Specific monoclonal antibody and application thereof in detection and neutralization of monkey pox virus

Abstract

The invention relates to a specific monoclonal antibody and application thereof. The amino acid sequence of a light chain CDR1 of the monoclonal antibody is as shown in SEQ ID No.1, the amino acid sequence of CDR2 of the monoclonal antibody is as shown in SEQ ID No.2, and the amino acid sequence of CDR3 of the monoclonal antibody is as shown in SEQ ID No.3; the amino acid sequence of a heavy chain CDR1 of the monoclonal antibody is as shown in SEQ ID No.4, the amino acid sequence of CDR2 of the monoclonal antibody is as shown in SEQ ID No.5, and the amino acid sequence of CDR3 of the monoclonal antibody is as shown in SEQ ID No.6. The recombinant monkey pox virus surface protein E8L is used as an antigen, the monoclonal antibody obtained through screening has good affinity with the recombinant monkey pox virus surface protein E8L at the molecular level and the cellular level, the infection efficiency of a VACV replication defective strain in VERO cells can be remarkably inhibited, and a good monkey pox virus in-vitro neutralization effect is achieved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a specific monoclonal antibody and its application in monkeypox virus detection and neutralization. Background Art

[0002] Monkeypox virus (MPXV) infection mainly damages multiple organs of the human body, such as the skin, lungs, and gastrointestinal tract, and can cause death in severe cases. Currently, clinical drugs for the treatment of monkeypox (Mpox) are mainly anti-smallpox virus drugs, and there is no effective treatment to control monkeypox infection. With the cessation of smallpox vaccination programs worldwide, more and more people lack the adaptive immunity to respond quickly to monkeypox infection. Therefore, there is an urgent need to develop new therapeutic drugs for monkeypox infection. Neutralizing antibodies have good clinical application prospects in inhibiting viral infection and clearing viruses from the body, such as Ebola hemorrhagic fever, respiratory syncytial infection, and COVID-19. At this stage, screening out high-affinity anti-monkeypox virus neutralizing antibodies has important clinical value for improving the treatment effect of monkeypox infected patients and preventing the spread of monkeypox.

[0003] E8L is one of the potential protective antigenic proteins of monkeypox virus and is a homologous protein of vaccinia virus (VACV) D8L. E8L is expressed on the surface of IMV-type viruses and can bind to cell surface chondroitin sulfate and mediate the adsorption of viruses to cells, such as Hsiao, JC, etc., "Vaccinia virus envelope D8L protein binds to cells surface chondroitin sulfate and mediates the adsorption of intracellular mature virions to cells." Journal of virology vol, 1999, 73 (10): 8750-61. In addition, studies have shown that six protective antigens of D8L, including A27L, A33R, B5R, H3R and L1R, can induce a strong immune response in mice and induce high levels of neutralizing antibodies, such as Heraud, JM et al., "Subunit recombinant vaccine protects against monkeypox." Journal of immunology, 2006, 177(4): 2552-64.

[0004] In terms of neutralizing antibody development, since homologous proteins have similar biological activities and immunogenicity, most of the antibodies currently used to fight monkeypox virus are induced and screened by VACV-related antigens. For example, Hadas Tamir et al. screened anti-D8L antibody MV33 and anti-A33R antibody EV42 through phage display technology. Both of them can effectively reduce the VACV titer in mice when used alone, and protect them from death under exposure to lethal doses of the virus (Tamir, H. et al., "Synergistic effect of two human-like monoclonal antibodies confers protection against orthopoxvirus infection." Nature communications, 2024, 15(1): 3265). At the same time, both have a significant protective effect against MPXV infection. Cross-immunity between members of the orthopoxvirus family suggests the broad-spectrum neutralization of anti-E8L antibodies.

[0005] Currently, there are few reports on directly using MPXV antigens for immunization or screening of monoclonal antibodies. Mengjun Li et al. prepared neutralizing antibodies that bind to MPXV surface proteins A29L and A35R (homologous to VACV A27L and A33R) through hybridoma technology. Among them, anti-A29L antibodies showed good ability to inhibit viral infection both in vivo and in vitro, while anti-A33 antibodies had a weaker neutralizing effect (Li, M. et al., "Three neutralizing mAbs induced by MPXV A29L protein recognizing different epitopes act synergistically against orthopoxvirus." "Emerging microbes & infections", 2023, 12(2): 2223669). Runchu Zhao et al. screened two neutralizing antibodies targeting MPXV surface protein B6R (homologous to VACV B5R) from VACV vaccine recipients. Both antibodies had a wide range of anti-orthopoxvirus infection effects in mouse models and have the potential to become antibody drugs for the treatment of monkeypox virus infection (Zhao, R. et al., "Two noncompeting human neutralizing antibodies targeting MPXV B6 show protective effects against orthopoxvirus infections." Nature communications, 2024, 15(1): 4660). However, there are no reports of monkeypox neutralizing antibodies prepared by immunization or screening against E8L. Summary of the invention

[0006] Based on the above-mentioned shortcomings and deficiencies in the prior art, one of the objects of the present invention is to at least solve one or more of the above-mentioned problems in the prior art. In other words, one of the objects of the present invention is to provide a specific monoclonal antibody that meets one or more of the above-mentioned needs and its application in the detection and neutralization of monkeypox virus.

[0007] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0008] A monoclonal antibody that specifically binds to monkeypox E8L protein, wherein the amino acid sequence of the light chain CDR1 of the monoclonal antibody is shown in SEQ ID No. 1, the amino acid sequence of CDR2 is shown in SEQ ID No. 2, and the amino acid sequence of CDR3 is shown in SEQ ID No. 3;

[0009] The amino acid sequence of the heavy chain CDR1 of the monoclonal antibody is shown in SEQ ID No.4, the amino acid sequence of CDR2 is shown in SEQ ID No.5, and the amino acid sequence of CDR3 is shown in SEQ ID No.6.

[0010] As a preferred embodiment, the amino acid sequence of the light chain variable region of the monoclonal antibody is shown as SEQ ID No.7, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID No.8.

[0011] As a preferred embodiment, the amino acid sequence of the light chain variable region of the monoclonal antibody is shown as SEQ ID No.9, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID No.10.

[0012] Among them, SEQ ID No. 1: QDVSNT;

[0013] SEQ ID No.2: WAS;

[0014] SEQ ID No. 3: QHHYNTPYT;

[0015] SEQ ID No.4: GYSITSDYA;

[0016] SEQ ID No.5: IMYSGNT;

[0017] SEQ ID No.6: ARSHYGSNFVY;

[0018] SEQ ID No.7:

[0019] DIVMTQSHKFMSTSVGDRVTITCKASQDVSNTVAWYQQRPGQSPKLLI YWASTRHTGVPDRFTGSGSGTDYTLTISSVQAEDLALYYCQHHYNTPYTFG GGTKLEIK;

[0020] SEQ ID No.8:

[0021] DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQSPGNKLEW MGYIMYSGNTNYNPSLKSRISITRDTSKNQLFLQLNSVTTEDTATYYCARSH YGSNFVYWGQGTLVTVSA;

[0022] SEQ ID No.9:

[0023] DIQMTQSPSSSLSASVGDRVTITCRASQDVSNTVAWYQQKPGKVPKLLI YWASTRHSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQHHYNTPYTFGG GTKLEIK;

[0024] SEQ ID No.10:

[0025] QVQLQESGPGLVKPSETLSLTCTVTGYSITSDYAWNWIRQPPGKGLEW MGYIMYSGNTNYNPSLKSRVTISSVDTSKNQFSLKLSSVTAADTATYYCARS HYGSNFVYWGQGTMVTVSS.

[0026] The present invention also provides use of the monoclonal antibody described in any of the above schemes in the preparation of a drug for neutralizing monkeypox virus.

[0027] As a preferred embodiment, the drug is a liquid injection or a dry powder.

[0028] The present invention also provides the use of the monoclonal antibody described in any of the above schemes in the preparation of an immunoassay tool for detecting monkeypox virus.

[0029] As a preferred embodiment, the immunoassay tool is a reagent, a test kit, a chip or a test paper.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The present invention uses the recombinant monkeypox virus Mpox virus surface protein E8L as an antigen, uses an immune library for screening, and obtains a specific monoclonal antibody against the target antigen, which has good affinity with the recombinant monkeypox virus surface protein E8L at the molecular level and the cellular level. At the same time, the antibody can significantly inhibit the infection efficiency of the VACV replication-deficient strain in VERO cells, has a good in vitro neutralization effect on orthopoxvirus, can inhibit the infection of monkeypox virus, and has potential medicinal and detection value. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is a mouse immunization flow chart of an embodiment of the present invention;

[0033] Figure 2 This is a curve diagram of the immune effect of recombinant E8L on mice detected by ELISA in an embodiment of the present invention;

[0034] Figure 3 This is a photo of the electrophoresis of E8-AmAb in ascites detected by SDS-PAGE electrophoresis in an embodiment of the present invention;

[0035] Figure 4 This is an affinity curve diagram of E8-AmAb and recombinant E8L detected by ELISA in an embodiment of the present invention;

[0036] Figure 5 This is a graph showing the affinity of E8-AmAb and recombinant E8L detected by ForteBio in an embodiment of the present invention;

[0037] Figure 6 This is a graph showing the affinity of E8-AmAb and cell surface recombinant E8L detected by flow cytometry in an embodiment of the present invention;

[0038] Figure 7 This is a schematic diagram of the E8-A mAb of an embodiment of the present invention inhibiting the infection of VACV-GFP in VERO cells;

[0039] Figure 8 is a bar graph showing that E8-A mAb of an embodiment of the present invention inhibits the infection of MPXV in VERO cells;

[0040] Fig. 9 It is an affinity curve diagram of huE8-A mAb and recombinant E8L detected by ELISA in an embodiment of the present invention. DETAILED DESCRIPTION

[0041] The technical solution of the present invention is further explained below through specific embodiments.

[0042] The screening of monoclonal antibodies that specifically bind to monkeypox E8L protein in the embodiment of the present invention includes the following process:

[0043] 1. Mouse Immunization

[0044] Six-week-old male BALB / c mice were used as immunization objects, and recombinant E8L was used as the antigen. The first immunization was performed after emulsification with Freund's complete adjuvant. Seven days later, the second, third, and fourth immunizations were performed after emulsification with Freund's incomplete adjuvant and antigen, with an interval of 7 days between each immunization. Three days after the fourth immunization, blood was collected to verify the immunization effect. The next step of the experiment can be performed after the anti-E8L antibody titer in the serum is greater than 8000-10000 times dilution.

[0045] 2. Hybridoma Fusion

[0046] The mice were killed after blood was collected after 4 immunizations, and the spleen was removed and a spleen B cell suspension was prepared. The spleen cells were evenly mixed with mouse myeloma cells Sp2 / 0 and fused under the mediation of polyethylene glycol 4000 (PEG-4000). The fused hybridoma cells were inoculated into a 96-well plate and selectively cultured with HAT medium [hypoxanthine H, aminopterin A and thymidine T]. Unfused cells, BB fusion cells and Sp2 / 0-Sp2 / 0 fusion cells will not survive.

[0047] 3. Monoclonal Antibody Screening

[0048] When the hybridoma cells grow to 1 / 3-1 / 2 of the bottom area of ​​the well, the ELISA technique is used to screen the wells that secrete the most anti-E8L antibodies, and the cells in the wells are monoclonalized by limiting dilution. When the monoclonal cells grow to 1 / 3-1 / 2 of the bottom area of ​​the well, the ELISA technique is used to screen the monoclonal hybridoma cells that secrete anti-E8L antibodies, and the positive clones are expanded and cultured.

[0049] 4. Antibody variable region gene sequencing

[0050] The positive monoclonal cells were subjected to antibody variable region gene sequencing, RNA extraction, 5'RACE reverse transcription, antibody light and heavy chain gene specific amplification, gene cloning and gene sequencing.

[0051] Embodiment 1:

[0052] The screening process of mouse monoclonal antibodies in this embodiment includes:

[0053] 1. Mouse immunization

[0054] like Figure 1 As shown, 6-week-old male BALB / c mice were used as immunization objects, and recombinant E8L was used as the antigen. For the first immunization, the antigen was ultrasonically emulsified with an equal volume of Freund's complete adjuvant, and immunization was performed by subcutaneous multi-point injection, with 100 μg of antigen injected per mouse. Seven days later, Freund's incomplete adjuvant and antigen were emulsified, and the second and third immunizations were performed with the same dose and the same method, with an interval of 7 days between each immunization. Three days after the third immunization, mouse serum was collected and the immunization effect was verified by enzyme-linked immunosorbent assay (ELISA). The fourth immunization could be performed after the anti-E8L antibody titer in the serum was greater than 8000-10000 times dilution. The fourth immunization was performed by intraperitoneal injection, with 300 μg of antigen injected per mouse. The spleen was collected on the third day after the fourth immunization for hybridoma fusion experiments.

[0055] 2. Hybridoma fusion

[0056] Before fusion, Sp2 / 0 cells were screened and cultured in a medium containing 8-azaguanine (20 μg / ml) for 14 days. One day before fusion, cells were taken from the peritoneal cavity of ordinary BALB / c mice and inoculated into 96-well plates as feeder cells. On the day of fusion, the mice that had been immunized four times were bled and killed, and the spleens were removed and spleen B cell suspensions were prepared. 1×10 8 Spleen cell suspension and 2×10 7 The Sp2 / 0 cell suspensions were mixed evenly, centrifuged at 1000rpm for 5min, and the supernatant was discarded. In a 37°C water bath, 700μl 50% PEG-4000 was slowly added to the mixed cells, followed by the slow addition of 30ml serum-free medium to terminate the fusion. The fused hybridoma cells were centrifuged at 1200rpm for 8min, resuspended in HAT medium, and inoculated into a 96-well plate containing feeder cells for selective culture. Unfused cells, BB fusion cells, and Sp2 / 0-Sp2 / 0 fusion cells will not survive.

[0057] 3. Monoclonal antibody screening

[0058] When the hybridoma cells grow to 1 / 3-1 / 2 of the bottom area of ​​the well, the ELISA technique is used to screen the wells that secrete the most anti-E8L antibodies, and the cells in the wells are monoclonalized by limiting dilution. When the monoclonal cells grow to 1 / 3-1 / 2 of the bottom area of ​​the well, the ELISA technique is used to screen the monoclonal hybridoma cells that secrete anti-E8L antibodies, and the positive clones are expanded and cultured.

[0059] 4. Antibody variable region gene sequencing

[0060] The positive monoclonal cells were sent to Nanjing Detai Biotechnology for antibody variable region gene sequencing. The technicians performed RNA extraction, 5'RACE reverse transcription, antibody light and heavy chain gene specific amplification, gene cloning and gene sequencing on the monoclonal cells. The results showed that the BCR light and heavy chain sequences of the hybridoma cells sent for testing were the same, and they were combined and named E8-A, and the antibodies they expressed were combined and named E8-A mAb.

[0061] The amino acid sequence of the light chain variable region (V region) of the above monoclonal antibody E8-A mAb is shown in SEQ ID No. 7, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 8. Among them, after bioinformatics analysis, the amino acid sequence of the light chain CDR1 is shown in SEQ ID No. 1, the amino acid sequence of CDR2 is shown in SEQ ID No. 2, and the amino acid sequence of CDR3 is shown in SEQ ID No. 3; the amino acid sequence of the heavy chain CDR1 of the monoclonal antibody is shown in SEQ ID No. 4, the amino acid sequence of CDR2 is shown in SEQ ID No. 5, and the amino acid sequence of CDR3 is shown in SEQ ID No. 6.

[0062] Among them, SEQ ID No. 1: QDVSNT;

[0063] SEQ ID No.2: WAS;

[0064] SEQ ID No. 3: QHHYNTPYT;

[0065] SEQ ID No.4: GYSITSDYA;

[0066] SEQ ID No.5: IMYSGNT;

[0067] SEQ ID No.6: ARSHYGSNFVY;

[0068] SEQ ID No.7:

[0069] DIVMTQSHKFMSTSVGDRVTITCKASQDVSNTVAWYQQRPGQSPKLLI YWASTRHTGVPDRFTGSGSGTDYTLTISSVQAEDLALYYCQHHYNTPYTFG GGTKLEIK;

[0070] SEQ ID No.8:

[0071] DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQSPGNKLEW MGYIMYSGNTNYNPSLKSRISITRDTSKNQLFLQLNSVTTEDTATYYCARSH YGSNFVYWGQGTLVTVSA.

[0072] Embodiment 2:

[0073] The preparation of mouse ascites and purification of E8-A mAb in this example specifically include the following steps:

[0074] 1. Preparation of mouse ascites

[0075] Six-week-old female BALB / c mice were intraperitoneally injected with 0.5 mL of paraffin oil. Fourteen days later, 5 × 10 6 E8-A cells. 9-11 days after the injection of cells, when the mice become slow in movement and have swollen abdomens, they are killed by dislocating the neck. Cut the skin of the mouse abdomen, bluntly expose the peritoneum, and use a syringe to aspirate the ascites in the abdominal cavity. After the ascites is placed at 4°C for 1 hour, the paraffin oil naturally coagulates, and it is centrifuged at 4°C, 4000rpm for 30 minutes, and the middle aqueous phase is taken for purification.

[0076] 2. E8-A mAb purification

[0077] The antibody in the ascites was purified using a Protein A (MabSelect SuRe LX) column, and the relevant purification operations were performed on an AKTA Avant chromatography system. The column was equilibrated with 10 column volumes of phosphate buffer (pH = 7.4), and the ascites was diluted 10 times with 20mM phosphate buffer and loaded at a speed of 1mL / min. After loading, the column was equilibrated again with 10 column volumes of phosphate buffer. After the post-equilibrium was completed, the target protein was eluted with 0.1M citric acid buffer (pH = 3.0), and the pH of the eluted product was immediately adjusted to 7.4 with 1MTris-HCl buffer (pH = 9.0). The eluted target protein was placed in a dialysis bag with a pore size of 30KD and dialyzed in PBS buffer (4°C). Fresh PBS buffer was replaced every 4-6h, and the target protein was finally obtained after three replacements.

[0078] Embodiment 3:

[0079] The affinity test of the above E8-A mAb to the antigen includes:

[0080] 1. Enzyme-linked immunosorbent assay (ELISA) to test the affinity of E8-A mAb to antigen

[0081] ELISA experiments were used to detect the specific binding ability of the purified E8-A mAb to recombinant E8L. Recombinant E8L was coated on the ELISA plate at a concentration of 2μg / mL and placed at 4°C overnight. After discarding the coating solution, wash three times with PBST and block with 3% BSA at 37°C for 30 minutes. Discard the liquid in the wells, add three-fold diluted E8-A mAb to the treatment group, and incubate at 37°C for 1 hour. Discard the liquid in the wells and wash three times with BST. Use HRP-labeled rabbit anti-mouse IgG (1:15000) as the secondary antibody for binding reaction, incubate at 37°C for 1 hour, discard the liquid in the wells and wash three times with PBST, add TMB substrate and place in the dark at room temperature, add ELISA stop solution after 5-10 minutes to stop the color reaction, and measure OD 450 .

[0082] 2. FoeteBio method to test the affinity of E8-A mAb to antigen

[0083] The equilibrium dissociation constant KD of E8-A mAb and recombinant E8L was detected by the ForteBio method. Recombinant E8L was biotinylated using a biotin labeling kit (Tongren Chemical) and then fixed on a pre-wetted streptavidin sensor (SA Sensor) at a concentration of 2 μg / mL. After the probe was equilibrated with PBST, it was combined with gradient dilutions of E8-A mAb (100 nmol / L, 25 nmol / L, 12.5 nmol / L, 6.25 nmol / L, 3.125 nmol / L). PBST was used as a blank control, and the binding time was set to 350 s and the dissociation time to 400 s. After deducting the background signal from the control sensor, a 1:1 binding model was used to fit the binding and dissociation curves, and the equilibrium dissociation constant KD was calculated.

[0084] 3. Flow cytometry to determine the binding affinity between E8-A mAb and E8L on the cell membrane

[0085] E8L stable transfected cells were collected by digestion and washed twice by FACS (PBS + 2% FBS) at 1100 rpm, 5 min centrifugation. 2 × 10 5 Cells were resuspended in three-fold diluted E8-A mAb and incubated at room temperature for 1 h. Washed three times by FACS centrifugation, diluted FITC-labeled goat anti-mouse IgG (1:20,000) was added and incubated at room temperature for 1 h in the dark. After incubation, the cells were washed three times by centrifugation, and the cell fluorescence intensity was analyzed by flow cytometry and data were analyzed by GraphPad Prism 9 software.

[0086] Embodiment 4:

[0087] The inhibition test of the above-mentioned E8-A mAb on VACV replication-deficient oncolytic virus is as follows:

[0088] An in vitro neutralization activity evaluation system was constructed using VACV (Vaccinia virus) replication-deficient oncolytic virus (VACV-GFP, Wuhan Shumi), and whether E8-A mAb has in vitro neutralization activity was explored. E8-A mAb was diluted to 600 μg / mL with neutralization medium (DMEM+3% FBS+5% complement), and diluted three times, and 50 μL was added to a 96-well plate for each concentration. Three replicate wells were set for each concentration, and a virus group (antibody concentration was 0 mg / mL) and a cell group (no antibody and virus) were set at the same time. VACV-GFP was diluted 5000 times with neutralization medium, 50 μL was added to each well and mixed thoroughly with the antibody diluent. The antibody-virus mixture was incubated at 37°C for 1 h. VERO-E6 cells were digested and collected, and the density was adjusted to 4×10 5cells / mL, 50 μL of cell suspension was added to each well, and the 96-well plate was incubated at 37°C, 5% CO2 for 24 h.

[0089] The cell culture medium was discarded and fixed with 4% paraformaldehyde at room temperature for 20 minutes. The liquid in the wells was discarded and washed twice with PBS, and 100 μL of diluted Hoechst dye solution (1:1000) was added. After incubation at room temperature for 30 minutes, the cells were washed twice with PBS. The cell images under each fluorescence channel were collected using a high-content microplate detection imaging system, and the green fluorescence area of ​​each well was calculated using harmony software. GraphPad Prism 9 was used for data analysis.

[0090] Embodiment 5:

[0091] The inhibition of the above E8-A mAb against MPXV was tested as follows:

[0092] MPXV was used to explore the in vitro neutralizing activity of E8-A mAb. In a biosafety level 3 laboratory, E8-A mAb was diluted two-fold with neutralization medium (DMEM + 3% FBS + 5% complement), and 50 μL was added to a 96-well plate for each concentration. Three replicate wells were set for each concentration, and a virus group (antibody concentration was 0 mg / mL) and a cell group (no antibody and virus) were set at the same time. MPXV was diluted to 100 PFU / mL with neutralization medium, 50 μL was added to each well and mixed thoroughly with the antibody diluent. The antibody-virus mixture was incubated at 37°C for 1 hour. VERO-E6 cells were digested and collected, and the density was adjusted to 2×10 5 cells / mL, add 100 μL of cell suspension to each well. Incubate the 96-well plate at 37°C, 5% CO2. Count the number of plaques in each well under a microscope after 24 hours and calculate PRNT 50 .

[0093] Embodiment 6:

[0094] Humanization of the above E8-A mAb:

[0095] Combining bioinformatics and genetic engineering techniques, the laboratory humanized the light and heavy chain sequences of E8-A mAb. The humanized antibody was named huE8-A mAb. The amino acid sequences of its light chain CDR1, CDR2, and CDR3 are the same as SEQ ID No.1, SEQ ID No.2, and SEQ ID No.3, and the amino acid sequences of its heavy chain CDR1, CDR2, and CDR3 are the same as SEQ ID No.4, SEQ ID No.5, and SEQ ID No.6.

[0096] Specifically, the amino acid sequence of the light chain variable region of the humanized antibody named huE8-A mAb is shown in SEQ ID No.9, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No.10.

[0097] SEQ ID No.9:

[0098] DIQMTQSPSSSLSASVGDRVTITCRASQDVSNTVAWYQQKPGKVPKLLI YWASTRHSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQHHYNTPYTFGG GTKLEIK;

[0099] SEQ ID No.10:

[0100] QVQLQESGPGLVKPSETLSLTCTVTGYSITSDYAWNWIRQPPGKGLEW MGYIMYSGNTNYNPSLKSRVTISSVDTSKNQFSLKLSSVTAADTATYYCARS HYGSNFVYWGQGTMVTVSS.

[0101] The various processes of the E8-A mAb of the above-mentioned embodiment are characterized as follows:

[0102] like Figure 2 As shown, after four immunizations, the maximum dilution of mouse serum that produced a positive reaction with the recombinant E8L antibody was 170100 (a positive reaction was defined as OD 450 The reading was greater than 2.1 times that of the negative control), indicating that the titer of anti-E8L antibodies in mice was 170100. After immunization, the mice produced a strong immune response, meeting the requirements for the preparation of monoclonal antibodies.

[0103] like Figure 3 As shown in the figure, after purification by Protein A affinity chromatography, there is a single band at 55kDa and 25kDA in the eluted fraction, which are the protein bands of the antibody heavy chain and light chain, respectively. The content of impurity proteins in the eluted fraction is low, and the antibody purity is high, which meets the requirements of subsequent experiments.

[0104] like Figure 4 As shown, the concentration-dependent binding relationship of E8-A mAb to recombinant E8L was analyzed by GraphPad Prism 9 software and a fitting curve was drawn. The EC50 value was calculated to be 16.30 ng / mL, indicating that E8-A mAb has good binding activity with the antigen.

[0105] like Figure 5As shown in Figure 2, the concentration-dependent binding relationship between E8-A mAb and recombinant E8L was analyzed by Fortebio software and a fitting curve was drawn. The equilibrium dissociation constant KD was measured to be 3.22×10 -11 M, belongs to high affinity antibody.

[0106] like Figure 6 As shown, the concentration-dependent binding relationship of E8-A mAb to cell surface recombinant E8L was analyzed by GraphPad Prism 9 software and a fitting curve was drawn. The EC50 value was calculated to be 136.5 ng / ml, indicating that E8-A mAb still had strong binding activity when the antigen was expressed on the cell surface.

[0107] like Figure 7 As shown in the figure, cells express green fluorescent protein after being infected by the virus, and the ability of antibodies to inhibit virus-infected cells at different concentrations can be detected by the green fluorescent area. The results show that the E8-A mAb has a concentration-dependent relationship with the GFP area ratio, and has a significant inhibitory effect within the tested concentration range.

[0108] like Figure 8 As shown in the figure, the maximum dilution factor of E8-A mAb to achieve 50% inhibition of MPXV was 40, and its PRNT 50 The value is 8.7μg / mL.

[0109] In addition, the affinity of the above huE8-A mAb to the antigen was tested as follows:

[0110] ELISA experiments were used to detect the specific binding ability of huE8-A mAb to recombinant E8L. Recombinant E8L was coated on the ELISA plate at a concentration of 2μg / mL and placed at 4°C overnight. After discarding the coating solution, wash three times with PBST and block with 3% BSA at 37°C for 30 minutes. Discard the liquid in the wells, add three-fold diluted huE8-A mAb to the treatment group, and incubate at 37°C for 1 hour. Discard the liquid in the wells and wash three times with BST. Use HRP-labeled goat anti-human IgG (1:15000) as the secondary antibody for binding reaction, incubate at 37°C for 1 hour, discard the liquid in the wells and wash three times with PBST, add TMB substrate and place in the dark at room temperature, add ELISA stop solution after 5-10 minutes to stop the color reaction, and measure OD 450 .

[0111] like Fig. 9 As shown, the concentration-dependent binding relationship of huE8-A mAb to recombinant E8L was analyzed by GraphPad Prism 9 software and a fitting curve was drawn, and its EC50 value was calculated to be 31.06 ng / mL, indicating that after humanization, huE8-A mAb still has a high binding activity to the antigen.

[0112] The adhesion of the virus to the cell surface glycosaminoglycan is an important step to enter the cell and cause viral infection. E8L is a binding molecule of chondroitin sulfate (a glycosaminoglycan). Based on its important role in the process of monkeypox virus entering cells, the E8-A mAb, a monkeypox neutralizing antibody targeting E8L of the present invention, has good antigen binding ability at both the molecular level and the cellular level, and can inhibit the infection of monkeypox virus by blocking the interaction between E8L and the cell surface. The antibody showed good ability to neutralize VACV in an in vitro neutralization model, and it is expected to be developed into a new antibody drug for the treatment of monkeypox virus infection.

[0113] Based on this, the present invention also provides the use of the above monoclonal antibody in the preparation of a drug for neutralizing monkeypox virus, wherein the drug is a liquid injection or a dry powder.

[0114] The present invention also provides the use of the above monoclonal antibody in preparing an immunoassay tool for detecting monkeypox virus, wherein the immunoassay tool is a reagent, a test kit, a chip or a test paper.

[0115] The above description is only a detailed description of the preferred embodiments and principles of the present invention. For ordinary technicians in this field, according to the ideas provided by the present invention, there will be changes in the specific implementation methods, and these changes should also be regarded as the protection scope of the present invention.

Claims

1. A monoclonal antibody that specifically binds to monkeypox E8L protein, characterized in that: The amino acid sequence of the light chain CDR1 of the monoclonal antibody is shown in SEQ ID No. 1, the amino acid sequence of CDR2 is shown in SEQ ID No. 2, and the amino acid sequence of CDR3 is shown in SEQ ID No. 3; The amino acid sequence of the heavy chain CDR1 of the monoclonal antibody is shown in SEQ ID No.4, the amino acid sequence of CDR2 is shown in SEQ ID No.5, and the amino acid sequence of CDR3 is shown in SEQ ID No.

6.

2. The monoclonal antibody according to claim 1, characterized in that The amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID No.7, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No.

8.

3. The monoclonal antibody according to claim 1, characterized in that The amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID No.9, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No.

10.

4. Use of the monoclonal antibody according to any one of claims 1 to 3 in the preparation of a medicament for neutralizing monkeypox virus.

5. The use according to claim 4, characterized in that: The medicine is a liquid injection or a dry powder.

6. Use of the monoclonal antibody according to any one of claims 1 to 3 in the preparation of an immunoassay tool for detecting monkeypox virus.

7. The use according to claim 6, characterized in that: The immunoassay tool is a reagent, a test kit, a chip or a test paper.

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