Hybridoma cell strain for recognizing 188th amino acid of highly pathogenic serum type 4 fowl adenovirus Hexon protein, monoclonal antibody and application of hybridoma cell strain

Abstract

The invention discloses a hybridoma cell strain capable of recognizing 188th amino acid of a highly pathogenic serum type 4 fowl adenovirus Hexon protein, a monoclonal antibody and application of the hybridoma cell strain. The preservation number of the cell strain 2C5 is CGMCC NO.45829, and the monoclonal antibody 2C5 capable of specifically recognizing the highly pathogenic serum type 4 fowl adenovirus hexon Hexon protein is secreted. The amino acid sequence of the antigen epitope recognized by the gene is Hexon protein GPGRNP, wherein the R188 amino acid in the Hexon protein is the key amino acid recognized by the gene. The monoclonal antibody 2C5 prepared by the invention has the specificity of high-pathogenicity FAdV-4, can specifically recognize the high-pathogenicity FAdV-4, has no cross reaction with non-pathogenicity FAdV-4 and FAdV-4 of other serotypes, and can be used as a diagnostic reagent for distinguishing the high-pathogenicity FAdV-4 and the non-pathogenicity FAdV-4.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology, and specifically relates to a hybridoma cell line for recognizing the 188th amino acid of Hexon protein of highly pathogenic serotype 4 avian adenovirus, a monoclonal antibody and application thereof. Background Art

[0002] Avian adenovirus (FAdV) belongs to the genus Avian adenovirus of the family Adenoviridae. According to restriction endonuclease digestion patterns and serum cross-neutralization tests, FAdV can currently be divided into 5 genotypes (FAdV-A, B, C, D, E) and 12 serotypes (FAdV-1-7, 8a, 8b, 9-11). In recent years, hepatitis-pericardial effusion syndrome (HHS) in chickens caused by infection with highly pathogenic serotype 4 avian adenovirus (FAdV-4) has been prevalent in chicken flocks, with a very high mortality rate, causing huge economic losses to the poultry industry. The genome size of FAdV-4 is about 45kb, encoding three major surface structural proteins, including hexon, penton and fiber.

[0003] At present, FAdV-4 is divided into highly pathogenic and non-pathogenic strains. The pathogenic FAdV-4 strains cause a chicken mortality rate of 30% to 90%. With the large-scale outbreak of highly pathogenic FAdV-4, the poultry industry has suffered huge economic losses. However, there are currently no relevant monoclonal antibodies and pathogen detection methods to distinguish between highly pathogenic strains and non-pathogenic strains. The existing methods mainly rely on gene sequencing to distinguish between strong and weak strains, which is time-consuming and labor-intensive. At present, the monoclonal antibodies for FAdV-4 are mainly prepared by immunizing mice with fusion proteins expressed by viruses and Fiber, Penton, and Hexon prokaryotes as immunogens. FAdV-4 and FAdV-10 belong to the same genotype C, and the homology of their genomes is very high. Therefore, most of the monoclonal antibodies for FAdV-4 currently obtained are FAdV broad-spectrum monoclonal antibodies. In the prior art, the two antibodies 3B5 and 6H9 prepared using GST-Fiber-1 as an immunogen can recognize FAdV-4 and FAdV-10, but cannot specifically recognize FAdV-4. Although Hexon proteins have important genus, species and serotype specific antigenic epitopes, Hexon proteins are mostly present in the form of inclusion bodies in prokaryotic expression systems, which are difficult to purify and have poor immunogenicity, which greatly limits the research on Hexon monoclonal antibodies and antigenic epitopes. In addition, Hexon proteins are highly conserved among FAdVs, with the homology of Hexon amino acids between FAdV-4 and FAdV-10 reaching 97%, and the homology of Hexon amino acids between highly pathogenic strains and non-pathogenic strains reaching 98.6%. Therefore, among the published Hexon monoclonal antibodies, most are FAdV broad-spectrum monoclonal antibodies and FAdV-C specific monoclonal antibodies, and it is difficult to distinguish FAdV-4 from FAdV-10. There are no reports of type-specific monoclonal antibodies targeting only FAdV-4 Hexon proteins, and there are no monoclonal antibodies that can distinguish highly pathogenic and non-pathogenic strains of FAdV-4. Summary of the invention

[0004] Purpose of the invention: In view of the deficiencies in the prior art, the present invention provides a monoclonal antibody 2C5 that recognizes the Hexon protein of the highly pathogenic FAdV-4 virus and its hybridoma cell line, so as to solve the problem that the prior art cannot effectively identify highly pathogenic and non-pathogenic FAdV-4 viruses. The monoclonal antibody 2C5 prepared by the present invention has a good specific reaction, and more importantly, it only specifically recognizes highly pathogenic FAdV-4, and has no cross-reaction with non-pathogenic FAdV-4 and other serotypes of FAdV, and can be used in the laboratory and clinical identification of highly pathogenic and non-pathogenic FAdV-4.

[0005] The present invention also provides the antigen epitope and key amino acids recognized by the monoclonal antibody.

[0006] Technical solution: In order to achieve the above-mentioned purpose, the hybridoma cell strain 2C5 secreting anti-serum type 4 avian adenovirus Hexon protein monoclonal antibody described in the present invention, the preservation number of the hybridoma cell strain is CGMCC NO.45829, the preservation date is March 21, 2024, and the classification name is mouse hybridoma cell, which is deposited in the China General Microbiological Culture Collection Center (CGMCC), address: Beijing, China.

[0007] The anti-serum type 4 avian adenovirus Hexon protein monoclonal antibody 2C5 of the present invention is obtained by secretion of the hybridoma cell line 2C5.

[0008] The hybridoma cell line is used to proliferate in the peritoneal cavity of mice, and the ascites is separated and purified to produce the monoclonal antibody 2C5 that recognizes the highly pathogenic FAdV-4.

[0009] Among them, the monoclonal antibody 2C5 specifically binds to the highly pathogenic FAdV-4 Hexon protein antigen, but does not undergo antigen-antibody reaction with the non-pathogenic FAdV-4 Hexon protein antigen.

[0010] Wherein, the heavy chain subtype of the monoclonal antibody 2C5 is IgG1.

[0011] The antigen epitope is located at amino acids 185 to 190 of the Hexon protein of the highly pathogenic serotype 4 fowl adenovirus strain SD2015, and the amino acid sequence of the antigen epitope is GPGRNP.

[0012] The key amino acid site of the antigenic epitope recognized by the monoclonal antibody 2C5 described in the present invention is the 188th amino acid R (Hexon R188) of the highly pathogenic FAdV-4 Hexon protein, that is, the amino acid sequence of the antigenic epitope is Hexon 185-GPGRNP-190.

[0013] Among them, the 188th amino acid R of the Hexon protein is a characteristic amino acid of the highly pathogenic serotype 4 avian adenovirus, that is, the key amino acid in the antigenic epitope recognized by the monoclonal antibody 2C5 is the R188 amino acid in the Hexon protein.

[0014] The monoclonal antibody 2C5 of the present invention is used in the preparation of a product for detecting and identifying highly pathogenic serotype 4 avian adenovirus.

[0015] Furthermore, the product includes a kit for diagnosing or detecting highly pathogenic FAdV-4.

[0016] Among them, the isolated strains of highly pathogenic serotype 4 avian adenovirus include SD2015, AHFY15, JH13, and JSCZ15.

[0017] Preferably, the product is based on one or more of the following detection technologies: Western blot technology and IFA technology.

[0018] The kit for detecting and identifying highly pathogenic serotype 4 avian adenovirus of the present invention comprises the monoclonal antibody 2C5 and auxiliary materials constituting the kit.

[0019] Based on the prediction of the B cell dominant epitope of Hexon protein, the present invention expressed the truncated protein, immunized mice with the expression product, and screened out a monoclonal antibody hybridoma cell line that can secrete Hexon protein against FAdV-4 using monoclonal antibody hybridoma technology, named hybridoma cell line 2C5. Characteristic detection found that the monoclonal antibody 2C5 secreted by the hybridoma cell line only reacts with highly pathogenic FAdV-4 strains, but does not react with other serotypes of avian adenovirus and non-pathogenic FAdV-4 strains. Epitope identification further found that the epitope recognized by monoclonal antibody 2C5 is located at 185GPGRNP190, and it was found that the 188th amino acid R (R188aa) in the Hexon protein is the key amino acid recognized by monoclonal antibody 2C5. The development of the monoclonal antibody 2C5 that reacts only with highly pathogenic FAdV-4 in the present invention provides molecular targets and biological materials for establishing a differential diagnosis technology that can distinguish highly pathogenic and non-pathogenic FAdV-4, and for conducting molecular epidemiology and pathogenic mechanism research on the highly pathogenic FAdV-4 prevalent in China.

[0020] The present invention provides a monoclonal antibody 2C5 for recognizing the hexon protein of highly pathogenic serotype 4 fowl adenovirus (FAdV-4). The heavy chain subtype is identified as IgG1, and the amino acid sequence of the antigen epitope recognized by the antibody is Hexon protein 185-GPGRNP-190, wherein the R188 amino acid in the Hexon protein is the key amino acid for recognition.

[0021] Hexon proteins have important genus, species and serotype specific antigenic epitopes, but because Hexon proteins appear in the form of inclusion bodies in prokaryotic expression systems, they are not easy to purify and have poor antigenicity, which hinders the research on Hexon monoclonal antibodies. In addition, due to the high homology between FAdV-4 and FAdV-10, there are currently no reports on FAdV-4 Hexon type-specific antibodies and type-specific antigenic epitopes. Previous detection methods and monoclonal antibodies cannot specifically identify FAdV-4, and there are no detection methods and antibodies that distinguish between highly pathogenic and non-pathogenic. In order to obtain FAdV-4 type-specific antibodies and more accurately prevent and control hepatitis-hydropericardium syndrome caused by FAdV-4 infection, the present invention first analyzes the dominant antigenic epitopes of Hexon proteins, and screens out the FAdV-4 type-specific dominant epitopes of Hexon proteins through epitope sequence alignment. Using the prokaryotic expression system, an antigen truncated protein containing a FAdV-4 type-specific dominant epitope was expressed. After immunizing mice, a hybridoma cell that only recognizes highly pathogenic serotype 4 and its secreted monoclonal antibody 2C5 were screened. Using the GST-fusion protein segmented expression pattern, the antigen epitope Hexon antigen epitope 185-GPGRNP-190 targeted by monoclonal antibody 2C5 was identified. Furthermore, the 188aa eukaryotic mutant plasmid was used to prove that the Hexon virulence site 188aaR is the key amino acid recognized by monoclonal antibody 2C5. The development of monoclonal antibody 2C5 can be used to develop a kit and detection method for detecting highly pathogenic serotype 4, which has a beneficial effect on the precise prevention and control of hepatitis-pericardial effusion syndrome caused by highly pathogenic serotype 4.

[0022] The existing monoclonal antibodies are mainly broad-spectrum monoclonal antibodies for avian adenovirus, or FAdV-C (FAdV-4 / 10) specific monoclonal antibodies. Hexon protein is the main structural protein of FAdV, accounting for 40% of the viral protein content. Compared with antibodies against other FAdV proteins, monoclonal antibodies against Hexon protein may have higher sensitivity in the establishment of detection methods. However, there are currently no reports on FAdV-4 Hexon protein-specific related antibodies and antigenic epitopes.

[0023] The present invention obtained a highly pathogenic strain-specific antibody 2C5, and clarified its recognized epitope and key amino acids, providing an ideal biomaterial for the precise prevention and control of highly pathogenic FAdV-4.

[0024] The present invention screens and prepares a monoclonal antibody 2C5 and a cell line thereof that can distinguish between highly pathogenic and non-pathogenic FAdV-4, and the key amino acid Hexon R188aa recognized by the monoclonal antibody 2C5 is the virulence site of FAdV-4, and Western blot and IFA experiments show that the monoclonal antibody 2C5 has good specificity. The monoclonal antibody 2C5 of the present invention can detect the pathogen of FAdV-4 in the double sandwich ELISA method, and can identify FAdV-4 in the detection methods of IFA and Western blot. However, there is currently no detection method or specific antibody that can distinguish or only identify highly pathogenic serotype 4 avian adenovirus.

[0025] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0026] 1) The monoclonal antibody of the present invention is produced by a specific single cell line and has the characteristics of high specificity and uniformity. The monoclonal antibody has high purity, strong specificity, and can be continuously and unlimitedly supplied. It has a wider research application value and commercial use value in immunoassays.

[0027] 2) The monoclonal antibody of the present invention has strong reactivity and good reactivity with the Hexon protein of highly pathogenic FAdV-4 in IFA, Western blot and ELISA, and can be used for the detection of highly pathogenic FAdV-4.

[0028] 3) The monoclonal antibody 2C5 prepared by the present invention has the specificity for highly pathogenic FAdV-4, can specifically identify highly pathogenic FAdV-4, and has no cross-reaction with non-pathogenic FAdV-4 and other serotypes of FAdV. It can be used as a diagnostic reagent to distinguish highly pathogenic FAdV-4 from non-pathogenic FAdV-4.

[0029] 4) The epitope recognized by the monoclonal antibody of the present invention is Hexon protein 185-GPGRNP-190, and the amino acid R188 in the Hexon protein of the FAdV-4 virulence-related site is the key amino acid recognized by it. Further three-dimensional structure prediction revealed that the Hexon R188I mutation caused the conformation of the SD2015 Hexon protein to change. These findings provide a very valuable new tool for further studying the biological functions and pathogenic mechanisms of Hexon proteins.

[0030] In summary, the present invention not only provides a new hybridoma cell or specific monoclonal antibody for establishing a detection method for highly pathogenic FAdV-4, but also provides molecular targets and biological materials for conducting molecular epidemiology and pathogenic mechanism research on highly pathogenic FAdV-4. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1The expression verification diagram of Hexon truncated protein. A, B: SDS-PAGE and Western blot verification of Hexon truncated protein expression and purification effect, lane M: protein marker; lane 1: pET-28a empty vector bacteria lysis precipitation; lane 2: recombinant bacteria lysis precipitation; lane 3: protein after inclusion body purification.

[0032] Figure 2 Western blot reactivity of monoclonal antibody 2C5 with avian adenovirus.

[0033] Figure 3 IFA reactivity of mAb 2C5 with avian adenovirus.

[0034] Figure 4 The localization of the amino terminus of the Hexon antigen epitope recognized by 2C5. A: Flow chart of the two rounds of localization of the amino terminus of the antigen epitope; B: Western blot results of the first round of preliminary localization; C: Western blot results of the second round of precise localization.

[0035] Figure 5 Identification of key amino acids for 2C5 to recognize Hexon antigen epitopes. A: IFA results of the monoclonal antibody and pathogenic or non-pathogenic FAdV-4 strains, FAdV-10 Hexon eukaryotic protein reaction; B: IFA results of the monoclonal antibody 2C5 and pathogenic or non-pathogenic FAdV-4 strains, FAdV-10 mutated eukaryotic protein reaction.

[0036] Figure 6 Conservation analysis of the epitope recognized by mAb 2C5.

[0037] Figure 7 Prediction of the three-dimensional conformation of Hexon protein. A: The antigen epitope recognized by 2C5 and the location of key amino acids on Hexon protein; B and C: Prediction of the effect of amino acid mutation at position 188 on the three-dimensional conformation of the protein. DETAILED DESCRIPTION

[0038] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0039] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources. Experimental methods without specific conditions specified in the examples are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0040] Highly pathogenic FAdV-4 strain SD2015, provided by Yangzhou University, literature: Outbreaks of serotype 4 fowl adenovirus with novel genotype, China, literature source: Emerging Microbes and Infections (2016) 5, e50; doi: 10.1038 / emi.2016.50; published online 25May2016.

[0041] Monoclonal antibody subclass identification kit, Beijing Biolong Biotechnology Co., Ltd., catalog number: KLB4015524.

[0042] Other strains were purchased from ATCC, FAdV-1 (ATCC -432 TM ), non-pathogenic FAV-4 (ON1) (ATCC -829 TM ),FAdV-5(ATCC -830 TM ),FAdV-6(ATCC -831 TM ),FAdV-7(ATCC -832 TM ),FAdV-9(ATCC -833 TM ), FAdV-10(ATCC -834 TM ). FAdV-8a, 8b, 2, 11 and other strains were provided by the Key Laboratory of Avian Preventive Medicine of Yangzhou University.

[0043] Preparation of LMH cells infected with SD2015 strain: LMH cells were plated in 96-well plates. When the cells grew for about 9 days and the cell clone density reached about 60% to 80% of the bottom area of ​​the well, FAdV4 (SD2015) was used to infect LMH cells at an MOI ratio of 0.01, and the cytopathic effect was observed every day. When the cytopathic effect was about 70%, the cells were fixed with pre-cooled acetone ethanol (acetone 3:2 anhydrous ethanol) at room temperature for 5-10 minutes, the fixative was poured off, and the cells were washed once with PBS, and then the cell plates were placed in a fume hood for ventilation for 3 hours to completely evaporate the residual acetone ethanol in the cell plates.

[0044] Example 1

[0045] Preparation of hybridoma cell lines and identification of monoclonal antibodies

[0046] 1. Prediction of dominant antigenic epitopes of serotype 4 avian adenovirus Hexon protein and preparation of immunogens

[0047] The applicant's preliminary analysis obtained a 104-291 amino acid fragment of the Hexon protein where the FAdV-4 type-specific dominant antigenic epitope is located (Hexon truncated protein, SEQ ID NO.2). Using the PCR primers with pET28a(+)15bp as shown in Table 1 and the FAdV4 strain SD2015 genome as a template, the hexon (309-873bp) PCR product (SEQ ID NO.3) was cloned and obtained. After gel purification, it was connected to pET28a(+) under the action of the homologous recombinase. After sequencing verification, the successfully recombined recombinant plasmid pET28a(+)-FAdV-Hexon (104-291aa) expression vector was transformed into Escherichia coli BL21 (DE3) for expression. The protein was purified by the inclusion body purification method, and its expression was verified by Western blot test to obtain the recombinant Hexon protein. Figure 1 As shown in A, the recombinant protein with His tag was successfully expressed. The purification effect was verified by SDS-PAGE. Figure 1 The purity and concentration of the purified antigen shown in B meet the requirements for mouse immunization.

[0048] Table 1 PCR primers used to construct immunogens

[0049]

[0050] 2. Screening of hybridoma cells and preparation of monoclonal antibodies

[0051] The purified recombinant Hexon truncated protein expressed in prokaryotes was mixed with an equal amount of Freund's adjuvant and emulsified. The mixture was subcutaneously injected into 5-week-old female BALB / c mice at multiple points, 50 μg / mouse. On days 21 and 35 after immunization, the recombinant Hexon protein was emulsified with Freund's incomplete adjuvant for booster immunization. Blood was collected 49 days after the first immunization to measure the antibody titer. The BALB / c mice with the highest antibody titer were selected and intraperitoneally injected with 50 μg of recombinant Hexon protein for shock immunization 3 days before cell fusion.

[0052] Aseptically take the spleen of the immunized mouse and put it into the cell sieve to make a spleen cell suspension. The spleen cells and myeloma cells SP2 / 0 were fused with PEG fusion agent at a ratio of 5:1. Resuspend the cells with HAT medium, dispense into 96-well cell culture plates, and culture them in a 37°C 5% carbon dioxide cell culture incubator. Collect the cell culture supernatant on the 12th day after fusion. LMH cells were plated into 96-well plates. When the cells grew for about 9 days and the cell clone density reached about 60% to 80% of the bottom area of ​​the well, FAdV4 (SD2015) was used to infect LMH cells, and the cell lesions were observed every day. When the cell lesions were about 70%, they were fixed at room temperature for 5-10 minutes with pre-cooled acetone ethanol (acetone 3:2 anhydrous ethanol), the fixative was poured off, washed once with PBS, and then the cell plate was placed in a fume hood for ventilation for 3 hours. After the residual acetone and ethanol in the cell plate evaporated, the hybridoma supernatant was added to the cell plate as the primary antibody, 50 μl / well; then the cell plate was placed in a 37°C constant temperature water bath for incubation for 45 minutes; the primary antibody was discarded and washed 3 times with PBS; FITC-labeled goat anti-mouse immunoglobulin was diluted 1:150 and added to the cell plate (protected from light), 50 μl / well; the cell plate was placed in a 37°C constant temperature water bath for incubation for 45 minutes; the secondary antibody was discarded and washed 3 times with PBS (protected from light); PBS was added and the results were analyzed using an inverted fluorescence microscope to obtain 3 IFA-positive cell lines. The positive hybridoma cells screened were subcloned three times to establish a hybridoma cell line that can stably secrete anti-FAdV-4, named hybridoma cell line 2C5. The hybridoma cell strain 2C5 was deposited in the China General Microbiological Culture Collection Center (CGMCC) on March 21, 2024, with the deposit number CGMCC NO.45829.

[0053] 3. Preparation of monoclonal antibodies against hybridomas

[0054] The monoclonal antibody 2C5 was isolated and plated in 25 cm 2 The cells were cultured in T25 cell culture flasks, and 6 mL of HT medium containing 10% serum was added. After 2 days of in vitro culture, the hybridoma cells formed a dense monolayer and the culture medium turned yellow. The culture supernatant was collected and centrifuged at 4°C, 12000 rpm for 10 min to remove the cells and their debris, and the 2C5 hybridoma monoclonal antibody was obtained.

[0055] 4. Identification of monoclonal antibody antigen subclasses

[0056] The subclass of monoclonal antibody 2C5 was identified using a monoclonal antibody subclass identification kit, and the result showed that the heavy chain subclass of monoclonal antibody 2C5 was IgG1.

[0057] 5. Preparation of Ascites

[0058] After the hybridoma cells were screened, they were expanded and cultured at 1×10 7 / 200μl was injected into BLAB / C mice with 200μl. After 7 days, when the abdominal circumference of the mice increased, the ascites was collected with a syringe needle, and the mixture was centrifuged at 4000r / min for 10min. The supernatant was filtered through 0.45μm to obtain the ascites mAb product.

[0059] 6. Determination of the titer of monoclonal antibodies in hybridoma cell supernatant and ascites

[0060] The specific steps of ELISA determination of supernatant and ascites titer are as follows: Hexon truncated protein is coated on the ELISA plate at 1 μg / well, incubated at 37℃ for 1 hour and 4℃ overnight. The next day, the liquid in the wells was discarded, and the wells were washed 3 times, each time for 3 minutes; 200 μl of 5% skim milk blocking solution was added to each well, and incubated at 37℃ for 1 hour; 1:10 1 ~10 9 The diluted 2C5 hybridoma supernatant and ascites were incubated at 37°C for 1 hour; washed 3 times and patted dry; HRP-labeled goat anti-mouse IgG (1:5000 dilution) was added and incubated at 37°C for 1 hour; washed 3 times and patted dry; 100 μl / well of color development solution was added and color was developed at room temperature in the dark for 15 minutes; the reaction was terminated, 50 μl of 2 mol / L sulfuric acid was added to each well, and the mixture was shaken and mixed. The OD450nm value was measured by an enzyme-labeled instrument. The titer of the monoclonal antibody secreted by the 2C5 hybridoma cells was 1:1600, and the titer of the prepared ascites antibody was 1:10 6 . Further, according to the IFA detection method, LMH cells infected with FAdV-4 (SD2015 strain) virus were fixed, and the ascites and supernatant were taken for 10 times dilution, and the titer was determined by indirect immunofluorescence. The IFA titer of 2C5 hybridoma cell supernatant was 1:800, and the ascites titer was 1:12500.

[0061] 7. Stability test of monoclonal antibodies secreted by hybridoma cell lines

[0062] After the hybridoma cells were cryopreserved for 12 months, the frozen hybridoma cell lines were taken out of liquid nitrogen for recovery, and then the indirect immunofluorescence experiment was performed to detect the secretion of monoclonal antibodies by the hybridoma cell lines. The results showed that the titer of the supernatant secreted by the hybridoma cell line 2C5 was consistent with that before cryopreservation, and the antibody secretion was stable. The detection results of the monoclonal antibodies secreted by the hybridoma after 30 generations also proved the stability of the secretion of monoclonal antibodies by the hybridoma cell lines.

[0063] Example 2

[0064] Western blot reactivity of monoclonal antibody 2C5 with different serotypes of avian adenovirus

[0065] The cells were infected with the above strains, and FAdV-1, highly pathogenic FAdV-4, non-pathogenic FAdV-4, FAdV-5, FAdV-7, FAdV-8b, FAdV-10 and FAdV-11 at an MOI of 0.01 were used to infect LMH cells. The LMH cells 3 days after infection were lysed with a lysis buffer (CST, MA, USA) containing PMSF (Beyotime, Shanghai China), and the supernatant was collected and added to the protein loading buffer. The samples were boiled at 98°C for 10 min, and the samples were separated by SDS-PAGE gel electrophoresis and transferred to the NC membrane; the NC membrane was blocked with skim milk and the ascites monoclonal antibody 2C5 prepared in Example 1 was used for PBST. 1:1000 dilution as primary antibody, incubate with NC membrane at room temperature for 2 hours; wash 3 times with PBST, incubate with NC membrane at room temperature for 1 hour with HRP-labeled goat anti-mouse IgG secondary antibody; after washing 3 times with PBST, add 500μl ECL colorimetric solution and detect with chemiluminescence instrument. First, use monoclonal antibody 1B5 that reacts with all serotypes of adenovirus to detect the infection of different serotypes of virus. The results are shown in Figure 2 As shown in the figure, there are Hexon protein-specific bands in the lanes where different serotypes of adenovirus are located, proving that the infection is successful. Figure 2 As shown in the figure, a clear specific band with a molecular weight of about 105 kDa can be seen in the lane of cells infected with highly pathogenic FAdV-4 (SD2015), while there is no such band in the lanes of other serotypes. This result shows that monoclonal antibody 2C5 can detect highly pathogenic FAdV-4 by Western blot technology, and does not cross-react with other serotypes.

[0066] Example 3

[0067] IFA reactivity of mAb 2C5 with avian adenovirus

[0068] Using a 96-well cell plate, cells were infected with the above strains, and LMH cells infected with 0.01 MOI virus of FAdV-1, FAdV-2, non-pathogenic FAdV-4 (ON1), highly pathogenic FAdV-4 (SD2015), FAdV-5, FAdV-6, FAdV-7, FAdV-8a, FAdV-8b, FAdV-10 and FAdV-11 were used as antigens. After being fixed with ice-cold acetone and ethanol (3:2) for 5 minutes, PBS was washed once, and the ascites monoclonal antibody 2C5 prepared in Example 1 was added and diluted with PBST 1:1000 to 100 μl / well. At the same time, LMH cells not infected with the virus were set as negative controls and incubated at 37°C for 45 minutes; washed 3 times with PBS, and FITC-labeled goat anti-mouse IgG secondary antibody was added and acted at 37°C for 45 minutes; washed 3 times with PBS and observed under a fluorescence microscope. The results are as follows Figure 3 As shown: In the wells incubated with mouse polyclonal antibodies against all serotypes, FAdV of each serotype has fluorescence. In the wells incubated with monoclonal antibody 2C5 and infected with highly pathogenic serotype 4, obvious bright green specific fluorescence can be seen ( Figure 3 ), while there was no specific green fluorescence in the wells infected with other types. This result shows that monoclonal antibody 2C5 can detect highly pathogenic FAdV-4 by IFA technology and does not cross-react with other serotypes.

[0069] Example 4

[0070] Analysis of epitope recognition and key amino acids of monoclonal antibody 2C5

[0071] 1. Analysis of monoclonal antibody recognition epitopes

[0072] In order to determine the hexon protein epitope recognized by mAb 2C5, two rounds of overlapping peptides fused to GST-Tag were designed using the primers shown in Table 2 ( Figure 4 A) and expressed in E. coli BL21 (DE3) competent cells. First, the 104-291 amino acid fragment of the immunogen Hexon was divided into three overlapping segments, namely H1 (104-178aa), H2 (170-231aa), H3 (223-291aa) and the FAdV-4 type specific antigen dominant region H4 (184-200aa). The results are shown in Figure 4 As shown in B, mAb 2C5 can react with H2 (170-231aa) and H4 (184-200aa), that is, the epitope targeted by mAb 2C5 is located between Hexon (Gln184-Gly200). In the second round, H4 (184-200aa) peptide was sequentially reduced from both ends. Finally, 185GPGRNP190 was identified as the B cell epitope recognized by mAb 2C5 ( Figure 4C).

[0073] Table 2 PCR primers used to identify epitopes recognized by monoclonal antibodies

[0074]

[0075]

[0076] 2. Identification of key amino acids in the epitope recognized by mAb 2C5

[0077] Preliminary comparison of the epitope sequences in step (1) revealed that pathogenic FAdV-4, non-pathogenic FAdV-4 and FAdV-10 had an amino acid difference at the 188th amino acid of the Hexon epitope (185GPGRNP190), with the highly pathogenic FAdV-4 188aa being R, the non-pathogenic FAdV-4 188aa being I, and the FAdV-10 188aa being V. The amino acid sequences of the three are shown in SEQ ID NO.4-6. IFA and Western blot experiments demonstrated that monoclonal antibody 2C5 did not react with non-pathogenic FAdV-4 and FAdV-10. In order to verify whether Hexon R188a is the key amino acid of monoclonal antibody 2C5, a eukaryotic expression vector with a mutation in the 188th amino acid of the Hexon protein was constructed using the primers listed in Table 3. First, the original Hexon genes (SEQ ID NO.7-9) in the three were cloned into pcDNA3.1 by homologous recombination. After sequencing verification, IFA verification was performed using monoclonal antibody 2C5 and Hexon mouse serum. Figure 5 As shown in A, the three constructed eukaryotic plasmids are all expressed normally, and the monoclonal antibody 2C5 only reacts with the highly pathogenic FAdV-4 Hexon eukaryotic protein (SEQ ID NO.1). Using these recombinant vectors as templates, the linearization primers containing mutation sites in Table 3 were used to linearize the recombinant plasmids constructed in the first step at Hexon188aa by PCR, and the linearized vectors were used for self-homologous recombination with homologous recombinase (NEB, USA, catalog number: M0203S). After sequencing verification, expression was performed and verified by IFA. Figure 5As shown in B, Hexon188aa of the recombinant pcDNA3.1 plasmid expressing the full-length Hexon of highly pathogenic FAdV-4 was mutated from R to I, and the corresponding non-pathogenic FAdV-4 188aa I was mutated to R, and FAdV-10 188aa V was mutated to R. After the 188R to I mutation, the highly pathogenic FAdV-4 Hexon protein no longer reacted with 2C5, while the eukaryotically expressed Hexon full-length proteins of the non-pathogenic FAdV-4 with 188aa I to R and the FAdV-10 strains with 188aa V toR reacted with 2C5, indicating that the difference in the 188aa of the Hexon of highly pathogenic and non-pathogenic FAdV-4 and FAdV-10 is the fundamental reason for the different reactivity to 2C5, and Hexon R188aa is the key amino acid recognized by the monoclonal antibody 2C5.

[0078] Table 3 PCR primers used to identify key amino acids of mAb 2C5

[0079]

[0080] 3. Analysis of epitope conservation of monoclonal antibody 2C5

[0081] By searching the sequences of FAdV strains published at home and abroad, DNAstar was used to compare the sequences. Figure 6 As shown, epitope 185GPGRNP190 is quite different in FAdV. Further comparison of pathogenic strains and non-pathogenic strains showed that the 188th amino acid of Hexon of domestic highly pathogenic strains was R, and that of non-pathogenic strains was I, while R188aa was the key amino acid recognized by monoclonal antibody 2C5. Therefore, the obtained monoclonal antibody 2C5 is an antibody only against highly pathogenic FAdV-4, and the epitope (185GPGRNP190) targeted by 2C5 is a unique epitope of highly pathogenic FAdV-4.

[0082] 4. Prediction of Hexon protein 3D structure

[0083] In order to identify the locations of the antigenic epitopes and key amino acids recognized by 2C5 on the Hexon protein, the three-dimensional structure of the SD2015 strain protein was predicted, and the antigenic epitopes and key amino acids were displayed on the Hexon protein structure.

[0084] like Figure 7 The results of A and 7B showed that the epitope 185-190aa recognized by mAb 2C5 was located in the HVR 1 region of the Hexon protein Loop 1 and protruded from the outside of the Hexon protein ( Figure 7 A). The key amino acid R188aa is located in the center of the epitope ( Figure 7B) This indicates that the region recognized by mAb 2C5 is the surface of the Hexon protein, making mAb 2C5 more likely to react with viral particles.

[0085] 5. Mutation of the key amino acid R188aa can lead to changes in the conformation of Hexon protein

[0086] In order to further explore why R188aa of Hexon protein can become the key amino acid recognized by monoclonal antibody 2C5, the amino acid sequence of Hexon protein with a single amino acid site mutation of R188aaI was designed. The original amino acid sequence of Hexon without amino acid mutation and the mutant sequence of Hexon with a single amino acid site mutation were submitted to SWISS-MODEOL for protein homology modeling. The three-dimensional model of Hexon protein with a single amino acid site mutation was compared with the three-dimensional model of Hexon protein without mutation by PyMOL software to locate the regions with differences in conformation.

[0087] The results are as follows Figure 7 As shown in C, after only mutation R188aaI, the predicted Hexon three-dimensional models before and after mutation cannot completely overlap, and the difference is mainly concentrated in the LOOP 1 region as shown in the white box. Further magnification of this region shows that the a, b, and c regions marked by the white box are the conformational change regions caused by amino acid mutations, of which a and c regions are located in Loop 1HVR 1, and b region is located in Loop 1HVR 2. Hexon R188I mutation causes the three-dimensional conformation of SD2015 Hexon to change in regions a, b, and c, of which the conformational change in region c occurs near the Hexon R188 site. It can be seen that the mutation of the key amino acid R188I can lead to changes in the conformation of the Hexon protein, thereby affecting the recognition of the epitope 185GPGRNP190 by the monoclonal antibody 2C5.

[0088] By the above Figure 7 A and B show that the region recognized by mAb 2C5 is the surface of Hexon protein, making mAb 2C5 more likely to react with viral particles. Figure C shows that the mutation of the key amino acid R188I can lead to a change in the conformation of Hexon protein, thereby affecting mAb 2C5's recognition of epitope 185GPGRNP190.

[0089] In summary, with the large-scale outbreak of highly pathogenic FAdV-4, a simple, rapid, and large-scale detection method for monitoring and epidemiological investigation of highly pathogenic FAdV-4 has not yet been established. The monoclonal antibody 2C5 prepared by the present invention and the epitopes and key amino acid sites recognized by it can be used to develop epidemiological and serological detection technologies for highly pathogenic FAdV-4, which is beneficial to the control of the spread of highly pathogenic FAdV-4. Therefore, monoclonal antibody 2C5 and the epitopes identified by it are expected to become materials and tools for detecting and diagnosing highly pathogenic FAdV-4 and further exploring the pathogenic mechanism of Hexon protein.

Claims

1. A hybridoma cell line 2C5 secreting monoclonal antibodies against serum type 4 avian adenovirus Hexon protein, characterized in that: The deposit number of the hybridoma cell line is CGMCC NO.45829, and the deposit date is March 21, 2024.

2. An anti-serum type 4 avian adenovirus Hexon protein monoclonal antibody 2C5, characterized in that: The protein is secreted by the hybridoma cell line 2C5 according to claim 1.

3. The monoclonal antibody 2C5 according to claim 2, characterized in that: The monoclonal antibody 2C5 specifically binds to the highly pathogenic FAdV-4 Hexon protein antigen, but does not react with the non-pathogenic FAdV-4 Hexon protein antigen.

4. The monoclonal antibody 2C5 according to claim 2, characterized in that: The heavy chain subtype of the monoclonal antibody 2C5 is IgG1.

5. An antigenic epitope recognized by the monoclonal antibody 2C5 according to claim 2, characterized in that: The antigen epitope is located at amino acids 185 to 190 of the Hexon protein of the highly pathogenic serotype 4 fowl adenovirus SD2015 strain, and the amino acid sequence of the antigen epitope is GPGRNP.

6. A key amino acid site of an antigen epitope recognized by the monoclonal antibody 2C5 according to claim 2, characterized in that: The key amino acid site of the antigen epitope is the 188th amino acid R (HexonR188) of the highly pathogenic FAdV-4 Hexon protein.

7. The key amino acid sites of the antigen epitope recognized by the monoclonal antibody 2C5 according to claim 6, characterized in that: The 188th amino acid R of the Hexon protein is a characteristic amino acid of highly pathogenic serotype 4 avian adenovirus.

8. Use of the monoclonal antibody 2C5 according to claim 2 in the preparation of a product for detecting and identifying highly pathogenic serotype 4 avian adenovirus.

9. The use according to claim 8, characterized in that: The isolated strains of the highly pathogenic serotype 4 avian adenovirus preferably include SD2015, AHFY15, JH13, and JSCZ15.

10. A kit for detecting and identifying highly pathogenic serotype 4 avian adenovirus, characterized in that: The kit comprises the monoclonal antibody 2C5 as claimed in claim 2 and auxiliary materials constituting the kit.

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