CN111138527B - Monoclonal antibody 4F1 for resisting subunit GP1 of Ebola virus glycoprotein and application thereof - Google Patents
Monoclonal antibody 4F1 for resisting subunit GP1 of Ebola virus glycoprotein and application thereof Download PDFInfo
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- CN111138527B CN111138527B CN201811310292.3A CN201811310292A CN111138527B CN 111138527 B CN111138527 B CN 111138527B CN 201811310292 A CN201811310292 A CN 201811310292A CN 111138527 B CN111138527 B CN 111138527B
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- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
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- C07K2317/00—Immunoglobulins specific features
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- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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Abstract
The invention discloses a monoclonal antibody 4F1 of an anti-Ebola virus glycoprotein GP1 subunit, which has a unique CDR region, has specific binding activity with an EBOV GP1 subunit, is stably bound, is not influenced by the low pH environment of a secondary endosome, and provides a foundation for the neutralization effect of the secondary endosome. 4F1 was effective in neutralizing EBOV pseudoviruses in vitro, as compared to control antibodies. The neutralizing activity of 4F1 increased with increasing antibody concentration, and 100% protection was achieved for EBOV at a concentration of 1. mu.g/mL. The monoclonal antibody 4F1 provided by the invention and a control antibody such as MIL77-3 and the like are bound to GP1 subunit and compete for binding, but 4F1 has in vitro neutralizing activity, and other antibodies do not have neutralizing activity, which suggests that 4F1 has the potential of composing cocktail combination therapy with other neutralizing antibodies bound to GP2 subunit.
Description
Technical Field
The invention discloses an antibody, and belongs to the technical field of polypeptides.
Background
Ebola virus (EBOV) can cause acute severe hemorrhagic fever in human and non-human primates, is one of the viruses with the highest fatality rate discovered so far, has the lethality rate as high as 90 percent, can be directly transmitted by contact, and has extremely strong infectivity and lethality rate. Glycoprotein (GP) spikes on the surface of the ebola virus envelope mediate almost all links of viral entry into cells, and thus ebola GP is an important target for virus-neutralizing antibodies. The ebola virus GP gene is processed into two proteins, one being secreted, non-structural GP (sGP); the other is a structural GP. The Ebola GP is synthesized as a polypeptide, and then is cut by Furin enzyme to become GP1 (amino acids 1-501) and GP2 (amino acids 502-676) subunits, the two subunits are connected by a disulfide bond, and the formed trimer is fixed on the surface of an envelope by a transmembrane region in GP 2. GP1 contains Mucin (Mucin), Glycan cap (Glycan cap), Head (Head), Base (Base), etc. domains. The GP structure formed after Furin enzyme digestion cannot directly stimulate the fusion process of the Ebola virus and the host cell membrane. After the ebola virus enters the body, the ebola virus is combined with receptors on the surface of cell membranes, but the adhesion factors on the surface of the host cell membranes are not the real receptors for the ebola virus to generate membrane fusion and enter the host cells. Upon binding of the virus to cell surface adhesion factors, endocytosis and pinocytosis are mediated by clathrin, and primary and secondary endosomal trafficking occurs. GP is cleaved by both histone B and histone L in endosomes, removing 60% of the amino acids including mucin and glycan cap from GP1, forming the active GP (GPcl), which activates the critical membrane fusion process. GPcl is combined with endosome membrane protein Niemann-Pick C1(NPC1) to generate membrane fusion, so that virus RNA enters cytoplasm to complete virus genome replication and transcription, and new virus protein is synthesized to assemble progeny virus particles and bud from the surface of a host cell. The endosomal membrane protein NPC1 is an essential host infectious agent for EBOV infection.
Ebola virus is classified as a class a biological/bioterrorism agent by NIH and CDC in the united states, and is classified as one of the pathogens that presents significant risks and risks to national safety and public health, and there is currently no approved drug for the prevention or treatment of ebola virus. After the ebola virus was discovered in 1967, a total of 12 larger scale transmissions were experienced. The western africa in 2014 has developed the largest-scale and most difficult-to-control ebola epidemic, which is identified as zaire type ebola virus, the epidemic causes more than 1 ten thousand deaths and more than 2.5 ten thousand infections (according to the ebola epidemic report issued at 2.17.2.2016. in WHO), the epidemic is spread out of the african continent for the first time, and great panic is caused in the world. The high level of public and authorities attention has rapidly pushed the search for ebola vaccines and antiviral drugs.
Several promising clinical trials of ebola vaccines have been rapidly conducted at home and abroad: gorilla adenovirus type 3 vector vaccines, adenovirus type 5 vector vaccines, vesicular stomatitis virus vector vaccines, and the like. Admittedly, the development of effective prophylactic-like drugs is an important goal, and prophylactic measures are considered to save a large number of lives, whereas vaccines are difficult to work in certain situations, such as: 1) the host individuals may have differences (e.g., weak immunity in the elderly, the young, and the immunocompromised); 2) the immune effect of the vaccine is gradually weakened after the vaccine is immunized for a long time; 3) vaccines elicit immunity in the host that is not resistant to high doses of ebola virus exposure; 4) most patients seek medical assistance only after the occurrence of ebola virus infection signals, and vaccines have difficulty eliciting an effective immune response in the host for a short treatment window. Therefore, the development of therapeutic drugs for ebola virus diseases in the process of developing vaccines is also not overlooked. Currently ebola virus neutralizing antibodies may exert protective effects through three pathways: 1) blocking GP from being digested and hydrolyzed by cathepsin; 2) block binding of the activated GP to the NPC1 receptor; 3) influencing the allosteric processes of GP before membrane fusion after binding to NPC1 receptor. Of these, the first two pathways have been documented, and the third pathway is a presumption in some documents, and there is no direct evidence.
Although there are no currently approved therapeutic agents for ebola virus, there are several experimental anti-ebola virus agents under investigation, including small interfering RNAs, antisense oligonucleotides, nucleotide analogs, antibodies, and the like. In 2018, in 5 months, the Ebola epidemic situation outbreaks again in the Congo fruit democratic republic, and by 12 days in 8 months, 57 people are infected and 41 people die. After this outbreak, the WHO approved 5 drugs in the study phase that could be used for the emergency treatment of ebola virus disease, including 3 antibody drugs (Zmapp, REGN3470-3471-3479 and mAb114) and 2 small molecule drugs (Remdesivir and Favipiravir). The antibody drug Zmapp formed by mixing three monoclonal antibodies and the optimized strain MIL77 successfully saves lives of a plurality of people during outbreak of West Africa Ebola epidemic situation in 2014, the effect and the treatment window period length of the antibody drug are far longer than those of other antiviral drugs, the drug safety and the patient cure rate are greatly improved, and people are greatly encouraged. The application of antibody drugs to treat filovirus diseases attracts worldwide attention and becomes a hotspot in the research field of medicines for treating ebola virus diseases.
Zmapp is a cocktail treatment strategy consisting of three human murine chimeric mabs (C2G4, C4G7, C13C6) expressed in the tobacco system, where C2G4 (epitopes C511, N550, G553, C556) and C4G7 (epitopes C511, D552, C556) bind to GP2 subunit with overlapping epitopes; C13C6 (epitope T270, K272) bound to the glycan cap region at an approximately perpendicular angle. MIL77 is an antibody combination optimized for Zmapp, MIL77-1/-2/-3 contains the variable regions of C2G4, C4G7 and C13C6, respectively, and the framework regions are humanized and engineered to be expressed in CHO cells. Cocktail combination therapy of two antibodies, MIL77-1 and ML77-3, was able to achieve 100% protective activity against nonhuman primates 72 hours after infection.
The excellent effect shown by the cocktail combination therapy brings new revelation to the treatment of the ebola virus, and the development of anti-ebola virus monoclonal antibodies capable of resisting unique epitopes becomes a great technical demand in the field, because if more anti-ebola virus monoclonal antibodies different from the anti-unique epitopes already appeared in the prior art are provided, more choices are provided for the treatment scheme of the ebola virus, especially the scheme design of the cocktail combination preparation. Therefore, the invention aims to provide an anti-Ebola virus glycoprotein monoclonal antibody capable of aiming at a unique epitope, and further provides application of the anti-Ebola virus glycoprotein monoclonal antibody in preparation of an Ebola virus treatment drug.
Disclosure of Invention
Based on the above purposes, the invention firstly provides a monoclonal antibody 4F1 for resisting the subunit GP2 of the Ebola virus glycoprotein, wherein the amino acid sequences of CDR1, CDR2 and CDR3 of the variable region of the light chain of the antibody are respectively shown as the amino acid sequences at positions 27-32, 50-52 and 89-97 of SEQ ID NO.1, and the amino acid sequences of CDR1, CDR2 and CDR3 of the variable region of the heavy chain of the antibody are respectively shown as the amino acid sequences at positions 26-33, 51-57 and 96-107 of SEQ ID NO. 5.
In a preferred embodiment, the amino acid sequence of the antibody light chain variable region is shown in SEQ ID NO.1, and the amino acid sequence of the antibody heavy chain variable region is shown in SEQ ID NO. 5.
In a more preferred embodiment, the amino acid sequence of the constant region of the antibody light chain is shown in SEQ ID NO.3 and the amino acid sequence of the constant region of the antibody heavy chain is shown in SEQ ID NO. 7.
Secondly, the invention also provides the gene coding sequences of the light chain and the heavy chain of the monoclonal antibody, wherein the gene coding sequence of the light chain variable region of the antibody is shown by SEQ ID NO.2, and the gene coding sequence of the heavy chain variable region of the antibody is shown by SEQ ID NO. 6.
In a preferred embodiment, the gene coding sequence for the light chain constant region of the antibody is represented by SEQ ID No.4 and the gene coding sequence for the heavy chain constant region of the antibody is represented by SEQ ID No. 8.
Furthermore, the present invention provides a functional element capable of expressing the coding sequence of the gene encoding the heavy and/or light chain of the monoclonal antibody described above.
In a preferred embodiment, the functional element is a linear expression cassette.
Still further, the present invention provides a host cell comprising the above linear expression cassette, wherein the cell is a 293T cell.
Finally, the invention provides the application of the monoclonal antibody in preparing the Ebola virus disease treatment medicine.
In a preferred embodiment, the monoclonal antibody is used as a component in an ebola virus cocktail formulation.
The monoclonal antibody 4F1 of the anti-Ebola virus envelope glycoprotein provided by the invention has a unique CDR region, has specific binding activity with EBOV GP1 subunit, is stable in binding, is not influenced by the low pH environment of a secondary endosome, and provides a foundation for the neutralization effect of the secondary endosome. 4F1 was effective in neutralizing EBOV pseudoviruses in vitro, as compared to control antibodies. The neutralizing activity of 4F1 increased with increasing antibody concentration, and 100% protection was achieved for EBOV at a concentration of 1. mu.g/mL. The control antibodies such as 4F1 and MIL77-3 provided by the invention are bound to GP1 subunit and compete for binding, but 4F1 has in vitro neutralizing activity, and other antibodies do not have neutralizing activity, which suggests that 4F1 has the potential to form cocktail combination therapy with other neutralizing antibodies bound to GP2 subunit.
Drawings
FIG. 1 is a graph of flow sort memory B cell results;
FIG. 2 shows the electrophoretic identification map of nested PCR amplified antibody gene;
FIG. 3 is a schematic diagram of the splicing sequence of the linear expression boxes;
FIG. 4 is a schematic diagram of antibody binding screening;
FIG. 5.4F 1 antibody binding activity profile with four antigens;
FIG. 6.4 graph of neutralizing activity of F1 antibody against different pseudoviruses;
FIG. 7 is a schematic diagram of a truncated GP construction;
FIG. 8.4 binding activity profile of F1 with each truncated antigen;
FIG. 9.4 binding activity profile of F1 with each truncated antigen;
FIG. 10 is a graph showing the values of competitive binding between antibodies;
FIG. 11.2G 1 binding activity profile with GP at different pH.
Detailed Description
The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are only illustrative and do not limit the scope of the present invention.
Example 1 antibody preparation
1. Collection of blood samples
After obtaining the informed consent, 5mL of blood sample of the recombinant ebola vaccine clinical trial subject after the second immunization for 28 days was collected for subsequent experiments.
FITC-labeled protein GPdM
Specific memory B cells need to be sorted by fluorescence labeled antigen, and the FITC labeled truncated antigen protein GPdM method is as follows:
1) fluoroescein Isothiocyanate _ FITC (SIGMA, F4274) was dissolved in DMSO at a concentration of 20 mg/mL.
2) mu.L of GPdM (3.3mg/mL) was taken and the buffer (pH9.6 carbonate buffer) was added to 400. mu.L.
3) Add 8. mu.L FITC to GPdM solution and incubate at 4 ℃ for 3h protected from light.
4) The solution was changed with PBS using a 50kD centrifuge tube until the filtrate was clear and colorless. And (4) wrapping the marked protein with tin foil paper, and storing at 4 ℃ for later use.
3. Flow sorting memory B cells
The collected blood samples were used for PBMC isolation by Ficoll density gradient centrifugation as follows:
1) taking fresh anticoagulated whole blood, and performing EDTA anticoagulation. Whole blood was diluted with an equal volume of PBS.
2) And (3) adding a certain volume of separation liquid into the centrifugal tube, flatly spreading the diluted blood sample above the liquid level of the separation liquid, and keeping the interface between the two liquid levels clear. The volume of the separating medium, anticoagulated undiluted whole blood and PBS (or physiological saline) is 1:1: 1.
3) Trim, room temperature, horizontal rotor 800g, acceleration 3acc, centrifuge for 30 min.
4) After centrifugation, the tube bottom is red blood cells, the middle layer is separation liquid, the uppermost layer is a plasma/tissue homogenate layer, and a thin and compact white membrane is arranged between the plasma layer and the separation liquid layer, namely: a layer of mononuclear cells (including lymphocytes and monocytes). Carefully aspirate the buffy coat into another centrifuge tube.
5) Dilute to volume with PBS and mix by inversion. At room temperature, the rotor was rotated horizontally at 300g, centrifuged for 10min, and the supernatant was discarded. The washing was repeated 2 times.
6) The lymphocytes were resuspended in PBS for use.
7) Counting the cells used for sorting, taking 1X 106The cells, 100. mu.L in volume, were incubated with 6 fluorochromes as recommended in the following table at 4 ℃ in the dark for 1 h.
8) The washing was repeated 2-3 times with PBS containing 2% FBS, the 400. mu.L FPBS was resuspended, the cell pellet was removed with a 40 μm cell sieve, and stored at 4 ℃ in the dark for sorting.
9) GP-specific individual memory B cells were sorted using a cell sorter (SONY, SH 800S). The sorting strategy is as follows: IgG +/CD3-/CD38-/CD19+/CD27+/GPdM +, single plasma cells were directly sorted into 96-well plates, each well of which contained 5U RNase inhibitor and 19.8. mu.L of RNase-free water and stored at-80 ℃. FIG. 1 is a graph of cell sorting results, with the cells in the boxed portion characterized by IgG +/CD3-/CD38-/CD19+/CD27+/GPdM +, i.e., the desired GP-specific memory B cells.
4. Single cell PCR amplification fully human source monoclonal antibody
1) Reverse transcription PCR
80 GPdM specific memory B cells are obtained by sorting, and the following specific primers aiming at each subtype of the heavy chain H, the kappa light chain and the lambda light chain are simultaneously added into each reaction system (the primer sequences are shown in a table 1).
TABLE 1 reverse transcription PCR primers
The PCR reaction system comprises: 5 Xbuffer 6 u L, dNTP 1.2.2 u L, reverse transcriptase 1.2 u L, primer, template for single cell, water to make up to 30L.
The PCR reaction conditions are as follows: reverse transcription at 50 deg.C for 30min, pre-denaturation at 95 deg.C for 15min, followed by 95 deg.C for 40s, 55 deg.C for 30s, 72 deg.C for 1min, 40 cycles, and final extension at 72 deg.C for 10 min.
2) Nested PCR
Taking the reverse transcription product as a template, and respectively carrying out nested PCR amplification on H, kappa and lambda for 3 times (the primer sequences are shown in Table 2), wherein the specific process is as follows:
TABLE 2 nested PCR primers
The PCR reaction system comprises: 2.5. mu.L of 10 Xbuffer, 0.5. mu.L of 10mM dNTP, 0.5. mu. L, DNA polymerase, 1. mu.L of the reverse transcription product as the template, and 25. mu.L of water.
The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 4min, followed by 94 ℃ for 30s, 57 ℃ for 30s, 72 ℃ for 45min, 40 cycles, and finally extension at 72 ℃ for 10 min.
3) Agarose gel electrophoresis
Clones with successful amplification of both heavy and light chain genes in a single cell were considered successful clones. And (3) carrying out 1% agarose gel electrophoresis on 5 mu L of nested PCR amplification products, sequencing the paired positive clones, analyzing the antibody variable region sequence obtained by sequencing by using Vector NTI software and an IMGT website, and carrying out antibody protein expression and function verification. FIG. 2 is a graph showing the identification of agarose gel electrophoresis after nested PCR amplification of three H, K, and lambda chain genes, which is considered to be naturally paired antibody genes only when heavy chain and light chain variable region genes are amplified from the same memory B cell, and the sequencing of the corresponding nested PCR products is further studied.
5. Linear expression cassette expression antibodies
Through the reverse transcription reaction, 13 matched antibody sequences are obtained from single cell cloning, and if the traditional cloning expression method is time-consuming and labor-consuming, the antibody can be quickly expressed by a method for constructing a linear expression frame. The basic principle of this method is to directly link a promoter sequence (GENBANK accession number: X03922.1), a coding sequence of an antibody leader peptide, an antibody variable region (amplified from a single cell), an antibody constant region (biosynthetic), and a poly-A tail (GENBANK accession number: X03896.1) by overlap extension PCR, and to transfect the linear form of DNA into cells for antibody expression.
The specific process is as follows:
1) a promoter-leader sequence fragment and a poly-A tail fragment were amplified using pSec Tag2(Invitrogen) as a template.
The PCR reaction system for amplifying the promoter-leader sequence fragment comprises: template plasmid pSec Tag2(Invitrogen)1ng, 10 Xbuffer 5. mu.L, 10mM dNTP 1. mu. L, DNA polymerase 0.5. mu.L, primer 5'CMV-FORWARD (CGATGTACGGGCCAGATATACGCGTTG), primer 3' leader-sequence (GTCACCAGTGGACCTGGAACCCCCA), water to 50. mu.L.
The PCR reaction system for amplifying poly-A tail fragments comprises: template plasmid pSec Tag2(Invitrogen)1ng, 10 Xbuffer 5. mu.L, 10mM dNTP 1. mu. L, DNA polymerase 0.5. mu.L, primer 5'POLY (A) (GCCTCGACTGTGCCTTCTAGTTGC), primer 3' POLY (A) (TCCCCAAGCATGCCTGCTATTGTCT), water to 50. mu.L.
The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 4min, followed by 94 ℃ for 30s, 60 ℃ for 30s, 72 ℃ for 1min, 30 cycles, and final extension at 72 ℃ for 10 min.
2) Amplification of antibody constant regions
The H chain constant region PCR system comprises: heavy chain constant region template 10ng, 10 x buffer 5u L, 10mM dNTP 1 u L, DNA polymerase 0.5 u L, primer 5'CH (ACCAAGGCCCATCGGGTCTTCCCC), primer 3' CH (GCAACTAGAGGCACAGGTCGAGGCTTTACCCGGAGACAGGGGA), water to 50 u L.
The kappa chain constant region PCR system comprises: mu.L of kappa chain constant region template 10ng, 5. mu.L of 10 Xbuffer, 0.5. mu.L of 10mM dNTP 1. mu. L, DNA polymerase, 5 'C.kappa.primer (ACTGTGGCTGCACCATCTGTCTTC), 3' C.kappa.primer (GCAACTAGAAGGCACAGTCGAGGCACACTCTCCCCTGTTGAAGCT), and water to 50. mu.L.
The lambda chain constant region PCR system comprises: lambda chain constant region template 10ng, 10 Xbuffer 5. mu.L, 10mM dNTP 1. mu. L, DNA polymerase 0.5. mu.L, primer 5 'C.lambda. (GAGGAGCTTCAAGCCAACAAGGCCACA), primer 3' C.lambda. (GCAACTAGAAGGCACAGTCGAGGCTGAACATTCTGTAGGGGCCAC), water to 50. mu.L.
The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 4min, followed by 94 ℃ for 30s, 60 ℃ for 60s, 72 ℃ for 3min, 30 cycles, and final extension at 72 ℃ for 10 min.
3) Amplification of antibody variable regions
The PCR system comprises: the template was 10ng of the reverse transcription PCR product, 5. mu.L of 10 Xbuffer, 1. mu.L of 10mM dNTP, 0.5. mu.L of DNA polymerase, primers shown in Table 3 (heavy chain and light chain primers were mixed and added to the system), and water was added to make up to 50. mu.L.
The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 4min, followed by 94 ℃ for 30s, 60 ℃ for 30s, 72 ℃ for 3min, 30 cycles, and final extension at 72 ℃ for 10 min.
TABLE 3 construction of PCR primers for Linear expression Frames
4) And (3) recovering and purifying PCR products: the PCR products were subjected to electrophoresis on a 1% agarose gel, cut and recovered using a recovery kit from OMEGA.
5) Amplification of Linear expression cassettes for heavy and light chains, respectively
The schematic diagram of the splicing sequence of the linear expression boxes is shown in FIG. 3. In FIG. 3, A is an H chain linear expression cassette, B is a kappa chain linear expression cassette, and C is a lambda chain linear expression cassette.
The PCR reaction system comprises:
template: 10ng of purified promoter-leader sequence fragment, 10ng of heavy chain/light chain variable region fragment, 10ng of heavy chain/light chain constant region fragment, 10ng of poly-A tail fragment, 2.5 μ L of 10 × buffer solution, 0.5 μ L of 10mM dNTP 0.5 μ L, DNA polymerase, 0.25 μ L of primer 5'CMV-FORWARD (CGATGTACGGGCCAGATATACGCGTTG) and 3' POLY (A) (TCCCCAGCATGCTATTGTCT), water-supplemented to 25 μ L.
The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 4min, followed by 94 ℃ for 30s, 60 ℃ for 30s, 72 ℃ for 3min, 30 cycles, and final extension at 72 ℃ for 10 min.
6) And (3) recovering and purifying PCR products: the PCR reaction product was recovered directly with the recovery kit of OMEGA.
7) DNA quantification: the PCR-recovered product was quantified using Nano (GE healthcare).
8) Cell inoculation: 293T cells at 2X 105Perml in 24-well cell culture plates in 5% CO2The cells were incubated at 37 ℃ overnight in an incubator.
9) Cell co-transfection: the next day, 1. mu.g each of the successfully constructed heavy and light chain linear expression cassette PCR products was added to 200. mu.L of serum-free MEM medium, mixed well, 4. mu.L of the transfection reagent Turbofect (Thermo Scientific, R0531) was added, incubated for 15-20min, and added dropwise to overnight-cultured 293T cell culture wells. In the presence of 5% CO2The cells were cultured at 37 ℃ for 48 hours in the cell incubator, and then the cell culture supernatant was collected for use.
ELISA screening for antibodies with binding Activity
1) Coating a 96-well enzyme-linked plate with 1 mu g/mL EBOV GP on the day before the experiment, and coating 100 mu L of EBOV GP in each well; the coated enzyme-linked plate was placed in a wet box at 4 ℃ overnight.
2) The experiment was washed 5 times on the day with a plate washer (BIO-TEK, 405_ LS).
3) Add 100. mu.L of blocking solution to each well and let stand at room temperature for 1 hour.
4) Washing with plate washing machine for 5 times.
5) 100. mu.L of the transfected cell culture supernatant was added and allowed to stand at room temperature for 1 hour.
6) Washing with plate washing machine for 5 times.
7) An HPR-labeled goat anti-human IgG secondary antibody (Abcam, ab97225) was diluted at 1:10000 in a diluent, 100. mu.L per well was added to the corresponding well of the ELISA plate, and the plate was incubated at room temperature for 1 hour.
8) Washing with plate washing machine for 5 times.
9) Adding 100 mu L of TMB single-component developing solution into each hole, developing for 6min, keeping the room temperature away from light, and then adding 50 mu L of stop solution into each hole to terminate the reaction.
10) Detecting OD value at the position of 450-630nm on a microplate reader, and storing and recording original data.
As a result: screening 13 monoclonal antibodies, and identifying the specificity of the combined antibody of the Ebola GP. There were 2 strains that were capable of specific binding to ebola GP, including 4C1 and 4F 1. FIG. 4 shows a control column of binding activity of 13 monoclonal antibodies.
7.4F1 antibody sequence description
The amino acid sequences of the light and heavy chains of the 4F1 antibody are described below:
the amino acid sequence of the light chain variable region is shown as SEQ ID NO.1, the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are respectively shown as amino acid sequences at positions 27-32, 50-52 and 89-97 of the SEQ ID NO.1, the gene coding sequence of the light chain variable region is shown as SEQ ID NO.2, the amino acid sequence of the light chain constant region is shown as SEQ ID NO.3, and the gene coding sequence of the light chain constant region is shown as SEQ ID NO. 4.
The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.5, the amino acid sequences of the CDR1, the CDR2 and the CDR3 of the heavy chain variable region are respectively shown as the amino acid sequences at positions 26-33, 51-57 and 96-107 of the SEQ ID NO.5, the gene coding sequence of the heavy chain variable region is shown as SEQ ID NO.6, the amino acid sequence of the heavy chain constant region is shown as SEQ ID NO.7, and the gene coding sequence of the heavy chain constant region is shown as SEQ ID NO. 8.
8. Expression plasmid construction and antibody preparation
4F1 is used for constructing an expression plasmid, and the monoclonal antibody is expressed and prepared. The method comprises the following steps:
1) the full-length genes of the linear expression cassettes of 4F1H and 4F1K were double digested with EcoRI (NEB, R3101) and NotI (NEB, R3189) and ligated into pcDNA3.4 expression plasmid.
2) 15. mu.g each of pcDNA3.4-4F1H and pcDNA3.4-4F1K was transfected into 30mL of Expi293 system (Life, A14524) at 125rpm with 5% CO2And culturing for 72 h.
3)3000 Xg, centrifugating for 10min to collect the expression supernatant, filtering with 0.22 μm needle filter, and affinity purifying with rProtein A.
4) The collected antibodies were subjected to a solution change with PBS, and then the antibody concentration was determined with BCA protein quantification kit (Thermo Scientific, 23225).
Example 2 ELISA detection of antibody binding Activity
1) One day before the experiment, 96-well enzyme-linked plates were coated with 1. mu.g/mL of EBOV GP, BDBV GP, SUDV GP and RESTV GP, and each well was coated with 100. mu.L. The coated enzyme-linked plate was placed in a wet box at 4 ℃ overnight.
2) The experiment was washed 5 times with a plate washer on the day.
3) Add 100. mu.L of blocking solution to each well and let stand at room temperature for 1 hour.
4) Washing with plate washing machine for 5 times.
5) 150 μ L of 4F1 mAb at 10 μ g/mL was added to the first well and 100 μ L of the dilution was added to the remaining wells. Aspirate 50 μ L from the first well and add to the second well and so on, dilute each well in a 1:3 gradient to a final volume of 100 μ L. The mixture was allowed to stand at room temperature for 1 hour.
6) Washing with plate washing machine for 5 times.
7) The HPR-labeled secondary goat anti-human IgG antibody was diluted at a ratio of 1:10000 in a diluent, 100. mu.L of each well was added to the corresponding well of the ELISA plate, and the plate was incubated at room temperature for 1 hour.
8) Washing with plate washing machine for 5 times.
9) Adding 100 mu L of TMB single-component developing solution into each hole, developing for 6min, keeping the room temperature away from light, and then adding 50 mu L of stop solution into each hole to terminate the reaction.
10) Detecting OD value at the position of 450-630nm on a microplate reader, and storing and recording original data.
As a result: the 4F1 monoclonal antibody and EBOV GP have good combination activity, EC50The value was 0.0057. mu.g/mL. 4F1 is weakly or non-bonded with BDBVGP, SUDV GP and RESTV GP, and has no broad-spectrum binding activity to the Ebola virus, which indicates that the binding epitope is not conserved on several Ebola virus GPs (the detection result is shown in figure 5).
Example 3 pseudovirus neutralization assay 4F1 neutralization Activity
EBOV pseudovirus packaging HIV backbone, 4F1 was evaluated for neutralizing activity in vitro as follows:
1) the 4F1 monoclonal antibody was diluted with DMEM medium, and 75. mu.L of the antibody diluent at a concentration of 100. mu.g/mL was added to the first well of the 96-well cell culture plate, and 50. mu.L of DMEM medium was added to the remaining wells.
2) Sucking 25 mul of liquid from the first hole, adding the liquid into the second hole, mixing uniformly, and diluting according to the ratio of 1:3 by analogy, wherein the final volume of each hole is 50 mul.
3) The pseudovirus was diluted 1:5 with DMEM medium (control well fluorescein readings were controlled between 20000-100000) and added to each antibody well at 50. mu.L per well. Mix well and incubate at 37 ℃ for 1 h.
4)293T cell count, 2X 105cells/mL, 100. mu.L per well.
5) And (3) putting the 96-hole cell culture plate into a 37 ℃ incubator for culturing for 36-48 hours.
6) The cell culture plate was removed and the culture broth carefully aspirated and discarded. Add 100. mu.L of cell lysate to each well and shake for 15min at 350rpm on a shaker.
7) Centrifuge at 3000rpm for 10min at room temperature.
8) A detection substrate lyophilizate of a luciferase detection system (Promega, E1501) and a detection buffer solution are mixed uniformly, and a detection pipeline of GLOMAX 96MICROPLATE LUMINOMETER (Promega) is filled.
9) And (4) sucking 20 mu L of reading values of the lysis supernatant, reading fluorescence values, and calculating the protection rate of the antibody on the cells.
As a result: 4F1 was effective in neutralizing EBOV pseudoviruses in vitro, as compared to control antibodies. The neutralizing activity of 4F1 increased with increasing antibody concentration, and 100% protection was achieved for EBOV at a concentration of 1. mu.g/mL (see FIG. 6 for assay results).
Example 4 truncated antigen analysis of the 4F1 binding region
4F1 exhibited good in vitro neutralization activity, and it was hoped to analyze the approximate binding region by constructing a truncated GP. Besides GPdTM, GPdM, GP1, sGP and GPcl which are common in the literature, GP is designed and constructed according to GP structural characteristics and analysis targets33-310;463-558、GP33-227、GP33-158、GP95-295、GP158-295And GP227-295。GP33-310;463-558The latter heptad repeat region is removed on the basis of GPdM; GP33-227The glycan cap is removed on the basis of sGP; GP33-158Most of the Base area and the Head area are included; GP95-295The Base region was deleted compared to sGP; GP158-295Compared with sGP, the Base region and the Head region are basically deleted; GP227-295The majority of the glycan cap region is contained. GPcl is obtained by cleaving the glycan cap with thermolysin (thermolysin) on the basis of GPdM, without constructing an expression plasmid. The schematic structure of each truncated GP is shown in FIG. 7.
Truncated GP analysis 4F1 binding region method was as follows:
1) according to the schematic diagram, each truncated GP gene segment is amplified from the GP full-length gene, and His is added at the carboxyl terminal6The label, the gene fragment is connected to pcDNA3.4 expression plasmid.
2) Each truncated GP was expressed in the Expi293 system and the protein of interest was obtained by NI-NTA purification.
3) The binding activity of 4F1 to each truncated GP was analyzed by ELISA as described in example 2.
As a result: 4F1 can be well combined with GPdM, sGP, GP1 and other truncated GPPs, and EC50The values were 0.0069. mu.g/mL, 0.0039. mu.g/mL and 0.0078. mu.g/mL, respectively, which is comparable to GPdTM. Although 4F1 is in contact with GPcl and GP95-295The binding activity of (A) is reduced by about 5 times compared with GPdTM, but the binding activity is relatively good, EC50The values were 0.032. mu.g/mL and 0.033. mu.g/mL, respectively. Thus, the binding epitope for 4F1 should be located in the region shared by these truncated GPs, amino acids 95-190. The binding activity of 4F1 to each truncated antigen was measured as shown in fig. 8-9.
Example 5 Competition experiments
4F1 was analyzed for epitopes of antigenic action using a competitive binding ELISA. Using MIL77-1/2/3 and the remaining several non-neutralizing antibodies known to bind epitopes as control antibodies, the presence of overlap in the binding epitopes of these antibodies was reflected by examining whether the binding of the test antibody to GP was blocked by competing antibodies incubated therewith. The method comprises the following steps:
1) 4mg of biotin (Thermo Scientific, 21335) was weighed and dissolved in 2mL of ultrapure water to a concentration of 2 mg/mL.
2) Antibodies 4F1, 2E5, 5D7, 5G11, 5B12, 1B3, MIL77-1, MIL77-2 and MIL77-3 are respectively taken at 200 mu G, and the volume is controlled to be about 200 mu L.
3) The antibodies were labeled at a molar ratio of biotin to antibody of 20: 1. The antibody and biotin were mixed, incubated at room temperature for 1 hour, and the solution was changed with a 50kD 0.5mL spin filter, about 400. mu.L of PBS each time, 3 more times.
4) The final PBS filter liquid to all the filter to about 100 u L (in order to make the antibody concentration similar), the antibody concentration determination.
5) The 96-well ELISA plates were coated with GPdM at a concentration of 1. mu.g/mL overnight at 4 ℃.
6) The plate washer washes the ELISA plates, adds 100. mu.L of blocking solution per well, and incubate for 1 hour at 37 ℃.
7) In the experiment, the detection antibody is a biotin-labeled antibody, and the competitive antibody is a non-biotin-labeled antibody. Diluting the competitive antibody to 2.5 mu g/mL by using the diluent; dilutions dilute the detection antibody to 25 ng/mL.
8) The 96-well plate was washed. Each well was loaded with 50. mu.L of detection antibody and 50. mu.L of competitor antibody, 100. mu.L per final well volume.
9) Washing, streptavidin antibody (Thermo scientific, 21126) diluted 1:1000, 100. mu.L per well, and incubation at 37 ℃ for 1 hour.
10) Washing, adding 100 mu L of TMB single-component color development liquid into each hole, developing for 6 minutes in a dark place, and adding 50 mu L of stop solution.
11) Read 450 and 630nm OD values.
As a result: a competition binding value of less than 30 is considered to be strong; greater than 30 and less than 60 are considered weak; above 60 no competition is considered. The antibodies compete well for binding with each other and can be divided into four groups according to the results. Two groups of the protein are combined with GP1 subunit and GP2 subunit. 4F1 did not compete with the non-neutralizing antibody 5B12, neutralizing antibodies MIL77-1 and MIL77-2, which bound to the GP2 subunit. There was more or less competition between 4F1 and control antibodies other than 5G11 that bound to the GP1 subunit, indicating that its binding epitope overlaps or is sterically hindered from that of these control antibodies. Both 4F1 and control antibodies such as MIL77-3 bound to GP1 subunit and competed for binding, but 4F1 had neutralizing activity in vitro, while the other antibodies did not, suggesting that 4F1 has the potential to constitute cocktail combination therapy with other neutralizing antibodies bound to GP2 subunit (see figure 10 for test results).
Example 6 detection of binding Activity of 4F1 with GPdM at Low pH
After binding to GP on the surface of the viral particle, the antibody enters the host cell together with the viral particle. In the acidic environment of the secondary endosome (pH about 5.5), GP is enzymatically hydrolyzed and then binds to the receptor, facilitating release of the viral genome. To further examine whether the binding activity of 4F1 to GP was affected by acidic environment, the binding activity of 4F1 to GP in vitro in pH 7.5, pH 6.5, pH 5.5 and pH 4.5 environments was tested, and the ELISA method was the same as that described in example 2.
As a result: the binding activity of 4F1 to GPdM was essentially unchanged in four environments with different pH. 4F1 is stable in binding with GP and is not influenced by the low pH environment of endosomes, thereby providing a basis for the neutralization (see figure 11).
Sequence listing
<110> military medical research institute of military science institute of people's liberation force of China
<120> monoclonal antibody 4F1 for resisting subunit GP1 of Ebola virus glycoprotein and application
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 107
<212> PRT
<213> Homo sapiens
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Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Gly Ser Ser
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Arg Ala Thr Gly Phe Pro Asp Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Gly Ser Pro Leu
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 2
<211> 321
<212> DNA
<213> Homo sapiens
<400> 2
gacatccaga tgacccagtc tccatcctcc ctgtctttgt ctccagggga aagagccacc 60
ctctcctgca gggccagtca gagtgttggc agttccttag cctggtacca gcagaaacct 120
ggccaggctc ccaggctcct catctatgct gcatccagca gggccactgg cttcccagac 180
aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcagcag actggagcct 240
gaagattttg cagtgtatta ctgtcagcag tatggtggct cacctctcac tttcggcgga 300
gggaccaagg tggaaatcaa a 321
<210> 3
<211> 106
<212> PRT
<213> Homo sapiens
<400> 3
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
1 5 10 15
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
20 25 30
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
35 40 45
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Gly Thr
50 55 60
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
65 70 75 80
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
85 90 95
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 4
<211> 318
<212> DNA
<213> Homo sapiens
<400> 4
actgtggctg caccatctgt cttcatcttc ccgccatctg atgagcagtt gaaatctgga 60
actgcctctg ttgtgtgcct gctgaataac ttctatccca gagaggccaa agtacagtgg 120
aaggtggata acgccctcca atcgggtaac tcccaggaga gtgtcacaga gcaggacagc 180
aaggacggca cctacagcct cagcagtacc ctgacgctga gcaaagcaga ctacgagaaa 240
cacaaagtct acgcctgcga agtcacccat cagggcctga gttcgcccgt cacaaagagc 300
ttcaacaggg gagagtgt 318
<210> 5
<211> 118
<212> PRT
<213> Homo sapiens
<400> 5
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Arg Ile Tyr
20 25 30
Asp Met His Trp Val Arg Gln Val Thr Gly Arg Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Gly Ser Ala Ser Asp Thr Tyr Tyr Arg Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ser Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Gly Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Ala Ser Phe Gly Glu Leu Met Phe Asp Leu Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 6
<211> 354
<212> DNA
<213> Homo sapiens
<400> 6
gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60
tcctgtgcag cctctggatt cagcttcagg atctacgaca tgcactgggt ccgccaagtt 120
acagggagag gtctggaatg ggtctcagct attggttctg ctagtgacac atactatcga 180
gactccgtga agggccgatt caccatctcc agagaaaatg ccaagaactc cttgtatctg 240
caaatgaaca gcctgagagc cggggacacg gctgtgtatt actgtgcaag agcgagtttc 300
ggggagttaa tgtttgactt atggggccag ggaaccctgg tcaccgtctc ctca 354
<210> 7
<211> 330
<212> PRT
<213> Homo sapiens
<400> 7
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Val Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Pro
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
225 230 235 240
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330
<210> 8
<211> 990
<212> DNA
<213> Homo sapiens
<400> 8
gcgtcgacca agggcccatc ggtcttcccc ctggcacctt cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300
aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360
ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480
tacgtggacg gcgtggaggt gcataatgtc aagacaaagc cgcgggagga gcagtacaac 540
agcacgtacc gtgtggtcag cgtcctcacc gtcccgcacc aggactggct gaatggcaaa 600
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660
aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 720
ctgaccaaga accaggtcag cctgacctgc ctggtcaagg gcttctatcc cagcgacatc 780
gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840
ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 900
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960
cagaagagcc tctccctgtc tccgggtaaa 990
Claims (9)
1. A monoclonal antibody for resisting the subunit GP1 of Ebola virus glycoprotein is characterized in that the amino acid sequences of CDR1, CDR2 and CDR3 in the variable region of a light chain of the antibody are respectively shown as the amino acid sequences at positions 27-32, 50-52 and 89-97 of SEQ ID NO.1, and the amino acid sequences of CDR1, CDR2 and CDR3 in the variable region of a heavy chain of the antibody are respectively shown as the amino acid sequences at positions 26-33, 51-57 and 96-107 of SEQ ID NO. 5.
2. The monoclonal antibody according to claim 1, wherein the amino acid sequence of the antibody light chain variable region is represented by SEQ ID No.1, and the amino acid sequence of the antibody heavy chain variable region is represented by SEQ ID No. 5.
3. The monoclonal antibody of claim 2, wherein the amino acid sequence of the antibody light chain constant region is set forth in SEQ ID No.3 and the amino acid sequence of the antibody heavy chain constant region is set forth in SEQ ID No. 7.
4. A polynucleotide encoding the light and heavy chains of the monoclonal antibody of any one of claims 1-3, wherein the polynucleotide encoding the light chain variable region of the monoclonal antibody has the sequence shown in SEQ ID No.2 and the polynucleotide encoding the heavy chain variable region of the monoclonal antibody has the sequence shown in SEQ ID No. 6.
5. The polynucleotide of claim 4, wherein the polynucleotide encoding the light chain constant region of the antibody has the sequence shown in SEQ ID No.4 and the polynucleotide encoding the heavy chain constant region of the antibody has the sequence shown in SEQ ID No. 8.
6. A linear expression cassette comprising polynucleotides encoding the heavy and light chains of a monoclonal antibody according to claim 5.
7. A host cell comprising the linear expression cassette of claim 6, wherein said cell is a 293T cell.
8. Use of a monoclonal antibody according to any one of claims 1 to 3 for the manufacture of a medicament for the treatment of ebola virus disease.
9. The use according to claim 8, wherein the monoclonal antibody is used as a component in an ebola virus cocktail formulation.
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