Recombinant mouse antibodies targeting adenovirus dbp protein and uses thereof

Abstract

The present application provides a monoclonal antibody targeting adenovirus DBP protein, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, and the light chain variable region comprising light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the amino acid sequences of HCDR1, HCDR2 and HCDR3 are respectively shown as SEQ ID NOs: 1-3, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are respectively shown as SEQ ID NOs: 4-6. The monoclonal antibody of the present application can be used for efficient detection of multiple types of HAdV DBP.

Description

Technical Field

[0001] This invention relates to the fields of immunology and molecular biology, and more particularly to monoclonal antibodies targeting adenovirus DBP protein and their uses. Background Technology

[0002] Human adenovirus (HAdV) belongs to the family Adenoviridae and the genus Mastadenovirus, comprising seven species: Human Adenovirus A (HAdV-A) to Human Adenovirus G (HAdV-G). As of March 2024, 116 types of HAdV have been identified. Human adenovirus is a non-enveloped, double-stranded DNA virus with a genome length of approximately 35 kb. The virus is spherical, and under negative-stain electron microscopy, the viral particle diameter is approximately 80 nm. The viral particle consists of a protein capsid and a DNA-containing core. The capsid is typically icosahedral in shape, composed of 252 capsomeres, containing 240 hexons and 12 pentons. The human adenovirus genome contains five early transcription units (E1A, E1B, E2, E3, and E4), four intermediate transcription units, and one late transcription unit. Late transcription units express capsid proteins, small capsid proteins, and core proteins, which are important antigenic components of the virus. Non-structural proteins mainly consist of preterminal proteins (pTP), viral DNA polymerase (Ad-Pol), viral single-stranded DNA-binding protein (DBP), and other early proteins, playing important roles in various stages of the viral life cycle.

[0003] Human adenoviruses can cause a variety of human diseases. Due to the different tissue tropisms of different adenovirus types, they can lead to infections and related diseases in multiple sites, including the respiratory, digestive, and urinary / reproductive systems. This virus is highly contagious, and outbreaks of respiratory infections caused by human adenovirus occur frequently in my country, with some areas experiencing clusters of severe cases. Furthermore, human adenoviruses can undergo genetic recombination, producing novel strains with higher pathogenicity and stronger infectivity. The virus's structural proteins are immunogenic and effectively stimulate the host's immune response. The adenovirus nonstructural protein DBP is encoded by the early gene E2A. Studies have shown that DBP is highly expressed in the early stages of viral infection and exhibits strong immunogenicity, inducing a strong antibody response in the early stages of infection. Furthermore, it shows strong conservation among different types of adenoviruses (see Guo L, Wu C, Zhou H, et al. Identification of a nonstructural DNA-binding protein (DBP) as an antigen with diagnostic potential for human adenovirus. PLoS One. 2013;8(3):e56708.), thus possessing potential application value as a diagnostic biomarker.

[0004] Hybridoma cells are formed by fusing B cells capable of producing specific antibodies with myeloma cells that can proliferate indefinitely in vitro. They possess both the ability to continuously secrete monoclonal antibodies and the characteristic of long-term passage. Monoclonal antibodies, due to their high specificity, high homogeneity, strong targeting, low toxicity, and mild side effects, have been widely used in the diagnosis and treatment of various diseases. Therefore, specific monoclonal antibodies against human adenovirus DBP have significant application value in the development of reagents for viral infection detection, etiology, and immunology. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a specific monoclonal antibody against human adenovirus DBP, which can be used for efficient detection of multiple types of HAdV DBP.

[0006] A first aspect of the present invention provides a monoclonal antibody targeting adenovirus DBP protein, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein:

[0007] The amino acid sequence of HCDR1 is shown in SEQ ID NO: 1, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 2, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 3; the amino acid sequence of LCDR1 is shown in SEQ ID NO: 4, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 5, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 6.

[0008] In a preferred embodiment of the present invention, the heavy chain variable region further includes heavy chain frame regions HFR1, HFR2, HFR3, and HFR4, and the light chain variable region further includes light chain frame regions LFR1, LFR2, LFR3, and LFR4, wherein:

[0009] The amino acid sequence of HFR1 is shown in SEQ ID NO: 7, the amino acid sequence of HFR2 is shown in SEQ ID NO: 8, the amino acid sequence of HFR3 is shown in SEQ ID NO: 9, and the amino acid sequence of HFR4 is shown in SEQ ID NO: 10; the amino acid sequence of LFR1 is shown in SEQ ID NO: 11, the amino acid sequence of LFR2 is shown in SEQ ID NO: 12, the amino acid sequence of LFR3 is shown in SEQ ID NO: 13, and the amino acid sequence of LFR4 is shown in SEQ ID NO: 14.

[0010] In a preferred embodiment of the present invention, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 16.

[0011] In a preferred embodiment of the present invention, the monoclonal antibody is a recombinant mouse antibody.

[0012] A second aspect of the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the above-described monoclonal antibody.

[0013] In a preferred embodiment of the invention, the nucleic acid molecule comprises a nucleotide sequence encoding the heavy chain variable region, as shown in SEQ ID NO: 17; and a nucleotide sequence encoding the light chain variable region, as shown in SEQ ID NO: 18.

[0014] A third aspect of the present invention provides a carrier comprising the above-described nucleic acid molecules.

[0015] A fourth aspect of the present invention provides a cell comprising the above-described nucleic acid molecule or the above-described carrier.

[0016] A fifth aspect of the present invention provides a kit for detecting human adenovirus, comprising the above-described monoclonal antibody.

[0017] In a preferred embodiment of the present invention, the kit is a Western Blot kit, which further comprises: a fluorescently labeled conjugated second antibody, a nitrocellulose membrane, a blocking solution, and a washing buffer.

[0018] In a more preferred embodiment of the present invention, the fluorescently labeled second antibody is an IRDye 800CW-conjugated goat anti-mouse IgG (H+L) antibody; the blocking solution is a 5% skim milk / PBS solution; and the washing buffer is a PBST solution.

[0019] In another preferred embodiment of the present invention, the kit is an immunofluorescence detection kit, which further comprises: a fluorescently labeled second antibody, a cell fixative, a blocking solution, a cell permeabilizer, and a washing buffer.

[0020] In a more preferred embodiment of the present invention, the fluorescently labeled second antibody is an Alexa 488-conjugated goat anti-mouse IgG (H+L) antibody; the cell fixation solution is a 4% paraformaldehyde solution; the blocking solution is a 5% BSA / PBS solution; the cell permeabilizer is Triton-X100; and the washing buffer is a PBS solution.

[0021] A sixth aspect of the present invention provides a method for preparing the above-described monoclonal antibody, comprising culturing the above-described cells and isolating and recovering the antibody therefrom.

[0022] The seventh aspect of the present invention provides the use of the above-described monoclonal antibody, the above-described nucleic acid molecule, the above-described vector, or the above-described cell for preparing a reagent for detecting human adenovirus in a sample.

[0023] The eighth aspect of the present invention provides a method for detecting human adenovirus in a sample, comprising: firstly binding the sample to the above-mentioned monoclonal antibody, then adding a labeled second antibody, and obtaining the detection result of human adenovirus in the sample by detecting the reaction signal.

[0024] In a preferred embodiment of the present invention, the human adenovirus is selected from HAdV-A12, HAdV-B3, HAdV-C5, HAdV-D8, HAdV-E4, HAdV-F41, HAdV-B7, HAdV-B11, HAdV-B14, and HAdV-B21.

[0025] In a preferred embodiment of the present invention, the method for detecting the reaction signal is Western blotting or immunofluorescence detection. Attached Figure Description

[0026] Figure 1 The results of HAdV DBP protein gel electrophoresis identification in Example 1.1 are as follows: (A) HAdV-A12; (B) HAdV-B3; (C) HAdV-C5; (D) HAdV-D8; (E) HAdV-E4; (F) HAdV-F41.

[0027] Figure 2 The results show the serum titers of HAdV DBP protein in mice immunized in Example 1.2. (A) HAdV-B3 DBP; (B) HAdV-E4 DBP; (C) HAdV-C5 DBP; (D) HAdV-D8 DBP; (E) HAdV-A12 DBP; (F) HAdV-F41 DBP.

[0028] Figure 3 The results show the titer of the mouse monoclonal antibody against different types of HADV DBP protein in Example 2.3.

[0029] Figure 4 The results of Western blot analysis of different types of HADV DBP protein using mouse recombinant monoclonal antibody 19E3 in Example 3.1 are shown.

[0030] Figure 5 The results are from Western blot analysis of mouse recombinant monoclonal antibody 19E3 used in Example 3.2 to detect different types of HADV-infected cell samples.

[0031] Figure 6 The results of immunofluorescence detection of mouse recombinant monoclonal antibody 19E3 in multiple HADV-infected cell samples in Example 4 are shown. NC: Uninfected control group; Scale bar: 50 mm. Detailed Implementation

[0032] It should be noted that, unless otherwise defined, the technical or scientific terms used in this application shall have the ordinary meaning as understood by one of ordinary skill in the art.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all reagents and materials used in the following examples are commercially available products.

[0034] Example 1: Screening of murine monoclonal antibodies against adenovirus DBP

[0035] Example 1.1 Preparation of Adv DBP protein

[0036] (1) Representative adenovirus strains infecting humans were selected, specifically HAdV-A12 (AdV12), HAdV-B3 (AdV3), HAdV-C5 (AdV5), HAdV-D8 (AdV8), HAdV-E4 (AdV4), and HAdV-F41 (AdV41). The DBP protein sequences of the above viral strains were codon-optimized, and after gene synthesis, they were constructed into the pFastBacHT A (Invitrogen) vector through Xho I-Hind III double restriction sites to obtain plasmids. The plasmids were transfected into sf9 cells, and after culturing for three days until obvious cytopathic effects were observed, the supernatant was harvested to obtain baculoviruses.

[0037] (2) Baculovirus was infected into sf9 cells in good growth condition. After 7-9 days, the cell pellet was collected, treated with cell lysis buffer, purified by Ni-NTA column, concentrated by 30kDa ultrafiltration tube and replaced with 0.01M PBS (pH 7.4), quantified by BCA protein, and aliquoted and stored at -80℃.

[0038] Taking the preparation of AdV12 DBP protein as an example, in short, baculoviruses containing the AdV12 DBP gene are used to infect well-growing sf9 cells, which are then placed in a 37℃ cell culture incubator and cultured by shaking for 7-9 days. After centrifugation at 350g for 10 minutes, the supernatant is discarded, and the cell pellet is collected into a centrifuge tube. Three times the cell volume of cell lysis buffer (containing 20mM Tris-HCl, 300mM NaCl, RNase I, DNase, and protease inhibitor, pH 8.0) is added, and the cells are sonicated on ice for lysis. After centrifugation at 12100g for 20 minutes, the supernatant is transferred, and the sample is loaded into a Ni-NTA column by gravity at a flow rate of 0.5-1mL / min. The flow-through is collected, and the loading is repeated once. Washing buffer (20mM Tris-HCl, 300mM NaCl, 10mM imidazole, pH 8.0) is added at a flow rate of 1mL / min for 15 column volumes. Elution buffer (20mM Tris-HCl, 300mM NaCl, 10mM imidazole, pH 8.0) is added. Elution was performed using Tris-HCl, 300mM NaCl, 500mM imidazole (pH 8.0), at a flow rate of 0.5 mL / min, with a column volume of 3. The eluted liquid was collected and added to the inner tube of a 30 kDa ultrafiltration tube. After centrifugation at 4000g for 20 minutes, the filtrate was discarded, and the inner tube was replenished with 0.01M PBS (pH 7.4). Centrifugation was repeated once, and the liquid in the inner tube was collected. Protein quantification was performed using a BCA quantitative kit (Pierce), and the resulting aliquots were stored at -80℃. The preparation procedure for DBP of other viral strains is the same as above.

[0039] (3) Thaw the prepared DBP proteins of each viral strain on ice. Take 1 μg of each and add it to the protein loading solution (containing DTT (dithiothreitol), reducing, R and without DTT, non-reducing, NR). After denaturation at 95℃ for 10 minutes, add it to a 4-20% SDS-PAGE protein gel and run at 200V for 35 minutes. Then, identify the results by Coomassie staining using an eBlot staining instrument. The results are as follows: Figure 1 As shown in the figure. Protein gel electrophoresis results showed that the molecular weight of each type of DBP protein prepared was 70-80 kDa, and the purity was greater than 85%, which can be used for subsequent animal immunization and immunoassay.

[0040] Example 1.2 Mouse Immunization and Serum Antibody Titer Detection

[0041] (1) Ten female Balb / C mice aged 6-8 weeks were randomly divided into two groups. The immunization program for group A (n=5) was as follows: the initial immunization consisted of 50 μg / mouse of AdV3, AdV4 and AdV5 DBP proteins, and booster immunizations were performed on days 14, 21 and 28, with each booster consisting of 25 μg / mouse of AdV3, AdV4 and AdV5 DBP proteins. The immunization program for group B (n=5) was as follows: the initial immunization consisted of 50 μg / mouse of AdV8, AdV12 and AdV41 DBP proteins, and booster immunizations were performed on days 14, 21 and 28, with each booster consisting of 25 μg / mouse of AdV8, AdV12 and AdV41 DBP proteins. After the immunization program was completed, peripheral blood was collected from the mice via orbital blood collection into centrifuge tubes, centrifuged at 400 g for 10 minutes, and the serum was aliquoted and frozen at -80℃.

[0042] (2) Each DBP protein was diluted with coating buffer (1.59 g / L Na2CO3, 2.94 g / L NaHCO3 aqueous solution, pH 9.6) and coated onto ELISA plates, 50 ng per well. The plates were incubated overnight at 4°C, the supernatant was discarded, and the plates were blocked with 3% BSA / PBS at 37°C for 2 hours. The serum from each mouse after immunization was serially diluted 1:2000 to 1:12800 with sample dilution buffer (0.5% BSA / PBS) and added to ELISA plates coated with different antigens. The plates were incubated at 37°C for 1 hour. After washing three times with washing buffer (0.03% Tween-80 / PBS), HRP (horseradish peroxidase)-conjugated goat anti-mouse antibody (1:10000 dilution) was added, and the mixture was incubated at 37°C for 1 hour. After washing three times with washing buffer, 100 μL / well of ELISA chromogenic solution (Solepro) was added, and the mixture was incubated at 37°C for 10 minutes. Finally, 50 μL / well of stop solution (Solepro) was added, and the absorbance was read at 450 nm using an Ensight microplate reader. The results are as follows. Figure 2 As shown in the ELISA results, all immunized mice in each group were able to produce anti-DBP antibodies with titers greater than 128,000, indicating that the immunization of mice was successful.

[0043] Example 2: Hybridoma cell preparation and hybridoma sequencing

[0044] Example 2.1 Hybridoma cell preparation

[0045] (1) After the last immunization, the spleen of the mouse was removed in a sterile environment and a spleen cell suspension was prepared using a 100μm cell filter. SP2 / 0 mouse myeloma cells in the logarithmic growth phase and in good growth condition were prepared. The spleen cells and myeloma cells were mixed at a ratio of 10:1. After centrifugation at 400g, the supernatant was discarded. Preheated 50% polyethylene glycol solution was slowly added to the cell pellet and stirred to promote cell membrane fusion. The cells were diluted with RPMI-1640 medium to terminate the PEG effect. The fused cells were collected by centrifugation at 110g.

[0046] (2) The fused cells were resuspended in HAT (hypoxanthine-aminopterin-thymine) selective medium and seeded into 96-well cell culture plates. They were cultured in a constant temperature incubator at 37°C and 5% CO2. After observing a distinct clonal cluster, single-clonal cells were obtained by infinite dilution. After culturing for 7-14 days, the supernatant was collected, and the antibody titer was detected according to the ELISA procedure in Example 1.2. Positive cell wells were marked, and after two subcloning processes, a single hybridoma cell line stably secreting highly specific monoclonal antibodies was obtained and transferred to 24-well plates for scale-up culture.

[0047] Example 2.2 Antibody purification from hybridoma supernatant

[0048] (1) After digestion and centrifugation, the hybridoma cell lines in good growth condition were adjusted to a concentration of 5 × 10⁻⁶ using serum-free hybridoma cell culture medium (Sino Techological). 5Cells / mL were transferred to shake flasks and cultured on a cell shaker at 125 rpm, 37°C, and 5% CO2 for 3-5 days. AmgMagbeads Protein A (Genescript) was added and incubated for 2 hours. Purification was then performed using the AmgMag S1 automated antibody purification system. The procedure was as follows: Wash buffer (0.01M PBS, pH 7.4; 5 volumes, 3 times), Elution buffer (0.1M Glycine-HCl, pH 3.0; 10 volumes, 1 time, incubation time 10 minutes). The eluted liquid was transferred to centrifuge tubes, and 0.1 volume of neutralization buffer (1M Tris-HCl, pH 8.0) was added. The mixture was then concentrated by centrifugation using a 50kDa ultrafiltration tube, and the solution was replaced with 0.01M PBS (pH 7.4). Antibody concentration was quantified by OD280 value and stored at -80°C.

[0049] Example 2.3 ELISA detection of hybridoma antibody titer

[0050] ELISA plates coated with each DBP protein were prepared as described in Example 1.2. The antibodies to be tested were diluted with sample dilution buffer (0.5% BSA / PBS) at an initial concentration of 10 μg / mL, and then diluted 5-fold for a total of 7 dilutions. After incubation at 37°C for 1 hour, the plates were washed with washing buffer (0.03% Tween-80 / PBS) and incubated with secondary antibody (HRP-conjugated goat anti-mouse antibody). After washing again, color development was performed and the incubation was terminated. Absorbance values ​​were read at 450 nm. Concentration-response curves for each antibody were plotted using Prism software, and the EC50 value was calculated. An EC50 value less than 1000 ng / mL was considered a positive reaction. The results are as follows: Figure 3 As shown, the 19E3 antibody was screened and found to have an EC50 concentration of 2-5 ng / mL against different types of HADV DBP protein, exhibiting high-titer broad-spectrum antibody performance.

[0051] Example 2.4 Hybridoma antibody variable region gene sequencing

[0052] (1) Collect the hybridoma cell pellet (approximately 1 × 10⁻⁶) after amplification of clone 19E3 in Example 2.1. 6After centrifuging at 400g for 5 minutes and discarding the supernatant, RNA was extracted using the Super FastPure Cell RNA Isolation Kit (Vazyme, Cat: RC102). 500 μl of Buffer CRL was added to the cell pellet, and the mixture was thoroughly vortexed until no obvious cell clumps remained. Subsequent procedures were performed according to the kit instructions. The cells were eluted with 50 μl of RNase-free H2O, quantified using a Nanodrop microspectrophotometer, and then subjected to reverse transcription and variable region amplification (including light and heavy chains) using the Mouse Monoclonal Antibody Hybridoma Cell Gene Variable Region Fragment Amplification (Sequencing) Kit (Frdbio Bioscience & Technology, Cat: MAC0080K). The amplified product (~450 bp) was recovered from the target band using a gel and purified using the FastPure gel DNA Extraction Kit (Novizan, Cat: DC301-01). The product was eluted with 50 μl of RNase-free H2O, and 2 μl of the nucleic acid product was used for ultra-universal TOPO cloning. The kit (Novazia, Cat: C603) was constructed into a cloning vector, and after transformation into DH5α competent cells, 10 clones were selected. Sequencing was performed using M13F / R universal primers, and the antibody variable region gene sequence was analyzed using the IgBlast website.

[0053] Example 2.5 Preparation of Recombinant Mouse Monoclonal Antibody

[0054] (1) Positive clone V gene was synthesized by Beijing Ruiboxing Biotechnology Co., Ltd. and then cloned into mouse antibody expression vector. The heavy chain V gene was digested into IgVec-mIgG2 vector by AgeI-SalI double enzyme digestion, and the light chain V gene was digested into IgVec-mIgK vector by AgeI-BswiI double enzyme digestion.

[0055] (2) Take Expi293 cells in good growth condition and adjust the concentration to 2.5 × 10⁻⁶. 6 The heavy chain and light chain antibody plasmids were mixed at a ratio of 2:1. The transfection reagent PEI-MAX and plasmid were mixed at a ratio of 3:1 in Opti-MEM medium and slowly added dropwise to the cell suspension. The mixture was then placed in a cell shaker and cultured at 37°C, 8% CO2, and 125 rpm for 5-7 days to obtain recombinant mouse monoclonal antibodies. The antibodies were purified according to the mouse antibody purification process in Example 2.2, and quantified by OD280 value and BCA before use.

[0056] Example 3: Recombinant mouse antibody 19E3 used for Western Blot detection

[0057] Example 3.1 Recombinant mouse antibody 19E3 for Western blot detection of adenovirus DBP protein

[0058] (1) Different types of HADV DBP proteins, including HADV-B3, HADV-E4, HADV-C5, HADV-D8, HADV-A12 and HADV-F41, were expressed using the baculovirus expression system in Example 1.1. 100 ng and 10 ng were taken respectively, and 4× loading buffer (reducing, Beijing Solarbio Science & Technology Co., Ltd.) was added, and ddH2O was added to a concentration of 1×.

[0059] (2) Place the sample in a boiling water bath and heat at 100°C for 15 min to denature it. Then, centrifuge at 12000 g for an instantaneous time and add YoungPAGE. TM In the protein gel (GenScript), 30 μL of sample was loaded into each well, and the electrophoresis program was run at 200V for 50 min.

[0060] (3) The protein on the gel was transferred to a nitrocellulose membrane (NC membrane) using the eBlot L1 wet protein transfer system. After blocking with 5% skim milk / PBS at room temperature for 1 h, the membrane was washed three times with 0.1% PBST (PBS containing 0.1% Tween 20) for 10 min each time. 1:1000 diluted 19E3 antibody was added, and the membrane was incubated at room temperature with shaking for 1 h. The membrane was then washed three times again with 0.1% PBST. 1:10000 diluted IRDye 800CW-conjugated goat anti-mouse IgG (H+L) antibody (LI-COR, Cat:925-32210) was added, and the membrane was incubated at room temperature with shaking for 1 h. After washing three times with 0.1% PBST, the images were scanned and analyzed using the Odyssey® imaging system. Figure 4 As shown, the results indicate that the mouse recombinant monoclonal antibody 19E3 can detect multiple types of AdV DBP proteins, including HAdV-B3, HAdV-E4, HAdV-C5, HAdV-D8, HAdV-A12 and HAdV-F41.

[0061] Example 3.2 Recombinant mouse monoclonal antibody 19E3 for Western Blot detection of adenovirus-infected samples

[0062] (1) HeLa cells in good growth condition were digested with trypsin and then resuspended in DMEM medium containing 10% fetal bovine serum (FBS) to adjust the cell concentration to 5 × 10⁻⁶ cells / year. 5 Cells / mL, take 3 mL of cell suspension and add it to a 6-well cell culture plate, and incubate overnight at 37°C in a 5% CO2 incubator.

[0063] (2) The next day, the culture medium was replaced with DMEM containing 2% FBS, and different types of adenovirus with an infection multiplicity (MOI) of 5, including HADV-B3, HADV-B7, HADV-B11, HADV-B14 and HADV-B21, were added and incubated for 1 h. After 1 h, the culture supernatant was aspirated, the cells were washed once with Versen buffer, and 3 mL of DMEM containing 2% FBS was added to each well. The cells were then incubated at 37°C in a 5% CO2 incubator for 24 h.

[0064] (3) The next day, the culture supernatant of different types of adenovirus was collected and transferred to centrifuge tubes. The tubes were centrifuged at 3000 g and 4℃ for 15 min. The supernatant was transferred to a new centrifuge tube. 100 μl of the supernatant was added to 25 μl of 4× loading buffer. After mixing thoroughly, the tubes were placed on ice for temporary storage.

[0065] Wash the adherent cells in the cell culture plate once with cold PBS, add 200 μL of 1× loading buffer, and gently pipette to detach all cells. Collect the cells and transfer them to a new centrifuge tube. Incubate on ice for 10 min, then centrifuge at 12000 g and 4°C for 15 min. Store the resulting sample on ice for later use.

[0066] (4) Place the above sample in a boiling water bath and heat it at 100°C for 10 min to denature it. Then centrifuge it at 12000 g at 4°C for 15 min and transfer the supernatant to a new centrifuge tube.

[0067] (5) Perform Western blot analysis according to the steps in Example 3.1, scan the images using the Odyssey® imaging system, and analyze the results. For example... Figure 5 As shown, the results indicate that recombinant antibody 19E3 can be used for Western blotting to detect cell samples infected with multiple HADV types, including HADV-B3, HADV-B7, HADV-B11, HADV-B14, and HADV-B21.

[0068] Example 4: Recombinant mouse antibody 19E3 used for cellular immunofluorescence detection after adenovirus infection.

[0069] (1) HeLa cells in good growth condition were taken, digested with trypsin, and the cell suspension concentration was adjusted to 5×10⁻⁶ cells using DMEM medium containing 10% FBS. 5 Cells / mL, take 3 mL of cell suspension and add it to a confocal dish, incubate overnight at 37°C in a 5% CO2 incubator.

[0070] (2) Same as the adenovirus infection process in Example 3.2 (2), process the cell samples in the confocal dish and incubate them in a 37°C, 5% CO2 incubator for 24 h.

[0071] (3) The next day, the culture supernatant was discarded, and the cells were rinsed once with PBS. Then, 4% paraformaldehyde was added, and the cells were incubated at room temperature for 20 min to fix the cell samples. The fixative was discarded, and the cells were rinsed three times with PBS for 10 min each time. 5% BSA / PBS solution containing 0.1% Triton-X100 was added, and the cells were incubated at room temperature for 1 h to perform cell permeabilization and blocking. After rinsing three times with PBS, mouse recombinant antibody 19E3 diluted 1:500 was added, and the cells were incubated at room temperature in the dark for 1 h. After rinsing three times with PBS, goat anti-mouse IgG (H+L) antibody (Invitrogen, Cat: A11029) conjugated with Alexa 488 diluted 1:1000 was added, and the cells were incubated at room temperature in the dark for 1 h. After rinsing three times with PBS, 10 μg / mL Hochest 33342 staining solution (Invitrogen, Cat: H3570) was added, and the mixture was incubated at room temperature in the dark for 10 min. Finally, the mixture was rinsed with PBS to remove residual liquid, and images were scanned and analyzed using a Zeiss LSM880 laser confocal imaging system. Figure 6 As shown, the results indicate that the mouse recombinant antibody 19E3 can be used for immunofluorescence detection of cell samples infected with multiple types of HADV, including HADV-B3, HADV-B7, HADV-B11, HADV-B14 and HADV-B21.

[0072] Sequence information

[0073] SEQ ID NO: 1-19E3 HCDR1 amino acid sequence

[0074] GFSLSTSGMG

[0075] SEQ ID NO: 2——19E3 HCDR2 amino acid sequence

[0076] IWWDDVR

[0077] SEQ ID NO: 3——19E3 HCDR3 amino acid sequence

[0078] ARTWFAY

[0079] SEQ ID NO: 4——19E3 LCDR1 amino acid sequence

[0080] SQHSTYT

[0081] SEQ ID NO: 5——19E3 LCDR2 amino acid sequence

[0082] LKKDGSH

[0083] SEQ ID NO: 6—19E3 LCDR3 amino acid sequence

[0084] GVGDTIKEQFVYV

[0085] SEQ ID NO: 7——19E3 HFR1 amino acid sequence

[0086] QVTLKESGPGILQPSQTLSLTCSFS

[0087] SEQ ID NO: 8—19E3 HFR2 amino acid sequence

[0088] VGWLRQPSGKALEWLAH

[0089] SEQ ID NO: 9——19E3 HFR3 amino acid sequence

[0090] YYNPALKSRLTISKDTSSSQVFLKIASVDTADSATYYC

[0091] SEQ ID NO: 10-19E3 HFR4 amino acid sequence

[0092] WGQGTLVTVSA

[0093] SEQ ID NO: 11-19E3 LFR1 amino acid sequence

[0094] QLVLTQSSSASFSLGASAKLTCTLS

[0095] SEQ ID NO: 12——19E3 LFR2 amino acid sequence

[0096] IEWYQQQPLKPPKYVME

[0097] SEQ ID NO: 13——19E3 LFR3 amino acid sequence

[0098] STGDGIPDRFSGSSSGADRYLSISNIQPEDEAIYIC

[0099] SEQ ID NO: 14——19E3 LFR4 amino acid sequence

[0100] FGGGTKVTVL

[0101] SEQ ID NO: 15 - Amino acid sequence of 19E3 VH

[0102] QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVGWLRQPSGKALEWLAHIWWDDVRYYNPALKSRLTISKDTSSSQVFLKIASVDTADSATYYCARTWFAYWGQGTLVTVSA

[0103] SEQ ID NO: 16 - Amino acid sequence of 19E3 VL

[0104] QLVLTQSSSASFSLGASAKLTCTLSSQHSTYTIEWYQQQPLKPPKYVMELKKDGSHSTGDGIPDRFSGSSSGADRYLSISNIQPEDEAIYICGVGDTIKEQFVYVFGGGTKVTVL

[0105] SEQ ID NO: 17 - Nucleotide sequence of 19E3 VH

[0106] caggttactctgaaagagtctggccctgggatattgcagccctcccagaccctcagtctgacttgttctttctctgggttttcactgagcacttctggtatgggtgtaggctggcttcgtcagccatcaggtaaggctctggagtggctggcacacatttggtgggatgatgtcaggtactataacccagccctgaagagccgactgactatctccaaggatacctccagtagccaggttttcctcaagatcgccagtgtggacactgcagattctgccacatactactgtgctcgaacctggtttgcttactggggccaagggactctggtcactgtctctgca

[0107] SEQ ID NO: 18 - Nucleotide sequence of 19E3 VL

[0108] Caacttgtgctcactcagtcatcttcagcctctttctccctgggagcctcagcaaaactcacgtgcaccttgagtagtcagcacagtacgtacaccattgaatggtatcagcaacagccactcaagcctcctaagtatgtgatggagcttaagaaagatggaagccacagcacaggtgatgggattcctgatcgcttctctggatccagctctggtgctgatcgctaccttagcatttccaacatccagcctgaagatgaagcaatatacatctgtggtgtgggtgatacaattaaggaacaatttgtgtatgttttcggcggtggaaccaaggtcactgtccta。

Claims

1. A monoclonal antibody targeting human adenovirus DBP protein, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein: The amino acid sequence of HCDR1 is shown in SEQ ID NO: 1, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 2, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:

3. The amino acid sequence of LCDR1 is shown in SEQ ID NO: 4, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 5, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:

6.

2. The monoclonal antibody according to claim 1, wherein the heavy chain variable region further comprises heavy chain framework regions HFR1, HFR2, HFR3, and HFR4, and the light chain variable region further comprises light chain framework regions LFR1, LFR2, LFR3, and LFR4, wherein: The amino acid sequence of HFR1 is shown in SEQ ID NO: 7, the amino acid sequence of HFR2 is shown in SEQ ID NO: 8, the amino acid sequence of HFR3 is shown in SEQ ID NO: 9, and the amino acid sequence of HFR4 is shown in SEQ ID NO:

10. The amino acid sequence of LFR1 is shown in SEQ ID NO: 11, the amino acid sequence of LFR2 is shown in SEQ ID NO: 12, the amino acid sequence of LFR3 is shown in SEQ ID NO: 13, and the amino acid sequence of LFR4 is shown in SEQ ID NO:

14.

3. The monoclonal antibody according to claim 1 or 2, wherein: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

16.

4. The monoclonal antibody according to any one of claims 1-3, wherein it is a recombinant mouse antibody.

5. A nucleic acid molecule comprising a nucleotide sequence encoding a monoclonal antibody according to any one of claims 1-4.

6. The nucleic acid molecule according to claim 5, comprising: The nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO: 17; and the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO:

18.

7. A vector comprising the nucleic acid molecule of claim 5 or 6.

8. A cell comprising the nucleic acid molecule of claim 5 or 6 or the vector of claim 7.

9. A kit for detecting human adenovirus, comprising the monoclonal antibody according to any one of claims 1-4.

10. The kit according to claim 9, which is a Western Blot kit, further comprising: a fluorescently labeled conjugated secondary antibody, a nitrocellulose membrane, a blocking solution, and a washing buffer.

11. The kit according to claim 10, wherein the fluorescently labeled second antibody is an IRDye 800CW-conjugated goat anti-mouse IgG (H+L) antibody; the blocking solution is 5% skim milk / PBS solution; and the washing buffer is PBST solution.

12. The kit according to claim 9, which is an immunofluorescence detection kit, further comprising: a fluorescently labeled second antibody, a cell fixative, a blocking solution, a cell permeabilizer, and a washing buffer.

13. The kit according to claim 12, wherein the fluorescently labeled second antibody is Alexa 488-conjugated goat anti-mouse IgG (H+L) antibody; the cell fixative is 4% paraformaldehyde solution; the cell permeabilizer is Triton-X100; the blocking solution is 5% BSA / PBS solution; and the washing buffer is PBS solution.

14. A method for preparing a monoclonal antibody according to any one of claims 1-4, comprising culturing the cells of claim 8 and isolating and recovering the antibody therefrom.

15. Use of the monoclonal antibody of any one of claims 1-4, the nucleic acid molecule of claim 5 or 6, the vector of claim 7, or the cell of claim 8 for the preparation of a reagent for detecting human adenovirus in a sample.

16. A non-diagnostic method for detecting human adenovirus in an isolated sample, comprising: First, the sample is bound to the monoclonal antibody as described in any one of claims 1-4, and then a labeled second antibody is added. The detection result of human adenovirus in the sample is obtained by detecting the reaction signal.

17. The method according to claim 16, wherein the human adenovirus is selected from HAdV-A12, HAdV-B3, HAdV-C5, HAdV-D8, HAdV-E4, HAdV-F41, HAdV-B7, HAdV-B11, HAdV-B14, and HAdV-B21.

18. The method according to claim 16 or 17, wherein the method for detecting the reaction signal is Western blotting or immunofluorescence detection.

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