Monoclonal antibody combined with monkey pox virus B6R protein and application thereof

Abstract

The invention belongs to the technical field of biological pharmacy, and particularly relates to a monoclonal antibody combined with monkey pox virus B6R protein and application of the monoclonal antibody. The antibody is any one or a combination of more of B6R-B5, B6R-F6 and B6R-F11, each of the B6R-B5, the B6R-F6 and the B6R-F11 comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region has three heavy chain CDRs, and the light chain variable region has three light chain CDRs. The invention provides three groups of monoclonal antibodies with excellent affinity with the monkey pox virus B6R and disclosed heavy chain and light chain variable region sequences, provides technical support for preparing a detection and diagnosis reagent of the monkey pox virus, and is expected to be developed into a special product for screening and diagnosing the monkey pox virus. High application values are realized on prevention, control and treatment of the monkey pox virus.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceuticals, and in particular relates to a monoclonal antibody binding to monkeypox virus B6R protein and an application thereof. Technical Background

[0002] In the 1980s, humans announced that smallpox had been eradicated worldwide. However, with the end of smallpox vaccination worldwide, monkeypox virus began to gradually appear in Central Africa, East Africa and West Africa. In recent years, it has shown a trend of global spread and has become an international public health emergency several times.

[0003] Monkeypox is caused by the monkeypox virus and is mainly transmitted from person to person through close contact with monkeypox patients (including family members). It can cause painful rashes, swollen lymph nodes, fever, headache, myalgia, back pain and fatigue, and in severe cases, death. Monkeypox virus is an enveloped double-stranded DNA virus of the genus Orthopoxvirus in the family Poxviridae, which includes smallpox, cowpox, vaccinia and other viruses. B6R is a membrane protein located on the membrane of the extracellular envelope virus particle (EEV). It is homologous to the complement control protein B5 of the cowpox virus, participates in the negative regulation of complement activation, and is a glycoprotein required for cell spread.

[0004] The global prevalence of monkeypox virus has led to an increase in the demand for diagnosis, and early screening, diagnosis or auxiliary diagnosis has become an urgent social need. The development of monoclonal antibodies against monkeypox virus B6R and the provision of technical support for the screening and diagnosis of monkeypox virus are currently urgent issues that need to be addressed. Summary of the invention

[0005] In order to solve the problems in the prior art, one of the objectives of the present invention is to provide a monoclonal antibody binding to monkeypox virus B6R protein and its application.

[0006] The present invention adopts the following technical solutions:

[0007] A monoclonal antibody that binds to the B6R protein of monkeypox virus, the antibody being a combination of any one or more of B6R-B5, B6R-F6 and B6R-F11, wherein B6R-B5, B6R-F6 and B6R-F11 all comprise a heavy chain variable region and a light chain variable region, the heavy chain variable region having three heavy chain CDRs, the light chain variable region having three light chain CDRs, wherein:

[0008] The three CDR amino acid sequences of the B6R-B5 heavy chain variable region are, in sequence, amino acid sequences having homologies greater than or equal to 80% with the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3; the three CDR amino acid sequences of the B6R-B5 light chain variable region are, in sequence, amino acid sequences having homologies greater than or equal to 80% with the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6;

[0009] The three CDR amino acid sequences of the B6R-F6 heavy chain variable region are, in sequence, amino acid sequences having homologies greater than or equal to 80% with the amino acid sequences shown in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9; the three CDR amino acid sequences of the B6R-F6 light chain variable region are, in sequence, amino acid sequences having homologies greater than or equal to 80% with the amino acid sequences shown in SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12;

[0010] The three CDR amino acid sequences of the B6R-F11 heavy chain variable region are, respectively, amino acid sequences with greater than or equal to 80% homology to the amino acid sequences shown in SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15; the three CDR amino acid sequences of the B6R-F11 light chain variable region are, respectively, amino acid sequences with greater than or equal to 80% homology to the amino acid sequences shown in SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18.

[0011] The homology sequence is preferably an amino acid sequence with at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology. The homology sequence also includes an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence shown in the sequence listing.

[0012] Preferably, the amino acid sequences of the six CDRs of the heavy chain variable region and the light chain variable region of B6R-B5 are shown as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively;

[0013] The amino acid sequences of the six CDRs of the heavy chain variable region and light chain variable region of B6R-F6 are shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, respectively;

[0014] The amino acid sequences of the six CDRs of the heavy chain variable region and the light chain variable region of B6R-F11 are shown in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively.

[0015] Specifically, the CDR amino acid sequences of the heavy chain variable region and light chain variable region of B6R-B5, B6R-F6, and B6R-F11 are shown in the following table:

[0016]

[0017]

[0018] In the table, H represents heavy chain, and K represents light chain.

[0019] Preferably, the amino acid sequence of the heavy chain variable region of B6R-B5 is shown as SEQ ID NO:19, and the amino acid sequence of the light chain variable region of B6R-B5 is shown as SEQ ID NO:20; the amino acid sequence of the heavy chain variable region of B6R-F6 is shown as SEQ ID NO:21, and the amino acid sequence of the light chain variable region of B6R-F6 is shown as SEQ ID NO:22; the amino acid sequence of the heavy chain variable region of B6R-F11 is shown as SEQ ID NO:23, and the amino acid sequence of the light chain variable region of B6R-F11 is shown as SEQ ID NO:24.

[0020] Preferably, the antibody is a murine antibody and / or a human-mouse chimeric antibody, and the human-mouse chimeric antibody comprises a constant region of a human antibody, including but not limited to a human IgG1 Fc domain, IgG2 Fc domain, IgG3 Fc domain or IgG4 Fc domain.

[0021] The present invention also provides a polynucleotide encoding the B6R-B5, B6R-F6, and B6R-F11 antibodies as described above. The nucleotide sequence encoding the B6R-B5 heavy chain variable region is shown in SEQ ID NO: 25, and the nucleotide sequence encoding the B6R-B5 light chain variable region is shown in SEQ ID NO: 26; the nucleotide sequence encoding the B6R-F6 heavy chain variable region is shown in SEQ ID NO: 27, and the nucleotide sequence encoding the B6R-F6 light chain variable region is shown in SEQ ID NO: 28; the nucleotide sequence encoding the B6R-F11 heavy chain variable region is shown in SEQ ID NO: 29, and the nucleotide sequence encoding the B6R-F11 light chain variable region is shown in SEQ ID NO: 30.

[0022] The present invention also provides an expression vector comprising the polynucleotide as described above, and a host cell comprising the expression vector as described above. Preferably, the host cell is a host cell for expressing foreign proteins, such as bacteria, yeast, insect cells, mammalian cells.

[0023] The present invention also provides a use of the above-mentioned antibody in preparing a detection reagent or a detection product for monkeypox virus.

[0024] The present invention provides a kit for detecting monkeypox virus, wherein the kit comprises the antibody as described above.

[0025] Preferably, the kit is a colloidal gold detection kit, an immunochromatographic detection kit, an enzyme immunoassay kit, a chemiluminescence kit or an immunoturbidimetric detection kit.

[0026] Preferably, the antibody further comprises an immune marker coupled thereto, and the immune marker is any one of an enzyme label, a fluorescent label, an isotope label, and a biotin label.

[0027] The present invention uses monkeypox B6R protein as an immunogen, prepares and obtains corresponding monoclonal antibodies by immunizing mice, and screens out 3 groups of monoclonal antibodies with excellent affinity to monkeypox virus B6R protein through ELISA combination experiments, providing technical support for the preparation of detection and diagnostic reagents for monkeypox virus, and has the potential to be developed into a special product for screening and diagnosis of monkeypox epidemics, which has high application value for the prevention, control and treatment of monkeypox virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Serum titer evaluation results.

[0029] Figure 2 Flow cytometric sorting of single B cells specific for the B6R antigen.

[0030] Figure 3This is the binding curve of the antibody and antigen B6R. DETAILED DESCRIPTION

[0031] For ease of understanding, the technical solution of the present invention is described in more detail below in conjunction with embodiments:

[0032] Example 1

[0033] Mouse antigen immunization and screening of antibodies binding to B6R glycoprotein

[0034] 1.1 Acquisition of antigen B6R

[0035] The B6R protein sequence was derived from the Uniprot database, with the sequence number Q8V4S2. The B6R extracellular domain (amino acids 1 to 279) and the C-terminal Strep-Tag II and 1×Flag tag were constructed into the vector pcDNA3.4 to form an expression plasmid. The plasmid was gently mixed with the transfection reagent PEI, incubated at room temperature for 20 minutes, and transfected into 293F suspension cells to obtain recombinant cells for protein expression. The recombinant cells were cultured in SMM 293-TII complete medium for 96 hours, then centrifuged at 4°C and 3000 rpm for 30 minutes, and the supernatant was collected. The target protein B6R was purified by affinity chromatography using a StrepTactin agarose gravity column. The specific purification scheme is as follows: Mix 200ml of supernatant with the balanced StrepTactin agarose filler, incubate at 4°C overnight; add the mixture to the gravity column, continuously flow through the collection solution, and leave the StrepTactin agarose filler in the gravity column; slowly add 30ml of PBS solution to the gravity column, flow through, and wash the filler; repeat the washing step three times. Add 30ml of elution buffer (5mM D-Desthiobiotin in PBS) to the gravity column, flip and mix at room temperature, and incubate for 20min. Collect the eluate using a gravity column, and flow the eluate containing the target protein into a clean centrifuge tube. Use a 10KDa cutoff ultrafiltration tube for concentration, and replace the solution with PBS buffer.

[0036] 1.2 Antigen immunization of mice

[0037] BALB / c mice were immunized according to the immunization scheme listed in Table 1 below:

[0038] Table 1 Mouse antigen immunization

[0039]

[0040] The results of serum ELISA for B6R are as follows Figure 1 As shown. Figure 1The results showed that after three immunizations with antigen B6R, the level of serum antibodies specific to antigen B6R was very high, and the mice can be used for subsequent experiments.

[0041] 1.3 Antigen-specific flow cytometry sorting of single B cells

[0042] Blood was collected from the cheeks of BALB / c mice, and the mice were killed by dislocating the neck. The spleen of the mice was removed using sterile surgical instruments and placed in a filter on a 50ml centrifuge tube. The spleen was ground with a grinding rod to collect B cells. Buffer (2% FBS in PBS) was added to 15ml, and the mixture was centrifuged at 1200rpm and 4℃ for 5min, and the supernatant was discarded. 1ml of red blood cell lysis buffer was added to resuspend the precipitate, and 15ml of buffer was added to terminate the process after 1min. The mixture was centrifuged at 1200rpm and 4℃ for 5min, and the supernatant was discarded. 1ml of buffer was added to resuspend the cells, and the cells were transferred to a 1.5ml centrifuge tube. After standing on ice, 300μl of the upper layer of cells were taken, and the B cells were stained by flow cytometry, and antigen-specific sorting was performed using a flow cytometer. Flow sorting and gating strategies are as follows. Figure 2 As shown, DAPI - B220 + CD38 + IgD - IgG1 + B6R + Memory B cells were sorted and single B cells were sorted into a 96-well plate containing cell lysate.

[0043] 1.4 Antibody variable region gene amplification

[0044] Using single B cell gene amplification technology, the cleaved mRNA was reverse transcribed to obtain cDNA, and single B cell antibody gene amplification primers were used to obtain the gene products of the heavy chain and light chain variable regions by PCR and sequenced. The sequences were analyzed using the IgBlast tool.

[0045] 1.5 Construction of recombinant plasmid

[0046] The heavy chain variable region DNA molecule was seamlessly cloned into the expression vector hIgG to obtain the heavy chain expression plasmid.

[0047] The light chain variable region DNA molecule was seamlessly cloned into the expression vector hIgκ to obtain the light chain expression plasmid.

[0048] The coding regions of the heavy chain and light chain each consist of three parts: a signal peptide, a variable region, and a constant region.

[0049] 1.6 ELISA binding assay to screen positive clones

[0050] Finally, multiple antibodies were cloned successfully and paired. The paired heavy chain expression plasmid and light chain expression plasmid were co-transfected into HEK-293T cells and cultured for 48 hours to express antibodies and obtain the cultured antibody supernatant. The antibody supernatant was used to screen positive clones.

[0051] 1) Antigen coating: Dilute the antigen B6R with PBS to a final concentration of 1 ng / μl. Coat each well of the ELISA plate with 100 μl at 4°C overnight.

[0052] 2) Blocking: Discard the solution, wash each well with 200 μl PBST for 3 times, add 200 μl blocking solution to each well, and block at 37°C for 2 h.

[0053] 3) Washing: discard the supernatant, add 200 μl PBST to each well, wash three times, and pat dry.

[0054] 4) Add the primary antibody to be tested: dilute the antibody supernatant 3-fold in a gradient manner, add 100 μl of antibody solution to each well, with the first well being the undiluted stock solution and the negative control being PBS. Incubate at 37°C for 1 hour.

[0055] 5) Add secondary antibody: discard the supernatant, add 200 μl PBST to each well, wash 3 times, pat dry. Add 100 μl HRP-labeled goat anti-human IgG (H+L) secondary antibody to each well. Incubate at 37°C for 1 hour.

[0056] 6) Color development: discard the supernatant, add 200 μl PBST to each well, wash 4 times, pat dry, add 100 μl TMB color development solution, and after the color changes, add 50 μl 10% sulfuric acid stop solution to each well to terminate the reaction.

[0057] 7) Reading: Read the absorbance at 450nm using an ELISA reader. Set up one set of duplicate wells for each cell culture antibody supernatant. Select the one with an absorbance higher than 0.5 as a positive antibody.

[0058] The experimental results are shown in Table 2. It can be seen that after the cell culture antibody supernatant was screened by ELISA binding experiment, 12 strains of positive antibodies against B6R were obtained.

[0059] Table 2 ELISA binding positive antibody screening

[0060]

[0061]

[0062] Example 2

[0063] Preparation of antibodies and EC 50 Determination of

[0064] 2.1 Antibody Preparation

[0065] 2.1.1 The heavy chain expression plasmids and light chain expression plasmids of the 12 antibodies screened above were co-transfected into 293F cells to obtain recombinant cells. The cells were cultured in SMM 293-TII complete medium for 96 h, then centrifuged at 4°C and 3000 rpm for 30 min, and the supernatant was collected.

[0066] 2.1.2 Protein A magnetic beads to purify antibodies

[0067] Filler for affinity chromatography: Protein A magnetic beads; elution buffer: 0.1 M glycine, pH 3.0; neutralization buffer: 1 M Tris, pH 8.5.

[0068] Mix 100 ml of supernatant with Protein A magnetic beads and incubate overnight at 4°C; separate the solution and beads using a magnetic stand. Add 30 ml of PBS solution to the magnetic beads, rotate to mix, wash the beads for 5 minutes, collect the beads using a magnetic stand and discard the supernatant; repeat the washing step three times. Add 18 ml of elution buffer to the magnetic beads, quickly resuspend, mix evenly, flip and mix at room temperature, and incubate for 5 minutes. Collect the magnetic beads using a magnetic separation stand and transfer the supernatant containing the eluted antibody to a clean 50 ml centrifuge tube. Immediately add 2 ml of neutralization buffer to the eluate to neutralize the pH to a weak alkaline to maintain the biological activity of the antibody and avoid antibody inactivation.

[0069] 2.1.3 Solution replacement

[0070] The antibody solution obtained above was concentrated using an ultrafiltration tube, and the solution was replaced with PBS buffer to obtain an antibody solution with a protein concentration of 1 mg / ml.

[0071] 2.2EC 50 Determination of

[0072] 2.2.1 Antigen coating: Dilute antigen B6R with PBS to a final concentration of 1 ng / μl. Coat each well of the ELISA plate with 100 μl at 4°C overnight.

[0073] 2.2.2 Blocking: Discard the solution and wash each well with 200μl PBST for 3 times. Add 200μl blocking solution to each well and block at 37℃ for 2h. Washing: Discard the supernatant and wash each well with 200μl PBST for 3 times and pat dry.

[0074] 2.2.3 Add primary antibody: antibody starting concentration 10 μg / ml, 3-fold gradient dilution, a total of 12 gradients, add 100 μl antibody dilution solution to each well, negative control is PBS. Incubate at 37℃ for 1 hour.

[0075] 2.2.4 Add secondary antibody: discard the supernatant, add 200 μl PBST to each well, wash 3 times, pat dry. Add 100 μl HRP-labeled goat anti-human IgG (H+L) secondary antibody to each well. Incubate at 37℃ for 1 hour.

[0076] 2.2.5 Color development: discard the supernatant, add 200μl PBST to each well, wash 4 times, pat dry. Add 100μl TMB color development solution, and after the color changes, add 50μl 10% sulfuric acid stop solution to each well to terminate the reaction.

[0077] 2.2.6 Reading: Read the absorbance value at 450nm using an ELISA reader. Set up one replicate well for each monoclonal antibody. Repeat the experiment twice independently.

[0078] 2.2.7EC 50 Calculation: GraphPad Prism 9 software was used to calculate the antibody concentration at which 50% binding occurred, i.e., the EC value of the antibody. 50 (μg / ml) values. The results are shown in Table 3 and Figure 3 .

[0079] Table 3 EC of antibodies 50 value

[0080]

[0081] It can be seen that B6R-B5, B6R-F6, and B6R-F11 have EC 50 The value is small, indicating that these three antibodies have high affinity for B6R and are worth further research and development as in vitro diagnostic reagents for monkeypox virus. The variable region sequences of each antibody are shown in the sequence table.

[0082] The above implementation modes are only used to illustrate the technical solutions of the present invention, but not to limit the present invention. Although the present invention has been described in detail with reference to the above implementation modes, those skilled in the art should understand that any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A monoclonal antibody that binds to monkeypox virus B6R protein, characterized in that: The antibody is a combination of any one or more of B6R-B5, B6R-F6 and B6R-F11, wherein B6R-B5, B6R-F6 and B6R-F11 all comprise a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has three heavy chain CDRs, and the light chain variable region has three light chain CDRs, wherein: The three CDR amino acid sequences of the B6R-B5 heavy chain variable region are, in sequence, amino acid sequences having homologies greater than or equal to 80% with the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3; the three CDR amino acid sequences of the B6R-B5 light chain variable region are, in sequence, amino acid sequences having homologies greater than or equal to 80% with the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6; The three CDR amino acid sequences of the B6R-F6 heavy chain variable region are, in sequence, amino acid sequences having homologies greater than or equal to 80% with the amino acid sequences shown in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9; the three CDR amino acid sequences of the B6R-F6 light chain variable region are, in sequence, amino acid sequences having homologies greater than or equal to 80% with the amino acid sequences shown in SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12; The three CDR amino acid sequences of the B6R-F11 heavy chain variable region are, respectively, amino acid sequences with greater than or equal to 80% homology to the amino acid sequences shown in SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15; the three CDR amino acid sequences of the B6R-F11 light chain variable region are, respectively, amino acid sequences with greater than or equal to 80% homology to the amino acid sequences shown in SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:

18.

2. A monoclonal antibody binding to monkeypox virus B6R protein according to claim 1, characterized in that: The amino acid sequences of the six CDRs of the heavy chain variable region and the light chain variable region of B6R-B5 are shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively; The amino acid sequences of the six CDRs of the heavy chain variable region and light chain variable region of B6R-F6 are shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, respectively; The amino acid sequences of the six CDRs of the heavy chain variable region and light chain variable region of B6R-F11 are shown in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively.

3. A monoclonal antibody binding to monkeypox virus B6R protein according to claim 2, characterized in that: The amino acid sequence of the heavy chain variable region of B6R-B5 is shown in SEQ ID NO:19, and the amino acid sequence of the light chain variable region of B6R-B5 is shown in SEQ ID NO:20; the amino acid sequence of the heavy chain variable region of B6R-F6 is shown in SEQ ID NO:21, and the amino acid sequence of the light chain variable region of B6R-F6 is shown in SEQ ID NO:22; the amino acid sequence of the heavy chain variable region of B6R-F11 is shown in SEQ ID NO:23, and the amino acid sequence of the light chain variable region of B6R-F11 is shown in SEQ ID NO:

24.

4. The antibody according to any one of claims 1 to 3, characterized in that The antibody is a mouse antibody and / or a human-mouse chimeric antibody.

5. A polynucleotide encoding the antibody according to any one of claims 1 to 3. An expression vector comprising the polynucleotide according to claim 5 . A host cell comprising the expression vector according to claim 6 .

8. Use of the antibody according to any one of claims 1 to 3 in the preparation of a detection reagent or a detection product for monkeypox virus.

9. A kit for detecting monkeypox virus, characterized in that: The kit comprises the antibody according to any one of claims 1 to 4.

10. The kit according to claim 9, characterized in that The kit is a colloidal gold detection kit, an immunochromatographic detection kit, an enzyme immunoassay detection kit, a chemiluminescence detection kit or an immunoturbidimetric detection kit.

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