Specific antibody binding to coronavirus HCoV-HKU1 spike protein and application thereof

Abstract

The invention discloses a specific antibody combined with coronavirus HCoV-HKU1 spike protein and application of the specific antibody. The sequences of the CDRs of the antibody disclosed by the invention are as shown in SEQ ID NO. 1 to SEQ ID NO. 6. The antibody provided by the invention comprises CDRs with a specific sequence, and can be specifically combined with HCoV-HKU1 spike protein. The antibody provided by the invention is strong in targeting property and high in binding activity, and the antibody prepared by the preparation method provided by the invention is high in purity and can be prepared on a large scale. On the whole, the antibody disclosed by the invention can be used for developing and optimizing a coronavirus HCoV-HKU1 infection detection method.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a specific antibody binding to the spike protein of coronavirus HCoV-HKU1 and a preparation method and application thereof. BACKGROUND

[0002] HCoV-HKU1 belongs to the Merbecovirus virus genus and is a low pathogenic coronavirus that can infect humans, mainly encoding four structural proteins, namely spike protein, membrane protein, envelope protein and nucleocapsid protein. The spike protein is a homotrimer, each monomer consisting of about 1200 amino acids, which is a highly glycosylated type I transmembrane glycoprotein anchored on the viral membrane, mediating viral invasion and fusion of the viral membrane with the host cell membrane. Since the spike protein of HCoV-HKU1 plays an important role in the process of viral invasion, it is an important target for therapeutic drugs or neutralizing antibodies. The development of antibodies against the spike protein of HCoV-HKU1 will help the development of infection detection methods. SUMMARY

[0003] Therefore, the purpose of the present application includes providing a specific antibody binding to the spike protein of coronavirus HCoV-HKU1, which can specifically bind to the spike protein of HCoV-HKU1 for the development of HCoV-HKU1 infection detection methods.

[0004] The above-mentioned purposes can be achieved by the following technical solutions:

[0005] In a first aspect, the present application provides a specific antibody binding to the spike protein of coronavirus HCoV-HKU1, comprising 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 respectively shown in SEQ ID NO: 1, SEQ ID NO. 2 and SEQ ID NO. 3; the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region of the antibody are respectively shown in SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6.

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

[0007] Preferably, the light chain variable region of the antibody is 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 an IgG1 constant region.

[0011] Preferably, the species origin 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 framework regions (FRs), and the amino acid sequences of the four FRs do not directly participate in the binding reaction.

[0013] The antibody provided by the present application can specifically bind to the spike protein of HCoV-HKU1.

[0014] In a second aspect, the present application provides the use of the specific antibody that binds to the spike protein of HCoV-HKU1 in the detection of HCoV-HKU1 or the preparation of an anti-HCoV-HKU1 drug.

[0015] In a third aspect, the present application provides a detection reagent, a detection kit or a drug, comprising the specific antibody that binds to the spike protein of HCoV-HKU1.

[0016] In a fourth aspect, the present application provides a nucleic acid comprising a nucleic acid sequence for encoding the specific antibody that binds to the spike protein of HCoV-HKU1.

[0017] In a fifth aspect, the present application provides a recombinant expression vector comprising the 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 application provides a host cell comprising the specific antibody, the nucleic acid or the recombinant expression vector.

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

[0022] In a seventh aspect, the present application provides a method for preparing the specific antibody binding to the spike protein of coronavirus HCoV-HKU1, comprising constructing the host cell as described above, culturing, and collecting the specific antibody binding to the spike protein of coronavirus HCoV-HKU1.

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

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

[0025] The antibody provided by the present application comprises CDRs with specific sequences, can specifically bind to the spike protein of coronavirus HCoV-HKU1, and is used for the development of an HCoV-HKU1 infection detection method. The antibody provided by the present application has high targeting and binding activity, and can specifically bind to the spike protein of HCoV-HKU1. Overall, the antibody of the present application can be used for the development of an HCoV-HKU1 infection detection method. The antibody prepared by the preparation method provided by the present application has high purity and can be prepared in large quantities. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 FIG. 2 is a graph showing the binding activity of the antibody prepared in Example 2 to the spike protein of HCoV-HKU1. DETAILED DESCRIPTION

[0027] The following examples are further illustrations of the present application and are not intended to limit the present application.

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

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

[0030] The monoclonal antibody prepared in this example has a heavy chain variable region comprising a heavy chain CDR1 with an amino acid sequence as shown in SEQ ID NO. 1, a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO. 2, and a heavy chain CDR3 with an amino acid sequence as shown in SEQ ID NO. 3, and a light chain variable region comprising a light chain CDR1 with an amino acid sequence as shown in SEQ ID NO. 4, a light chain CDR2 with an amino acid sequence as shown in SEQ ID NO. 5, and a light chain CDR3 with an amino acid sequence as shown in SEQ ID NO. 6. Specifically, the heavy chain variable region is as shown in SEQ ID NO. 7, the light chain variable region is as shown in SEQ ID NO. 8, the light chain constant region is as shown in SEQ ID NO. 9, and the heavy chain constant region is as shown in SEQ ID NO. 10.

[0031] The preparation method of the monoclonal antibody of the present embodiment comprises the following steps:

[0032] I. BALB / c mice were immunized with HCoV-HKU1 spike protein purchased from Beijing Yiqiao God State Company (item number: 40606-V08B) with aluminum hydroxide adjuvant, and the mice were immunized by intramuscular injection, a total of 4 times, with an interval of 2 weeks each time, and the dose of each immunization was 5 μg. After the completion of the immunization cycle, the immunized 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 chain and light chain of the antibody were respectively PCR amplified using the method 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 2016 Sep 15. PMID: 27658009.

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

[0034] III. Transfection of cells, expression and purification of monoclonal antibodies

[0035] 1. Transfection

[0036] Gibco Expi293F expression system (Cat. No. A14635) was used for transfection according to the instructions. The steps are briefly described as follows:

[0037] (1) 30 μg of DNA of two recombinant expression vectors expressing heavy chain and light chain of the antibody (15 μg of each) was mixed with 80 μL of the matching transfection reagent ExpiFectamine TM , and incubated at room temperature for 20 minutes to form a stable complex;

[0038] (2) Then added to 25.5 mL of Expi293F cell culture solution with a concentration of 2.9 x 10 6 cells / mL;

[0039] (3) Incubated at 37°C, 8% (v / v) CO2, 125 rpm shaker for 20 hours;

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

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

[0042] 2. Purification

[0043] Centrifuged at 3000 rpm for 15 minutes to collect the supernatant, and then purified 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 at room temperature on a shaker at 210 RPM for 4 hours;

[0046] (2) Absorb the magnetic beads with a magnetic stand, discard the cell supernatant, and wash the magnetic beads with 10 mL of 1 x PBS with 0.1% (v / v) Tween 20 at pH 7.0 for 5 times;

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

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

[0049] (5) The balanced monoclonal antibody was desalted and solvent-exchanged using a PD-10 Desalting Column (Cytiva, item number 17085101). After washing the desalting column with 25 mL of DPBS, 2.5 mL of the balanced antibody solution was passed through the column, and then 3.5 mL of DPBS was used for elution, obtaining a monoclonal antibody solution with DPBS as the solvent.

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

[0051] Thus, a monoclonal antibody was obtained, the amino acid sequence of the heavy chain variable region of which is shown in SEQ ID NO. 7, the amino acid sequence of the light chain variable region of which is shown in SEQ ID NO. 8, the amino acid sequence of the light chain constant region of which is shown in SEQ ID NO. 9, and the amino acid sequence of the heavy chain constant region of which is shown in SEQ ID NO. 10.

[0052] Example 2: Functional analysis of the monoclonal antibody

[0053] 1. Detection of the binding activity of the specific antibody binding to the HCoV-HKU1 spike protein to the antigen

[0054] The binding ability of the monoclonal antibody obtained in Example 1 to the HCoV-HKU1 spike protein was determined by ELISA.

[0055] The steps are briefly described as follows:

[0056] (1) 25 ng of HCoV-HKU1 spike protein (the amino acid sequence is shown in SEQ ID NO. 11) was coated on an ELISA plate as the coating liquid, and the plate was incubated at 4°C overnight;

[0057] (2) 10% (v / v) calf serum in DPBS was used as the blocking liquid, and the plate was incubated at 37°C for 2 hours; the prepared monoclonal antibody to be tested was diluted in a gradient (10 -4 , 10 -2 , 10 0 , 10 2 ) and added to the plate, and the plate was incubated at 37°C for 2 hours;

[0058] (3) 1:40000 diluted HRP-conjugated Goat anti-human IgG (H+L) antibody (Jackson ImmunoResearch) was added as the secondary antibody, and the plate was incubated at 37°C for 1 hour;

[0059] (4) After color development with TMB one-component color developing solution, the reaction was terminated with 2M sulfuric acid, and the absorbance A450 value was detected by an enzyme-labeled instrument.

[0060] 2. Results

[0061] The results of the detection of the binding activity of the monoclonal antibody to the antigen are shown in Table 1 below. Figure 1 The binding activity against the spike protein of HCoV-HKUl was EC50= 0.002178 μg / mL (Table 1). Figure 1

[0062] SEQ ID NO. 1

[0063] SYWIH

[0064] SEQ ID NO. 2

[0065] RIYPGTVTTYYNDNFKD

[0066] SEQ ID NO. 3

[0067] GDDLRGFGY

[0068] SEQ ID NO. 4

[0069] KASQSVTDDVA

[0070] SEQ ID NO. 5

[0071] SASNRYT

[0072] SEQ ID NO. 6

[0073] QQDYSPVFT

[0074] SEQ ID NO. 7

[0075] QVQLKQSGAELVRPGASVKLSCKTSGYIFTSYWIHWLKQRSGQGLEWIARIYPGTVTT YYNDNFKDKATLTADKSSSTAYMQLSSLKSEDSAVYFCARGDDLRGFGYWGQGTLVTVSA SEQ ID NO. 8

[0076] SIVMTQTPKFLLVSAGDRVTIACKASQSVTDDVAWYQQKPGQSPRLLIFSASNRYTGVP DRFSGSGYGTDFTFTINTVQAEDLAVYFCQQDYSPVFTFGSGTKLEMK

[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 application, which is not used to limit the patent scope of the present application. Any equivalent implementation or modification made without departing from the present application shall be included in the patent scope of the present application.

Claims

1. A specific antibody that binds to the spike protein of coronavirus HCoV-HKU1, 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 binding to the spike protein of coronavirus HCoV-HKU1 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 binding to the spike protein of coronavirus HCoV-HKU1 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 binding to the spike protein of coronavirus HCoV-HKU1 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 that binds to the spike protein of coronavirus HCoV-HKU1 as described in any one of claims 1-4 in the detection of HCoV-HKU1.

6. A detection reagent, detection kit, or drug, characterized in that, Contains a specific antibody that binds to the spike protein of coronavirus HCoV-HKU1 as described in any one of claims 1-4.

7. A nucleic acid, characterized in that, Includes a nucleic acid sequence encoding a specific antibody that binds to the spike protein of coronavirus HCoV-HKU1 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-HKU1 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-HKU1.

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