A specific antibody binding to the spike protein of coronavirus HCoV-229E

Abstract

The application discloses a specific antibody combined with a coronavirus HCoV-229E spike protein. The sequences of CDRs of the antibody are shown in SEQ ID NO. 1 to SEQ ID NO. 6. The antibody provided by the application can be combined with the HCoV-229E spike protein in a specific manner due to the CDRs with specific sequences. The antibody provided by the application has high targeting property and high binding activity. The antibody prepared by the preparation method provided by the application has high purity and can be prepared in a large amount. Overall, the antibody provided by the application can be used for development and optimization of a coronavirus HCoV-229E infection detection method.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a specific antibody that binds to the spike protein of coronavirus HCoV-229E, its preparation method, and its application. Background Technology

[0002] HCoV-229E belongs to the Alpha coronavirus genus and is a low-pathogenic coronavirus that can infect humans. It primarily encodes four structural proteins: spike protein, membrane protein, envelope protein, and nucleocapsid protein. The spike protein is a homotrimer, with each monomer composed of approximately 1200 amino acids. It is a highly glycosylated type I transmembrane glycoprotein, anchored to the viral membrane, mediating viral invasion and the fusion of the viral membrane with the host cell membrane. Because the HCoV-229E spike protein plays a crucial role in viral invasion, it is an important target for therapeutic drugs or neutralizing antibodies. The development of antibodies targeting the HCoV-229E spike protein will contribute to the development of infection detection methods. Summary of the Invention

[0003] Accordingly, the present invention aims to provide a specific antibody that binds to the spike protein of coronavirus HCoV-229E, which is capable of specifically binding to the spike protein of HCoV-229E, for the development of a method for detecting HCoV-229E infection.

[0004] The above-mentioned objective can be achieved through the following technical solution:

[0005] In a first aspect, the present invention provides a specific antibody that binds to the spike protein of coronavirus HCoV-229E, comprising a heavy chain and a light chain; the amino acid sequences of CDR1, CDR2, and CDR3 of the variable region of the heavy chain of the antibody are shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; the amino acid sequences of CDR1, CDR2, and CDR3 of the variable region of the light chain of the antibody are shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

[0006] Preferably, the heavy chain variable region of the antibody is as shown in SEQ ID NO.7.

[0007] Preferably, the light chain variable region of the antibody is as shown in SEQ ID NO.8.

[0008] Preferably, the light chain constant region of the antibody is as shown in SEQ ID NO.9.

[0009] Preferably, the heavy chain constant region of the antibody is as shown in SEQ ID NO.10.

[0010] Preferably, the sequence of the constant region of the antibody is the sequence of the IgG1 constant region.

[0011] Preferably, the species source of the constant region of the antibody is human.

[0012] The heavy chain variable region shown in SEQ ID NO.7 and the light chain variable region shown in SEQ ID NO.8 also contain a framework region (FR), and the amino acid sequences of the four FRs do not directly participate in the binding reaction.

[0013] The antibody provided by this invention can specifically bind to the HCoV-229E spike protein.

[0014] Secondly, the present invention provides the application of the above-mentioned specific antibody that binds to the spike protein of coronavirus HCoV-229E in the detection of HCoV-229E or in the preparation of anti-HCoV-229E drugs.

[0015] Thirdly, the present invention provides a detection reagent, detection kit, or drug comprising the aforementioned specific antibody that binds to the spike protein of coronavirus HCoV-229E.

[0016] Fourthly, the present invention provides a nucleic acid containing a nucleic acid sequence for encoding the above-mentioned specific antibody that binds to the spike protein of coronavirus HCoV-229E.

[0017] Fifthly, the present invention provides a recombinant expression vector comprising the above-mentioned nucleic acid.

[0018] Preferably, the recombinant expression vector is an antibody expression vector.

[0019] Preferably, the recombinant expression vector is AbVec2.0-IGHG1 or AbVec1.1-IGKC.

[0020] In a sixth aspect, the present invention provides a host cell comprising the above-described specific antibody, or the above-described nucleic acid, or the above-described recombinant expression vector.

[0021] Preferably, the host cell is a 293T cell or an Expi293F cell.

[0022] In a seventh aspect, the present invention provides a method for preparing the above-mentioned specific antibody binding to the spike protein of coronavirus HCoV-229E, comprising constructing the above-mentioned host cell, culturing it, and collecting the specific antibody binding to the spike protein of coronavirus HCoV-229E.

[0023] Preferably, the host cell is a 293T cell or an Expi293F cell.

[0024] Compared with the prior art, this application has the following beneficial effects:

[0025] The antibody provided by this invention contains specific CDR sequences that can specifically bind to the spike protein of coronavirus HCoV-229E, and can be used for the development of methods for detecting HCoV-229E infection. The antibody provided by this invention has strong targeting and high binding activity, and can specifically bind to the spike protein of HCoV-229E. Overall, the antibody of this invention can be used for the development of methods for detecting HCoV-229E infection. The antibody prepared using the method provided by this invention has high purity and can be prepared in large quantities. Attached Figure Description

[0026] Figure 1 This is a graph showing the detection results of the antibody's binding activity against the HCoV-229E spike protein in Example 2 of the present invention. Detailed Implementation

[0027] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0028] In the following embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0029] Example 1: Construction, expression, and purification of monoclonal antibody expression vectors

[0030] The monoclonal antibody prepared in this embodiment has a heavy chain variable region comprising heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 as shown in SEQ ID NO.1, and a light chain variable region comprising light chain CDR1, light chain CDR2, and light chain CDR3 as shown in SEQ ID NO.4, SEQ ID NO.5, and light chain CDR3 as shown in SEQ ID NO.6. Specifically, its heavy chain variable region is shown in SEQ ID NO.7, its light chain variable region is shown in SEQ ID NO.8, its light chain constant region is shown in SEQ ID NO.9, and its heavy chain constant region is shown in SEQ ID NO.10.

[0031] The method for preparing monoclonal antibodies in this embodiment includes the following steps:

[0032] I. BALB / c mice were immunized with HCoV-229E spike protein (catalog number: 40605-V08B) purchased from Beijing Sinocare Pharmaceutical Co., Ltd., in combination with aluminum hydroxide adjuvant. The mice were immunized intramuscularly four times, with each immunization occurring two weeks apart. The dose for each immunization was 5 μg. After the immunization cycle was completed, hybridoma cells were prepared using the method described in the reference Zhang C. Hybridoma technology for the generation of monoclonal antibodies. Methods Mol Biol. 2012; 901:117-35. doi:10.1007 / 978-1-61779-931-0_7.PMID:22723097. The heavy and light chains of the antibody were amplified by PCR using the method described in the reference von Boehmer L, Liu C, Ackerman S, Gitlin AD, Wang Q, Gazumyan A, Nussenzweig MC. Sequencing and cloning of antigen-specific antibodies from mouse memory B cells. Nat Protoc. 2016 Oct;11(10):1908-1923. doi:10.1038 / nprot.2016.102. Epub 2016Sep 15.PMID:27658009. to obtain the variable region sequences of the antibody heavy and light chains.

[0033] 2. Nucleotide sequences encoding the antibody heavy chain (SEQ ID NO.7) and light chain variable region (SEQ ID NO.8) were integrated into AbVec2.0-IGHG1 and AbVec1.1-IGKC (vectors purchased from AddGene website, catalog numbers: #80795 and #80796, respectively) containing the heavy and light chain constant region sequences of human IgG1 antibody. The restriction enzyme cloning sites used for AbVec2.0-IGHG1 were AgeI and SalI, and the restriction enzyme cloning sites used for AbVec1.1-IGKC were AgeI and BsiWI. After restriction enzyme digestion and ligation, recombinant expression vectors capable of expressing the heavy chain and light chain of the target antibody were obtained.

[0034] III. Cell transfection, monoclonal antibody expression and purification

[0035] 1. Transfection

[0036] The Gibco Expi 293F expression system (catalog number A14635) was used, and transfection was performed according to the instructions. The steps are briefly described as follows:

[0037] (1) Mix 30 μg of DNA from two recombinant expression vectors expressing the heavy and light chains of the antibody (15 μg each of the heavy and light chains) with 80 μL of the matching transfection reagent ExpiFectamine. TM Mix and let stand at room temperature for 20 minutes to allow it to form a stable complex.

[0038] (2) Then add to 25.5 mL of the solution, which has been adjusted to a concentration of 2.9 × 10⁻⁶. 6 In Expi293F cell culture medium at cells / mL;

[0039] (3) Incubate at 37℃, 8% (v / v) CO2, and 125 rpm for 20 hours in a shaker;

[0040] (4) Add transfection enhancer 1 (150 μL) and transfection enhancer 2 (1.5 mL) provided with the Expi293F expression system;

[0041] (5) Continue to incubate at 37℃, 8% (v / v) CO2, and 125 rpm for 4 days.

[0042] 2. Purification

[0043] Centrifuge at 3000 rpm for 15 minutes to collect the supernatant, and purify the antibody using Protein A magnetic beads from Genscript.

[0044] The purification steps are briefly described as follows:

[0045] (1) Mix 500 μL of Protein A magnetic beads with 30 mL of cell supernatant and incubate on a shaker at room temperature for 4 hours at a speed of 210 RPM.

[0046] (2) Adsorb the magnetic beads with a magnetic rack, discard the cell supernatant, and wash the magnetic beads 5 times with 10 mL of 1×PBS with pH 7.0 0.1% (v / v) Tween 20.

[0047] (3) Elute with 2.5 mL of pH 2.0 0.1 M glycine Elution buffer;

[0048] (4) Equilibrate to pH 7.0 using 1M Tris buffer at pH 8.5;

[0049] (5) The equilibrated monoclonal antibody was desalted and replaced with DPBS solvent using a PD-10 Desalting Column (Cytiva, catalog number 17085101). After washing the desalting column with 25 mL of DPBS, the equilibrated antibody solution was passed through 2.5 mL of DPBS and then eluted with 3.5 mL of DPBS to obtain a monoclonal antibody solution with DPBS solvent replacement.

[0050] (6) The purified antibody was stored in a -80°C freezer.

[0051] Monoclonal antibodies were thus obtained, the amino acid sequence of which is shown in SEQ ID NO.7, the amino acid sequence of which is shown in SEQ ID NO.8, the amino acid sequence of which is shown in SEQ ID NO.9, and the amino acid sequence of which is shown in SEQ ID NO.10.

[0052] Example 2: Functional analysis of monoclonal antibodies

[0053] 1. Detection of the binding activity of specific antibodies to the HCoV-229E spike protein and antigen.

[0054] The binding ability of the monoclonal antibody obtained in Example 1 to the HCoV-229E spike protein was determined by ELISA.

[0055] The steps are briefly described as follows:

[0056] (1) 25 ng of HCoV-229E spike protein (amino acid sequence as shown in SEQ ID NO.11) was coated onto each well of an ELISA plate using DPBS (Dubor's phosphate buffer) as the coating solution and incubated overnight at 4°C.

[0057] (2) Using 10% (v / v) fetal serum in DPBS as the blocking solution, block at 37°C for 2 hours; then add serially diluted (10) -4 10 -2 10 0 10 2 The monoclonal antibody to be tested, prepared in Example 1, was incubated at 37°C for 2 hours;

[0058] (3) Add HRP-conjugated Goat anti-human IgG (H+L) antibody (Jackson ImmunoResearch) diluted 1:40000 as a secondary antibody and incubate at 37°C for 1 hour;

[0059] (4) After developing the color with TMB single-component colorimetric solution, terminate the reaction with 2M sulfuric acid and detect the absorbance A450 value with an enzyme-linked immunosorbent assay (ELISA) reader.

[0060] 2. Results

[0061] The results of the monoclonal antibody-antigen binding activity assay are shown in [the table below]. Figure 1 The binding activity against the HCoV-229E spike protein was EC50 = 0.003064 μg / mL. Figure 1 ).

[0062] SEQ ID NO.1

[0063] GYTFPTYT

[0064] SEQ ID NO.2

[0065] IIPSSGYT

[0066] SEQ ID NO.3

[0067] CAISYFYGSSHDYW

[0068] SEQ ID NO.4

[0069] SSVTY

[0070] SEQ ID NO.5

[0071] ATS

[0072] SEQ ID NO.6

[0073] CQQWSSNPWTF

[0074] SEQ ID NO.7

[0075] QVQLQQSAAELARPGASVKMSCKASGYTFPTYTIQWIKQRPGQGLEWIGYIIPSSGYTEYNQKFKDKTSMTADKSSSTSYMQLSSLTSEDSAIYYCAISYFYGSSHDYWGQGTTLTVSSSEQ ID NO.8

[0076] QIVLSQSPAILSASPGEKVTMTCRASSSVTYMHWYQQKPGSSPKPWIYATSNLASGIPGRFSGSGSGTSYSLTISRVEAEDAATYYCQQWSSNPWTFGGGTKLEIK

[0077] SEQ ID NO.9

[0078] TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0079] SEQ ID NO.10

[0080] STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0081] SEQ ID NO.11

[0082]

[0083] The above detailed description is a specific description of the embodiments of the present invention. These embodiments are not intended to limit the patent scope of the present invention. All equivalent implementations or modifications that do not depart from the present invention should be included in the patent scope of this case.

Claims

1. A specific antibody that binds to the spike protein of coronavirus HCoV-229E, characterized in that, It includes a heavy chain and a light chain; the amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region of the antibody are shown in SEQ ID NO:1, SEQ ID NO.2, and SEQ ID NO.3, respectively; The amino acid sequences of CDR1, CDR2, and CDR3 of the light chain variable region of the antibody are shown in SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively.

2. The specific antibody against the spike protein of coronavirus HCoV-229E according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO.7; the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO.

8.

3. The specific antibody against the spike protein of coronavirus HCoV-229E according to claim 1, characterized in that, The sequence of the constant region of the antibody is the sequence of the IgG1 constant region; the species source of the constant region of the antibody is human.

4. The specific antibody against the spike protein of coronavirus HCoV-229E according to claim 1, characterized in that, The amino acid sequence of the light chain constant region of the antibody is shown in SEQ ID NO.9; the amino acid sequence of the heavy chain constant region of the antibody is shown in SEQ ID NO.

10.

5. The use of the specific antibody binding to the spike protein of coronavirus HCoV-229E as described in any one of claims 1-4 in the preparation of HCoV-229E detection products.

6. A detection reagent, detection kit, or drug, characterized in that, Contains a specific antibody that binds to the spike protein of coronavirus HCoV-229E as described in any one of claims 1-4.

7. A nucleic acid, characterized in that, The nucleic acid is a nucleic acid sequence encoding the specific antibody that binds to the spike protein of coronavirus HCoV-229E as described in any one of claims 1-4.

8. A recombinant expression vector, characterized in that, It includes the nucleic acid as described in claim 7.

9. A host cell, characterized in that, It comprises the specific antibody as described in any one of claims 1-4, the nucleic acid as described in claim 7, or the recombinant expression vector as described in claim 8.

10. A method for preparing a specific antibody against the spike protein of coronavirus HCoV-229E as described in any one of claims 1-4, characterized in that, This includes constructing the host cell as described in claim 9, culturing it, and collecting specific antibodies that bind to the spike protein of coronavirus HCoV-229E.

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