substitution and/or deletion and/or addition of one or more amino acid residues to obtain the antibody or antigen binding fragment with the same function.
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 prokaryotic cells, yeast cells, insect cells, plant cells, animal cells.
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 an antibody or antigen-binding fragment according to claims 1-2, a genetically engineered antibody according to claim 3, a fusion protein according to claim 4 or a conjugate according to claim 5, comprising isolating/recovering the protein or polypeptide of interest from the tissue sample or culture according to claim 9.
12. A pharmaceutical composition comprising as an active ingredient an antibody or antigen-binding fragment of claim 1 or 2 or a genetically engineered antibody of claim 3 or a fusion protein of claim 4 or a conjugate of 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 pharmaceutical composition 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.
16. Epitope located in the conserved region of the surface Spike protein RBD domain of SARS-CoV-2 virus and the combination thereof, wherein the epitope has the following amino acid sequence:
1) the TR amino acid sequence is positioned at 345-346 th site of the surface Spike protein RBD structural domain of the SARS-CoV-2 virus;
2) NLDSKVGGNYN amino acid sequence located at 440-450 site of the surface Spike protein RBD structural domain of SARS-CoV-2 virus; and
3) PT amino acid sequence located at 499 nd 500 th site of surface Spike protein RBD structure domain of SARS-CoV-2 virus.
17. Use of the epitope of claim 16 for the preparation of a product for:
1) developing broad-spectrum coronavirus antigen or vaccine, especially antigen vaccine aiming at SARS-CoV-2 virus original strain and mutant strain thereof;
2) preparing antibodies, antigen-binding fragments, medicaments or products for preventing, treating and/or diagnosing broad-spectrum coronavirus infection, in particular to SARS-CoV-2 virus original strains and mutant strains thereof;
3) basic scientific research related to coronavirus, especially basic scientific research related to SARS-CoV-2 virus original strain and mutant strain thereof is carried out.
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| 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 |
| 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 |
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