Nanobodies against Ebola virus VP40 protein and their application in virus detection

Abstract

The present invention discloses nanobodies against the VP40 protein of the Ebola virus and their use in Ebola virus detection. The nanobodies against the VP40 protein of the Ebola virus include 3E07 and 3F06 antibodies. The nanobodies against the VP40 protein of the Ebola virus prepared by the present invention have the characteristics of high sensitivity and broad binding spectrum. A double-antibody sandwich enzyme-linked immunosorbent assay constructed with the 3E07 and 3F06 antibodies can efficiently detect the VP40 protein and virus-like particles of multiple subtypes of Ebola virus, including Zaire, Sudan, and Bundibugyo. The detection sensitivity for the VP40 protein of the Zaire subtype of Ebola virus can reach 0.039 ng / mL. This antibody combination has broad application prospects in the diagnosis of Ebola virus disease and pathogen detection.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunology, and particularly relates to antibodies against Ebola virus VP40 protein and applications thereof in Ebola virus detection. Background Art

[0002] Ebola virus, a member of the Filoviridae family, is an enveloped, non-segmented, single-stranded, negative-sense RNA virus with a long, filamentous appearance. The Ebola virus genome is encapsidated by the nucleoprotein NP, which, together with polymerase L and viral proteins VP35 and VP30, forms the nucleocapsid responsible for transcription and replication. VP40 interacts with VP24 and is encapsidated by the matrix protein VP40, which is attached to the inner viral membrane. VP40 is the most abundant protein in Ebola virus and the most important target for immunological detection of Ebola virus. Currently, six subtypes of Ebola virus have been reported: Zaire ebolavirus (EBOV), Sudan ebolavirus (SUDV), Bundibugyo ebolavirus (BDBV), Tai Forest ebolavirus (TAFV), Reston ebolavirus (RESTV), and Bombali ebolavirus (BOMV). With the exception of BOMV, all five subtypes of Ebola virus are capable of infecting humans. Among them, EBOV is the subtype that has caused the most Ebola epidemics, with an overall mortality rate of over 65%.

[0003] Given the short incubation period and high mortality rate of Ebola virus infection, early diagnosis plays a crucial role in Ebola prevention and control. Polymerase chain reaction (PCR) is the most important method for detecting Ebola virus infection, but due to strict laboratory conditions, it cannot be widely used in outbreak areas. Immunological diagnostic methods, such as enzyme-linked immunosorbent assay (ELISA), lateral flow immunochromatography (LFIA), indirect immunofluorescence assay (IFA), and immunohistochemistry, are also commonly used for diagnosing Ebola virus infection due to their simplicity, rapidity, and low cost.

[0004] Camelids harbor naturally absent light chain antibodies, comprising a heavy chain variable region (VH) as well as constant regions 2 (CH2) and 3 (CH3). This homodimer is known as a heavy chain antibody (HCAb), and its VH domain is the heavy chain variable region fragment (VHH). VHHs have a relatively low molecular weight of approximately 15 kDa, earning them the designation "nanobody." Compared to human VHs, the complementarity-determining region 3 (CDR3) of VHHs has a longer amino acid chain, forming a bulge-like loop structure that allows them to specifically bind to enzyme active sites and specific epitopes on viral particles, enhancing recognition of hidden antigenic epitopes. The present invention aims to leverage the unique advantages of nanobodies to screen for high-affinity anti-Ebola virus antibodies from alpacas, thereby developing immunological detection methods for Ebola virus with improved specificity and sensitivity. Summary of the Invention

[0005] Based on the above purpose, the present invention first provides a nanobody against Ebola virus VP40 protein, wherein the amino acid sequences of the CDR1, CDR2 and CDR3 regions of the heavy chain variable region of the nanobody against Ebola virus VP40 protein are as shown in positions 26-33, 51-57 and 96-106 of SEQ ID NO: 1, respectively; or

[0006] The amino acid sequences of the CDR1, CDR2 and CDR3 regions of the heavy chain variable region of the anti-Ebola virus VP40 protein nanobody are shown in positions 26-33, 51-58 and 97-122 of SEQ ID NO: 2, respectively.

[0007] The Nanobodies described in this invention are heavy chain-only antibodies, comprising a variable region and a constant region. The variable region has three Complementarity Determining Regions (CDRs): CDR1, CDR2, and CDR3, which are highly variable and diverse. The sequence diversity of the CDR regions determines the specificity and affinity of the antibody, as they recognize and bind to specific antigenic determinants through interaction with the antigen.

[0008] In a preferred embodiment, the amino acid sequence of the heavy chain variable region of the anti-Ebola virus VP40 protein Nanobody is as shown in SEQ ID NO: 1. In the present invention, the Nanobody having this variable region is named "3E07"; or,

[0009] The amino acid sequence of the heavy chain variable region of the nanobody against Ebola virus VP40 protein is shown in SEQ ID NO: 2. In the present invention, the nanobody having this variable region is named "3F06".

[0010] Secondly, the present invention provides a polynucleotide encoding the above-mentioned anti-Ebola virus VP40 protein Nanobody, the sequence of the polynucleotide encoding the heavy chain variable region of the anti-Ebola virus VP40 protein Nanobody is shown in SEQ ID NO: 3. In the present invention, Nanobody 3E07 is an antibody having this coding sequence, or,

[0011] The sequence of the polynucleotide encoding the heavy chain variable region of the nanobody against Ebola virus VP40 protein is shown in SEQ ID NO: 4. In the present invention, nanobody 3F06 is an antibody having this coding sequence.

[0012] Third, the present invention provides a vector containing a polynucleotide encoding the heavy chain variable region of the above-mentioned Nanobody against Ebola virus VP40 protein. The vector is used to clone and / or express the gene encoding the Nanobody against Ebola virus VP40 protein. In a specific embodiment of the present invention, the vector is pcDNA3.4. Other vectors known to those skilled in the art, in particular eukaryotic expression vectors, can also be used for cloning and expressing the coding genes of the present invention.

[0013] Fourth, the present invention provides a host cell containing a vector containing a polynucleotide encoding the heavy chain variable region of the above-mentioned Nanobody against Ebola virus VP40 protein. The host cell is used to express the above-mentioned Nanobody against Ebola virus VP40 protein. In a specific embodiment of the present invention, the host cell is an Expi293F cell. Other host cells known to those skilled in the art, especially eukaryotic host cells, can also be used for the expression of the Nanobody of the present invention.

[0014] Fifth, the present invention provides the use of the above-mentioned nanoantibodies against the VP40 protein of Ebola virus in the preparation of drugs for treating and / or preventing Ebola virus disease. The nanoantibodies provided by the present invention have excellent affinity and binding activity with the VP40 protein of Ebola virus, and can specifically target the VP40 protein of Ebola virus. Therefore, these properties of the nanoantibodies are used as a specific inhibitor of the binding of Ebola virus to host cells, or to specifically target therapeutic drugs to infected lesions or pathogens to play a role in clinical treatment or prevention of infection. Therefore, the present invention provides the use of the above-mentioned nanoantibodies against the VP40 protein of Ebola virus in the preparation of drugs for treating and / or preventing Ebola virus disease.

[0015] Sixth, the present invention provides a detection kit containing the aforementioned anti-Ebola virus VP40 nanobody. Based on the excellent affinity of the nanobody provided by the present invention for the Ebola virus VP40 protein, it can be used to detect Ebola virus particles containing the VP40 protein that may be present in a sample. The detection antigen can be detected using a single antibody, i.e., the nanobody acts as a primary antibody and specifically binds to the pathogen, and the binding is then detected using a secondary antibody. Alternatively, a dual antibody combination can be used.

[0016] In a preferred embodiment, the detection kit is a double-antibody sandwich immunoassay kit. The double-antibody sandwich method described herein is a commonly used immunological detection technique, typically used to detect macromolecular antigens such as proteins and peptides. It is primarily based on the principle of specific binding between antigens and antibodies. Two specific antibodies, a capture antibody and a detection antibody, are used to sandwich the target antigen. The capture antibody is first immobilized on a solid-phase support (such as a microplate or membrane), and then the sample to be tested is added. The target antigen in the sample specifically binds to the capture antibody and becomes immobilized on the solid-phase support. Next, a labeled detection antibody is added and specifically binds to another site of the antigen bound to the solid-phase support, forming an antibody-antigen-antibody complex. Finally, the labeled detection antibody is detected using a specific detection method (such as a colorimetric reaction in an enzyme-linked immunosorbent assay), thereby indirectly determining the target antigen content in the sample.

[0017] In the present invention, the combination of capture and detection antibodies in the detection kit is a Nanobody (3E07) against Ebola virus VP40 protein whose heavy chain variable region amino acid sequence is shown in SEQ ID NO: 1, and a Nanobody (3F06) against Ebola virus VP40 protein whose heavy chain variable region amino acid sequence is shown in SEQ ID NO: 2. In the technical solutions provided by the present invention, both 3E07 and 3F06 can be used as capture or detection antibodies. When one antibody is used as a capture antibody, the other is used as a detection antibody, i.e., the combination of capture and detection antibodies described in the present invention.

[0018] The immunoassay described in the present invention is not limited to the disclosed enzyme-linked immunosorbent assay, but may also include but is not limited to technical fields such as radioimmunoassay and chemiluminescence immunoassay.

[0019] In one specific embodiment, the anti-Ebola virus VP40 Nanobody (3E07) with the amino acid sequence of the heavy chain variable region shown in SEQ ID NO:1 is used as the detection antibody, and the anti-Ebola virus VP40 Nanobody (3F06) with the amino acid sequence of the heavy chain variable region shown in SEQ ID NO:2 is used as the capture antibody. In another alternative embodiment of the present invention, 3E07 can be used as the capture antibody and 3F06 as the detection antibody.

[0020] In specific implementations of the present invention, fusion proteins in which the C-termini of the aforementioned Nanobody 3E07 and Nanobody 3F06 are fused to a human Fc protein are used as detection or capture antibodies. VHH-Fc conjugation is a common method for constructing expression vectors. The addition of an Fc facilitates affinity purification, while Fc dimers enhance the antibody's ability to bind to the antigen. Those skilled in the art may also choose to add other purification tags to achieve the same technical purpose.

[0021] Finally, the present invention provides an antibody composition against Ebola virus VP40 protein, comprising an anti-Ebola virus VP40 protein Nanobody (3E07) having an amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 1 and an anti-Ebola virus VP40 protein Nanobody (3F06) having an amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 2. The present invention's double-antibody sandwich ELISA assay for Ebola virus VP40 protein demonstrates that the two Nanobodies target different antigen-binding epitopes on the VP40 protein. Therefore, when used as an antibody composition, the two antibodies can simultaneously bind to the same Ebola virus, further enhancing their impact on the three-dimensional structure of the Ebola virus and the binding potency of the Ebola virus to its receptor. Alternatively, different or the same therapeutic drugs can be targeted to the virus, serving as a reference for cocktail therapy. Therefore, the present invention provides a composition containing the two anti-Ebola virus VP40 protein antibodies.

[0022] The present invention screened two nanobodies against Ebola virus VP40 protein through alpaca immunization and phage antibody library. Both nanobodies have unique heavy chain variable region CDR regions, which have excellent binding activity to Ebola virus VP40 protein. 50 The affinity KD of 10-13.16 ng / mL and 19.85 ng / mL for Ebola virus VP40 protein were 2.55×10 -9 M and 1.79×10 -9 M, excellent binding activity and affinity show the use of the nanobody provided by the present invention in the preparation of drugs for treating and / or preventing Ebola virus disease.

[0023] The two nanobodies against Ebola virus VP40 protein provided by the present invention can bind to different antigenic epitopes in the Ebola virus VP40 protein. Therefore, the present invention provides a combination of the two nanobodies as a detection antibody and a capture antibody for double-antibody sandwich immunoassay. In the embodiment of the double-antibody sandwich ELISA method constructed by the present invention for detecting the Zaire type Ebola virus VP40 protein, the detection sensitivity is 0.039 ng / mL, which can efficiently detect the VP40 proteins and virus-like particles of multiple subtypes of Ebola virus such as Zaire, Sudan, and Bundibugyo, and has broad application prospects in the diagnosis of Ebola virus disease and pathogen detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The SDS-PAGE results of antibodies 3E07 and 3F06 are shown. Lane 1 is the marker, lane 2 is the antibody 3E07, and lane 3 is the antibody 3F06.

[0025] Figure 2 ELISA binding assay results for antibody 3E07;

[0026] Figure 3 ELISA binding test results for antibody 3F06;

[0027] Figure 4 The BLI affinity test results of antibody 3E07;

[0028] Figure 5 The BLI affinity test results of antibody 3F06;

[0029] Figure 6 This is the standard curve for detecting EBOV-VP40 protein using the double-antibody sandwich ELISA method;

[0030] Figure 7 Western Blot analysis of the packaging of multi-subtype Ebola virus VP40 virus-like particles;

[0031] Figure 8 Results of double antibody sandwich ELISA for detecting multi-subtype Ebola virus VP40 virus-like particles. DETAILED DESCRIPTION

[0032] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of protection defined by the claims of the present invention.

[0033] The reagents, methods and equipment used in the examples of the present invention are conventional reagents, methods and equipment in the present technical field. Unless otherwise specified, all reagents and materials used are commercially available.

[0034] Example 1. Expression and identification of anti-Ebola virus VP40 antibodies

[0035] 1. Acquisition, Expression, and Purification of Anti-Ebola Virus VP40 Antibodies

[0036] Alpacas were immunized subcutaneously at multiple sites on the back with 1 mg of eukaryotically expressed Zaire Ebola virus VP40 protein (genbank ID: 911825) mixed with complete Freund's adjuvant (CFA) at a 1:1 ratio. Booster immunizations were performed every 14 days, with the complete Freund's adjuvant replaced with incomplete Freund's adjuvant. Serum titers were monitored by ELISA. Peripheral blood was collected from the alpacas, and lymphocytes were isolated by density gradient centrifugation. RNA was extracted and reverse-transcribed into cDNA. Using the cDNA as a template, the VHH gene was amplified by PCR, followed by restriction digestion and ligation to construct the phagemid pComb3X-VHH. The phagemid was transformed into Escherichia coli XL1-Blue by electroporation. Helper phage VCSM13 was added during the logarithmic phase, and phages were harvested after overnight culture to generate a phage-displayed nanoantibody library against Ebola virus VP40 protein. Solid-phase affinity panning was used to select anti-Ebola virus VP40 protein nanoantibodies from the prepared antibody library. After multiple rounds of panning, clones were selected for phage-ELISA identification, and the positive clones were sequenced. Two anti-VP40 specific nanoantibodies were screened and named "3E07" and "3F06" respectively.

[0037] The sequencing results showed that the amino acid sequence of the heavy chain variable region of Nanobody 3E07 was as shown in SEQ ID NO: 1, wherein the amino acid sequences of the CDR1, CDR2 and CDR3 regions of the heavy chain variable region were as shown in positions 26-33, 51-57 and 96-106 of SEQ ID NO: 1, respectively.

[0038] The amino acid sequence of the heavy chain variable region of antibody 3F06 is shown in SEQ ID NO: 2, wherein the amino acid sequences of the CDR1, CDR2 and CDR3 regions of the heavy chain variable region are shown in positions 26-33, 51-58 and 97-122 of SEQ ID NO: 1, respectively.

[0039] 2. Antibody Eukaryotic Expression and Purification

[0040] The nanobody sequences obtained by sequencing in step 1 were codon-optimized for human expression. The codon-optimized sequences for nanobody 3E07 and 3F06 are shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively. The codon-optimized sequences were fused to the human Fc protein gene (sequence shown in SEQ ID NO. 5, where bp 1-12 encode the linker peptide GGGS) at their 3' ends. The constructs were then inserted into the pcDNA3.4 vector and transfected into Expi293F cells using PEI transfection reagent (Yisheng Bio, 40816ES01) for expression. On the second day of expression, 5% protein-free feed (Aupumai, F081918) and 2 mM sodium valproate (VPA) (MCE, HY-10585A), a protein expression enhancer, were added. Six days after transfection, the cell supernatant was harvested. Cell debris was removed by centrifugation, and the antibodies were purified by protein A affinity chromatography. The purified antibodies were then ultrafiltered and exchanged into PBS buffer for storage.

[0041] 3. SDS-PAGE to check antibody purity

[0042] Mix 5 μg of purified antibody with protein reducing buffer, add PBS to 30 μL, heat at 100°C for 10 minutes to fully denature the protein, take 9 μL and add it to the wells of polyacrylamide gel, separate by electrophoresis, stain and destain the gel using a staining and destaining instrument, and then scan the gel image and save it.

[0043] The results of SDS-PAGE detection were as follows Figure 1 As shown in the figure, the two bands corresponding to the antibody 3E07 and 3F06 fused with human Fc protein under reducing conditions have a molecular weight of about 40 kDa, which is consistent with the expected protein size, and no obvious miscellaneous bands appear, indicating good purity.

[0044] Example 2. Analysis of Binding Activity of Anti-Ebola Virus VP40 Antibodies

[0045] 1. Dilute Zaire Ebola virus VP40 protein to 1 μg / mL using carbonate coating solution, add 100 μL per well to the ELISA plate, seal the plate with sealing film, and incubate at 4°C overnight.

[0046] 2. Discard the liquid in the wells and wash the plate with PBST solution, 300 μL / well, for a total of three washes. Pat the plate dry.

[0047] 3. Add 100 μL of blocking solution (PBST containing 3% BSA) to each well, seal the plate with sealing film, and incubate at 37°C for 1 hour.

[0048] 4. After blocking, discard the liquid in the wells and wash the plate with PBST solution, 300 μL / well, for a total of three washes. Pat the plate dry.

[0049] 5. During this time, add 100 μL of 3E07 and 3F06 antibodies diluted 4-fold to each well at a concentration of 2 μg / mL in the first well to the ELISA plate and incubate at 37°C for 1 hour.

[0050] 6. Discard the liquid in the wells and wash the plate with PBST solution, 300 μL / well, for a total of three washes. Pat the plate dry.

[0051] 7. Add 100 μL of diluted anti-human IgG (HRP) antibody to the corresponding wells, seal the plate with sealing film, and incubate at 37°C for 1 hour;

[0052] 8. Discard the liquid in the wells and wash the plate with PBST solution, 300 μL / well, for a total of three washes. Pat the plate dry.

[0053] 9. Add 100 μL of color development solution to each well and incubate at room temperature for 15 minutes.

[0054] 10. Add 50 μL of stop solution to each well and gently shake the plate until mixed.

[0055] 11. Measure the absorbance of each well at 450 nm and 630 nm using a microplate reader and read the reading within 10 minutes after termination.

[0056] 12. The results of ELISA binding experiments showed that antibody 3E07 ( Figure 2 )、antibody 3F06( Figure 3 ) can specifically bind to Zaire Ebola virus VP40 protein, EC 50 13.16 ng / mL and 19.85 ng / mL respectively.

[0057] Example 3. Affinity Analysis of Anti-Ebola Virus VP40 Antibodies

[0058] The Ni-NTA sensor was rinsed in buffer for 10 minutes; the baseline was stabilized in buffer for 60 seconds. The sensor was then exposed to Ebola virus VP40 protein diluted in buffer for 60 seconds to stabilize the baseline. Antibodies 3E07 and 3F06 were then exposed to a 250 nM concentration in the first well and a two-fold serial dilution for 300 seconds of binding and 300 seconds of dissociation in buffer. All raw data were fitted using Fortebio data analysis software.

[0059] BLI analysis results are as follows Figure 4 and Figure 5As shown in the figure, the fitted lines represent the binding and dissociation patterns of antibodies 3E07 and 3F06 with VP40 protein over time. The results show that the affinity KD of antibodies 3E07 and 3F06 for binding to Ebola virus VP40 protein are 2.55×10 -9 M and 1.79×10 -9 M.

[0060] Example 4. Establishment of a double antibody sandwich enzyme-linked immunosorbent assay (ELISA)

[0061] An ELISA method for detecting Ebola virus VP40 protein was established using antibody 3F06 as the capture antibody and antibody 3E07 as the detection antibody.

[0062] 1. Biotinylation of Antibodies Against Ebola Virus VP40 Protein

[0063] NHS-esterified biotin EZ-link NHS-PEG4-biotin and the antibody 3E07 of the present invention were mixed at a molar ratio of 20:1 and reacted in 500 μL PBS at 4°C overnight for labeling. After the reaction, the solution was centrifuged using a desalting column to collect the biotin-labeled antibody 3E07, and the antibody concentration was detected using the BCA method.

[0064] 2. Double Antibody Sandwich ELISA for Detection of Ebola Virus VP40 Protein

[0065] (1) Dilute the antibody 3F06 to 2 μg / mL with carbonate coating solution, add 100 μL per well to the ELISA plate, seal the plate with sealing film, and place at 4°C overnight;

[0066] (2) Discard the liquid in the wells and wash the plate with PBST solution, 300 μL / well, wash the plate three times in total, and pat the plate dry;

[0067] (3) Add 100 μL of blocking solution (PBST containing 3% BSA by mass) to each well, seal the plate with a sealing film, and incubate at 37°C for 2 h;

[0068] (4) During this period, the serially diluted Ebola virus VP40 protein standard (final concentrations of 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98, 0 ng / mL) was mixed with the biotinylated antibody 3E07 (final concentration of 1 μg / mL) and incubated at 37°C for 1 h;

[0069] (5) After blocking, discard the liquid in the wells and wash the plate with PBST solution, 300 μL / well, for a total of 3 washes, and pat the plate dry;

[0070] (6) Add the above serially diluted standards and biotinylated 3E07 antibody premix to the corresponding wells and incubate at 37°C for 1 h;

[0071] (7) Discard the liquid in the wells and wash the plate with PBST solution, 300 μL / well, for a total of 3 washes, and pat the plate dry;

[0072] (8) Add 100 μL of diluted Streptavidin-HRP to the corresponding wells, seal the plate with a sealing film, and incubate at 37°C for 1 h;

[0073] (9) Discard the liquid in the wells and wash the plate with PBST solution, 300 μL / well, wash the plate three times in total, and pat the plate dry;

[0074] (10) Add 100 μL of color development solution to each well and incubate at room temperature for 15 min;

[0075] (11) Add 50 μL of stop solution to each well and gently shake the plate until mixed evenly;

[0076] (12) Use an enzyme-labeled instrument to measure the absorbance of each well at 450 nm and 630 nm, and read the value within 10 minutes after termination.

[0077] 3. Drawing of the standard curve

[0078] Absorbance OD 450nm - 630nm The results are shown in Table 1. The ELISA standard curve was drawn based on the results in Table 1. 450nm - 630nm The standard curve was prepared by using linear regression to obtain the formula of the standard curve with the concentration of the standard (1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, and 0.98 ng / mL) as the vertical axis and the concentration of the standard (1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, and 0.98 ng / mL) as the horizontal axis.

[0079] 4. Sensitivity calculation

[0080] The detection sensitivity is negative sample (OD 450nm - 630nm ) is the Ebola virus VP40 protein concentration corresponding to the mean + 2 times SD value.

[0081] 5. Experimental Results

[0082] Table 1 Quantitative detection of Ebola virus VP40 protein OD by double antibody sandwich method 450 -OD 630 Reading

[0083]

[0084] Select the readings at concentrations of 31.25, 15.63, 7.81, 3.91, 1.95, and 0.98 ng / mL to draw a standard curve, and use linear regression to calculate the standard curve formula and R 2 , the results are as follows Figure 6 As shown, the standard curve formula is: Y=0.04170*X+0.03746, R 2 =0.9942. After calculation, the sensitivity of the double-antibody sandwich ELISA method of the present invention is 0.039 ng / mL.

[0085] Example 5. Detection of Ebola virus VP40 virus-like particles by double antibody sandwich ELISA

[0086] 1. Preparation of Multi-subtype Ebola Virus VP40 Virus-like Particles

[0087] HEK293T cells were plated at 5 × 10 5 / mL, 2 mL per well was inoculated into a 6-well cell culture plate, and the cells were cultured at 37°C in a carbon dioxide incubator for 12 h before cell transfection;

[0088] 2 μg of EBOV VP40 (Genbank ID: 911825)-His, SUDV VP40 (Genbank ID: 3160775)-His, BDBV VP40 (Genbank ID: 9487264)-His, TAFV VP40 (Genbank ID: 9487534)-His, and RESTV VP40 (Genbank ID: 955192)-His eukaryotic expression plasmids were transfected into adherent HEK 293T cells using Lipofectamine 3000 transfection reagent (Thermo Fisher, L3000015) according to the manufacturer's instructions. The cells were then cultured in a CO2 incubator at 37°C for 20 h.

[0089] The cell supernatant was collected 20 hours after transfection, centrifuged at 2000 rpm for 5 minutes to remove cell debris, and the supernatant was collected. The supernatant contained Ebola virus VP40 virus-like particles.

[0090] 2. Western blot verification of Ebola virus VP40 virus-like particles in the supernatant

[0091] The supernatant of the prepared virus-like particles was mixed with reducing protein loading buffer, separated by SDS-PAGE, and transferred to a NC membrane using a membrane transfer apparatus. The membrane was incubated with mouse anti-His tag pAb as the primary antibody and goat anti-mouse IgG (HRP) as the secondary antibody. After development with developer, the gel was photographed and analyzed using a gel imager.

[0092] Western Blot identification results Figure 7 As shown, the band size is about 40 kDa, which is consistent with the theoretical molecular weight of the VP40 protein monomer of each serotype of Ebola virus. There is no obvious band in the untransfected plasmid group, which proves that Ebola virus VP40 virus-like particles are clearly present in the supernatant and can be used as a detection sample for the double antibody sandwich ELISA method.

[0093] 3. Detection of Ebola virus VP40 virus-like particles by double antibody sandwich ELISA

[0094] (1) Dilute the antibody 3F06 to 2 μg / mL with carbonate coating solution, add 100 μL per well to the ELISA plate, seal the plate with sealing film, and place at 4°C overnight;

[0095] (2) Discard the liquid in the wells and wash the plate with PBST solution, 300 μL / well, wash the plate three times in total, and pat the plate dry;

[0096] (3) Add 100 μL of blocking solution (PBST containing 3% BSA by mass) to each well, seal the plate with a sealing film, and incubate at 37°C for 2 h;

[0097] (4) During this period, the collected virus-like particle packaging supernatant was mixed with NP40 lysis buffer at a volume ratio of 1:4, lysed at room temperature for 5 minutes, and then mixed with biotinylated antibody 3E07 at a volume ratio of 1:1 and incubated at 37°C for 1 hour;

[0098] (5) After blocking, discard the liquid in the wells and wash the plate with PBST solution, 300 μL / well, for a total of 3 washes, and pat the plate dry;

[0099] (6) Add the above-mentioned serially diluted Ebola virus VP40 virus-like particle lysate and biotin-labeled 3E07 antibody premix into the corresponding well plate and incubate at 37°C for 1 h;

[0100] (7) Discard the liquid in the wells and wash the plate with PBST solution, 300 μL / well, for a total of 3 washes, and pat the plate dry;

[0101] (8) Add 100 μL of diluted Streptavidin-HRP to the corresponding wells, seal the plate with a sealing film, and incubate at 37°C for 1 h;

[0102] (9) Discard the liquid in the wells and wash the plate with PBST solution, 300 μL / well, wash the plate three times in total, and pat the plate dry;

[0103] (10) Add 100 μL of color development solution to each well and incubate at room temperature for 10 min;

[0104] (11) Add 50 μL of stop solution to each well and gently shake the plate until mixed evenly;

[0105] (12) Use an enzyme-labeled instrument to measure the absorbance of each well at 450 nm and 630 nm, and read the value within 10 minutes after termination.

[0106] 4. Experimental Results

[0107] The results of double antibody sandwich ELISA for identification of Ebola virus VP40 virus-like particles are as follows Figure 8 As shown, the OD value of the non-transfected VP40 plasmid group was extremely low (lower than the detection limit of the standard curve of the double-antibody sandwich ELISA method) because the supernatant did not contain VP40 virus-like particles. The VP40 virus-like particles of various genera of Ebola virus released VP40 protein after lysis, so the OD value was higher. The above data indicate that the double-antibody sandwich ELISA method of the present invention can identify all Ebola virus VP40 proteins and virus-like particles that are infectious to humans, and has important application prospects in the early diagnosis of Ebola virus.

Claims

1. A nanobody against Ebola virus VP40 protein, characterized in that: The amino acid sequences of the CDR1, CDR2 and CDR3 regions of the heavy chain variable region of the anti-Ebola virus VP40 protein nanobody are shown in positions 26-33, 51-58 and 97-122 of SEQ ID NO: 2, respectively.

2. The nanobody against Ebola virus VP40 protein according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the nanobody against Ebola virus VP40 protein is shown in SEQ ID NO:

2.

3. A polynucleotide encoding the nanobody against Ebola virus VP40 protein according to claim 1 or 2, characterized in that The sequence of the polynucleotide encoding the heavy chain variable region of the anti-Ebola virus VP40 protein nanobody is shown in SEQ ID NO:

4.

4. A vector containing the polynucleotide encoding the heavy chain variable region of the nanobody against Ebola virus VP40 protein according to claim 3.

5. A host cell containing a vector of a polynucleotide encoding the heavy chain variable region of the nanobody against Ebola virus VP40 protein according to claim 4.

6. Use of the nanobody against Ebola virus VP40 protein according to claim 1 or 2 in the preparation of a medicament for treating and / or preventing Ebola virus disease.

7. A detection kit containing the nanobody against Ebola virus VP40 protein according to claim 1 or 2.

8. The detection kit according to claim 7, characterized in that The detection kit is a double antibody sandwich immunoassay kit, wherein the combination of the capture antibody and the detection antibody in the detection kit is a nanobody against the Ebola virus VP40 protein whose amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1 and a nanobody against the Ebola virus VP40 protein whose amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:

2.

9. The detection kit according to claim 8, characterized in that The anti-Ebola virus VP40 protein Nanobody with the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 1 is the detection antibody, and the anti-Ebola virus VP40 protein Nanobody with the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 2 is the capture antibody.

10. An antibody composition against Ebola virus VP40 protein, characterized in that: The antibody composition includes an anti-Ebola virus VP40 protein nanobody having an amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 1 and an anti-Ebola virus VP40 protein nanobody having an amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 2.

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