CN116102643B

CN116102643B - Monoclonal antibody for monkey poxvirus A35 protein and application thereof

Info

Publication number: CN116102643B
Application number: CN202211658203.0A
Authority: CN
Inventors: 张政; 鞠斌; 周兵; 刘磊; 程林; 葛向阳
Original assignee: Shenzhen National Clinical Research Center For Infectious Diseases; Third Peoples Hospital of Shenzhen
Current assignee: Shenzhen National Clinical Research Center For Infectious Diseases; Third Peoples Hospital of Shenzhen
Priority date: 2022-12-22
Filing date: 2022-12-22
Publication date: 2023-10-13
Anticipated expiration: 2042-12-22
Also published as: CN116102643A

Abstract

The application discloses a monoclonal antibody for a monkey pox virus A35 protein and application thereof. The monoclonal antibodies of the application include MPXV-mAb 975 and/or MPXV-mAb 981; CDRs 1 to 3 of the mab 975 heavy chain are in sequence "RASISSSNW", "IFHSGST" and "ARSQGGSGLFRAKWFDP", and CDRs 1 to 3 of the light chain are in sequence "QSVSSSY", "GAS" and "QQYGSSPRT"; monoclonal antibodies 981 heavy chain CDRs 1-3 are in sequence "GGSISSNNDY", "IYYSGRT" and "ARLRLGSLLHRYRAVDP", and light chain CDRs 1-3 are in sequence "qrvsty", "GAS" and "QHFDSSLLT". The monoclonal antibody can specifically bind with the A35 protein of the monkey pox virus, and provides a new scheme and approach for the detection and prevention of the monkey pox virus.

Description

Monoclonal antibody for monkey poxvirus A35 protein and application thereof

Technical Field

The application relates to the technical field of monkey pox virus detection, in particular to a monoclonal antibody aiming at a monkey pox virus A35 protein and application thereof.

Background

Human monkey pox is a rare human and livestock co-disease caused by infection of monkey pox virus (MPXV), which poses a serious threat to human health. Congo found that the first cases of infection in monkeys were also largely unreported in western africa and in 1970, for a long time; however, since the first diagnosis was found in the uk at 5.7 of 2022, more than 3 tens of thousands of diagnosis cases have been made in more than 70 countries and regions worldwide by 8.11 of the current year.

MPXV belongs to a member of the genus orthopoxvirus (OrthoPoxvirus, OPXV), one of the largest and most complex DNA viruses among all animal viruses. Is close to smallpox virus, two important pathogens with potential bioterrorism threat; monkey pox is considered the most important orthopoxvirus infection in humans since eradication of smallpox. For most people, monkey pox is a self-limiting disease with mild symptoms after infection, usually with complete recovery for 2-4 weeks. However, the severity of the symptoms is also closely related to the route of transmission, the extent of viral exposure and the underlying disease of the patient itself. Epidemiological studies of infection with human monkey pox have shown that children, pregnant women and non-smallpox vaccine people may have more severe symptoms after infection and are prone to complications such as secondary bacterial infection, respiratory distress, pneumonia, septicemia, gastrointestinal infection, encephalitis, corneal infection and vision loss. The mortality rate of monkey pox is 0% -11% in the general population and higher in infants. The monkey poxvirus outbreak originates from the western branch, which has a mortality rate of 3.6% and the death rate of the medium non-evolving branch of 10.6%, which is of further interest. The monkey pox virus contains soluble antigen, nucleoprotein antigen and hemagglutinin, and has cross immunogenicity with other three human-infected orthopoxviruses, smallpox virus, vaccinia virus and vaccinia virus. Monkey poxviruses share structural and soluble antigens with other orthopoxviruses and are difficult to distinguish from smallpox and poxviruses in complement binding assays and agar diffusion assays.

At present, no specific therapy is available for the infection of the monkey pox virus, mainly for the treatment of disease support and complications, and early diagnosis is very important for early discovery of infected patients and prevention and control of the epidemic situation of the monkey pox. OPXV contains two virions, mature Virions (MV) and Enveloped Virions (EV), carrying different surface antigens. Taking vaccinia virus (VACV) as an example, several neutralizing antibody targets have been identified at present, such as a13, a17, a27, etc. on MV, a33, B5, etc. on EV; however, there is currently no technique for early diagnosis of monkey poxviruses.

Recently, monkey pox has become a global focus of attention. Monkey pox is an acute eruptive zoonotic disease caused by the monkey pox virus, and has been mainly caused in african areas in the past. This monkey pox case is unusually outbreak in a number of non-endemic countries or regions and has a tendency to spread interpersonally, attracting great attention. In order to prevent further spread of the monkey pox epidemic, it is highly desirable to solve the problem of effective early detection and diagnosis of monkey pox virus.

Disclosure of Invention

The application aims to provide a novel monoclonal antibody aiming at a monkey pox virus A35 protein and application thereof.

In order to achieve the above purpose, the present application adopts the following technical scheme:

in a first aspect the application discloses a monoclonal antibody directed against the monkey poxvirus a35 protein, the monoclonal antibody comprising MPXV-mAb 975 and/or MPXV-mAb 981; monoclonal antibody MPXV-mAb 975 consists of a heavy chain and a light chain paired therewith; the sequences of CDR1, CDR2 and CDR3 of the heavy chain of monoclonal antibody MPXV-mAb 975 were "RASISSSNW", "IFHSGST" and "ARSQGGSGLFRAKWFDP" in sequence, and the sequences of CDR1, CDR2 and CDR3 of the light chain were "QSVSSSY", "GAS" and "QQYGSSPRT" in sequence; monoclonal antibody MPXV-mAb981 consists of a heavy chain and a light chain paired therewith; the sequences of CDR1, CDR2 and CDR3 of the heavy chain of monoclonal antibody MPXV-mAb981 were "GGSISSNNDY", "IYYSGRT" and "ARLRLGSLLHRYRAVDP" in sequence, and the sequences of CDR1, CDR2 and CDR3 of the light chain were "qrvsty", "GAS" and "QHFDSSLLT" in sequence.

The key point of the application is that the monkey pox virus A35 protein is used as bait, the specific single memory B cells are separated from peripheral blood by flow cytometry, and 2 fully human monoclonal antibodies, namely MPXV-mAb 975 and MPXV-mAb981, are cloned and identified from the specific single memory B cells by utilizing RT-PCR and nested PCR technologies. The 2-strain monkey pox virus monoclonal antibody has high affinity with MPXV-A35 protein, has dissociation constants (KDS) smaller than 1pM, has the characteristics of high efficiency, broad spectrum and multiple purposes, can be used as a detection antibody for serodiagnosis of MPXV, and provides a powerful tool for detecting the monkey pox virus.

It should be noted that, the monkey pox virus monoclonal antibodies MPXV-mAb 975 and/or MPXV-mAb981 of the present application can be used alone, and are used for the detection of monkey pox virus respectively; can also be combined together for use according to the requirements.

In one implementation of the application, the heavy chain variable region sequence of the monoclonal antibody MPXV-mAb 975 is the sequence shown by Seq ID No.1, and the light chain variable region sequence is the sequence shown by Seq ID No. 2; the heavy chain variable region sequence of monoclonal antibody MPXV-mAb981 is the sequence shown by Seq ID No.3, and the light chain variable region sequence is the sequence shown by Seq ID No. 4.

It should be noted that the heavy chain variable region and the light chain variable region of the above specific sequences are only monoclonal antibody sequences specifically employed in one implementation of the present application; it will be appreciated that for monoclonal antibodies, the regions that affect their exact complementarity to an epitope are complementarity determining regions (abbreviated CDRs), specifically, CDR1, CDR2 and CDR3 of the heavy chain, and CDR1, CDR2 and CDR3 of the light chain; therefore, the function and effect of the monkey pox virus monoclonal antibody of the application can be basically realized as long as the CDR1, CDR2 and CDR3 sequences of the heavy chain and the light chain of the application are ensured to be unchanged. That is, the specific sequences of the monkey poxvirus monoclonal antibodies of the present application are not limited to the heavy chain variable region and the light chain variable region of the above specific sequences.

In a second aspect, the application discloses a nucleic acid fragment encoding a monkey poxvirus monoclonal antibody, which encodes the heavy and light chains of the monoclonal antibody MPXV-mAb 975 of the application.

In one embodiment of the present application, the nucleic acid fragment comprises a nucleic acid fragment encoding a heavy chain variable region shown in Seq ID No.1, the sequence of which is shown in Seq ID No. 5; a nucleic acid fragment encoding the light chain variable region shown in Seq ID No.2, which has the sequence shown in Seq ID No. 6.

In a third aspect the application discloses another nucleic acid fragment encoding a monkey poxvirus monoclonal antibody encoding the heavy and light chains of the monoclonal antibody MPXV-mAb981 of the application.

In one embodiment of the present application, the nucleic acid fragment comprises a nucleic acid fragment encoding a heavy chain variable region shown in Seq ID No.3, the sequence of which is shown in Seq ID No. 7; a nucleic acid fragment encoding the light chain variable region shown in Seq ID No.4, which has the sequence shown in Seq ID No. 8.

It is to be noted that the above specific nucleic acid sequences are only those specifically employed in one embodiment of the present application, and it is understood that a plurality of codons may be present for one amino acid; thus, depending on the degeneracy of the codons, there may be several nucleic acid sequences encoding the same heavy or light chain, in addition to the nucleic acid sequences defined above, while guaranteeing that the coding amino acid sequence is unchanged, all falling within the scope of the application.

In a fourth aspect of the application, a recombinant plasmid comprising a nucleic acid fragment of the application is disclosed.

It should be noted that the recombinant plasmid of the present application is intended to be capable of efficiently expressing the nucleic acid fragment of the present application, thereby obtaining a corresponding heavy chain, light chain or monkey pox virus monoclonal antibody; thus, in principle, vectors which are capable of transfecting nucleic acid fragments into host cells for expression of nucleic acids may be used in the present application.

In a fifth aspect, the application discloses a recombinant cell comprising a nucleic acid fragment of the application or a recombinant plasmid of the application.

The recombinant cell of the present application refers to a host cell transfected with the nucleic acid fragment or the recombinant plasmid of the present application; the heavy, light or monkey poxvirus monoclonal antibodies of the application can generally be obtained by direct culture of such host cells. It can be understood that the monkey pox virus monoclonal antibody of the application can be obtained by carrying out protein expression on the recombinant plasmid of the application by adopting the recombinant cell of the application, extracting and purifying the expressed protein.

In a sixth aspect, the application discloses the use of the monoclonal antibody directed against the A35 protein of the monkey pox virus of the application, or the nucleic acid fragment of the application, or the recombinant plasmid of the application, or the recombinant cell of the application, in the preparation of a detection reagent for the monkey pox virus.

It should be noted that, the monkey pox virus monoclonal antibody of the application has strong specific binding capacity and high affinity to the monkey pox virus A35 protein; thus, can be used for preparing corresponding monkey pox virus detection reagents. As for the nucleic acid fragments, recombinant plasmids and recombinant cells, these can be used as raw materials for preparing monoclonal antibodies to the monkey pox virus, and thus can be used for preparing reagents for detecting the monkey pox virus.

It will be appreciated that the monkey poxvirus detection reagent or kit of the present application may contain other auxiliary detection buffers or enzymes in addition to the monkey poxvirus monoclonal antibodies MPXV-mAb 975 and/or MPXV-mAb981 of the present application, depending on the detection method employed, and is not particularly limited herein.

In a seventh aspect, the application discloses a monkey pox virus detection reagent comprising the monoclonal antibody of the application directed against the monkey pox virus A35 protein.

Due to the adoption of the technical scheme, the application has the beneficial effects that:

the monoclonal antibodies aiming at the monkey pox virus A35 protein comprise the monkey pox virus monoclonal antibodies MPXV-mAb 975 and/or MPXV-mAb981, have specific binding capacity to the monkey pox virus A35 protein, and provide a new scheme and path for the detection and prevention of the monkey pox virus.

Drawings

FIG. 1 is a graph showing the results of flow cytometry sorting monkey pox virus A35 protein specific IgG+ memory B cells in an embodiment of the application;

FIG. 2 is a graph showing ELISA results for binding activities of the monoclonal antibodies MPXV-mAb 975 and MPXV-mAb981 to the monkey poxvirus A35 protein in the examples of the present application;

FIG. 3 is a graph showing SPR results of the monoclonal antibodies MPXV-mAb 975 and MPXV-mAb981 against the monkey poxvirus A35 protein in the examples of the present application;

FIG. 4 is a graph showing the results of the detection of vaccinia virus by IFA using the monoclonal antibodies MPXV-mAb 975 and MPXV-mAb981 in the examples of the present application.

Detailed Description

Since the monkey pox virus is closely related to smallpox virus, vaccination with smallpox vaccine has cross-immunity against infection with other orthopoxviruses, including monkey pox. Studies have shown that smallpox vaccines are up to 85% effective against monkey pox virus. Monkey poxvirus a35 is homologous to vaccinia outer membrane specific protein a33, and thus a35 can be used as a target for serodiagnosis of MPXV infection.

The application uses monkey pox virus A35 protein as bait, separates specific single memory B cells from 2 cases of human peripheral blood (MPXV-donor 42 and MPXV-donor 3) born before 1980 by flow cytometry, and clones and identifies 2 strains of fully human monoclonal antibodies from the monoclonal antibodies by RT-PCR and nested PCR technologies, namely MPXV-mAb 975 and MPXV-mAb 981.

The application further adopts ELISA and SPR technology to test the affinity of two monoclonal antibodies of MPXV-mAb 975 and MPXV-mAb981 and monkey pox virus A35 protein, and the result shows that the 2 monoclonal antibodies have high affinity with the MPXV-A35 protein, and the dissociation constants (KDS) of the 2 monoclonal antibodies are less than 1pM. The ability of MPXV-mAb 975 and MPXV-mAb981 to recognize live virus was tested in the present application, which uses vaccinia virus (VACV) as a model due to limited resources of monkey pox live virus. The A33 protein of VACV is closely related to the A35 protein of MPXV, has 95.6% amino acid sequence homology, and also has homology to the A36 protein of smallpox virus (VARV) and the A34 protein of vaccinia virus (CPXV), suggesting that monoclonal antibodies targeting this region may have broad recognition activity. Cell localization of the A33 protein in vaccinia virus infected Vero E6 cells was clearly seen by immunofluorescence experiments (IFA) using MPXV-mAb 975 and MPXV-mAb 981. The results show that the two monoclonal antibodies have strong binding affinity and can be used as good detection antibodies of MPXV virus A35 and vaccinia A33.

The 2 monoclonal antibodies aiming at the monkey pox virus A35 protein, namely MPXV-mAb 975 and MPXV-mAb981, obtained by screening have the characteristics of high efficiency, broad spectrum and multiple purposes. The application further provides various schemes for detecting the monkey pox virus by using 2 monoclonal antibodies, and the results show that the 2 monoclonal antibodies have strong specific binding capacity to the monkey pox virus A35 protein, can be used as detection antibodies for serodiagnosis MPXV, and provide a powerful tool for detecting the monkey pox virus.

The application will be described in further detail below with reference to the drawings by means of specific embodiments. The following examples are merely illustrative of the present application and should not be construed as limiting the application. Unless otherwise specified, the instruments and materials used in the examples below are those conventionally used in laboratories.

Examples

1. Materials and methods

1. Study approval and biological samples

The study was approved by the ethical committee of the third people hospital in Shenzhen City (approval number: 2021-030). The participants provided written informed consent for sample collection and subsequent analysis. Peripheral Blood Mononuclear Cell (PBMCs) samples were stored in Shenzhen third people hospital biological sample banks. PBMCs were stored in a freezing medium and in liquid nitrogen prior to use.

In this example, 2 samples were collected specifically, and the sample collection method was as follows:

peripheral blood mononuclear cell isolation: 10mL of venous blood is collected by an anticoagulation tube, transferred into a 50mL centrifuge tube, diluted by 10mL of PBS solution and gently mixed; two 15mL centrifuge tubes were taken and 5mL Ficoll separate solution was added to each tube. Then 10mL of diluted blood is added to the upper layer of the Ficoll separating liquid; centrifuging at 2000rpm for 20 minutes; sucking the leukocyte layer into a clean 15mL centrifuge tube; PBS was added to 10mL, centrifuged at 1500rpm for 10 min, the supernatant was removed, resuspended in 3mL of cell cryopreservation solution, 1mL of cells were each taken into 3 cryopreservation tubes, placed in a cryopreservation cassette and placed in a-80℃refrigerator overnight, and the cells were transferred into liquid nitrogen the next day for long-term storage.

2. Isolation of monoclonal antibodies from B cells of Peripheral Blood Mononuclear Cell (PBMCs) samples

The frozen peripheral blood mononuclear cells were resuscitated and washed 2 times with 10mL of PBS. CD19-PE-Cy7, CD3-Pacific Blue, CD8-Pacific Blue, CD14-Pacific Blue, CD27-APC/Cy7, igG-FITC (all from BD Biosciences) and His-tagged monkey pox virus A35 protein (Sino Biological) probe mixtures were added to 100. Mu.L staining buffer (PBS+2% fetal bovine serum), peripheral blood mononuclear cells were resuspended and stained at 4℃for 30 minutes. After washing 2 times with PBS, APC and PE-labeled anti-His tag secondary antibody (Abcam) was added to 100. Mu.L of staining buffer (PBS+2% fetal bovine serum), and cells were stained at 4℃for 30 minutes. After 2 washes in PBS, monkey poxvirus a35 protein specific igg+b cells were sorted by BD FACSymphony S6 sorting flow cytometer.

Individual B cells were sorted into 96-well PCR plates containing lysis buffer, and then RT-PCR and nested PCR were performed according to the methods of literature (Liao HX, levesque MC, nagel A, dixon A, zhang R, walter E, et al, high-throughput isolation of immunoglobulin genes from single human B cells and expression as monoclonal anti-bodies. Journal of biological methods 2009; 158:171-9.) to amplify heavy and light chain variable regions, respectively. The PCR amplified products were sent to the Probiotechnological engineering (Shanghai) Co., ltd for sequencing. The sequence of the antibody variable region obtained by sequencing was synthesized by the company of the Kirschner Biotechnology Co., ltd, and the variable regions of the heavy and light chains of the antibody were cloned into the full-length IgG1 heavy and light chain expression vector pCDNA3.4 (the company of the Kirschner Biotechnology Co., ltd.) respectively, and plasmids of the heavy and light chains of the antibody were prepared in large quantities. After obtaining plasmids prepared by aurey corporation, 293F cells (500 mL, for example) were co-transfected with pairs of heavy and light chain expression plasmids using PEI transfection reagent to express monoclonal antibodies from the culture supernatant using a protein A adsorption column. The method comprises the following specific steps:

293F cells were cultured in 8% CO ₂ The 293F cell concentration was adjusted to 1.2X10 in an incubator at 37 ℃ ⁶ Culturing for 2 hours at a concentration of one mL/mL; preparing solution A: to 12.5mL of opti-MEM (31985070, gibco) was added 250. Mu.g of antibody heavy chain plasmid and 250. Mu.g of antibody light chain plasmid; preparing a solution B: to 12.5mL of opti-MEM was added 2.5mL of 1mg/mL PEI transfection reagent (24885-2, polyscices) and allowed to stand for 5 minutes; mixing the solution A and the solution B, and standing for 20 minutes to obtain an AB mixed solution; 25mL of AB mixture was added dropwiseIn 500mL 293F cells, dripping and shaking uniformly; culturing the cells for 5 days; then, the 293F cells were centrifuged at 3000g for 20 minutes, and the supernatant was collected and filtered with a 0.45 μm filter; opening a cover in the Protein A gravity column, completely flowing out 20% ethanol solution in the column by utilizing gravity, and balancing the Protein A gravity column by using 10mM PBS solution with 5 times of column volume; adding the filtered cell supernatant into a Protein A gravity column, and flowing out by virtue of gravity; the Protein a gravity column was washed with 3 column volumes of PBS solution and eluted with 5 volumes of 0.1M glycine-hydrochloric acid solution (ph=3.0); placing the eluent into an ultrafiltration concentration tube with 30KD, supplementing with PBS, centrifuging at 3500rpm for 40 min at 4deg.C, discarding the waste liquid in the collection tube, adding 20mL of PBS solution, centrifuging at 3500rpm for 40 min at 4deg.C, absorbing the concentrated and displaced antibody solution, and determining the concentration of antibody protein.

3. Enzyme-linked immunosorbent assay (ELISA) to verify the binding capacity of the A35 antibody

The specific binding capacity of the obtained A35 monoclonal antibodies MPXV-mAb 975 and MPXV-mAb981 to monkey poxvirus A35 was examined by ELISA experiments. MPXV A35 protein (40886-V08H, sino Biological) was coated overnight at 2. Mu.g/mL at 4℃in 96-well ELISA plates, 100. Mu.L per well. PBST was washed 5 times (PBS solution containing 0.5% Tween-20); sealing with sealing liquid for 1 hour at normal temperature, 150 mu L of sealing liquid is used for each hole, and then PBST is used for washing 5 times, wherein the sealing liquid comprises the following formula: 5% skim milk+2% BSA (PBS formulation), all following antibody dilutions were formulated as blocking solution; the antibodies to be detected are serially diluted by a 5-fold ratio from the highest concentration of 10 mug/mL, the total dilution is 7, 100 mug of each well is incubated for 1 hour at 37 ℃, and then PBST is used for washing 5 times; HRP-goat anti-human IgG (ZB-2304, zhonghuperzia) was added at 1:5000, incubated at 37℃for 1 hour, and then washed 5 times with PBST; mixing the color development solution A and solution B (in the process) according to a ratio of 1:1, and reacting for 20 minutes in a dark place at normal temperature with 100 mu L of each hole; then 50 mu L of 2M H ₂ SO ₄ The reaction was terminated. Optical density was measured at 450nm (OD) using a Varioskan LUX multimode microplate reader (Thermo Scientific).

4. Surface Plasmon Resonance (SPR) analysis of affinity of antibodies to a35 protein

Binding assays of the monkey poxvirus A35 protein (Sino Biological) to the two antibodies obtained MPXV-mAb 975 and MPXV-mAb981 were performed using the Biacore 8K system (GE Healthcare). The method comprises the following steps:

a10 mM sodium acetate buffer (pH 5.0) was used to dissolve the monkey poxvirus A35 protein (Sino Biological), covalently coat one flow cell of the CM5 sensor chip, and a final Response Unit (RU) of about 120 was obtained, while the other flow cell remained uncoated, and was blocked as a control. All assays were performed in HBS-EP buffer (10 mM HEPES pH 7.4, 150mM NaCl, 3mM EDTA and 0.05% Tween-20) at a flow rate of 30. Mu.L/min. Serial dilutions of antibody were injected for 60 seconds, respectively, and the resulting data were fitted in a 1:1 binding model using Biacore assessment software (GE Healthcare). Each measurement was performed twice and a single value was used to generate the average affinity constant.

5. Verification of monoclonal antibody function by immunofluorescence experiment

In this example, two monoclonal antibodies, MPXV-mAb 975 and MPXV-mAb981, were obtained for IFA detection of vaccinia virus A33 protein, and the experimental procedure specifically included:

the experiment was performed in a certified biosafety secondary laboratory. Vero E6 cell suspensions (1.6X10) were seeded in 96-well plates ⁴ Cell wall-attached growth of cells after pre-culturing the culture plates in an incubator for 12 hours; the Vero E6 cells were infected with vaccinia virus MOI 0.001 and incubated at 37℃for 1 hour before replacement in fresh medium containing 2% fetal bovine serum. After 16 hours incubation at 37℃cells were fixed with 4% paraformaldehyde solution for 30 min at room temperature, discarded and the cells were permeabilized with buffer containing 0.2% Triton X-100 for 10 min. After washing, it was stained with 1. Mu.g/mL MPXV-mAb 975, MPXV-mAb981 or VRC01 for 2 hours at room temperature, then Alexa at room temperature488-coat anti-Human IgG (H+L) Secondary Antibody (A-11013,Thermo Fisher) for 2 hours. After washing, heochst staining solution (33342, heochst AG) was added and allowed to act for 20 minutes, and microscopic images were obtained under a digital inverted microscope EVOS (Life Technologies) after 2 times of PBST washing.

2. Results and analysis

1. Isolation of monoclonal antibodies

The monkey pox virus A35-specific IgG+ memory B cells were isolated by flow cytometry from 2 peripheral blood of humans born before 1980 (MPXV-donor 42 and MPXV-donor 3) using the A35 protein of MPXV as a bait. As shown in FIG. 1, the ratio of the memory B cells specific for the monkey pox virus A35 in peripheral blood of MPXV-donor 42 and MPXV-donor 3 was 0.046% and 0.12%, respectively. Finally, 2 strains of fully human monoclonal antibodies, namely MPXV-mAb 975 and MPXV-mAb981, are obtained.

The heavy chain variable region and light chain variable region sequences of the monoclonal antibodies MPXV-mAb 975 and MPXV-mAb981 obtained by the isolation of this example are shown in tables 1 and 2, and the sequencing results are shown. Wherein, table 1 is a monoclonal antibody and its sequence, and table 2 is a monoclonal antibody heavy chain variable region and light chain variable region nucleotide sequencing result.

TABLE 1 monoclonal antibody sequences

TABLE 2 monoclonal antibody nucleic acid sequencing results

The analysis showed that the sequences of CDR1, CDR2 and CDR3 of the heavy chain of MPXV-mAb 975 were "RASISSSNW", "IFHSGST" and "ARSQGGSGLFRAKWFDP" in sequence, and the sequences of CDR1, CDR2 and CDR3 of the light chain were "QSVSSSY", "GAS" and "QQYGSSPRT" in sequence. The sequences of CDR1, CDR2 and CDR3 of the heavy chain of MPXV-mAb981 were "GGSISSNNDY", "IYYSGRT" and "ARLRLGSLLHRYRAVDP" in sequence, and the sequences of CDR1, CDR2 and CDR3 of the light chain were "qrvsty", "GAS" and "QHFDSSLLT" in sequence.

2. ELISA results

The binding activity of the monoclonal antibodies MPXV-mAb 975 and MPXV-mAb981 isolated from peripheral blood of MPXV-donor 42 and MPXV-donor 3 on the monkey poxvirus A35 protein was analyzed by ELISA experiments, and the results are shown in FIG. 2.

The results in FIG. 2 show that both 2 monoclonal antibodies specifically bind to A35, EC of the monkey poxvirus ₅₀ 0.0015. Mu.g/mL and 0.0016. Mu.g/mL, respectively, showed that the 2 antibodies had good binding activity to the A35 protein of MPXV.

3. Surface Plasmon Resonance (SPR) results

The affinity of MPXV-mAb 975 and MPXV-mAb981 to the monkey poxvirus A35 protein was detected by surface plasmon resonance techniques and the results are shown in FIG. 3.

The results in FIG. 3 show that MPXV-mAb 975 and MPXV-mAb981 have high affinity for the A35 protein of MPXV, and have dissociation constants (KDS) of less than 1pM.

4. Immunofluorescence assay (IFA) results

The function of the two monoclonal antibodies MPXV-mAb 975 and MPXV-mAb981 was verified by immunofluorescence assay (IFA), and the results are shown in FIG. 4.

The results in FIG. 4 show that the cellular localization of the A33 protein in vaccinia virus infected Vero E6 cells can be clearly seen using MPXV-mAb 975 and MPXV-mAb 981. The 2-strain monoclonal antibody can be used as a good detection antibody of the monkey pox virus A35 and the vaccinia virus A33.

The experiment and the result show that the 2 monoclonal antibodies obtained by screening in the example have strong binding capacity with the A35 protein of the monkey pox virus, and can detect the A35 protein of the monkey pox virus by adopting various detection modes, and meanwhile, can identify the live vaccinia virus, and has good broad spectrum and sensitivity.

The foregoing is a further detailed description of the application in connection with specific embodiments, and it is not intended that the application be limited to such description. It will be apparent to those skilled in the art that several simple deductions or substitutions can be made without departing from the spirit of the application.

Claims

1. A monoclonal antibody directed against the monkey poxvirus a35 protein, characterized in that: the monoclonal antibodies include MPXV-mAb 975 and/or MPXV-mAb 981;

monoclonal antibody MPXV-mAb 975 consists of a heavy chain and a light chain paired therewith;

the sequences of CDR1, CDR2 and CDR3 of the heavy chain of monoclonal antibody MPXV-mAb 975 were "RASISSSNW", "IFHSGST" and "ARSQGGSGLFRAKWFDP" in sequence, and the sequences of CDR1, CDR2 and CDR3 of the light chain were "QSVSSSY", "GAS" and "QQYGSSPRT" in sequence;

monoclonal antibody MPXV-mAb981 consists of a heavy chain and a light chain paired therewith;

the sequences of CDR1, CDR2 and CDR3 of the heavy chain of monoclonal antibody MPXV-mAb981 were "GGSISSNNDY", "IYYSGRT" and "ARLRLGSLLHRYRAVDP" in sequence, and the sequences of CDR1, CDR2 and CDR3 of the light chain were "qrvsty", "GAS" and "QHFDSSLLT" in sequence.

2. The monoclonal antibody of claim 1, wherein: the heavy chain variable region sequence of the monoclonal antibody MPXV-mAb 975 is a sequence shown by a Seq ID No.1, and the light chain variable region sequence is a sequence shown by a Seq ID No. 2;

the heavy chain variable region sequence of the monoclonal antibody MPXV-mAb981 is the sequence shown in the Seq ID No.3, and the light chain variable region sequence is the sequence shown in the Seq ID No. 4.

3. A nucleic acid fragment encoding a monkey poxvirus monoclonal antibody, characterized in that: the nucleic acid fragment encodes the heavy and light chains of monoclonal antibody MPXV-mAb 975 in the monoclonal antibodies of claim 1 or 2.

4. A nucleic acid fragment according to claim 3, wherein: a nucleic acid fragment comprising a heavy chain variable region encoding the sequence shown in Seq ID No.1, the sequence of which is the sequence shown in Seq ID No. 5; a nucleic acid fragment encoding the light chain variable region shown in Seq ID No.2, which has the sequence shown in Seq ID No. 6.

5. A nucleic acid fragment encoding a monkey poxvirus monoclonal antibody, characterized in that: the nucleic acid fragment encodes the heavy and light chains of monoclonal antibody MPXV-mAb981 in the monoclonal antibodies of claim 1 or 2.

6. The nucleic acid fragment of claim 5, wherein: a nucleic acid fragment comprising a heavy chain variable region encoding the sequence shown in Seq ID No.3, the sequence of which is the sequence shown in Seq ID No. 7; a nucleic acid fragment encoding the light chain variable region shown in Seq ID No.4, which has the sequence shown in Seq ID No. 8.

7. A recombinant plasmid comprising the nucleic acid fragment of any one of claims 3-6.

8. A recombinant cell comprising the nucleic acid fragment of any one of claims 3-6 or the recombinant plasmid of claim 7.

9. Use of the monoclonal antibody of claim 1 or 2, or the nucleic acid fragment of any one of claims 3-6, or the recombinant plasmid of claim 7, or the recombinant cell of claim 8, in the preparation of a monkey pox virus detection reagent.

10. A monkey poxvirus detection reagent, characterized in that: the monkey pox virus detection reagent comprises the monoclonal antibody according to claim 1 or 2.