Nano antibody for resisting Ebola virus VP40 protein and application of nano antibody in virus detection

Abstract

The invention discloses an anti-Ebola virus VP40 protein nano antibody and an application of the anti-Ebola virus VP40 protein nano antibody in Ebola virus detection. The anti-Ebola virus VP40 protein nano antibody comprises a 3E07 antibody and a 3F06 antibody. The anti-Ebola virus VP40 protein nano antibody prepared by the invention has the characteristics of high sensitivity, wide binding spectrum and the like. A double-antibody sandwich enzyme-linked immunosorbent assay method constructed by the 3E07 and 3F06 antibodies can be used for efficiently detecting VP40 proteins and virus-like particles of various subtype Ebola viruses such as Zaire, Sudan, Bandong and the like, and the detection sensitivity for the VP40 proteins of the Zaire type Ebola viruses can reach 0.039 ng / mL; the antibody combination has a wide application prospect in diagnosis and pathogen detection of Ebola virus diseases.

Description

Technical Field

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

[0003] Ebola virus belongs to the Filoviridae family. It is a non-segmented single-stranded negative-sense RNA virus with an envelope. The genome of Ebola virus is wrapped by the nucleoprotein NP, which together with polymerase L, viral proteins VP35 and VP30 form a nucleocapsid with transcription and replication functions, interacts with VP24, and is wrapped by the matrix protein VP40 attached to the inner membrane of the virus. VP40 is the most abundant protein in Ebola virus and is the most important target in the immunological detection of Ebola virus. There are currently 6 subtypes of Ebola virus reported: Zaire Ebola virus (EBOV), Sudan Ebola virus (SUDV), Bundibugyo Ebola virus (BDBV), Tai Forest Ebola virus (TAFV), Reston Ebola virus (RESTV) and Bombali Ebola virus (BOMV). Except for BOMV, the other five subtypes of Ebola virus are all capable of infecting humans. Among them, EBOV is the subtype that causes the most Ebola outbreaks, with an overall mortality rate of over 65%.

[0004] Given the short incubation period and high mortality rate of Ebola virus infection, early diagnosis plays an important role in the prevention and control of Ebola. Polymerase chain reaction (PCR) is the most important detection method for Ebola virus, but it is limited by strict laboratory conditions and cannot be used on a large scale in outbreak areas. Immunological diagnostic methods are also commonly used in the diagnosis of Ebola virus infection due to their simplicity, rapidity and low cost, including enzyme-linked immunosorbent assay (ELISA), lateral flow immunochromatography (LFIA), indirect immunofluorescence (IFA), immunohistochemistry, etc.

[0005] There is an antibody that naturally lacks light chains in the peripheral blood of camelids. The antibody includes a heavy chain variable region (VH) and constant region 2 (CH2) and constant region 3 (CH3). The homologous dimer composed of this structure is called a heavy chain antibody (HCAb), and its VH domain is the heavy chain antibody variable region fragment (VHH). VHH has a small molecular weight of about 15 kDa, so it is also called a nanobody. The complementary determining region 3 (CDR3) of VHH has a longer amino acid chain than that of human VH, which can form a convex ring structure, and can specifically bind to the enzyme active site and the special epitope of the virus particle, enhancing the recognition ability of the hidden antigen epitope. The purpose of the present invention is to screen high-affinity anti-Ebola virus antibodies from alpacas based on the unique advantages of nanoantibodies, and then develop an immunological detection method for Ebola virus with better specificity and higher sensitivity. Summary of the invention

[0006] Based on the above purpose, the present invention first provides a nanobody against Ebola virus VP40 protein, 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 shown in positions 26-33, 51-57 and 96-106 of SEQ ID NO: 1, respectively; or 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 nanobody described in the present invention is an antibody with only a heavy chain, wherein the heavy chain includes a variable region and a constant region, wherein the variable region has three complementary determining regions (CDRs): CDR1, CDR2 and CDR3, which are highly variable and diverse. The sequence diversity of the CDR region determines the specificity and affinity of the antibody, because they recognize and bind to specific antigenic determinants by interacting 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, 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".

[0009] 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 the coding sequence, or, The sequence of the polynucleotide encoding the heavy chain variable region of the nanobody against the Ebola virus VP40 protein is shown in SEQ ID NO: 4. In the present invention, nanobody 3F06 is an antibody having this coding sequence.

[0010] Third, the present invention provides a vector containing a polynucleotide encoding the heavy chain variable region of the above-mentioned nanobody against the Ebola virus VP40 protein. The vector is used to clone and / or express the coding gene of the nanobody against the 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, especially eukaryotic cell expression vectors, can also be used for cloning and expression of the coding gene of the present invention.

[0011] Fourth, the present invention provides a host cell containing a vector of a polynucleotide encoding the heavy chain variable region of the above-mentioned anti-Ebola virus VP40 protein nanobody. The host cell is used to express the above-mentioned anti-Ebola virus VP40 protein nanobody. 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.

[0012] Fifth, the present invention provides the use of the above-mentioned nano-antibodies against the VP40 protein of the Ebola virus in the preparation of drugs for treating and / or preventing Ebola virus disease. The nano-antibodies provided by the present invention have excellent affinity and binding activity with the VP40 protein of the Ebola virus, and can specifically target the VP40 protein of the Ebola virus. Therefore, these characteristics of the nano-antibodies are used as specific inhibition of the binding of the Ebola virus to host cells, or therapeutic drugs are specifically targeted 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 nano-antibodies against the VP40 protein of the Ebola virus in the preparation of drugs for treating and / or preventing Ebola virus disease.

[0013] Sixth, the present invention provides a detection kit containing the above-mentioned nano antibody against Ebola virus VP40 protein. Based on the excellent affinity of the nano antibody provided by the present invention to the Ebola virus VP40 protein, it can be used to detect Ebola virus particles with VP40 protein that may be present in a sample. The detection antigen is a single antibody detection, that is, the nano antibody specifically binds to the pathogen as a primary antibody, and then the binding is detected by a secondary antibody; it can also be a combination of double antibodies.

[0014] In a preferred embodiment, the detection kit is a double antibody sandwich immunoassay kit. The double antibody sandwich method described in the present invention is a commonly used immunological detection technology, which is usually used to detect macromolecular antigens, such as proteins, polypeptides, etc. It is mainly based on the principle of specific binding between antigens and antibodies. Two specific antibodies, namely capture antibodies and detection antibodies, are used to clamp the target antigen. The capture antibody is first fixed on a solid phase carrier (such as a microplate, a membrane, etc.), and then the sample to be tested is added. The target antigen in the sample specifically binds to the capture antibody and is fixed on the solid phase carrier. Next, a labeled detection antibody is added, which specifically binds to another part of the antigen that has been bound to the solid phase carrier to form an antibody-antigen-antibody complex. Finally, the labeled detection antibody is detected by a specific detection method (such as a color reaction in an enzyme-linked immunosorbent assay), thereby indirectly determining the content of the target antigen in the sample.

[0015] In the present invention, the combination of the capture antibody and the detection antibody in the detection kit is a nano antibody (3E07) 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 nano antibody (3F06) against the Ebola virus VP40 protein whose amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 2. In the technical solution provided by the present invention, both 3E07 and 3F06 can be used as a capture antibody or a detection antibody. When one antibody is used as a capture antibody, the other antibody is used as a detection antibody, that is, the combination of the capture antibody and the detection antibody described in the present invention.

[0016] 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 chemiluminescent immunoassay.

[0017] In a specific embodiment, the anti-Ebola virus VP40 protein nanobody (3E07) whose amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1 is a detection antibody, and the anti-Ebola virus VP40 protein nanobody (3F06) whose amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 2 is a capture antibody. In another optional technical solution of the present invention, 3E07 can be used as a capture antibody and 3F06 can be used as a detection antibody.

[0018] In the specific implementation of the present invention, the fusion protein in which the C-terminus of the above-mentioned nanobody 3E07 and nanobody 3F06 is fused with human Fc protein is used as the detection antibody or capture antibody. VHH coupling Fc is a conventional expression vector construction method. On the one hand, the addition of Fc facilitates affinity purification. On the other hand, Fc forms a dimer, which is beneficial to improve the binding ability of the antibody to the antigen. Those skilled in the art can also choose to add other purification tags to achieve the same technical purpose.

[0019] Finally, the present invention provides an antibody composition against Ebola virus VP40 protein, the antibody composition comprising a nano antibody (3E07) against Ebola virus VP40 protein whose amino acid sequence of heavy chain variable region is shown as SEQ ID NO: 1 and a nano antibody (3F06) against Ebola virus VP40 protein whose amino acid sequence of heavy chain variable region is shown as SEQ ID NO: 2. The embodiment of the double antibody sandwich ELISA detection of Ebola virus VP40 protein of the present invention verifies that the two nano antibodies target different antigen binding epitopes of VP40 protein, so when the two antibodies are used as an antibody composition, they can bind to the same Ebola virus at the same time, further increasing the influence on the three-dimensional structure of Ebola virus and the influence on the binding efficiency of Ebola virus to its receptor, or different or the same therapeutic drugs can be targeted to the virus, which can be used as a reference scheme for cocktail therapy, therefore, the present invention provides a composition containing the two antibodies against Ebola virus VP40 protein.

[0020] The present invention screened two nanoantibodies against Ebola virus VP40 protein through alpaca immunization and phage antibody library. Both of the two nanoantibodies have unique heavy chain variable region CDR regions, which have excellent binding activity with Ebola virus VP40 protein. 50 The affinity KD of 10-10 and 10-10 were 13.16 ng / mL and 19.85 ng / mL, respectively, and the affinity KD of 10-10 and 10-10 were 2.55×10 -9 M and 1.79×10 -9 M, excellent binding activity and affinity show the use of the nanoantibodies provided by the present invention in the preparation of drugs for treating and / or preventing Ebola virus disease.

[0021] 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 the two nanobodies as a combination of detection antibodies and capture antibodies 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, Bundibugyo, etc., and has broad application prospects in the diagnosis of Ebola virus disease and pathogen detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The SDS-PAGE identification results of antibodies 3E07 and 3F06, lane 1 is the marker, lane 2 is the antibody 3E07, and lane 3 is the antibody 3F06; Figure 2 ELISA binding test results of antibody 3E07; Figure 3 The ELISA binding test results of antibody 3F06; Figure 4 The BLI affinity test results of antibody 3E07; Figure 5 The results of BLI affinity determination of antibody 3F06; Figure 6 This is the standard curve for detecting EBOV-VP40 protein by double antibody sandwich ELISA; Figure 7 Western Blot was used to identify the packaging results of multi-subtype Ebola virus VP40 virus-like particles; Figure 8 The results of double antibody sandwich ELISA for detecting multi-subtype Ebola virus VP40 virus-like particles. DETAILED DESCRIPTION

[0023] The present invention will be further described below in conjunction with 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.

[0024] The reagents, methods and equipment used in the examples of the present invention are conventional reagents, methods and equipment in the technical field. Unless otherwise specified, the reagents and materials used are commercially available.

[0025] Example 1. Expression and identification of anti-Ebola virus VP40 antibodies 1. Acquisition, expression and purification of anti-Ebola virus VP40 antibodies 1 mg of eukaryotically expressed Zaire Ebola virus VP40 protein (genbank ID: 911825) was mixed with Freund's complete adjuvant at a ratio of 1:1 and immunized alpacas at multiple points on the back subcutaneously. After booster immunization every 14 days, Freund's complete adjuvant was replaced with Freund's incomplete adjuvant, and serum titer was monitored by ELISA. Peripheral blood was collected from alpacas, lymphocytes were separated by density gradient centrifugation, RNA was extracted and reversely transcribed into cDNA. Using cDNA as a template, VHH gene was amplified by PCR, and phagemid pComb3X-VHH was constructed after enzyme digestion and ligation. The phagemid was transformed into Escherichia coli XL1-Blue by electroporation, and helper phage VCSM13 was added in the logarithmic phase. Phages were collected after overnight culture to obtain a phage display 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.

[0026] 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.

[0027] 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 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.

[0028] 2. Eukaryotic Expression and Purification of Antibodies The sequence of the nanobody obtained by sequencing in step 1 was optimized for human codons. The codon-optimized sequence of nanobody 3E07 is shown in SEQ ID NO.3, and the codon-optimized sequence of nanobody 3F06 is shown in SEQ ID NO.4. The 3' end of the nanobody was fused with the human Fc protein gene (sequence shown in SEQ ID NO.5, wherein the 1-12 bp is the encoding linker peptide GGGS), constructed 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 protein expression enhancer sodium valproate (VPA) (MCE, HY-10585A) were added, and the cell supernatant was collected on the sixth day after transfection. The collected supernatant was centrifuged to remove cell debris, and the antibody was purified by protein A affinity chromatography. After purification, the antibody was replaced by ultrafiltration and stored in PBS buffer.

[0029] 3. SDS-PAGE to detect antibody purity Mix 5 μg of purified antibody with protein reducing buffer, fill the system to 30 μL with PBS, 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 decolorize the gel using a staining and decolorizing instrument, and then scan the gel image and save it.

[0030] The results of SDS-PAGE were as follows: Figure 1 As shown in the figure, the molecular weights of the two bands corresponding to the antibody 3E07 and 3F06 fused human Fc protein under reducing conditions are both about 40 kDa, which is consistent with the expected protein size, and no obvious mixed bands appear, indicating good purity.

[0031] Example 2. Analysis of Binding Activity of Anti-Ebola Virus VP40 Antibodies 1. Dilute the Zaire Ebola virus VP40 protein to 1 μg / mL with carbonate coating solution, add 100 μL to each well of the ELISA plate, seal the plate with a sealing film, and place at 4°C overnight; 2. Discard the liquid in the wells and wash the plate with PBST solution, 300 μL / well, wash the plate 3 times in total, and pat the ELISA plate dry; 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 1 hour; 4. After blocking, discard the liquid in the wells, wash the plate with PBST solution, 300 μL / well, wash the plate 3 times in total, and pat the ELISA plate dry; 5. During this period, add 100 μL of 3E07 and 3F06 antibodies diluted 4 times with a concentration of 2 μg / mL in the first well to each well of the ELISA plate and incubate at 37°C for 1 h. 6. Discard the liquid in the wells and wash the plate with PBST solution, 300 μL / well, wash the plate 3 times in total, and pat the ELISA plate dry; 7. Add 100 μL of diluted anti-human IgG (HRP) antibody to the corresponding wells, seal the plate with a sealing film, and incubate at 37°C for 1 h; 8. Discard the liquid in the wells and wash the plate with PBST solution, 300 μL / well, wash the plate 3 times in total, and pat the ELISA plate dry; 9. Add 100 μL of color development solution to each well and incubate at room temperature for 15 min; 10. Add 50 μL of stop solution to each well and gently shake the ELISA plate until mixed evenly; 11. Use an ELISA reader to measure the absorbance of each well at 450 nm and 630 nm, and read the value within 10 min after termination; 12. The results of ELISA binding experiments showed that antibody 3E07 ( Figure 2 )、Antibody 3F06( Figure 3 ) can specifically bind to the VP40 protein of Zaire Ebola virus, EC 50 13.16 ng / mL and 19.85 ng / mL respectively.

[0032] Example 3. Affinity analysis of anti-Ebola virus VP40 antibodies The Ni-NTA sensor was rinsed in buffer for 10 min; the baseline was stabilized in buffer for 60 seconds, the sensor was exposed to Ebola virus VP40 protein diluted in buffer for 60 seconds to stabilize the baseline, and then the antibodies 3E07 and 3F06 with a first well concentration of 250 nM and a 2-fold gradient dilution were exposed to binding for 300 seconds and dissociation in buffer for 300 seconds. All raw data were fitted by Fortebio data analysis software.

[0033] BLI analysis results Figure 4 and Figure 5 As 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.

[0034] Example 4. Establishment of double antibody sandwich enzyme-linked immunosorbent assay (ELISA) method An ELISA method for detecting Ebola virus VP40 protein was established using antibody 3F06 as capture antibody and antibody 3E07 as detection antibody.

[0035] 1. Biotinylation of Ebola virus VP40 protein antibody The 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 the reaction was carried out in 500 μL PBS at 4°C overnight for labeling; after the reaction, the desalting column was used for centrifugation and liquid exchange, the biotin-labeled antibody 3E07 was collected, and the antibody concentration was detected by the BCA method.

[0036] 2. Double Antibody Sandwich ELISA for Detection of Ebola Virus VP40 Protein (1) Dilute antibody 3F06 to 2 μg / mL with carbonate coating solution, add 100 μL to each well of the ELISA plate, seal the plate with a sealing film, and place at 4°C overnight; (2) Discard the liquid in the wells and wash the plate with PBST solution, 300 μL / well, wash the plate 3 times in total, and pat the plate dry; (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; (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 biotin-labeled antibody 3E07 (final concentration of 1 μg / mL) and incubated at 37°C for 1 h. (5) After blocking, discard the liquid in the wells and wash the plate with PBST solution, 300 μL / well, wash the plate 3 times in total, and pat the ELISA plate dry; (6) Add the above serially diluted standards and biotin-labeled 3E07 antibody premix to the corresponding wells of the plate and incubate at 37°C for 1 h. (7) Discard the liquid in the wells and wash the plate with PBST solution, 300 μL / well, wash the plate 3 times in total, and pat the plate dry; (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. (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; (10) Add 100 μL of color development solution to each well and incubate at room temperature for 15 min. (11) Add 50 μL of stop solution to each well and gently shake the plate until mixed. (12) Use an ELISA reader to measure the absorbance of each well at 450 nm and 630 nm and read the value within 10 min after termination.

[0037] 3. Drawing of standard curve 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 With 0.05 ng / mL as the ordinate and the concentration of the standard (1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 ng / mL) as the abscissa, the formula of the standard curve was obtained by linear regression, and the standard curve was prepared.

[0038] 4. Sensitivity calculation The detection sensitivity is negative sample (OD 450nm - 630nm ) is the Ebola virus VP40 protein concentration corresponding to the mean value + 2 times SD value.

[0039] 5. Experimental Results Table 1 Quantitative detection of Ebola virus VP40 protein OD by double antibody sandwich method 450 -OD 630 Reading value

[0040] Select the readings at 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.

[0041] Example 5. Detection of Ebola virus VP40 virus-like particles by double antibody sandwich ELISA 1. Preparation of Multi-subtype Ebola Virus VP40 Virus-like Particles HEK293T cells were plated at 5 × 10 5 / mL density, 2 mL per well was inoculated in a 6-well cell culture plate, and the cells were transfected after being cultured at 37 °C in a carbon dioxide incubator for 12 h; Lipofectamine 3000 transfection reagent (Thermo Fisher, L3000015) was used to transfect 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 RESTVVP40 (Genbank ID: 955192)-his eukaryotic expression plasmids into adherent HEK 293T cells according to the instructions, and the cells were cultured in a CO2 incubator at 37 °C for 20 h. The cell supernatant was collected 20 h 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.

[0042] 2. Western blot verification of Ebola virus VP40 virus-like particles in the supernatant The prepared virus-like particle supernatant was mixed with reducing protein loading buffer, separated by SDS-PAGE, and the protein was transferred to the NC membrane using a membrane transfer apparatus, and 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 in a gel imager.

[0043] Western Blot 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 obviously present in the supernatant and can be used as a detection sample for the double antibody sandwich ELISA method.

[0044] 3. Detection of Ebola virus VP40 virus-like particles by double antibody sandwich ELISA (1) Dilute antibody 3F06 to 2 μg / mL with carbonate coating solution, add 100 μL to each well of the ELISA plate, seal the plate with a sealing film, and place at 4°C overnight; (2) Discard the liquid in the wells and wash the plate with PBST solution, 300 μL / well, wash the plate 3 times in total, and pat the plate dry; (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; (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; (5) After blocking, discard the liquid in the wells and wash the plate with PBST solution, 300 μL / well, wash the plate 3 times in total, and pat the ELISA plate dry; (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; (7) Discard the liquid in the wells and wash the plate with PBST solution, 300 μL / well, wash the plate 3 times in total, and pat the plate dry; (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. (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; (10) Add 100 μL of color development solution to each well and incubate at room temperature for 10 min. (11) Add 50 μL of stop solution to each well and gently shake the plate until mixed. (12) Use an ELISA reader to measure the absorbance of each well at 450 nm and 630 nm and read the value within 10 min after termination.

[0045] 4. Experimental Results 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 relatively high. The above data show 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, 51-57 and 96-106 of SEQ ID NO: 1, respectively; or 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: 1, or, 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 nanobody against Ebola virus VP40 protein is shown in SEQ ID NO: 3, or, 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 a polynucleotide encoding the heavy chain variable region of the nanobody against Ebola virus VP40 protein as described in 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 as claimed in claim 4.

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

7. A detection kit containing the nanoantibody 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 nanobody against Ebola virus VP40 protein whose amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1 is the detection antibody, and the nanobody against Ebola virus VP40 protein whose amino acid sequence of the heavy chain variable region is 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 a nanobody against 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 Ebola virus VP40 protein whose amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 2.

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