A specific antibody MR-34 targeting the RBD of the spike protein of the coronavirus MERS-CoV

Abstract

The application discloses a specific antibody MR-34 targeting a coronavirus MERS-CoV spike protein RBD. 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 specifically combine with the MERS-CoV spike protein RBD domain by containing the specific sequence of CDRs. The antibody provided by the application has high targeting property and high combining activity. The antibody prepared by the preparation method provided by the application has high purity and can be prepared in large quantities. Overall, the antibody of the application can be used for development and optimization of a MERS-CoV infection detection method.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a specific binding antibody targeting the RBD domain of the spike protein of the coronavirus MERS-CoV, its preparation method, and its application. Background Technology

[0002] MERS-CoV is a single-stranded positive-sense RNA virus belonging to the subgenus Merbecovirus of the genus Beta coronaviruses. Its genome is approximately 30.1 kb in length and encodes four structural proteins (Spike / S, Envelope / E, Membrane / M, and Nucleocapsid / N). MERS-CoV invades cells by binding to the dipeptidyl peptidase 4 (DPP4) receptor on the host cell surface via its spike protein (S protein) trimer. The S protein consists of the S1 subunit (containing the receptor-binding domain RBD) and the S2 subunit. Camels are the primary natural host, and human-to-human transmission is mainly through droplet or contact transmission within hospitals. The case fatality rate is approximately 35% (WHO data). Pathological features include alveolar damage, cytokine storm, and delayed innate immune response. Binding antibodies targeting the MERS-CoV receptor-binding domain RBD can inhibit the binding of the virus to susceptible cells, thus serving as an important target for antibody development. Summary of the Invention

[0003] Based on this, the present invention aims to provide a specific binding antibody targeting the RBD domain of the spike protein of the coronavirus MERS-CoV, which specifically binds to the RBD domain of the MERS-CoV spike protein for the development of a method for detecting MERS-CoV 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 binding antibody targeting the spike protein RBD of the coronavirus MERS-CoV, comprising a heavy chain and a light chain; the amino acid sequences of the 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 the 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 RBD domain of the MERS-CoV spike protein, which is highly conserved in MERS-CoV.

[0014] Secondly, the present invention provides a specific binding antibody targeting the spike protein RBD of the coronavirus MERS-CoV for MERS-CoV detection.

[0015] Thirdly, the present invention provides a detection reagent, detection kit, or drug comprising the aforementioned specific binding antibody targeting the spike protein RBD of the coronavirus MERS-CoV.

[0016] Fourthly, the present invention provides a nucleic acid containing a nucleic acid sequence for encoding a specific binding antibody targeting the spike protein RBD of the coronavirus MERS-CoV.

[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 binding antibody targeting the spike protein RBD of the coronavirus MERS-CoV, comprising constructing the above-mentioned host cells, culturing them, and collecting the specific binding antibody targeting the spike protein RBD of the coronavirus MERS-CoV.

[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 sequenced CDRs that can specifically bind to the RBD domain of the MERS-CoV spike protein. The antibody provided by this invention exhibits strong targeting and high binding activity, specifically binding to the RBD domain of the MERS-CoV spike protein. Overall, the antibody of this invention can be used for the development of methods for detecting MERS-CoV infection. The antibody prepared using the method provided by this invention has high purity. Attached Figure Description

[0026] Figure 1 This is a graph showing the detection results of the antibody binding activity against the RBD domain of the MERS-CoV 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. Referring to the invention patent published by the inventor, "A Method for Preparing High-Titer EB Virus, a Method and Application of EB Virus Immortalized Memory B Cells", patent number: 20190975849.3, positive cell clones binding to the spike protein of the coronavirus MERS-CoV were screened, and RNA was extracted from the positive cell clones. The reference used was Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning. Tiller T, Meffre E, Yurasov S, Tsuiji M, Nussenzweig MC, Wardemann HJ Immunol Methods. 2008 Jan 1; 329(1-2):112-24. Epub 2007 Oct 31.10.1016 / j.jim.2007.09.017 PubMed The method in 17996249 involves PCR amplification of the heavy and light chains of the antibody, respectively, to obtain the variable region sequences of the 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 the two recombinant expression vectors (15 μg each of the heavy and light chains) containing the antibody 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 binding antibodies to the RBD domain of the MERS-CoV spike protein and antigen.

[0054] The binding ability of the monoclonal antibody obtained in Example 1 to the RBD domain of the MERS-CoV spike protein was determined by ELISA.

[0055] The steps are briefly described as follows:

[0056] (1) 25 ng of the RBD domain of the MERS-CoV 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 bovine 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 RBD domain of the MERS-CoV spike protein was EC50 = 5.388 μg / mL. Figure 1 )。

[0062] SEQ ID NO.1

[0063] GGSFSNY

[0064] SEQ ID NO.2

[0065] NHSGS

[0066] SEQ ID NO.3

[0067] CAGKGYCTGGGCYSGWYFDLW

[0068] SEQ ID NO.4

[0069] RASQGISNYLA

[0070] SEQ ID NO.5

[0071] AASTLQS

[0072] SEQ ID NO.6

[0073] CQKYNSAPRTF

[0074] SEQ ID NO.7

[0075] QVQLQQWGAGLLKPSETLSLTCAVNGGSFSNYYWSWVRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISEDTSKNQFSLKLTSVTAADTAVYYCAGKGYCTGGGCYSGWYFDLWGRGTLVTVSS

[0076] SEQ ID NO.8

[0077] DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIYAASTLQSGVP SRFSGSGSGTDFTLTINGLQPEDFATYYCQKYNSAPRTFGQGTKVEIK

[0078] SEQ ID NO.9

[0079] TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0080] SEQ ID NO.10

[0081] STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO.11

[0082] AKPSGSVVEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQSFSNP TCLILATVPHNLTTITKPLKYSYINKCSRLLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCP

[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 binding antibody targeting the RBD of the coronavirus MERS-CoV spike protein, 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 binding antibody targeting the RBD of the coronavirus MERS-CoV spike protein 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 binding antibody targeting the RBD of the coronavirus MERS-CoV spike protein 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 binding antibody targeting the RBD of the coronavirus MERS-CoV spike protein 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 antibody specifically binding to the spike protein RBD of the coronavirus MERS-CoV as described in any one of claims 1-4 in the preparation of MERS-CoV detection products.

6. A detection reagent or a detection kit, characterized by, The present invention comprises a specific binding antibody targeting the spike protein RBD of the coronavirus MERS-CoV as described in any one of claims 1-4.

7. A nucleic acid, characterized in that, Includes a nucleic acid sequence encoding a specific binding antibody targeting the spike protein RBD of the coronavirus MERS-CoV 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 binding antibody targeting the spike protein RBD of the coronavirus MERS-CoV 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 binding antibodies targeting the spike protein RBD of the coronavirus MERS-CoV.

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