An indirect ELISA kit and method for detecting avian adenovirus serum type 4 antibodies

Abstract

This invention relates to the application of the avian adenovirus serotype 4 multi-epitope recombinant chimeric protein rcF2 in the preparation of a kit for detecting avian adenovirus serotype 4 antibodies. The amino acid sequence of the avian adenovirus serotype 4 multi-epitope recombinant chimeric protein rcF2 is shown in SEQ ID NO.1. This invention utilizes the multi-epitope recombinant chimeric protein rcF2 to establish an indirect ELISA detection method and kit for detecting avian adenovirus serotype 4. This recombinant chimeric protein can be recognized by avian adenovirus serotype 4 positive sera, and the detection sensitivity of positive sera samples can reach 1:16000. It shows no cross-reactivity with antibodies against other pathogens, indicating that the ELISA detection method based on this multi-epitope recombinant chimeric protein rcF2 is simple to operate, enhances detection sensitivity, and has good specificity and repeatability.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of rcF2, a multi-epitope recombinant chimeric protein of avian adenovirus serotype 4, in the preparation of a kit for detecting avian adenovirus serotype 4 antibodies, an indirect ELISA kit for detecting avian adenovirus serotype 4, and its detection method. Background Technology

[0002] Adenovirus serotype 4 (FAdV-4) primarily causes pericardial effusion and hepatitis syndrome in broilers and laying hens, also known as "Ankara disease." It is a member of the genus Avian adenovirus in the family Avian adenoviridae. Since 2015, a new genotype of highly virulent FADV-4 has been prevalent in my country, becoming a significant pathogen in poultry farms. The new genotype FADV-4 spreads rapidly and exhibits strong tissue tropism, quickly invading multiple organs and tissues, including the liver, kidneys, spleen, and lungs. Affected chickens typically exhibit stunted growth and low humoral immunity, making them susceptible to co-infection with other diseases such as infectious bursal disease or infectious anemia virus (IAVV), causing severe economic losses to the poultry industry, with mortality rates ranging from 30% to 80%.

[0003] Currently, rapid detection methods for FADV-4 pathogens mainly include real-time quantitative PCR, polymerase chain reaction (PCR), and loop-mediated isothermal amplification (LAM). These methods are accurate and rapid, but costly and require a high level of operator skill. Serological diagnostic methods for FADV-4 infection include agar diffusion assay (AGP), neutralization assay (SN), and enzyme-linked immunosorbent assay (ELISA). However, AGP and SN are time-consuming, have low sensitivity, are easily affected by individual variability, and are difficult to quantitatively detect in large quantities. ELISA is simple to operate, low in cost, and has good specificity; therefore, establishing a rapid and accurate ELISA method for detecting FADV-4 antibodies is crucial for the prevention and control of this disease. However, existing commercially available antibody kits detect FADV-I group-specific antibodies, but their specificity is not strong enough. This study used structural biology techniques to screen for dominant antigenic epitopes, then synthesized positive recombinant expression plasmids using chemical methods. After induction and purification, the avian adenovirus serological type 4 multi-epitope recombinant chimeric protein rcF2 was obtained. Based on this protein, a highly specific and sensitive indirect ELISA detection method for detecting FAdV-4 antibodies was established. This method is suitable for large-scale serological testing in poultry farms, aiming to provide a simple and efficient technique for FAdV-4 vaccine development, serological monitoring, and infection status screening. Summary of the Invention

[0004] The purpose of this invention is to provide the application of avian adenovirus serotype 4 multi-epitope recombinant chimeric protein rcF2 in the preparation of a kit for detecting avian adenovirus serotype 4 antibodies, an indirect ELISA kit for detecting avian adenovirus serotype 4 antibodies, and a detection method thereof. The purified avian adenovirus serotype 4 multi-epitope recombinant chimeric protein rcF2 can be recognized by avian adenovirus serotype 4 positive serum. An indirect ELISA detection method for detecting avian adenovirus serotype 4 is established, thereby achieving specific diagnosis of avian adenovirus serotype 4. By using avian adenovirus serotype 4 multi-epitope recombinant chimeric protein rcF2 as a coating antigen to establish an indirect ELISA detection method, not only can the detection sensitivity be enhanced, but the detection efficiency can also be improved.

[0005] The objective of this invention is achieved through the following technical solution: This invention provides the application of avian adenovirus serotype 4 multi-epitope recombinant chimeric protein rcF2 in the preparation of a kit for detecting avian adenovirus serotype 4 antibodies, the amino acid sequence of which is shown in SEQ ID NO.1.

[0006] SEDLQQFISATGEAAAKFDATSVAPTTYEYMNEAAAKYVNFHIQVEAAAKQLDLVYPFDYVADPVGGLNPPFLGGSGPLVDQGGQLTLNVTDPIIIKNRSVDLAHDPSLDVNAQGQLAVAVDPE GALDITPDGLDVKVDGVTVMVNDDWELAVKVDPSGGLDSTAGGLGVSVDDTLLVDQGELGVHLNQQGPITADSGIDLEINPNMFTVNTSTGSGVLELNLKAQGGIQADSSGVGVSVDESLQIV NNTLEVKPDPSGPLTVSANGLGLKYDTNTLAVTAGALTVVGGGSVSTPIATFVSSGSPSLNTYNATTVNSSANAFSCAYYLQQWNIQGLLVTSLYLKLDSATMGNRPGDLNSANAKWFTFWVSAY LQQCNPSGIQAGTVSPSTATLTDFEPMANRSVTSPWTYSANGYYEPSIGEFQVFSPVVTGAWNPGNIGIRVLPVPVSASGERYTLLCYSLQCTNASIFNPNNSGTMIVGPVLYSCPAASLP (SEQ ID NO.1).

[0007] This invention provides an indirect ELISA kit for detecting avian adenovirus serotype 4 antibodies. The indirect ELISA kit uses avian adenovirus serotype 4 multi-epitope recombinant chimeric protein rcF2 as the coating antigen. The amino acid sequence of avian adenovirus serotype 4 multi-epitope recombinant chimeric protein rcF2 is shown in SEQ ID NO.1.

[0008] The preparation method of the avian adenovirus serum type 4 multi-epitope recombinant chimeric protein rcF2 is as follows: (1) Screening and construction of multi-epitope antigens: The amino acid sequence of Hexon protein from avian adenovirus serum type 4 was selected, and peptides with good hydrophilicity, flexibility and accessibility were selected as candidate epitope regions; then, the secondary and tertiary structures of the protein were predicted, and peptides rich in β-turns and random coils were selected. The protein was analyzed using a coil structure, with amino acid residues having low α-helix and β-sheet content as candidate epitope regions. B-cell linear epitopes were then predicted. Ten candidate antigenic epitopes were selected and named H1~H10 peptides, and Dot-blot was used to screen for epitopes with good antigenicity. Finally, three peptides, H1, H5, and H9, were selected, with amino acid sequences SEDLQQFISATG, FDATSVAPTTYEYMN, and YVNFHIQV, respectively. Subsequently, the nucleotides of the selected antigenic epitope short peptide sequences H1, H5, and H9 were linked to an EAAAK linker and then further linked to the Fiber2 gene sequence (as shown in SEQ ID NO.2) via the EAAAK linker. Following the H1-linker-H9-linker-H5-linker-Fiber2 linking sequence, a multi-epitope recombinant chimeric rcF2 nucleotide sequence was chemically synthesized, with the nucleotide sequence shown in SEQ ID NO.3. (2) Construction of multi-epitope recombinant chimeric rcF2 plasmid: The multi-epitope recombinant chimeric rcF2 nucleotide sequence was double-digested and then purified by gel extraction to obtain the double-digested rcF2 nucleotide sequence; then the vector plasmid pET32a-TEV was digested and then purified by gel extraction to obtain the digested vector backbone; the digested vector backbone and the double-digested rcF2 nucleotide sequence were ligated to obtain the pET32a-rcF2 ligation product; the obtained pET32a-rcF2 ligation product was added to competent cells BL21(DE3), cultured, colonies were picked and cultured, plasmid was extracted and double-digested and sequenced for verification, and finally the positive recombinant bacteria pET32a-rcF2 was obtained and frozen; (3) Expression and purification of avian adenovirus serum type 4 multi-epitope recombinant chimeric protein: The positive recombinant bacteria pET32a-rcF2 was induced to express and purified to obtain the avian adenovirus serum type 4 multi-epitope recombinant chimeric protein rcF2.

[0009] The Fiber2 gene sequence (codon optimized) SEQ ID NO.2, the multi-epitope recombinant chimeric rcF2 nucleotide sequence SEQ ID NO.3, and the Fiber2 protein amino acid sequence are as follows: The amino acid sequence of the Fiber2 protein is as follows: QLDLVYPFDYVADPVGGLNPPFLGGSGPLVDQGGQLTLNVTDPIIIKNRSVDLAHDPSLDVNAQGQLAVAVDPEGALDITPDGLDVKVDGVTVMVNDDWELAVKVDPSGGL DSTAGGLGVSVDDTLLVDQGELGVHLNQQGPITADSSGIDLEINPNMFTVNTSTGSGVLELNLKAQGGIQADSSGVGVSVDESLQIVNNTLEVKPDPSGPLTVSANGLGLK YDTNTLAVTAGALTVVGGGSVSTPIATFVSGSPSLNTYNATTVNSSANAFSCAYYLQQWNIQGLLVTSLYLKLDSATMGNRPGDLNSANAKWFTFWVSAYLQQCNPSGIQA GTVSPSTATLTDFEPMANRSVTSPWTYSANGYYEPSIGEFQVFSPVVTGAWNPGNIGIRVLPVPVSASGERYTLLCYSLQCTNASIFNPNNSGTMIVGPVLYSCPAASLP.

[0010] The indirect ELISA kit also includes HRP-labeled secondary antibody (goat anti-chicken IgG), blocking buffer, washing buffer, diluent, positive control sample, negative control sample, chromogenic solution, and stop solution.

[0011] A method for detecting avian adenovirus serological type 4 antibodies using the aforementioned indirect ELISA kit, which is not for diagnostic purposes, includes the following steps: The avian adenovirus serum type 4 multi-epitope recombinant chimeric protein rcF2, used as the coating antigen, was diluted and coated onto an ELISA plate, which was then washed and blocked. After blocking, diluted chicken serum to be tested was added as the primary antibody and incubated. After incubation, wash, add diluted HRP-labeled secondary antibody (goat anti-chicken IgG), and incubate. After incubation, wash, add color developer, and incubate in the dark; Add stop solution and read OD using a microplate reader. 492nm value.

[0012] The coating concentration of the avian adenovirus serum type 4 multi-epitope recombinant chimeric protein rcF2 was 2 μg / mL, and the blocking time was 1 h.

[0013] The chicken serum to be tested was diluted 1:500 and incubated for 1 hour.

[0014] The HRP-labeled secondary antibody (goat anti-chicken IgG) was diluted at a ratio of 1:10000, and the incubation time for the HRP-labeled secondary antibody (goat anti-chicken IgG) was 1 hour.

[0015] Compared with the prior art, the advantages of the present invention are as follows: 1. This invention screens dominant antigenic epitopes based on structural biology analysis and further utilizes the prokaryotically expressed avian adenovirus serotype 4 multi-epitope recombinant chimeric protein rcF2 as the coating antigen to establish an indirect ELISA method for detecting avian adenovirus serotype 4 antibodies.

[0016] 2. This invention establishes an indirect ELISA detection method using avian adenovirus type 4 multi-epitope recombinant chimeric protein rcF2 as the coating antigen. This method can rapidly detect avian adenovirus type 4 antibodies. The rcF2 protein can be recognized by avian adenovirus type 4 positive sera, and the detection sensitivity for positive sera samples can reach 1:16000. It shows no cross-reactivity with positive sera antibodies from other pathogens. The ELISA detection method based on this rcF2 protein has advantages such as simple operation, high specificity, high sensitivity, and good reproducibility. It can be used to detect a large number of clinical samples, and the results show that this method can be applied to practical production for the detection of avian adenovirus type 4 antibodies. Attached Figure Description

[0017] Figure 1 This is a diagram of the Ni-NTA purification of the recombinant chimeric multi-epitope protein rcF2 of the present invention; wherein, Marker; 1: 300 mM eluted protein; 2: 300 mM eluted protein + TEV enzyme; 3: recombinant multi-epitope protein rcF2 obtained by second nickel column pass-through buffer; 4: recombinant multi-epitope protein rcF2 obtained by second nickel column pass-through buffer with 10 mM imidazole.

[0018] Figure 2 These are the specific detection results of the indirect ELISA detection method established in this invention; where NC represents the negative serum control; Positive represents the positive serum control; and the dashed line represents the cut-off value.

[0019] Figure 3 These are the sensitivity test results of the indirect ELISA detection method established in this invention.

[0020] Figure 4These are the clinical serum sample detection results of the indirect ELISA detection method established in this invention; where NC represents the negative serum control; Positive represents the positive serum control; and the dashed line represents the cut-off value. Detailed Implementation

[0021] The present invention will now be described in detail with reference to the accompanying drawings and embodiments: Example 1: Preparation of avian adenovirus serum type 4 multi-epitope recombinant chimeric protein rcF2 (abbreviated as: multi-epitope recombinant chimeric protein rcF2) The preparation method of rcF2, a multi-epitope recombinant chimeric protein of avian adenovirus serum type 4, is roughly as follows: (1) Screening and construction of multi-epitope antigens: The amino acid sequence of Hexon protein from avian adenovirus serum type 4 was selected, and peptides with good hydrophilicity, flexibility and accessibility were selected as candidate epitope regions; then, the secondary and tertiary structures of the protein were predicted, and peptides rich in β-turns and random coils were selected. The protein was analyzed using a coil structure, with amino acid residues having low α-helix and β-sheet content as candidate epitope regions. B-cell linear epitopes were then predicted. Ten candidate antigenic epitopes were selected and named H1~H10 peptides, and Dot-blot was used to screen for epitopes with good antigenicity. Finally, three peptides, H1, H5, and H9, were selected, with amino acid sequences SEDLQQFISATG, FDATSVAPTTYEYMN, and YVNFHIQV, respectively. Subsequently, the nucleotides of the selected antigenic epitope short peptide sequences H1, H5, and H9 were linked to an EAAAK linker and then further linked to the Fiber2 gene sequence via the EAAAK linker. Following the H1-linker-H9-linker-H5-linker-Fiber2 linking sequence, a multi-epitope recombinant chimeric rcF2 nucleotide sequence was chemically synthesized, as shown in SEQ ID NO.3. (2) Construction of multi-epitope recombinant chimeric rcF2 plasmid: The multi-epitope recombinant chimeric rcF2 nucleotide sequence was double-digested and then purified by gel extraction to obtain the double-digested rcF2 nucleotide sequence; then the vector plasmid pET32a-TEV was digested and then purified by gel extraction to obtain the digested vector backbone; the digested vector backbone and the double-digested rcF2 nucleotide sequence were ligated to obtain the pET32a-rcF2 ligation product; the obtained pET32a-rcF2 ligation product was added to competent cells BL21(DE3), cultured, colonies were picked and cultured, plasmid was extracted and double-digested and sequenced for verification, and finally the positive recombinant bacteria pET32a-rcF2 was obtained and frozen; (3) Expression and purification of avian adenovirus serum type 4 multi-epitope recombinant chimeric protein: The positive recombinant bacteria pET32a-rcF2 was induced to express and purified to obtain the avian adenovirus serum type 4 multi-epitope recombinant chimeric protein rcF2.

[0022] The preparation of rcF2, a multi-epitope recombinant chimeric protein of avian adenovirus serum type 4, is described in detail below: 1.1 Screening and Construction of Multiepitope Antigens The amino acid sequence of the hexon protein from FAdV-4 GDMZ strain (GenBank ID: MG856954) was selected. Peptides with good hydrophilicity, flexibility, and accessibility were screened using the Expasy database and selected as candidate epitope regions. The secondary and tertiary structures of the protein were predicted using the SOPMA online database and PyMOL software, respectively. Amino acid residues rich in β-turns and random coils, but with low α-helix and β-sheet content, were selected as candidate epitope regions. The Kolaskar and Bepipred methods in the IEBD online tool were used to predict B-cell linear epitopes of the protein, and regions with a threshold of not less than 0.5 were selected as candidate epitopes. The cytotoxicity of the protein was predicted using ToxinPred software, and non-cytotoxic peptides were selected as candidate epitopes. Using the aforementioned bioinformatics tools, 10 candidate antigenic epitopes were screened out and named H1~H10 peptides, respectively. These were then used by Sangon Biotech (Shanghai) Co., Ltd. for short peptide synthesis, and further Dot-blot screening was conducted to identify epitopes with good antigenicity.

[0023] Among them, the FAdV-4 GDMZ strain (GenBank ID: MG856954) was preserved by the Poultry Disease Laboratory of the Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences.

[0024] 1.2 Dot-blot detection of antigenic epitopes Sample preparation: The synthesized short peptide powder was dissolved in ddH2O to form a 1 mg / mL short peptide solution H1~H10, aliquoted and placed in an ultra-low temperature freezer for use as a coating antigen.

[0025] 1) Activation: Take a polyvinylidene fluoride (PVDF) membrane and draw a circle with a pencil for sample spotting. Activate the PVDF membrane in 15 mL of analytical grade methanol solution for 60 seconds. After activation, place the PVDF membrane in ddH2O and rinse it three times on a decolorizing shaker for 2 minutes each time, then transfer it to PBST solution for equilibration for 5 minutes.

[0026] 2) Coating: After air drying at room temperature for 5 minutes, add 4 μL of short peptide to the center of the PVDF membrane circle. Fiber2 protein is set as a positive control and sterile PBS as a negative control. Three replicates are set for each sample.

[0027] 3) Blocking: After drying the PVDF membrane in a 37℃ incubator for 10 minutes, place the membrane in a 3% BSA / PBST solution and gently shake at room temperature for 2 hours to block. After blocking, wash the membrane twice with PBST solution for 3 minutes each time.

[0028] 4) Primary antibody: Add chicken positive serum diluted 1:500 to the PVDF membrane and incubate at 37°C for 1.5 h.

[0029] 5) Secondary antibody: Discard the primary antibody, wash the membrane 3 times with PBST solution for 3 minutes each time, add horseradish peroxidase-labeled goat anti-chicken IgG antibody diluted 1:5000 to the PVDF membrane, and incubate at 37°C for 1 hour.

[0030] 6) Color development: Discard the secondary antibody, wash the membrane three times with PBST solution for 3 minutes each time, air dry at room temperature for 5 minutes, then add an appropriate amount of DAB color development solution to evenly cover the membrane surface. Incubate at 37°C in the dark for 10 minutes, then add ddH2O to rinse three times to terminate the reaction. Air dry the membrane at room temperature until the spots are clear, then take a picture to record the results.

[0031] 1.3 Construction of Recombinant Chimeric rcF2 Plasmid Alphafold predicted the three-dimensional spatial structure of the Fiber2 protein. Combined with immunoinformatics analysis, the major antigenic epitopes on its backbone were preserved. The selected Hexon epitopes were then recombined into the 5' end of the Fiber2 gene sequence via linker linkage, and the resulting nucleotide sequence was chemically synthesized. The specific methods are as follows: Based on the spatial structure analysis of the antigenic epitope short peptide sequence, the construction sequence was determined. The screened antigenic epitope short peptide sequence and the Fiber2 gene sequence were then fused together via an intermediate linker peptide EAAAK to chemically synthesize the recombinant chimeric rcF2 nucleotide sequence, which was then subjected to double enzyme digestion. The total volume of the digestion system was 50 μL, including 5 μL of 10×Cutsmart buffer, 1 μL of HindIII, 1 μL of XhoI, 20 μL of purified PCR product, and 23 μL of ddH2O. Digestion was performed at 37℃ for 60 min, followed by gel purification to obtain the double-digested rcF2 nucleotide sequence.

[0032] The pET32a-TEV vector was then digested with enzymes. The pET32a-TEV digestion system was as follows: total volume 50 μL, including 5 μL of 10×Cutsmart buffer, 1 μL of HindIII, 1 μL of XhoI, 5 μL of vector plasmid, and 38 μL of ddH2O. After gel recovery, the digested vector backbone was obtained.

[0033] The double-digested vector backbone and the double-digested rcF2 nucleotide sequence were ligated. The ligation reaction system consisted of 1 μL of 10×T4 DNA Ligase Buffer, 1 μL of T4 DNA Ligase, 1 μL of vector backbone, and 7 μL of gene. Ligation was carried out overnight at 16°C to obtain the pET32a-rcF2 ligation product.

[0034] The obtained pET32a-rcF2 ligation product was added to competent BL21(DE3) cells, placed on ice, and then heat-shocked in a 42°C water bath for 45 seconds. Immediately afterward, the cells were transferred and cooled on ice for 2 min. 500 μL of antibiotic-free liquid LB medium was added, and the cells were incubated on a shaker for 1 h before being plated and cultured at 37°C for 14 h. Colonies were picked from the plates, and plasmids were extracted for double enzyme digestion and sequencing verification. Finally, the positive recombinant bacteria pET32a-rcF2 were cryopreserved.

[0035] 1.4 Expression and purification of multi-epitope recombinant chimeric protein rcF2 After thawing the positive recombinant bacteria pET32a-rcF2, 10 μL was transferred to 1 mL of liquid LB medium (containing 100 μg / mL ampicillin) and incubated overnight at 37 °C and 220 rpm to allow the recombinant bacteria to fully recover. Then, it was inoculated at a 1:1000 ratio into 10 mL of liquid LB medium (containing 100 µg / mL ampicillin) and cultured continuously at 37 °C and 200 rpm for 6 h. Next, it was inoculated at a 1:1000 ratio into 1 L of liquid LB medium and cultured continuously at 37 °C and 200 rpm for 3.5 h. Afterward, the shaker temperature was set to 16 °C for overnight induction (12-16 h). After cooling, IPTG (final concentration 0.3 mM) was added to the bacterial culture and incubated overnight at 190 rpm. The inducing culture was then poured into a collection bottle and centrifuged at 4 °C and 8000 rpm for 10 min to collect the precipitate. The precipitate was resuspended in protein buffer (25 mM Tris pH 8.0, 500 mM NaCl, 0.5% Tween, 2 mM β-mercaptoethanol, 5 mM imidazole). The cells were then disrupted using a high-pressure cell disruptor. Before disruption, the cells were washed once with ddH2O and twice with protein buffer to equilibrate the cell disruptor. The disruption conditions were 5 °C, 800 bar, and 2 min. After high-pressure disruption, the cells were centrifuged at 12000 × g for 45 min, and the supernatant was collected. This supernatant was added to a nickel column equilibrated with protein buffer at a flow rate of 4 s / drop to ensure the target protein was fully bound to nickel. Then, the cells were eluted with 20 mM imidazole and 40 mM imidazole to remove contaminating proteins, followed by elution with 300 mM imidazole at a rate of 10 s / drop. The collected protein suspension was then dialyzed with TEV enzyme at 4 °C overnight. After enzyme digestion and dialysis, the protein was passed through a nickel column a second time, and the flow-through was collected. After the flow-through was complete, protein buffer containing 10 mM imidazole was added to elute the protein on the column. Then, the multi-epitope recombinant chimeric protein rcF2 was concentrated to 4.43 mg / mL.

[0036] 1.5 Experimental Results: 1.5.1 Screening and Construction of Multiepitope Antigens Ten candidate antigenic epitopes, named H1–H10, were screened using structural biology and bioinformatics techniques. Further screening using Dot-blot method revealed strong positive results for H1, H5, and H9; weak positive results for H2, H7, and H10, indicating that H1, H5, and H9 were the dominant peptides. Their corresponding amino acid sequences are SEDLQQFISATG, FDATSVAPTTYEYMN, and YVNFHIQV, respectively. Based on spatial structure analysis of the short peptide sequences, the construction sequence was determined to be H1-linker-H9-linker-H5. The selected short peptide sequences were then linked to an EAAAK linker peptide and subsequently linked to the Fiber2 gene sequence in the following order: H1-linker-H9-linker-H5-linker-Fiber2. A multi-epitope recombinant chimeric rcF2 nucleotide sequence (SEQ ID NO. 3) was chemically synthesized. The multi-epitope recombinant chimeric rcF2 nucleotide sequence was further constructed into the pET-32a vector to obtain positive recombinant bacteria pET32a-rcF2, which were then expressed as proteins.

[0037] 1.5.2 Expression and purification of multi-epitope chimeric proteins SDS-PAGE identified the Ni-NTA purified multi-epitope recombinant chimeric protein rcF2, which was found to be soluble in the supernatant of the lysate after sonication. Impurities were removed by elution with 40 mM imidazole, followed by elution with 300 mM imidazole to obtain a Trx+His-tagged protein with a molecular weight of approximately 80 kDa. TEV digestion overnight separated the target and tag proteins. After dialysis overnight, the protein was passed through a nickel column again to remove the Trx+His tag protein, finally yielding a 60 kDa protein. Figure 1 ).

[0038] Example 2: Establishment of an Indirect ELISA Detection Method 2.1 Establishment of Indirect ELISA Detection Method The multi-epitope recombinant chimeric protein rcF2 was diluted to 5 μg / mL with carbonate buffer (pH 9.6) and coated onto ELISA plates, 50 μL per well. The plates were incubated overnight at 4°C. The next day, the wells were washed once with 250 μL PBST. 100 μL of 2% BSA / PBST solution was added to each well, and the plates were blocked at 37°C for 1 h. Chicken serum diluted with PBST containing 1% BSA was used as the primary antibody, diluted at dilutions of 1:250, 1:500, 1:1000, 1:2000, 1:4000, 1:8000, 1:1600; 1:32000, 1:64000, 1:128000, 1:256000, and 1:512000, and added to each well, 50 μL per well, and incubated at 37°C for 1 h. A blank control group was set up. After incubation, wash each well three times with 250 μL PBST. Add 50 μL of HRP-labeled secondary antibody (goat anti-chicken IgG), diluted 1:10000, to each well and incubate at 37°C for 1 h. After incubation, wash three times with 250 μL PBST and once with ddH2O. Add 50 μL of OPD chromogenic solution (freshly prepared) to each well and incubate at 37°C in the dark for 10-15 min. Stop the reaction by adding 50 μL of 2M H2SO4 to each well. Read the OD values ​​using a microplate reader. 492nm value.

[0039] 2.2 Determination of Critical Values An indirect ELISA method based on the multi-epitope recombinant chimeric protein rcF2 as the coating antigen was used to detect the results of 30 SPF chicken negative serum samples. The average value was calculated based on the absorbance at 492 nm. - x and variance s, - x±3s determines the negative critical value.

[0040] 2.3 Optimization of Indirect ELISA Reaction Conditions The optimal dilutions for coating multi-epitope recombinant chimeric protein rcF2 and chicken serum primary antibody were determined using a square matrix assay: rcF2 was serially diluted to 5 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, and 0.25 μg / mL, with each concentration gradient coated in one row. Primary antibody-positive and negative chicken serum were diluted at 1:250, 1:500, 1:1000, 1:2000, 1:4000, 1:8000, 1:1600; 1:32000, 1:64000, 1:128000, and 1:256000, with each dilution added to one column. The ELISA procedure was followed as described above, with three replicates. OD values ​​of positive serum were selected. 492nmThe optimal reaction conditions are those corresponding to wells with a P / N ratio close to 1. The dilution of goat anti-chicken enzyme-labeled secondary antibody, antigen coating conditions, blocking time, primary and secondary antibody incubation time, and color development time were optimized and screened to determine the optimal reaction procedure for the established indirect ELISA.

[0041] 2.4 Specificity test The established ELISA method was used to detect positive serum samples for Newcastle disease (NDV), infectious bronchitis (IBV), infectious bursal disease (IBDV), Marek's disease (MDV), egg drop syndrome (EDSV), avian leukosis (ALV), and H5N1 (AIV-H5) and H9N2 (AIV-H9) influenza. Positive and negative controls were set up to determine the specificity of the indirect ELISA method.

[0042] 2.5 Sensitivity Test Three FAdV-4 positive sera were randomly selected and diluted at ratios of 1:250, 1:500, 1:1000, 1:2000, 1:4000, 1:8000, 1:1600; 1:32000, 1:64000, 1:128000, and 1:256000, respectively, and then added to an ELISA plate. The established optimal ELISA reaction procedure was used for detection to determine the sensitivity of the established indirect ELISA method.

[0043] 2.6 Repeatability Test Five FadV-4-infected chicken positive serum samples and one SPF chicken negative serum sample were randomly selected. Using antigen-coated plates prepared from the same batch, samples were tested at three different time points, with three replicate wells per test, for intra-batch repeatability testing. Samples were also tested simultaneously at three different batches of antigen-coated plates for inter-batch repeatability testing. The coefficient of variation (coefficient of variation = variance / mean × 100%) of the indirect ELISA method based on the multi-epitope recombinant chimeric protein rcF2 was less than 10%, indicating good intra-batch and inter-batch repeatability of the established ELISA method.

[0044] 2.7 Clinical Sample Testing Twenty-five serum samples were tested using an indirect ELISA method based on the multi-epitope recombinant chimeric protein rcF2. These serum samples were obtained from laying hens in a chicken house that were infected with avian adenovirus serotype 4. The agar diffusion assay (AGP) was then used to further test these serum samples, and the concordance rates of the two detection methods were compared.

[0045] 2.8 Experimental Results 2.8.1 Optimization of ELISA Detection Conditions The concentration of the coating antigen, serum dilution, and enzyme-labeled secondary antibody dilution were optimized using the above method. The optimal conditions were as follows: the concentration of the multi-epitope recombinant chimeric protein rcF2 was 2 μg / mL (0.1 μg / well), the serum was diluted 1:500, and the goat anti-chicken enzyme-labeled secondary antibody was diluted 1:10000. The OD of 30 negative sera from SPF chickens was detected using the established ELISA method. 492 Value, calculate the average - x is 0.213 and the standard deviation s is 0.035. The mean is added to three times the standard deviation as the threshold for judging positive and negative results. The critical value is 0.318. That is, if the OD value of the serum to be tested is >0.318, it is judged as positive for avian adenovirus serum type 4 antibody, and if the OD value is <0.318, it is judged as negative.

[0046] 2.8.2 Specificity analysis of the ELISA method The ELISA method established above was used to simultaneously detect NDV, IBV, IBDV, MDV, EDSV, ALV, and AIV-H5 and AIV-H9 positive sera, with FADV-4 positive serum and a negative control also included. Results showed that the indirect ELISA method established using the multi-epitope recombinant chimeric protein rcF2 as the coating antigen reacted only with FADV-4 positive serum, and showed no cross-reactivity with exogenous positive sera of NDV, IBV, IBDV, MDV, EDSV, ALV, and AIV-H5 and AIV-H9. This indicates that the indirect ELISA method established using the multi-epitope recombinant chimeric protein rcF2 has good specificity. Figure 2 ).

[0047] 2.8.3 Sensitivity Test Three FAdV-4 positive serum samples were serially diluted from 1:250 to 1:256000 and subjected to ELISA detection under the optimal reaction conditions determined in this experiment. The results showed that even when the FAdV-4 positive serum was diluted to 1:16000, the detection result was still positive, indicating that the ELISA detection method based on the multi-epitope recombinant chimeric protein rcF2 has high sensitivity. Figure 3 ).

[0048] 2.8.4 Repeatability Test The established ELISA method was used to perform intra-assay and inter-assay replicates on 5 positive sera and 1 negative sera. The results showed that the coefficient of variation for intra-assay replicates was 2.62%–4.17%, while the coefficient of variation for inter-assay replicates was 2.58%–4.47%, both less than 5% (Table 1). This indicates that the indirect ELISA detection method based on the multi-epitope recombinant chimeric protein rcF2 has good reproducibility.

[0049] Table 1 Results of intra-batch and inter-batch repeatability tests

[0050] 2.8.5 Clinical serum sample testing The ELISA and AGP antibody detection methods established in this experiment were used to detect serum samples from poultry farms clinically infected with FADV-4. The results showed that 23 out of 25 chicken serum samples were positive by ELISA, with AGP titers ranging from 1:8 to 1:32. Two serum samples, numbered 7 and 19, were double-negative. Figure 4 The results showed that the ELISA test results were 100% consistent with the AGP test results, indicating that FADV-4 infection had occurred in the farm. Furthermore, the indirect ELISA detection method based on the multi-epitope recombinant chimeric protein rcF2 can be used for serological detection of avian adenovirus serotype 4 clinical samples.

[0051] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. The application of avian adenovirus serotype 4 multi-epitope recombinant chimeric protein rcF2 in the preparation of a kit for detecting avian adenovirus serotype 4 antibodies, characterized in that: The amino acid sequence of the avian adenovirus serum type 4 multi-epitope recombinant chimeric protein rcF2 is shown in SEQ ID NO.

1.

2. An indirect ELISA kit for detecting avian adenovirus serum type 4 antibodies, characterized in that: The indirect ELISA kit uses avian adenovirus serum type 4 multi-epitope recombinant chimeric protein rcF2 as the coating antigen; the amino acid sequence of the avian adenovirus serum type 4 multi-epitope recombinant chimeric protein rcF2 is shown in SEQ ID NO.

1.

3. The indirect ELISA kit according to claim 2, characterized in that: The preparation method of the avian adenovirus serum type 4 multi-epitope recombinant chimeric protein rcF2 is as follows: (1) Screening and construction of multi-epitope antigens: The amino acid sequence of Hexon protein of avian adenovirus serum type 4 was selected, and peptides with good hydrophilicity, flexibility and accessibility were selected as candidate epitope regions; then, the secondary and tertiary structures of the protein were predicted, and amino acid residues rich in β-turns and random coil structures, and low in α-helices and β-sheets were selected as candidate epitope regions; then, the B-cell linear epitopes of the protein were predicted; 10 candidate antigenic epitopes were selected and named H1~H10 peptides, and Dot-blot was used to further screen epitopes with good antigenicity; finally, H1, H5 and H1 were selected. The three peptide segments 9 have amino acid sequences of SEDLQQFISATG, FDATSVAPTTYEYMN, and YVNFHIQV, respectively. Subsequently, the nucleotides of the selected antigenic epitope short peptide sequences H1, H5, and H9 are linked to the EAAAK intermediate linker peptide and then linked to the Fiber2 gene sequence through the EAAAK intermediate linker peptide. Following the linker sequence H1-linker-H9-linker-H5-linker-Fiber2, a multi-epitope recombinant chimeric rcF2 nucleotide sequence is chemically synthesized, and its nucleotide sequence is shown in SEQ ID NO.

3. (2) Construction of multi-epitope recombinant chimeric rcF2 plasmid: The multi-epitope recombinant chimeric rcF2 nucleotide sequence was double-digested and then purified by gel extraction to obtain the double-digested rcF2 nucleotide sequence; then the vector plasmid pET32a-TEV was digested and then purified by gel extraction to obtain the digested vector backbone; the digested vector backbone and the double-digested rcF2 nucleotide sequence were ligated to obtain the pET32a-rcF2 ligation product; the obtained pET32a-rcF2 ligation product was added to competent cells BL21(DE3), cultured, colonies were picked and cultured, plasmid was extracted and double-digested and sequenced for verification, and finally the positive recombinant bacteria pET32a-rcF2 was obtained and frozen; (3) Expression and purification of avian adenovirus serum type 4 multi-epitope recombinant chimeric protein: The positive recombinant bacteria pET32a-rcF2 was induced to express and purified to obtain the avian adenovirus serum type 4 multi-epitope recombinant chimeric protein rcF2.

4. The indirect ELISA kit according to claim 2, characterized in that: The indirect ELISA kit also includes an HRP-labeled secondary antibody (goat anti-chicken IgG), blocking solution, washing solution, diluent, positive control sample, negative control sample, chromogenic solution, and stop solution.

5. A method for detecting avian adenovirus serum type 4 antibodies using the indirect ELISA kit as described in claim 2, wherein the method is not for diagnostic purposes, characterized in that: It includes the following steps: The avian adenovirus serum type 4 multi-epitope recombinant chimeric protein rcF2, used as the coating antigen, was diluted and coated onto an ELISA plate, which was then washed and blocked. After blocking, diluted chicken serum to be tested was added as the primary antibody and incubated. After incubation, wash, add diluted HRP-labeled secondary antibody (goat anti-chicken IgG), and incubate. After incubation, wash, add color developer, and incubate in the dark; Add stop solution and read OD using a microplate reader. 492nm value.

6. The detection method according to claim 5, characterized in that: The coating concentration of the avian adenovirus serum type 4 multi-epitope recombinant chimeric protein rcF2 was 2 μg / mL, and the blocking time was 1 h.

7. The detection method according to claim 5, characterized in that: The chicken serum to be tested was diluted 1:500 and incubated for 1 hour.

8. The detection method according to claim 5, characterized in that: The HRP-labeled secondary antibody (goat anti-chicken IgG) was diluted at a ratio of 1:10000, and the incubation time for the HRP-labeled secondary antibody (goat anti-chicken IgG) was 1 hour.

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