A monoclonal antibody binding to monkeypox virus B6R protein and its application

Abstract

The present invention belongs to the field of biopharmaceutical technology, and specifically relates to a monoclonal antibody that binds to the monkeypox virus B6R protein and its application. The antibody is a combination of any one or more of B6R-B5, B6R-F6, and B6R-F11, and the B6R-B5, B6R-F6, and B6R-F11 all contain a heavy chain variable region and a light chain variable region, the heavy chain variable region having three heavy chain CDRs, and the light chain variable region having three light chain CDRs. The present invention provides three groups of monoclonal antibodies with excellent affinity for monkeypox virus B6R and disclosed sequences of each heavy chain and light chain variable region, providing technical support for the preparation of monkeypox virus detection and diagnostic reagents, and is expected to be developed into a specialized product for screening and diagnosis of monkeypox virus, with high application value for the prevention, control, and treatment of monkeypox 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] Humans announced that smallpox had been eradicated worldwide in the 1980s, but 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 primarily transmitted from person to person through close contact with individuals infected with the virus (including family members). It can cause a painful rash, lymphadenopathy, 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 extracellular envelope of virions (EEVs). It is homologous to the cowpox virus complement control protein B5, participates in the negative regulation of complement activation, and is a glycoprotein essential for cellular spread.

[0004] The growing global prevalence of monkeypox virus (MPXV) has led to an increased demand for diagnostics, creating a pressing need for early screening, diagnosis, or even auxiliary diagnosis. Developing monoclonal antibodies targeting MPV B6R to provide technical support for MPV screening and diagnosis is a pressing issue. 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 monkeypox virus B6R protein, 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 each comprise a heavy chain variable region and a light chain variable region, the heavy chain variable region having three heavy chain CDRs, and the light chain variable region having three light chain CDRs, wherein:

[0008] The amino acid sequences of the three CDRs of the B6R-B5 heavy chain variable region are, in order, amino acid sequences that have 80% or greater homology to the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3; the amino acid sequences of the three CDRs of the B6R-B5 light chain variable region are, in order, amino acid sequences that have 80% or greater homology to the amino acid sequences set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6;

[0009] The amino acid sequences of the three CDRs of the B6R-F6 heavy chain variable region are, in order, amino acid sequences that are greater than or equal to 80% identical to the amino acid sequences set forth in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9; the amino acid sequences of the three CDRs of the B6R-F6 light chain variable region are, in order, amino acid sequences that are greater than or equal to 80% identical to the amino acid sequences set forth 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, in turn, 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, in turn, 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 homologous sequences are preferably amino acid sequences with at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology. The homologous sequences also include amino acid sequences having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequences shown in the sequence listing.

[0012] Preferably, the amino acid sequences of the six CDRs of the heavy chain variable region and 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 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 the constant region of a human antibody, including but not limited to 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 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 described above, and a host cell comprising the expression vector described above. Preferably, the host cell is a host cell for expressing foreign proteins, such as bacteria, yeast, insect cells, or 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, which comprises the antibody 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 fluorescein 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 three groups of monoclonal antibodies with excellent affinity to the monkeypox virus B6R protein through ELISA combined experiments, providing technical support for the preparation of detection and diagnostic reagents for monkeypox virus. It is expected to be developed into a specialized product for screening and diagnosis of monkeypox epidemics, and 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 with reference to 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 obtained from the Uniprot database, with sequence number Q8V4S2. The B6R extracellular domain (amino acids 1 to 279) along with a C-terminal Strep-Tag II and 1× Flag tag were constructed into the pcDNA3.4 vector to generate 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 generate recombinant cells for protein expression. Recombinant cells were cultured in SMM 293-TII complete medium for 96 hours, then centrifuged at 3000 rpm for 30 minutes at 4°C, 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: 200 ml of supernatant was mixed with the equilibrated StrepTactin agarose filler and incubated at 4°C overnight to bind; the mixture was added to a gravity column and the solution was continuously flowed through to collect the solution, leaving the StrepTactin agarose filler in the gravity column; 30 ml of PBS solution was slowly added to the gravity column, and the solution was washed through; the washing step was repeated three times. 30 ml of elution buffer (5 mM D-Desthiobiotin in PBS) was added to the gravity column, inverted and mixed at room temperature, and incubated for 20 minutes. The eluate was collected using a gravity column, and the eluate containing the target protein was flowed through into a clean centrifuge tube. Concentrated using an ultrafiltration tube with a 10 kDa cutoff, and the solution was replaced 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 could 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 cervical dislocation. The spleens of the mice were removed using sterile surgical instruments and placed on a filter on a 50ml centrifuge tube. The spleens were ground with a grinding rod to collect B cells. Buffer (2% FBS in PBS) was added to 15ml, and the cells were 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 stop the process after 1min. The cells were 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 96-well plates containing cell lysate.

[0043] 1.4 Antibody variable region gene amplification

[0044] Using single B cell gene amplification technology, lysed mRNA was reverse transcribed to generate cDNA. Using single B cell antibody gene amplification primers, PCR was performed to obtain the heavy and light chain variable region gene products, which were then sequenced. 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 both the heavy and light chains 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 antibody strains were successfully cloned and paired. The paired heavy and light chain expression plasmids were co-transfected into HEK-293T cells and cultured for 48 hours to express the antibodies. The antibody supernatant was then obtained and used to screen positive clones.

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

[0052] 2) Blocking: Discard the solution and wash each well three times with 200 μl of PBST. Add 200 μl of 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 series, adding 100 μl of antibody solution to each well, with the first well containing the undiluted stock solution and the negative control containing PBS. Incubate at 37°C for 1 hour.

[0055] 5) Add secondary antibody: Discard the supernatant, add 200 μl of PBST to each well, wash three times, and pat dry. Add 100 μl of 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 four times, and pat dry. Add 100 μl TMB color development solution. After color development, add 50 μl 10% sulfuric acid stop solution to each well to terminate the reaction.

[0057] 7) Reading: Read the absorbance at 450 nm using a microplate reader. Set up one replicate well for each cell culture supernatant containing the antibody. Positive antibodies are identified as those with an absorbance greater than 0.5.

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

[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 Co-transfect 293F cells with the heavy and light chain expression plasmids of the 12 antibodies screened above to obtain recombinant cells. Culture the cells in SMM 293-TII complete medium for 96 hours, then centrifuge at 3000 rpm at 4°C for 30 minutes and collect the supernatant.

[0066] 2.1.2 Antibody purification using Protein A magnetic beads

[0067] Affinity chromatography filler: 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 rack. Add 30 ml of PBS solution to the beads, vortex to mix, wash the beads for 5 minutes, collect the beads using a magnetic rack, and discard the supernatant; repeat the wash step three more times. Add 18 ml of elution buffer to the beads, quickly resuspend, mix thoroughly, invert and mix at room temperature, and incubate for 5 minutes. Collect the beads using a magnetic separation rack 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 slightly alkaline pH to maintain antibody bioactivity and prevent 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 the B6R antigen in PBS to a final concentration of 1 ng / μl. Coat each well of the ELISA plate with 100 μl of the antigen at 4°C overnight.

[0073] 2.2.2 Blocking: Discard the solution and wash each well three times with 200 μl of PBST. Add 200 μl of blocking solution to each well and block at 37°C for 2 h. Washing: Discard the supernatant and wash each well three times with 200 μl of PBST. Pat dry.

[0074] 2.2.3 Add primary antibody: Starting at 10 μg / ml, dilute the antibody in a 3-fold series for a total of 12 dilutions. Add 100 μl of antibody diluent to each well. Use PBS as a negative control. Incubate at 37°C for 1 hour.

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

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

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

[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 values ​​are relatively small, indicating that these three antibodies have high affinity for B6R and are valuable for further research and development as in vitro diagnostic reagents for monkeypox virus. The variable region sequences of each antibody are listed in the sequence listing.

[0082] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the above embodiments, 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 scope of protection of the present invention.

Claims

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

2. The monoclonal antibody that binds to monkeypox virus B6R protein according to claim 1, wherein 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.

3. The antibody according to any one of claims 1 to 2, wherein The antibody is a mouse antibody or a human-mouse chimeric antibody.

4. A polynucleotide encoding the antibody according to any one of claims 1-2. An expression vector comprising the polynucleotide according to claim 4 . A host cell comprising the expression vector according to claim 5 .

7. Use of the antibody according to any one of claims 1 to 2 in the preparation of a detection reagent for monkeypox virus.

8. A kit for detecting monkeypox virus, characterized in that: The kit comprises the antibody according to any one of claims 1 to 3.

9. The kit according to claim 8, wherein The kit is a colloidal gold detection kit, an immunochromatographic detection kit, an enzyme immunoassay kit, a chemiluminescence kit or an immunoturbidimetric detection kit.

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