Nanobody 3e07 against ebola virus vp40 protein and use in virus detection

Abstract

The application discloses a nano antibody against Ebola virus VP40 protein and application thereof in Ebola virus detection, wherein the nano antibody against Ebola virus VP40 protein is a 3E07 antibody. The nano antibody against Ebola virus VP40 protein prepared by the application has the characteristics of high sensitivity and wide binding spectrum. A double antibody sandwich enzyme-linked immunosorbent assay method constructed by 3E07 and 3F06 antibodies can efficiently detect VP40 proteins and virus-like particles of various Ebola virus subtypes such as Zaire, Sudan and Beni, wherein the detection sensitivity of the Ebola virus VP40 protein of Zaire type can reach 0.039 ng / mL, and the antibody combination has a wide application prospect in the diagnosis and pathogen detection of Ebola virus disease.

Description

TECHNICAL FIELD

[0001] The present application is a divisional application of Chinese Patent Application CN202510059894X.

[0002] The present application belongs to the technical field of immunology, and specifically relates to an antibody against Ebola virus VP40 protein and application thereof in Ebola virus detection. BACKGROUND

[0003] Ebola hemorrhagic fever is a severe infectious disease caused by Ebola virus infection. Since it was first discovered in 1976, there have been more than 40 Ebola outbreaks worldwide, infecting more than 34,000 people and killing about 15,000 people. Ebola virus is one of the most deadly viruses in the world, with a very high mortality rate after infection. WHO has listed it as one of the potential biological warfare agents, posing a great threat to human health and national security.

[0004] Ebola virus belongs to the Filoviridae family and has a long filamentous shape. It is an enveloped, non-segmented, single-stranded negative-sense RNA virus. The genome of Ebola virus is wrapped by nucleoprotein NP, which, together with polymerase L, viral protein VP35, and VP30, forms a nucleocapsid with transcription and replication functions, and interacts with VP24, which is wrapped by matrix protein VP40 attached to the inner membrane. VP40 is the most abundant protein in Ebola virus and is the most important target for immunological detection of Ebola virus. There are currently six 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 capable of infecting humans. Among them, EBOV is the most frequently occurring subtype of Ebola epidemic, with an overall mortality rate of over 65%.

[0005] Given the short incubation period and high mortality rate of Ebola virus infection, early diagnosis plays an important role in Ebola epidemic prevention and control. Polymerase chain reaction (PCR) is the most important method for detecting Ebola virus, but it is limited by strict laboratory conditions and cannot be widely used in epidemic areas. Immunological diagnosis methods are also commonly used for the diagnosis of Ebola virus infection due to their simplicity, speed, and low cost, including enzyme-linked immunosorbent assay (ELISA), lateral flow immunoassay (LFIA), indirect immunofluorescence (IFA), and immunohistochemistry.

[0006] There is a natural lack of light chain antibody in the peripheral blood of Camelidae, which includes a heavy chain variable region (VH) and constant region 2 (CH2) and constant region 3 (CH3), and the homodimer of this structure is called heavy chain antibody (HCAb), and the VH domain is the heavy chain antibody variable region fragment (VHH). The molecular weight of VHH is small, about 15 kDa, so it is also called Nanobody. The complementarity determining region 3 (CDR3) of VHH has a longer amino acid chain than human VH, which can form a structure similar to a convex ring, and can specifically bind to the active site of the enzyme and the special epitope of the virus particle, and enhance the recognition ability of the hidden antigen epitope. The purpose of the present application is to screen high-affinity anti-Ebola virus antibodies from llamas based on the unique advantages of Nanobody, and then develop a more specific and more sensitive immunological detection method for Ebola virus. SUMMARY

[0007] Based on the above purpose, the present application first provides a Nanobody against Ebola virus VP40 protein, wherein the amino acid sequences of CDR1, CDR2 and CDR3 regions of the heavy chain variable region of the Nanobody against Ebola virus VP40 protein are respectively shown as SEQ ID NO: 1 at positions 26-33, 51-57 and 96-106; or

[0008] The amino acid sequences of CDR1, CDR2 and CDR3 regions of the heavy chain variable region of the Nanobody against Ebola virus VP40 protein are respectively shown as SEQ ID NO: 2 at positions 26-33, 51-58 and 97-122.

[0009] The Nanobody provided by the present application is a heavy chain-only antibody, which includes a variable region and a constant region, and the variable region has three complementarity determining regions (CDRs): CDR1, CDR2 and CDR3, which have high variability and diversity. The sequence diversity of CDR regions determines the specificity and affinity of the antibody, because they recognize and bind to specific antigenic determinants by interacting with antigens.

[0010] In a preferred embodiment, the amino acid sequence of the heavy chain variable region of the Nanobody against Ebola virus VP40 protein is shown as SEQ ID NO: 1, and in the present application, the Nanobody with this variable region is named "3E07"; or,

[0011] The amino acid sequence of the heavy chain variable region of the Nanobody against Ebola virus VP40 protein is shown as SEQ ID NO: 2, and in the present application, the Nanobody with this variable region is named "3F06".

[0012] Secondly, the present application provides a polynucleotide encoding the anti-Ebola virus VP40 protein nanobody described above, the sequence of the polynucleotide encoding the heavy chain variable region of the anti-Ebola virus VP40 protein nanobody is shown as SEQ ID NO: 3, in the present application, nanobody 3E07 is an antibody with the coding sequence, or,

[0013] the sequence of the polynucleotide encoding the heavy chain variable region of the anti-Ebola virus VP40 protein nanobody is shown as SEQ ID NO: 4, in the present application, nanobody 3F06 is an antibody with the coding sequence.

[0014] Thirdly, the present application provides a vector containing the polynucleotide encoding the heavy chain variable region of the anti-Ebola virus VP40 protein nanobody described above. The vector is used for cloning and / or expressing the coding gene of the anti-Ebola virus VP40 protein nanobody, in a specific embodiment of the present application, 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 in the present application.

[0015] Fourthly, the present application provides a host cell containing the vector containing the polynucleotide encoding the heavy chain variable region of the anti-Ebola virus VP40 protein nanobody described above. The host cell is used for expressing the anti-Ebola virus VP40 protein nanobody described above. In a specific embodiment of the present application, the host cell is Expi293F cell. Other host cells known to those skilled in the art, especially eukaryotic host cells, can also be used for expression of the nanobody in the present application.

[0016] Fifthly, the present application provides the use of the anti-Ebola virus VP40 protein nanobody described above in the preparation of a medicament for treating and / or preventing Ebola virus disease. The nanobody provided by the present application has excellent affinity and binding activity with Ebola virus VP40 protein, and can specifically target Ebola virus VP40 protein, therefore, by using these characteristics of the nanobody, it can be used as a specific inhibitor of Ebola virus and host cell binding, or a therapeutic drug specifically targeting infectious lesions or pathogens to play a role in clinical treatment or prevention of infection, therefore, the present application provides the use of the anti-Ebola virus VP40 protein nanobody described above in the preparation of a medicament for treating and / or preventing Ebola virus disease.

[0017] Sixth, the present application provides a detection kit containing the above-mentioned anti-Ebola virus VP40 protein nanobody. Based on the excellent affinity of the nanobody provided by the present application to the Ebola virus VP40 protein, it can be used to detect the Ebola virus particles with VP40 protein that may exist in the sample. The detection antigen is a single antibody detection, that is, the nanobody specifically binds to the pathogen as a primary antibody, and the secondary antibody is used to detect the binding; it can also be a double antibody combination detection.

[0018] In a preferred embodiment, the detection kit is a double antibody sandwich immunoassay kit. The double antibody sandwich method described in the present application is a commonly used immunological detection technique, which is usually used to detect macromolecular antigens such as proteins, polypeptides, etc. It is mainly based on the principle of specific binding of antigen and antibody. Two specific antibodies, i.e. capture antibody and detection antibody, are used to sandwich the target antigen. The capture antibody is first immobilized on a solid support (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 immobilized on the solid support. Then, the labeled detection antibody is added, which specifically binds to another part of the antigen that has been bound to the solid support to form an antibody-antigen-antibody complex. Finally, the labeled detection antibody is detected by a specific detection method (such as the color development reaction in enzyme-linked immunosorbent assay) to indirectly determine the content of the target antigen in the sample.

[0019] In the present application, the combination of capture antibody and detection antibody in the detection kit is the anti-Ebola virus VP40 protein nanobody (3E07) with the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 1 and the anti-Ebola virus VP40 protein nanobody (3F06) with the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 2. In the technical solutions provided by the present application, the 3E07 and 3F06 can be used as capture antibodies or detection antibodies, and when one antibody is used as a capture antibody, the other antibody is used as a detection antibody, i.e. the combination of capture antibody and detection antibody described in the present application.

[0020] The immunoassay described in the present application is not limited to the disclosed enzyme-linked immunoassay, but can also include but is not limited to the technical field of radioimmunoassay, chemiluminescent immunoassay, etc.

[0021] In one specific embodiment, the nanobody against Ebola virus VP40 protein (3E07) with the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 1 is used as a detection antibody, and the nanobody against Ebola virus VP40 protein (3F06) with the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 2 is used as a capture antibody. In another alternative technical solution of the present application, 3E07 can be used as a capture antibody, and 3F06 can be used as a detection antibody.

[0022] In the specific implementation of the present application, the fusion protein with the C-terminus of the nanobody 3E07 and the nanobody 3F06 described above fused to the human Fc protein is used as a detection antibody or a capture antibody. VHH coupled with Fc is a conventional expression vector construction method. On one hand, the addition of Fc facilitates affinity purification. On the other hand, Fc forms a dimer, which is conducive to improving the binding capacity of the antibody to the antigen. Those skilled in the art can also select other purification tags to achieve the same technical purpose.

[0023] Finally, the present application provides an antibody composition against Ebola virus VP40 protein, which comprises the nanobody against Ebola virus VP40 protein (3E07) with the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 1 and the nanobody against Ebola virus VP40 protein (3F06) with the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 2. The example of the double-antibody sandwich ELISA method for detecting Ebola virus VP40 protein in the present application verifies that the two nanobodies are directed against different antigen binding epitopes of the VP40 protein. Therefore, when the two antibodies are used as an antibody composition, they can simultaneously bind to the same Ebola virus, further increasing the influence on the three-dimensional structure of the Ebola virus and the influence on the binding efficiency of the Ebola virus and 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 application provides a composition containing the two antibodies against Ebola virus VP40 protein.

[0024] The present application screens two nanobodies against Ebola virus VP40 protein through immunization of a llama and screening of a phage antibody library. The two nanobodies both have a unique heavy chain variable region CDR region, which has excellent binding activity to Ebola virus VP40 protein, EC 50 50, and the affinity KD to Ebola virus VP40 protein is 2.55×10 -9 M and 1.79×10 -9 M, respectively. The excellent binding activity and affinity show that the nanobodies provided by the present application can be used in the preparation of a medicine for treating and / or preventing Ebola virus disease.

[0025] The two nanobodies against Ebola virus VP40 protein provided by the application can bind to different antigen epitopes in the Ebola virus VP40 protein, therefore, the application provides a combination of the two nanobodies as detection antibodies and capture antibodies for double antibody sandwich method immunodetection. In the embodiment of the double antibody sandwich ELISA method for detecting Zaire Ebola virus VP40 protein constructed by the application, the detection sensitivity is 0.039 ng / mL, and the VP40 protein and virus-like particles of various subtypes of Ebola virus such as Zaire, Sudan and Bundibugyo can be efficiently detected, which has a wide application prospect in the diagnosis and pathogen detection of Ebola virus disease. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 SDS-PAGE identification results of antibodies 3E07 and 3F06, lane 1 is Marker, lane 2 is antibody 3E07, and lane 3 is antibody 3F06;

[0027] Figure 2 ELISA binding experiment results of antibody 3E07;

[0028] Figure 3 ELISA binding experiment results of antibody 3F06;

[0029] Figure 4 BLI affinity determination results of antibody 3E07;

[0030] Figure 5 BLI affinity determination results of antibody 3F06;

[0031] Figure 6 Standard curve of double antibody sandwich ELISA method for detecting EBOV-VP40 protein;

[0032] Figure 7 Western Blot identification of multi-subtype Ebola virus VP40 virus-like particle packaging results;

[0033] Figure 8 Results of double antibody sandwich ELISA method for detecting multi-subtype Ebola virus VP40 virus-like particles. DETAILED DESCRIPTION

[0034] The application will be further described below in conjunction with specific embodiments, and the advantages and characteristics of the application will be more apparent as the description proceeds. However, these embodiments are only exemplary and do not constitute any limitation on the protection scope defined by the claims of the application.

[0035] The reagents, methods and devices used in the embodiments of the application are conventional reagents, methods and devices in the technical field, and unless otherwise specified, the reagents and materials used are commercially available.

[0036] Example 1. Expression and identification of anti-Ebola virus VP40 antibodies

[0037] 1. Acquisition and expression purification of anti-Ebola virus VP40 antibodies

[0038] The eukaryotic expression of 1 mg of Zaire Ebola virus VP40 protein (genbank ID: 911825) was mixed with Freund's complete adjuvant at 1:1, and the alpaca was immunized subcutaneously on the back with multiple points, and the immunization was boosted every 14 days, and the Freund's complete adjuvant was replaced with Freund's incomplete adjuvant, and the serum titer was monitored by ELISA. The peripheral blood of the alpaca was collected, the lymphocytes were separated by density gradient centrifugation, and the RNA was extracted and reverse transcribed into cDNA. The VHH gene was amplified by PCR using the cDNA as a template, and the phagemid pComb3X-VHH was constructed by enzyme digestion and ligation. The phagemid was transformed into E. coli XL1-Blue by electroporation, and the helper phage VCSM13 was added in the logarithmic phase, and the phage was collected after overnight culture, and the anti-Ebola virus VP40 protein phage display nanobody library was obtained. The anti-Ebola virus VP40 protein nanobodies were selected from the prepared antibody library by solid-phase affinity panning method, and after several rounds of selection, clones were selected for phage-ELISA identification, and the positive clones were sequenced, and 2 strains of anti-VP40 specific nanobodies were obtained, which were named as “3E07” and “3F06”, respectively.

[0039] The sequencing results show that the amino acid sequence of the heavy chain variable region of the nanobody 3E07 is shown as SEQ ID NO: 1, wherein the amino acid sequences of CDR1, CDR2 and CDR3 regions of the heavy chain variable region are shown as SEQ ID NO: 1 26-33, 51-57 and 96-106, respectively.

[0040] The amino acid sequence of the heavy chain variable region of the antibody 3F06 is shown as SEQ ID NO: 2, wherein the amino acid sequences of CDR1, CDR2 and CDR3 regions of the heavy chain variable region are shown as SEQ ID NO: 1 26-33, 51-58 and 97-122, respectively.

[0041] 2. Eukaryotic expression and purification of antibodies

[0042] The nanobody sequence obtained by sequencing in step 1 was codon-optimized for human, the codon-optimized sequence of nanobody 3E07 is shown as SEQ ID NO. 3, and the codon-optimized sequence of nanobody 3F06 is shown as SEQ ID NO. 4. The 3' end of the sequence was fused with the human Fc protein gene (the sequence is shown as SEQ ID NO. 5, wherein the first to 12th bp is a linker peptide GGGS encoding), and constructed into a pcDNA3.4 vector. The PEI transfection reagent (Yoxing Biological, 40816ES01) was used to transfect Expi293F cells for expression. The next day, 5% protein-free feed (Aupharma, F081918) and 2 mM protein expression enhancer sodium valproate (VPA) (MCE, HY-10585A) were added. 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 into PBS buffer by ultrafiltration for storage.

[0043] 3. SDS-PAGE detection of antibody purity

[0044] 5 μg of the purified antibody was mixed with a protein reducing buffer, the system was supplemented with PBS to 30 μL, and the protein was denatured by heating at 100°C for 10 minutes. 9 μL was added to a polyacrylamide gel well, and electrophoresis was performed for separation. The gel was dyed and decolored by a dyeing and decoloring instrument, and the gel image was scanned and saved.

[0045] The SDS-PAGE detection result is shown in Figure 1 The molecular weight of the two bands corresponding to the antibody 3E07 and 3F06 fusion human Fc protein under reducing conditions is about 40 kDa, which is consistent with the expected protein size, and no obvious impurity band is present, and the purity is good.

[0046] Example 2. Analysis of the binding activity of anti-Ebola virus VP40 antibody

[0047] 1. The Zaire Ebola virus VP40 protein was diluted to 1 μg / mL with carbonate coating solution, added to the enzyme-labeled plate well, 100 μL per well, sealed with sealing film, and placed at 4°C overnight;

[0048] 2. Discard the liquid in the well, wash the plate with PBST washing solution, 300 μL / well, soak for a total of 3 times, and pat the enzyme-labeled plate dry;

[0049] 3. Add 100 μL of blocking solution (containing 3% BSA by mass fraction in PBST) to each well, seal with sealing film, and incubate at 37°C for 1 h;

[0050] 4. After the end of the closure, the liquid in the wells was discarded, the plate was washed with PBST solution, 300 μL / well was soaked, and the plate was washed a total of 3 times, and the enzyme-labeled plate was patted dry;

[0051] 5. During the period, 100 μL of 2 μg / mL of the first hole concentration of 4-fold diluted 3E07 and 3F06 antibodies was added to the enzyme-labeled plate, and incubated at 37°C for 1 h;

[0052] 6. The liquid in the wells was discarded, the plate was washed with PBST solution, 300 μL / well was soaked, and the plate was washed a total of 3 times, and the enzyme-labeled plate was patted dry;

[0053] 7. 100 μL of diluted anti-human IgG (HRP) antibody was added to the corresponding plate wells, and the plate was sealed with a sealing film and incubated at 37°C for 1 h;

[0054] 8. The liquid in the wells was discarded, the plate was washed with PBST solution, 300 μL / well was soaked, and the plate was washed a total of 3 times, and the enzyme-labeled plate was patted dry;

[0055] 9. 100 μL of color developing solution was added to each well, and incubated at room temperature for 15 min;

[0056] 10. 50 μL of stop solution was added to each well, and the enzyme-labeled plate was gently shaken to mix evenly;

[0057] 11. The absorbance values of each well at 450 nm and 630 nm wavelengths were measured by an enzyme-labeled instrument, and the readings were taken within 10 min after termination;

[0058] 12. The results of the ELISA binding experiment showed that the antibodies 3E07 ( Figure 2 ) and 3F06 ( Figure 3 ) could specifically bind to Ebola virus VP40 protein, and the EC 50 values were 13.16 ng / mL and 19.85 ng / mL, respectively.

[0059] Example 3. Affinity analysis of anti-Ebola virus VP40 antibodies

[0060] The Ni-NTA sensor was rinsed in buffer for 10 min; the baseline was stabilized for 60 seconds with buffer, the sensor was passed through Ebola virus VP40 protein diluted with buffer, the baseline was stabilized for 60 seconds with buffer, and then the antibodies 3E07 and 3F06 were passed through at a first hole concentration of 250 nM with a 2-fold gradient dilution for 300 seconds of binding and 300 seconds of dissociation in buffer, and all raw data were fitted by Fortebio data analysis software.

[0061] The results of BLI analysis are shown in Figure 4 and Figure 5The fitting lines represent the binding and dissociation of antibodies 3E07 and 3F06 with VP40 protein over time, respectively, and the results show that the affinity of antibodies 3E07 and 3F06 to bind to Ebola virus VP40 protein is 2.55 x 10 -9 M and 1.79 x 10 -9 M, respectively.

[0062] Example 4. Establishment of a double antibody sandwich ELISA method

[0063] 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.

[0064] 1. Biotinylation of Ebola virus VP40 protein antibodies

[0065] NHS esterified biotin EZ-link NHS-PEG4-biotin was mixed with antibody 3E07 of the application at a molar ratio of 20:1, and the reaction labeling was carried out overnight at 4°C in 500 μL PBS; after the reaction was completed, the desalting column was centrifuged to replace the liquid, and the biotin-labeled antibody 3E07 was collected, and the antibody concentration was detected using the BCA method.

[0066] 2. Double antibody sandwich ELISA method for detecting Ebola virus VP40 protein

[0067] (1) Dilute antibody 3F06 to 2 μg / mL with carbonate coating solution, add 100 μL per well to the enzyme-labeled plate, seal with a sealing film, and place at 4°C overnight;

[0068] (2) Discard the liquid in the wells, wash the plate with PBST washing solution, immerse 300 μL per well, and wash the plate for a total of 3 times, and pat dry the enzyme-labeled plate;

[0069] (3) Add 100 μL of blocking solution (containing 3% BSA by mass fraction in PBST) per well, seal with a sealing film, and incubate at 37°C for 2 h;

[0070] (4) During this period, mix the serially diluted Ebola virus VP40 protein standard (final concentration: 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98, 0 ng / mL) with biotin-labeled antibody 3E07 (final concentration: 1 μg / mL), and incubate at 37°C for 1 h;

[0071] (5) After blocking, discard the liquid in the wells, wash the plate with PBST washing solution, immerse 300 μL per well, and wash the plate for a total of 3 times, and pat dry the enzyme-labeled plate;

[0072] (6) Add the above-mentioned series of diluted standards and biotin-labeled 3E07 antibody premix liquid into the corresponding hole plate, and incubate at 37°C for 1 h;

[0073] (7) Discard the liquid in the hole, wash the plate with PBST, soak for 300 μL / hole, and wash the plate for a total of 3 times, and pat dry the enzyme-labeled plate;

[0074] (8) Add 100 μL of diluted Streptavidin-HRP into the corresponding plate hole, seal the plate with a sealing film, and incubate at 37°C for 1 h;

[0075] (9) Discard the liquid in the hole, wash the plate with PBST, soak for 300 μL / hole, and wash the plate for a total of 3 times, and pat dry the enzyme-labeled plate;

[0076] (10) Add 100 μL of color developing liquid to each hole, and incubate at room temperature for 15 min;

[0077] (11) Add 50 μL of termination liquid to each hole, and gently shake the enzyme-labeled plate to mix evenly;

[0078] (12) Measure the absorbance values of each hole at 450 nm and 630 nm wavelengths with an enzyme-labeled instrument, and read the values within 10 min after termination.

[0079] 3. Drawing of standard curve

[0080] Absorbance value OD 450nm - 630nm The results are shown in Table 1. According to the results in Table 1, the ELISA standard curve is drawn with the absorbance value OD 450nm - 630nm as the vertical coordinate, and the standard concentration (1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 ng / mL) as the horizontal coordinate, the formula of the standard curve is obtained by linear regression, and the standard curve is prepared.

[0081] 4. Sensitivity calculation

[0082] The detection sensitivity is the average value of the negative sample (OD 450nm - 630nm ) + 2 times the SD value corresponding to the Ebola virus VP40 protein concentration.

[0083] 5. Experimental results

[0084] Table 1 OD 450 -OD 630 reading of double antibody sandwich method for quantitative detection of Ebola virus VP40 protein

[0085]

[0086] The read values at the concentrations of 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 ng / mL were selected to draw a standard curve, and the standard curve formula and R were calculated by linear regression 2 . The results are shown in Table 1, and the standard curve formula is Y = 0.04170 * X + 0.03746, and R = 0.9942. It is calculated that the sensitivity of the bispecific antibody sandwich ELISA method of the application is 0.039 ng / mL. Figure 6 2

[0087] Example 5. Detection of Ebola virus VP40 virus-like particles by bispecific antibody sandwich ELISA method

[0088] 1. Preparation of multi-subtype Ebola virus VP40 virus-like particles

[0089] HEK293T was inoculated in a 6-well cell culture plate at a density of 5 x 10 5 / mL, 2 mL per well, and incubated at 37 °C in a carbon dioxide incubator for 12 h before cell transfection;

[0090] 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 instructions, and incubated at 37 °C in a carbon dioxide incubator for 20 h.

[0091] 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 Ebola virus VP40 virus-like particles were contained in the supernatant.

[0092] 2. Western blot verification of Ebola virus VP40 virus-like particles in supernatant

[0093] ​​The prepared virus-like particle supernatant was mixed with a reducing protein loading buffer, separated by SDS-PAGE, transferred to an NC membrane using a transmembrane instrument, and incubated with an antibody using a Mouse anti-His tag pAb as a primary antibody and a Goat anti-mouse IgG (HRP) as a secondary antibody. After development with a developing solution, the gel was imaged in a gel imager.

[0094] The Western Blot identification result is shown in Figure 7 The band size is about 40 kDa, which is consistent with the theoretical molecular weight of the Ebola virus VP40 protein monomer of each serotype. There is no obvious band in the group without transfected plasmid, which proves that the Ebola virus VP40 virus-like particles are obviously present in the supernatant and can be used as a detection sample for double antibody sandwich ELISA.

[0095] 3. Double antibody sandwich ELISA method for detecting Ebola virus VP40 virus-like particles

[0096] (1) Dilute the antibody 3F06 to 2 μg / mL with a carbonate coating solution, add 100 μL per well to the enzyme-labeled plate, seal the plate with a sealing film, and place it at 4°C overnight;

[0097] (2) Discard the liquid in the wells, wash the plate with PBST, immerse 300 μL per well, and wash the plate for a total of 3 times, and dry the enzyme-labeled plate;

[0098] (3) Add 100 μL of blocking solution (containing 3% BSA by mass fraction in PBST) per well, seal the plate with a sealing film, and incubate at 37°C for 2 h;

[0099] (4) During this period, mix the collected virus-like particle packaging supernatant with NP40 lysis buffer according to a volume ratio of 1:4, lyse at room temperature for 5 min, and then mix with biotinylated antibody 3E07 according to a volume ratio of 1:1, and incubate at 37°C for 1 h;

[0100] (5) After blocking, discard the liquid in the wells, wash the plate with PBST, immerse 300 μL per well, and wash the plate for a total of 3 times, and dry the enzyme-labeled plate;

[0101] (6) Add the above series of diluted Ebola virus VP40 virus-like particle lysates and biotin-labeled 3E07 antibody premix to the corresponding wells, and incubate at 37°C for 1 h;

[0102] (7) Discard the liquid in the wells, wash the plate with PBST, immerse 300 μL per well, and wash the plate for a total of 3 times, and dry the enzyme-labeled plate;

[0103] (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;

[0104] (9) Discard the liquid in the hole, wash the plate with PBST solution, 300 μL / hole soak, a total of 3 times of plate washing, and dry the enzyme-labeled plate;

[0105] (10) Add 100 μL of color developing solution to each hole, and incubate at room temperature for 10 min;

[0106] (11) Add 50 μL of termination solution to each hole, and gently shake the enzyme-labeled plate until it is mixed evenly;

[0107] (12) Measure the absorbance values of each hole at 450 nm and 630 nm wavelengths with an enzyme-labeled instrument, and read within 10 min after termination.

[0108] 4. Experimental results

[0109] The results of identifying Ebola virus VP40 virus-like particles by double antibody sandwich ELISA method are shown in Table 1. Figure 8 As shown in Table 1, the OD value of the group without transfected VP40 plasmid is extremely low (lower than the detection lower limit of the standard curve of double antibody sandwich ELISA method) because the supernatant does not contain VP40 virus-like particles, and the OD value of Ebola virus VP40 virus-like particles of various genera is higher because the VP40 protein is released after lysis. The above data show that the double antibody sandwich ELISA method of the present application can identify all Ebola virus VP40 proteins and virus-like particles that have the ability to infect humans, and has important application prospects in early diagnosis of Ebola virus.

Claims

1. A Nanobody against the Ebola virus VP40 protein, characterized in that, The amino acid sequences of CDR1, CDR2 and CDR3 regions of the heavy chain variable region of the anti-Ebola virus VP40 protein nanobody are respectively shown in SEQ ID NO: 1 at positions 26-33, 51-57 and 96-106.

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

1.

3. A polynucleotide encoding the Nanobody against the Ebola virus VP40 protein of 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:

3.

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

5. A host cell containing the vector of claim 4 containing the polynucleotide of claim 4 encoding the heavy chain variable region of the anti-Ebola virus VP40 protein nanobody.

6. A detection kit containing the anti-Ebola virus VP40 protein nanobody of claim 1 or 2.

Images