FAdV-11 Fiber recombinant protein as well as preparation and application thereof

Abstract

The invention discloses an FAdV-11Fiber recombinant protein as well as preparation and application thereof. The amino acid sequence of the recombinant protein is as shown in SEQ ID NO. 1. The invention provides a nucleotide sequence of a gene for coding the FAdV-11Fiber recombinant protein, wherein the nucleotide sequence of the gene is as shown in SEQ ID NO.2 or SEQ ID NO.3. The invention also provides a gene for coding the FAdV-11Fiber recombinant protein. According to the invention, the problem that the FAdV-11 infection cannot be rapidly and quantitatively detected in the prior art is solved. According to the invention, FAdV-11Fiber protein expressed by an escherichia coli expression system and a mammalian cell expression system is utilized, two ELISA detection methods with strong specificity, high sensitivity and good repeatability are constructed, and a new technical means is provided for specific detection of FAdV-11.

Description

Technical Field

[0001] This invention relates to a FAdV-11 protein, specifically to a recombinant FAdV-11 Fiber protein and its preparation and application. Background Technology

[0002] Avian adenovirus (FAdV) is one of the most common pathogenic pathogens in poultry, belonging to the genus Adenovirus in the family Adenoviridae. It is a major cause of hepatitis and hydropericardium syndrome (HHS) and inclusion body hepatitis (IBH). Avian adenovirus is mainly found in commercial poultry (chickens, turkeys, ducks, geese, etc.) and wild birds, and is distributed worldwide. With the rise of intensive industrialized poultry farming, highly pathogenic mixed or singular infections of IBH and HHS caused by avian adenovirus types 4 (FAdV-4), 8 (FAdV-8), and 11 (FAdV-11) have been reported worldwide. Avian adenovirus is no longer considered an opportunistic pathogen that can rapidly cause disease and even death in large numbers of poultry. In recent years, cases of IBH caused by FAdV-11 have appeared in various regions of my country, mainly showing local epidemics, causing serious economic losses to my country's poultry industry. The FAdV-11 pathogen can also be detected in healthy live poultry in some areas.

[0003] Existing technologies involve the following detection methods: Reference 1 (CheemaAE, AhmadJ, Afzal M. Anadenovirus infection of poultry in Pakistan[J]. Rev Sci Tech, 1989, 8(3): 789-795) involves necropsy of poultry suspected of avian adenovirus infection, homogenization of the liver, and observation of virus particles using an electron microscope. Simultaneously, the liver homogenate suspected of adenovirus infection is filtered and sterilized before being inoculated into chicken embryos or susceptible cells, and the presence of avian adenovirus infection is confirmed by observing characteristic lesions. However, due to factors such as mixed infection and the time-consuming and laborious process of virus isolation, the conventional microbiological diagnostic process is lengthy and cannot provide an effective and rapid diagnosis. Reference 2 (Raue R, Hess M. Hexon-based PCRs combined with restriction enzyme analysis for rapid detection and differentiation of fowl adenoviruses and egg drop syndrome virus[J]. J Virol Methods. 1998, 73(2): 211-217) points out that PCR detection is rapid, simple, and highly sensitive, and is widely used in primary care clinical diagnosis. Conventional PCR identification of the FADV genome is one of the most commonly used diagnostic methods. However, conventional PCR cannot perform quantitative detection and can only be used for qualitative detection of FADV. Reference 3 (Guo Y, Xie S, Xu Z, et al. An Efficient and Rapid Assay for Detecting Neutralizing Antibodies Against Serotype 4 fowl Adenovirus[J]. Front Vet Sci, 2022, 9: 867697-867703) suggests that this type of VNT method can be used to determine antibody neutralization titers after FADV vaccine immunization. Reference 4 (Erny K, Pallister J, Sheppard M. Immunological and molecular comparison of fowl adenovirus serotypes 4 and 10[J]. Arch Virol. 1995, 140(3): 491-501) demonstrates the use of this type of VNT method for viral infection diagnosis and typing. However, the VNT tests mentioned in references 3 and 4 exhibit cross-protection and have long testing cycles, making them unsuitable for large-scale testing.Enzyme-linked immunosorbent assay (ELISA) involves attaching antigens to a solid-phase carrier through incubation. Reference 5 (Lu H, Wang W, Zhang J, et al. An efficient fiber-based ELISA for detection of antibody against fowl adenovirus serotypes 7 and 8[J]. J Vet Diagn Invest. 2020, 32(3): 444-449) describes an ELISA constructed using GST-Fiber protein that can effectively detect antibodies produced by natural infection and immunization of FAdV-7 and FAdV-8, but does not react with other serotypes such as FAdV-1, FAdV-4, and FAdV-10. Therefore, this ELISA method cannot be used for the serological detection of FAdV-11. Reference 6 (Wu Xiaoqian. Expression of recombinant protein of avian adenovirus type 4 and establishment of indirect ELISA antibody detection method [D]. Guangxi University, 2022) successfully constructed five indirect ELISA methods to evaluate the antibody titers of FAdV-4 infection and FAdV-4 inactivated vaccine immunization, using FAdV-4 structural and non-structural proteins as coating antigens. The results showed that the 100K-ELISA and 22K-ELISA of non-structural proteins could only detect FAdV-4 infection. The Fiber-1-ELISA, Fiber-2-ELISA and Penton-ELISA established with structural proteins could detect antibodies produced by natural infection and antibodies produced by FAdV-4 inactivated vaccine immunization, but could not detect FAdV-11 infection.

[0004] Therefore, existing technologies cannot provide rapid and quantitative detection of FAdV-11 infection, nor do they cover serological detection methods specifically for FAdV-11. Summary of the Invention

[0005] The purpose of this invention is to provide a recombinant FAdV-11 Fiber protein, its preparation and application, which solves the problem that existing technologies cannot perform rapid and quantitative detection of FAdV-11 infection.

[0006] To achieve the above objectives, the present invention provides a recombinant FAdV-11 Fiber protein, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] The present invention also provides a gene encoding the FAdV-11 Fiber recombinant protein as described above, the nucleotide sequence of which is shown in SEQ ID NO.2 or SEQ ID NO.3.

[0008] The present invention also provides a recombinant vector containing the gene as described above.

[0009] Preferably, the recombinant vector is pGEX-4T-1 or pCAGGS.

[0010] This invention also provides a method for preparing the FAdV-11 Fiber recombinant protein as described above. When prokaryotic cell expression is used, the method comprises:

[0011] (1) Homology comparison analysis was performed using the FAdV-11 Fiber gene sequence already available in the GenBank database. One strain was selected and codon optimization was performed to suit the Escherichia coli Rosetta expression system. EcoRI and Xhol sequences were introduced at the 5' and 3' ends, respectively. Gene synthesis was performed to obtain the target sequence shown in SEQ ID NO.2. The target sequence was cloned into the pGEX-4T-1 plasmid vector with the GST tag to construct the prokaryotic expression vector pGEX-GST-Fiber.

[0012] (2) The pGEX-GST-Fiber expression vector was transformed into Rosetta(DE3) competent cells and cultured at 37℃ in an inverted state. Single colonies were picked and inoculated into LB liquid medium and cultured until the OD600 of the bacterial culture was 0.6-0.8. The bacterial culture was mixed with glycerol and stored at -80℃ to obtain Rosetta(DE3) strain containing the pGEX-GST-Fiber expression vector.

[0013] (3) Rosetta (DE3) strain was inoculated into LB liquid medium and activated at 37°C. The activated bacterial solution was then inoculated into LB liquid medium and cultured until the OD600 of the bacterial solution was 0.6-0.8. IPTG was added and the solution was induced at 20°C. The induced bacterial solution was centrifuged at 8000 r / min at 4°C, the supernatant was discarded, and the bacterial cells were collected. Sterile PBS was added, and the bacterial cells were repeatedly resuspended by pipetting. The solution was centrifuged at 8000 r / min at 4°C, the supernatant was discarded, and the operation was repeated to resuspend the bacterial cells with PBS. Lysozyme was added and the solution was lysed on ice. The solution was then sonicated. The lysate was centrifuged at 10000 r / min at 4°C, and the supernatant and precipitate were collected to obtain the protein sample. The protein sample was mixed with protein loading buffer at a volume ratio of 1:4.

[0014] When using eukaryotic cell expression, this method includes:

[0015] (1) Based on the FAdV-11 Fiber gene sequence in the GenBank database, codon optimization was performed to suit the mammalian cell expression system. EcoRI sequence, kozak sequence and IL-10 signal peptide were introduced sequentially at the 5' end, and 6×His and Xhol sequences were added at the 3' end. Gene synthesis was performed to obtain the target sequence as shown in SEQ ID NO.3. The target sequence was ligated into the pCAGGS plasmid vector to construct the eukaryotic expression vector pCAGGS-His-Fiber.

[0016] The nucleotide sequence of the kozak is shown in SEQ ID NO.4; the amino acid sequence of the IL-10 signal peptide is shown in SEQ ID NO.5;

[0017] (2) Transform the pCAGGS-His-Fiber expression vector into DH5α competent cells, incubate at 37℃ in an inverted state, pick single colonies, inoculate them into LB liquid medium, and incubate until the OD600 of the bacterial culture is 0.6-0.8. Mix the bacterial culture with glycerol and store at -80℃ to obtain DH5α bacteria containing the pCAGGS-His-Fiber expression vector. Inoculate the DH5α bacteria into LB liquid medium and incubate at 37℃ to activate them. Inoculate the activated bacterial culture into LB liquid medium and incubate at 37℃ and 220 r / min. Collect the bacterial culture, centrifuge at 8000 r / min, discard the supernatant, and collect the bacterial cells. Repeat the steps until all bacterial cells are collected. Extract the pCAGGS-His-Fiber expression vector and transfect it into 293F cells.

[0018] More preferably, when prokaryotic cell expression is used, in steps (2) and (3), the LB liquid culture medium contains 50 μg / mL Amp. + .

[0019] More preferably, when prokaryotic cells are used for expression, in step (3), the final concentration of IPTG is 0.8 mM; the disruption is performed in groups of 5 seconds followed by a 5-second pause, for a total of 20 groups; and the final concentration of lysozyme is 1 mg / mL.

[0020] More preferably, when eukaryotic cell expression is used, the transfection conditions include: a mass ratio of PEI to pCAGGS-His-Fiber expression vector of 4:1, and a transfection time of 120 h.

[0021] More preferably, when using eukaryotic cell expression, the method for purifying the obtained FAdV-11 Fiber recombinant protein is as follows:

[0022] (1) Sample processing: The collected culture medium was centrifuged at 4℃ and 10000r / min, and the supernatant was filtered to remove cell debris from the culture medium to obtain the sample;

[0023] (2) Peristaltic pump cleaning: Extend the pipe at the inlet end below the liquid surface, add sterile ddH2O to submerge the pipe; turn on the switch, adjust the flow rate to 2mL / min, quickly clean the flow pipe, and remove air bubbles in the pipe at the same time;

[0024] (3) Preparation of chromatography column: His Trap TM The Excel pre-packed column is connected to one end of the peristaltic pump pipeline. The flow rate is adjusted to 1 mL / min, allowing ddH2O to slowly pass through the pre-packed column to wash the matrix in the pre-packed column. Then, the inlet end is connected to the sample loading volumetric flask of the binding buffer, allowing the binding buffer to pass through the pre-packed column to equilibrate the matrix in the column. The volume of ddH2O is 5 times that of the pre-packed column.

[0025] (4) Sample loading: Add the sample obtained in step (1) into the sample loading volumetric flask, and let the culture medium slowly pass through the pre-packed column. When the His-Fiber protein flows through the chromatography column, it binds with nickel in the matrix; collect the flow liquid with a sterile collection bottle.

[0026] (5) Elution: The proteins bound in the matrix were eluted in a gradient using elution buffers containing different concentrations of imidazole through a pre-packed column. At the same time, the protein samples eluted from the outlet were collected in sterile centrifuge tubes. The His-Fiber protein in the recovered imidazole elution buffer was determined by SDS-PAGE.

[0027] (6) Cleaning: The matrix in the column is cleaned sequentially with binding buffer, sterile ddH2O and 20% ethanol.

[0028] The present invention also provides the use of the FAdV-11 Fiber recombinant protein as described or the recombinant vector as described in the preparation of reagents for detecting FAdV-11 virus.

[0029] This invention discloses a recombinant FAdV-11 fiber protein, its preparation, and its application, which solves the problem that existing technologies cannot rapidly and quantitatively detect FAdV-11 infection, and has the following advantages:

[0030] This invention constructs a FAdV-11 Fiber protein expression vector, expresses the FAdV-11 Fiber protein using an E. coli expression system and a mammalian cell expression system, and then purifies, dialyzes, and concentrates the protein. The Fiber protein is used as an immunogen to immunize New Zealand white rabbits, preparing FAdV-11 Fiber protein rabbit hyperimmune serum. Using the FAdV-11 Fiber protein as a coating antigen, two highly specific, sensitive, and reproducible ELISA detection methods are constructed, providing new technical means for the specific detection of FAdV-11.

[0031] 1. The optimal induction conditions for the recombinant strain pGEX-GST-Fiber / Rosetta(DE3) of this invention are an IPTG concentration of 0.8 mmol / L, an induction time of 8 h, and an induction temperature of 16 °C. A large amount of GST-Fiber protein can be detected and purified from the supernatant.

[0032] 2. The optimal conditions for the GST-Fiber-ELISA method of this invention are as follows: protein coating concentration of 0.5 μg / mL, coating at 4℃ for 12 h; blocking buffer of 1% BSA, blocking at 37℃ for 1 h; serum dilution of 8-fold, incubation at 37℃ for 2 h; and Anti-His-Fiber serum dilution of 2 × 10⁻⁶. 5 The enzyme-labeled secondary antibody was diluted 2500 times and incubated at 37℃ for 2 hours. The reaction time was 1 hour at 37℃, and the TMB color development time was 10 minutes.

[0033] 3. The optimal conditions for the His-Fiber-ELISA method of this invention are as follows: protein coating concentration of 1 μg / mL, coating at 4℃ for 12 h; blocking buffer of 5% skim milk powder, blocking at 37℃ for 1 h; serum dilution of 8-fold, incubation at 37℃ for 1 h; and Anti-GST-Fiber serum dilution of 2 × 10⁻⁶. 5 The two methods were incubated at 37°C for 1 hour, followed by incubation at 37°C for 1 hour with a 5000-fold dilution of the enzyme-labeled secondary antibody, and a TMB color development time of 10 minutes. Both methods exhibited high specificity and did not cross-react with other avian disease-positive sera; they were also easy to operate, had good repeatability, and both inter- and intra-group coefficients of variation were less than 10%.

[0034] 4. This invention is based on the FAdV-11 fiber protein, expressed using both prokaryotic and eukaryotic expression systems. GST-Fiber-ELISA and His-Fiber-ELISA detection methods were constructed, and clinical sample testing and sensitivity experiments were performed. Results showed that the two ELISA methods had a high concordance rate of 98.75% for detecting clinical samples; the positive detection rate for 80 clinical samples was 8.75%; and both methods exhibited high sensitivity, reaching 1:320. The antibody titers for both Anti-GST-Fiber and Anti-His-Fiber were higher than 1:10. 5 It also exhibits good reactivity. Attached Figure Description

[0035] Figure 1 This is an SDS-PAGE diagram of different expression forms of the GST-Fiber protein of this invention.

[0036] Figure 2 This is a Western blotting diagram of different expression forms of the GST-Fiber protein of this invention.

[0037] Figure 3 This is an SDS-PAGE image of GST-Fiber protein eluted with different elution solutions according to the present invention.

[0038] Figure 4 This is a Western blotting image of different expression forms of His-Fiber protein in this invention.

[0039] Figure 5 This is a Western blot image of different transfection mass ratios of PEI∶pCAGGS-His-Fiber according to the present invention.

[0040] Figure 6 This is an SDS-PAGE image of the His-Fiber protein at different harvest times according to the present invention.

[0041] Figure 7 This is an SDS-PAGE image of His-Fiber protein eluted with different imidazole concentrations according to the present invention.

[0042] Figure 8 This is a titer chart of the Anti-GST-Fiber antibody of the present invention.

[0043] Figure 9 This is a titer diagram of the Anti-His-Fiber antibody of the present invention.

[0044] Figure 10 This is a reactivity diagram of the Anti-GST-Fiber antibody of the present invention.

[0045] Figure 11This is a diagram showing the reactivity of the Anti-His-Fiber antibody in this invention. Detailed Implementation

[0046] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0047] The main carrier, cells, and serum used in this invention are as follows:

[0048] The pGEX-4T-1 vector was purchased from Beijing Qingke Biotechnology Co., Ltd.

[0049] The pCAGGS vector was purchased from Suzhou Genewiz Biotechnology Co., Ltd.

[0050] Rosetta (DE3), DH5α competent cells, 293F cells, rabbit negative serum, and SPF chicken negative serum were preserved by the Poultry Important Disease Prevention and Control Laboratory of the College of Veterinary Medicine, Shanxi Agricultural University.

[0051] Experimental Example 1: Prokaryotic Expression and Purification of FAdV-11 Fiber Protein

[0052] 1. Construction of pGEX-GST-Fiber expression vector

[0053] 1.1 Optimization and Synthesis of Target Genes

[0054] Homology comparison analysis was performed using the FAdV-11 Fiber gene sequence (GenBank accession number: UPO24982.1) already available in the GenBank database. A highly conserved strain was selected, and codon optimization was performed to suit the Escherichia coli (Rosetta) expression system. EcoRI and Xhol sequences were introduced at the 5' and 3' ends, respectively. The gene was synthesized by Beijing Qingke Biotechnology Co., Ltd., and the target sequence (SEQ ID NO.2) was cloned into the pGEX-4T-1 plasmid vector with a GST tag to construct the prokaryotic expression vector pGEX-GST-Fiber.

[0055] 2. Expression of GST-Fiber protein

[0056] 2.1 Transformation of pGEX-GST-Fiber expression vector

[0057] The correctly identified pGEX-GST-Fiber expression vector was transformed into Rosetta(DE3) competent cells and cultured at 37°C inverted mode for 12 hours. Single colonies were picked and inoculated into 5 mL of solution containing 50 μg / mL LAmp. + In LB liquid medium, culture until the bacterial culture OD 600 The concentration is 0.6–0.8. Mix 700 μL of bacterial culture with glycerol at a volume ratio of 1:1 and store at -80°C.

[0058] 2.2 Solubility analysis of GST-Fiber protein

[0059] Take 20 μL of the preserved Rosetta (DE3, containing the pGEX-GST-Fiber expression vector) strain and inoculate it into 2 mL of solution containing 50 μg / mL Amp + After activation by culturing in LB liquid medium at 37°C and 220 rpm for 12 h, 100 μL of the activated bacterial solution was inoculated into 10 mL of medium containing 50 μg / mL Amp at a volume ratio of 1:100. + In LB liquid medium, culture until the bacterial culture OD 600 The concentration was 0.6–0.8, and IPTG was added to bring the final concentration to 0.8 mM. The mixture was induced at 20°C and 220 rpm for 8 h. The induced bacterial culture was transferred to a centrifuge tube and centrifuged at 4°C and 8000 rpm for 3 min. The supernatant was discarded, and the bacterial cells were collected. 3 mL of sterile PBS was added to the centrifuge tube, and the cells were repeatedly resuspended by pipetting. The tube was then centrifuged at 4°C and 8000 rpm for 3 min, and the supernatant was discarded. This process was repeated three times, and the cells were finally resuspended in 3 mL of sterile PBS. Lysozyme was added to a concentration of 1 mg / mL, and the mixture was placed on ice for 30 min to lyse. The mixture was then sonicated 20 times using a 5-second interval between sonication cycles. The lysate was centrifuged at 10000 rpm for 20 min at 4°C. The supernatant and precipitate were collected to obtain the protein sample. The protein sample was mixed with a protein loading buffer at a volume ratio of 1:4, denatured, and then analyzed by SDS-PAGE.

[0060] like Figure 1 As shown, the SDS-PAGE images of different expression forms of the GST-Fiber protein of this invention are as follows: M: protein molecular weight standard; 1: lysate after induction by pGEX empty vector expression bacteria; 2: lysate supernatant before induction by recombinant expression bacteria; 3: lysate after induction by recombinant expression bacteria; 4: lysate supernatant after induction by recombinant expression bacteria; 5: lysate precipitate after induction by recombinant expression bacteria. Figure 1 As can be seen, a distinct target band, approximately 100 kDa in size, is visible in lanes 3-5. This indicates that the GST-Fiber protein exists in the expressing bacteria in two forms: soluble protein and inclusion bodies.

[0061] 2.3 Western Blot Validation of GST-Fiber Protein

[0062] Take 2.2 denatured protein samples and perform protein gel electrophoresis and Western blot detection. Rabbit anti-GST antibody was used as the primary antibody (diluted with 2% skim milk powder at a volume ratio of 1:7500), and goat anti-rabbit IgG-HRP was used as the secondary antibody (diluted with 2% skim milk powder at a volume ratio of 1:20000). After washing with PBST solution three times, chemiluminescence color development was performed.

[0063] like Figure 2 The image shows Western blotting plots of different expression forms of the GST-Fiber protein of this invention, where M: protein molecular weight standard; 1: lysate after induction by pGEX empty vector expression bacteria; 2: lysate supernatant before induction by recombinant expression bacteria; 3: lysate after induction by recombinant expression bacteria; 4: lysate supernatant after induction by recombinant expression bacteria; 5: lysate precipitate after induction by recombinant expression bacteria. Figure 2 As can be seen, a single band representing the GST tag protein is visible in lane 1, while distinct target bands of approximately 100 kDa are visible in lanes 3, 4, and 5. This indicates that the GST-Fiber protein exists in the expressing bacteria in two forms: soluble protein and inclusion bodies, with inclusion bodies being the predominant form.

[0064] 3. Large-scale expression and purification of GST-Fiber protein

[0065] Rosetta (DE3: containing pGEX-GST-Fiber expression vector) was cultured and activated according to step 2.2 above. 1 mL of the activated strain was inoculated into 100 mL of LB liquid medium and cultured until the bacterial culture reached OD500. 600 The concentration was 0.6–0.8. After induction under the optimal expression conditions (induction concentration of 0.8 mmol / L, induction time of 8 h, and induction temperature of 16 °C), the bacterial culture was centrifuged at 4 °C and 8000 r / min for 3 min, the supernatant was discarded, and 100 mL of bacterial culture was collected as a pellet. 4 mL of lysis buffer was added, and the pellet was resuspended thoroughly by pipetting. Simultaneously, an appropriate amount of protease inhibitor mixture was added. Protein purification was performed according to the instructions of the GST-tagged protein purification kit (Beyotime Biotechnology Co., Ltd.). After purification, the sample was collected for analysis, denatured, and then analyzed by SDS-PAGE.

[0066] like Figure 3 The image shows SDS-PAGE images of GST-Fiber protein eluted with different elution buffers according to the present invention, where M: protein molecular weight standard; 1: lysis buffer; 2: flow buffer; 3: washing buffer 1; 4: washing buffer 2; 5: elution buffer 1; 6: elution buffer 2; 7: elution buffer 3. Figure 3As can be seen, a clear and single target band with a size of approximately 100 kDa is visible in lanes 5-7. This indicates that the GST-Fiber protein has been successfully purified.

[0067] 4. Dialysis and concentration of GST-Fiber protein

[0068] Dialysis: The purified GST-Fiber was placed into a dialysis bag with a pore size of 30000 MWCO, sealed with a dialysis clamp, leaving approximately 3 mm of head space. Then, the dialysis sample was immersed in 100 times its volume of PBS solution and dialyzed at 4°C. The dialysis solution was changed every 4 hours, for a total of 4 changes.

[0069] Concentration: Transfer the dialyzed protein sample to an ultrafiltration tube (molecular weight cutoff: 30000 MWCO), balance the liquid, and centrifuge at 2500 rpm for 30 min at 4°C. Observe the concentration until the protein is concentrated 10 times its original volume, then collect the GST-Fiber protein sample.

[0070] Experimental Example 2: Eukaryotic Expression and Purification of FAdV-11 Fiber Recombinant Protein

[0071] 1. Construction of pCAGGS-His-Fiber expression vector

[0072] 1.1 Target gene optimization and synthesis

[0073] Based on the FAdV-11 Fiber gene sequence (GenBank: UPO24982.1) in the GenBank database, codon optimization suitable for mammalian cell expression systems was performed. EcoRI, kozak (GCCACC), and IL-10 signal peptide (MHSSALLCCLVLLTGVRA) were sequentially introduced at the 5' end, and 6×His and Xhol sequences were added at the 3' end. The gene was synthesized by Suzhou Genewise Biotech Co., Ltd., and the target sequence (SEQ ID NO.3) was ligated into the pCAGGS plasmid vector to construct the eukaryotic expression vector pCAGGS-His-Fiber.

[0074] 1.2 Large-scale extraction of pCAGGS-His-Fiber expression vector

[0075] The bacterial culture containing the pCAGGS-His-Fiber expression vector was inoculated into 200 mL of LB liquid medium (containing Amp). +(Final concentration: 50 μg / mL) Incubate at 37℃ on a shaker at 220 rpm for 12–16 h. Collect the bacterial culture in a 50 mL centrifuge tube, centrifuge at 8000 rpm for 15 min, discard the supernatant, and collect the bacterial cells. Repeat the above steps until all bacterial cells are collected. Extract the pCAGGS-His-Fiber expression vector according to the instructions of the endotoxin-free plasmid large-scale extraction kit (Beijing Tiangen Biotech Co., Ltd.).

[0076] 2. Expression of His-Fiber recombinant protein

[0077] 2.1pCAGGS-His-Fiber transfection

[0078] Following the PEI transient transfection instructions for suspension cells, 293F cells were transfected using the following specific steps:

[0079] (1) Cell counting: On the day of transfection, cell density and viability were calculated, and the cell density was adjusted to 1×10⁶. 6 cell / mL, total 20mL.

[0080] (2) Preparation of the transfection complex:

[0081] A. Dilute 20 μg pCAGGS-His-Fiber expression vector with 500 μL of 150 mM NaCl solution and let stand for 5 min.

[0082] B. Dilute 80 μL of PEI with 500 μL of 150 mM NaCl solution and let stand for 5 min.

[0083] C. Add A to B, mix gently, the total volume is about 1 mL, and incubate at room temperature for 20 min.

[0084] The ratio of PEI volume to pCAGGS-His-Fiber plasmid transfection mass was 4 μL: 1 μg.

[0085] (3) Add the transfection complex dropwise to each well, gently shaking the culture flask while adding the complex. After mixing, continue to incubate in an incubator.

[0086] (4) 24 hours after transfection, loosen the bottle mouth to release the CO2 produced by cell metabolism, to prevent the pH value of the culture medium from being too low due to excessive CO2, which would affect cell growth.

[0087] (5) 48–72 h after transfection, collect the sample, centrifuge at 10,000 r / min at 4℃ for 5 min to separate the cells from the culture medium, lyse the cells on ice with high efficiency RIPA for 30 min, centrifuge at 10,000 r / min at 4℃ for 10 min to separate the cell supernatant and debris, collect the cell lysate supernatant and cell culture medium and store at -80℃.

[0088] 2.2 Western Blot Validation of His-Fiber Recombinant Protein

[0089] The protein samples collected 48–72 h after transfection in step 2.1 were subjected to Western blotting analysis. Rabbit anti-His antibody was used as the primary antibody, and goat anti-rabbit IgG-HRP was used as the secondary antibody. The remaining steps were the same as those described above (step 2.3 in a method for preparing FAdV-11 Fiber protein).

[0090] like Figure 4 The image shows Western blotting plots of different expression forms of His-Fiber protein in this invention, where M: protein molecular weight standard; 1: untransfected 293F cell culture medium; 2: untransfected 293F cell lysate supernatant; 3: transfected cell culture medium; 4: transfected cell lysate supernatant. Figure 4 It was observed that, 48 hours after transfection, a clear target band was visible at 100 kDa in the cell culture medium and cell lysate supernatant, while no target band was observed in the untransfected cell culture medium and cell lysate. This indicates that the His-Fiber recombinant protein was successfully expressed.

[0091] 3. Selection of optimal expression conditions for His-Fiber recombinant proteins

[0092] 3.1 Determination of the optimal transfection quality ratio of PEI:pCAGGS-His-Fiber expression vector

[0093] The mass ratio of PEI:pCAGGS-His-Fiber expression vector was adjusted to 2:1, 3:1 and 4:1. Transient transfection of 293F cells was performed according to the transfection procedure in 2.1. Protein samples were harvested after 48 h for Western blotting analysis.

[0094] like Figure 5 The image shows Western blotting plots of different transfection mass ratios of PEI:pCAGGS-His-Fiber according to this invention, where M: protein molecular weight standard; 1: PEI:pCAGGS-His-Fiber mass ratio is 2:1; 2: PEI:pCAGGS-His-Fiber mass ratio is 3:1; 3: PEI:pCAGGS-His-Fiber mass ratio is 4:1. (The last sentence appears to be a fragment and doesn't translate directly.) Figure 5 It can be seen that the transfection complex formed by the mass ratio of EI:pCAGGS-His-Fiber expression vector at 4:1 has a thicker band compared to the transfection complexes formed at mass ratios of 2:1 or 3:1. This indicates that the mass ratio of PEI:pCAGGS-His-Fiber expression vector to the transfection complex formed at 4:1 has a higher transient transfection efficiency.

[0095] 3.2 Determination of the optimal harvest time for recombinant protein

[0096] Transient transfection of 293F cells was performed according to the transfection procedure in 2.1 with the optimal mass ratio of PEI:pCAGGS-His-Fiber expression vector. Protein samples were harvested at 48h, 60h, 72h, 84h, 96h, 108h, 120h, and 132h post-transfection and analyzed by SDS-PAGE electrophoresis.

[0097] like Figure 6 The image shows SDS-PAGE images of the His-Fiber protein at different harvest times, where M: protein molecular weight standard; 1: 48 h post-transfection; 2: 60 h post-transfection; 3: 72 h post-transfection; 4: 84 h post-transfection; 5: 96 h post-transfection; 6: 108 h post-transfection; 7: 120 h post-transfection; 8: 132 h post-transfection; 9: untransfected cell culture medium. Figure 6 It was found that the protein content in protein samples harvested 120 hours after transfection was higher than that harvested at other time points. Therefore, 120 hours after transfection is the optimal time for protein harvesting.

[0098] 4. Large-scale expression and purification analysis of His-Fiber recombinant protein

[0099] Following the optimal transfection procedure in 3.1, transfect 100 μg pCAGGS-His-Fiber expression vector into 293F cells for recombinant protein expression. Cell culture medium containing the His-Fiber recombinant protein was collected at the optimal protein harvest time, referring to the His Trap protocol. TM Use the pre-loaded column protein affinity chromatography instructions in Excel to purify His-Fiber recombinant proteins. The steps are as follows:

[0100] (1) Sample processing: The collected culture medium was placed in a 50 mL sterile centrifuge tube and centrifuged at 10,000 r / min at 4℃ for 30 min. The supernatant was then filtered using a 0.22 μm vacuum pump to remove cell debris from the culture medium.

[0101] (2) Peristaltic pump cleaning: Connect the inlet and outlet pipes of the peristaltic pump, extend the inlet end of the pipe to the bottom of the sample bottle, and add sterile ddH2O to submerge the pipe. Turn on the switch and adjust the flow rate to 2 mL / min to quickly clean the flow pipe and remove air bubbles in the pipe (Note: keep the inlet end of the pipe below the liquid surface at all times).

[0102] (3) Preparation of chromatography column: His Trap TMConnect the Excel pre-packed column to one end of the peristaltic pump tubing and adjust the flow rate to 1 mL / min. Slowly pass 5 column volumes of ddH2O through the pre-packed column to wash the matrix within. Then connect the inlet end to the volumetric flask containing the binding buffer and slowly pass 5 column volumes of binding buffer through the pre-packed column to equilibrate the matrix.

[0103] (4) Sample loading: Add the sample prepared in (1) to the volumetric flask and allow the culture medium to slowly pass through the pre-packed column. When the His-Fiber protein flows through the chromatography column, it binds to nickel in the matrix. Collect the flow-through in a sterile collection bottle for subsequent purification and identification.

[0104] (5) Elution: Proteins bound to the matrix were eluted in a gradient using elution buffers containing different concentrations of imidazole through a pre-packed column. Protein samples eluted from the outlet were collected in sterile centrifuge tubes. His-Fiber protein recovered from the imidazole eluent was determined by SDS-PAGE.

[0105] (6) Washing: The matrix in the column is washed sequentially with about 5 column volumes of binding buffer, sterile ddH2O and 20% ethanol.

[0106] like Figure 7 The image shows SDS-PAGE images of His-Fiber protein eluted with different imidazole concentrations, where M: protein molecular weight standard; 1: loading buffer; 2: flow buffer; 3-5: 10, 20, and 30 mmol / L imidazole elution buffers; 6-9: 40, 50, 60, and 100 mmol / L imidazole elution buffers. Figure 7 It was found that when eluted with 10, 20, and 30 mmol / L imidazole, the harvested protein samples contained contaminating proteins. However, when eluted with 40, 50, 60, and 100 mmol / L imidazole, the bands were single and no contaminating protein bands were observed. Therefore, the target protein eluted with 40, 50, 60, and 100 mmol / L imidazole should be harvested.

[0107] 5. Dialysis and concentration of His-Fiber recombinant protein

[0108] The purified His-Fiber recombinant protein was placed in a dialysis bag with a pore size of 30,000 MWCO and dialyzed according to step 1.5. After dialyzing, the protein sample was transferred to an ultrafiltration tube (molecular weight cutoff of 30,000 MWCO) and concentrated according to step 1.5.

[0109] Example 3: Preparation method of Anti-FAdV-11 Fiber protein hyperimmune serum

[0110] After determining the concentrations of concentrated GST-Fiber and His-Fiber proteins, 1 mg of each was emulsified with Freund's adjuvant at a 1:1 volume ratio and used to immunize New Zealand white rabbits. Rabbit serum was collected 7 days post-immunization, and serum titers were determined using indirect ELISA. Western blotting was used to detect the reactivity of the hyperimmune rabbit serum to anti-fiber proteins. The specific method is as follows:

[0111] 1. Animal immunization

[0112] The nine 2kg New Zealand White rabbits were kept in cages for 5 days to avoid stress that could cause the rabbits to die or affect the experimental results.

[0113] Initial immunization: Rabbit serum was collected before the initial immunization as negative serum. Then, purified GST-Fiber and His-Fiber proteins (1 mg) were emulsified with Freund's complete adjuvant at a volume ratio of 1:1 and administered via multiple subcutaneous injections in the neck. Three rabbits were immunized with each of the two methods, each receiving 1 mg. A control group was also established (three rabbits were immunized with sterile PBS emulsified with an equal volume of Freund's complete adjuvant).

[0114] Second immunization: 14 days after the first immunization, 1 mg of GST-Fiber and His-Fiber protein were emulsified with Freund's incomplete adjuvant at a volume ratio of 1:1 and then injected subcutaneously at multiple points in the neck for the second immunization; the control group was immunized with an equal volume of sterile PBS and a mixture of Freund's incomplete adjuvant.

[0115] Three-stage immunization: Seven days after the second immunization, 1 mg of GST-Fiber and His-Fiber protein were emulsified with Freund's incomplete adjuvant at a volume ratio of 1:1 and then injected subcutaneously at multiple points in the neck for the third immunization; the control group was immunized with an equal volume of sterile PBS and a mixture of Freund's incomplete adjuvant.

[0116] Fourth immunization: Seven days after the third immunization, 1 mg of GST-Fiber and His-Fiber protein were injected subcutaneously at multiple points in the neck for the fourth immunization. The control group was immunized with an equal volume of sterile PBS.

[0117] High-immune serum separation: Blood was collected from the heart 7 days after the fourth immunization and the serum was separated and stored at -20°C.

[0118] 2. Determination of serum antibody titers for Anti-GST-Fiber and Anti-His-Fiber

[0119] Anti-GST-Fiber serum, Anti-His-Fiber serum, and negative serum were diluted 10,000, 20,000, 40,000, 80,000, 160,000, 320,000, 640,000, and 1,280,000 times, respectively, and antibody titers were determined using indirect ELISA (see results). Figure 8 Table 2 Figure 9 (See Table 3), and the specific operation is as follows:

[0120] (1) Coating: The purified fiber protein was diluted to 1 μg / mL with carbonate buffer at pH 9.6 and added to an ELISA plate at 100 μL / well. The plate was then coated under certain conditions.

[0121] (2) Washing: Discard the liquid components in the microplate, add 200 μL of PBST washing solution, wash for 3 min, repeat 3 times, and pat the plate dry on a clean disposable absorbent paper after each wash.

[0122] (3) Blocking: Add 200 μL of 5% skim milk blocking solution to each well and incubate at 37°C for 2 hours.

[0123] (4) Washing: Same as step (2) above.

[0124] (5) Primary antibody incubation: Anti-GST-Fiber serum, Anti-His-Fiber and negative serum were diluted with blocking buffer at appropriate ratios, and 100 μL was added to each well and incubated at 37°C for 1 h.

[0125] (6) Washing: Same as step (2) above.

[0126] (7) Secondary antibody incubation: Dilute goat anti-rabbit IgG-HRP 5000 times with blocking solution, add 100 μL to each well, and incubate at 37°C for 1 h.

[0127] (8) Washing: Same as step (2) above.

[0128] (9) Color development: Add 100 μL of TMB color development solution to each well and develop color for 10 min in the dark.

[0129] (10) Termination of color development: Add 50 μL of 1 mol / L H2SO4 to each well to terminate the reaction.

[0130] (11) Microplate reader reading: OD is measured using a microplate reader. 450 value.

[0131] (12) Potency assessment: Select P (OD measured in positive serum) 450 Serum titers are defined as those with a value close to 1 and a P / N value greater than 2.1.

[0132] Table 2 Anti-GST-Fiber antibody titer

[0133]

[0134] Table 3 Anti-His-Fiber antibody titer

[0135]

[0136] Depend on Figures 8-9 As shown in Tables 2 and 3, when Anti-GST-Fiber serum was serially diluted to 640,000 times, the OD of rabbit negative serum was measured. 450 The value was 0.074, and the serum OD value of Anti-GST-Fiber rabbit 1 was... 450 The value was 1.060, Anti-GST-Fiber rabbit serum OD2 450 The value was 1.174, and the P1 / N ratios were 14.289 and 15.819, both greater than 2.1. When the Anti-GST-Fiber serum was serially diluted to 320,000-fold, the OD value of the rabbit negative serum was measured. 450 The value was 0.064, and the serum OD value of Anti-GST-Fiber rabbits was 0.064. 450 The value was 0.928, and the P3 / N ratio was 14.500, greater than 2.1; when the Anti-His-Fiber serum was serially diluted to 640,000 times, the OD value of the rabbit negative serum was measured. 450 The value was 0.146, Anti-His-Fiber rabbit serum OD3 450 The value was 1.043, and the P3 / N ratio was 7.144, greater than 2.1. When the Anti-His-Fiber serum was serially diluted to 320,000-fold, the OD value of the rabbit negative serum was measured. 450 The value was 0.146. The OD450 value of Anti-His-Fiber rabbit 1 serum was 0.944, and the P1 / N ratio was 6.465, which is greater than 2.1. When Anti-His-Fiber rabbit 2 serum was serially diluted to 160,000 times, the OD450 value of the rabbit negative serum was measured. 450 The value was 0.063, Anti-His-Fiber rabbit serum OD. 450 The value was 0.814, and the P2 / N ratio was 12.888, which is greater than 2.1. Therefore, the serum titers of both Anti-GST-Fiber and Anti-His-Fiber were higher than 1:10. 5 .

[0137] 3. Determination of Anti-GST-Fiber and Anti-His-Fiber Antibody Reactivity

[0138] After denaturing His-Fiber and GST-Fiber proteins, 2 μg was loaded onto each well for electrophoresis. The prepared Anti-GST-Fiber and Anti-His-Fiber hyperimmune serum were diluted 1:10000 (v / v) with PBS as primary antibodies and incubated. Simultaneously, commercially available rabbit anti-His-tagged protein antibody (GenScript Biotechnology Co., Ltd.) and rabbit anti-GST-tagged protein antibody (GenScript Biotechnology Co., Ltd.) were diluted 1:1000 as positive controls and incubated. The remaining steps were the same as step 2.3 in the above method (a method for preparing FAdV-11 Fiber protein), and reactivity analysis was performed based on the grayscale value after exposure.

[0139] Depend on Figures 10-11 The grayscale value of the reaction between 10,000-fold diluted Anti-GST-Fiber serum and His-Fiber protein was 32157. This is significantly higher than the grayscale value of rabbit anti-His antibody diluted more than 4,000 times (27866). Similarly, the grayscale value of the reaction between 10,000-fold diluted Anti-His-Fiber serum and GST-Fiber protein was 33084. This is significantly higher than the grayscale value of rabbit anti-GST antibody diluted more than 1,000 times (32699). Therefore, this indicates that both Anti-GST-Fiber and Anti-His-Fiber antibodies in serum exhibit good reactivity with fiber proteins and can be used for Western blotting detection of fiber proteins.

[0140] Experimental Example 4: ELISA Detection Method for FAdV-11 Fiber Antibody Blockage

[0141] (1) Coating: The purified fiber protein was diluted to a certain concentration with carbonate buffer and added to the microplate at 100 μL / well. Coating was carried out under certain conditions.

[0142] (2) Washing: Discard the liquid components in the microplate, add 200 μL of PBST washing solution, wash for 3 min, repeat 3 times, and pat the plate dry on a clean disposable absorbent paper after each wash.

[0143] (3) Sealing: Add 200 μL of sealing solution to each well and seal at 37°C for a certain period of time.

[0144] (4) Washing: Same as step (2) above.

[0145] (5) Primary antibody incubation: Dilute Anti-GST-Fiber antibody or Anti-His-Fiber antibody with blocking buffer according to appropriate ratio, add 100 μL to each well, and incubate at 37°C for a certain period of time.

[0146] (6) Washing: Same as step (2) above.

[0147] (7) Secondary antibody incubation: Dilute goat anti-rabbit IgG-HRP with blocking solution at a certain ratio, add 100 μL to each well, and incubate at 37°C for a certain time.

[0148] (8) Washing: Same as step (2) above.

[0149] (9) Color development: Add 100 μL of TMB color development solution to each well and develop color in the dark.

[0150] (10) Termination of color development: Add 50 μL of 1 mol / L H2SO4 to each well to terminate the reaction.

[0151] (11) Microplate reader reading: OD is measured using a microplate reader. 450 value.

[0152] Experimental Example 5: Determination of the positive and negative cutoff values ​​for blocking ELISA

[0153] Following the steps in Example 3, the optimal reaction conditions for GST-Fiber-ELISA and HIS-Fiber-ELISA were determined as follows: Optimal experimental conditions for the GST-Fiber-ELISA method: GST-Fiber protein coating concentration of 0.5 μg / mL, coating at 4℃ for 12 h; blocking buffer of 1% BSA, blocking at 37℃ for 1 h; serum dilution of 8-fold, incubation at 37℃ for 2 h; Anti-His-Fiber serum dilution of 2 × 10⁻⁶. 5 The optimal experimental conditions for the His-Fiber-ELISA method are: His-Fiber protein coating concentration of 1 μg / mL, coating at 4℃ for 12 h; blocking buffer of 5% skim milk powder, blocking at 37℃ for 1 h; serum dilution of 8-fold, incubation at 37℃ for 1 h; and Anti-GST-Fiber serum dilution of 2 × 10⁻⁶. 5 The enzyme-labeled secondary antibody was diluted 5000 times and incubated at 37°C for 1 hour. The TMB color development time was 10 minutes. Sixty-four negative sera and SPF control sera were tested under optimal blocking ELISA conditions, and their OD values ​​were measured using an ELISA reader. 450 Values. Based on the OD values ​​of 64 negative serum samples (control group). 450 The value is used to calculate the blocking rate PI, thereby determining the positive and negative threshold values. PI = (OD of control group) / (OD of control group) * ... 450 - Serum OD to be tested 450 ) / Control group OD 450Or PI = 1 - (OD of the serum sample to be tested) 450 / Control group OD 450 ).

[0154] Table 4. Determination of Critical Values ​​for GST-Fiber-ELISA

[0155]

[0156] Table 5. Determination of the critical value for His-Fiber-ELISA

[0157]

[0158]

[0159] As shown in Tables 4 and 5, the PI values ​​of 64 negative serum samples measured using the GST-Fiber protein-coated ELISA plate (Table 4) were calculated as follows: average inhibition rate of 10.30%, standard deviation of 4.62%, positive cutoff value of 24.17%, and negative cutoff value of 19.55%. Similarly, the PI values ​​of 64 negative serum samples measured using the His-Fiber protein-coated ELISA plate (Table 5) were calculated as follows: average inhibition rate of 10.06%, standard deviation of 4.35%, positive cutoff value of 23.10%, and negative cutoff value of 18.75%. Therefore, when using the GST-Fiber protein-coated ELISA plate to detect the PI of clinical samples, a PI greater than 24.17% is considered positive; a PI between 19.55% and 24.17% is considered suspicious; and a PI less than 19.55% is considered negative. The inhibition rate (PI) of clinical samples was detected using an enzyme-linked immunosorbent assay (ELISA) plate coated with His-Fiber protein. A PI greater than 23.10% was considered positive; a PI between 18.75% and 23.10% was considered suspicious; and a PI less than 18.75% was considered negative.

[0160] Experimental Example 6: Blocking ELISA Specificity Assay

[0161] The established GST-Fiber-ELISA and His-Fiber-ELISA methods were used to detect positive sera for FAdV-4, FAdV-8b, AIVH9, AIV H5, AIV H7, IBDV, IBV, and NDV. Triple replicates were performed for each serum sample to measure its OD value. 450 The value is used to calculate the PI. This determines whether the established ELISA method has cross-reactivity.

[0162] Table 6 ELISA Specificity Tests

[0163]

[0164] Table 6 shows that the PI values ​​for positive serological tests for common avian diseases were all within the negative cutoff range. This indicates that the established ELISA detection method does not exhibit cross-reactivity with other viral antibodies.

[0165] Experimental Example 7: Blocking ELISA Repeatability Test

[0166] 1. Intra-group repeatability test

[0167] The same batch of GST-Fiber and His-Fiber proteins were coated, and eight negative sera from different sources were tested under optimal experimental conditions. Each serum sample was performed in quadruplicate, and OD was measured. 450 OD values ​​were then calculated for each serum sample. 450 The mean (X), standard deviation (SD), and intra-batch coefficient of variation (CV) are calculated.

[0168] Table 7. Intra-group repeatability tests of GST-Fiber-ELISA

[0169]

[0170]

[0171] Table 8. His-Fiber-ELISA Intragroup Repeatability Tests

[0172]

[0173] As shown in Tables 7 and 8, the intra-assay coefficient of variation for the GST-Fiber-ELISA method was the highest at 5.39%, which is less than 10%; the intra-assay coefficient of variation for the His-Fiber-ELISA method was the highest at 4.36%, which is also less than 10%. This indicates that the established blocking ELISA methods have good intra-assay reproducibility.

[0174] 2. Intergroup repeatability test

[0175] Three batches of GST-Fiber and His-Fiber proteins were coated and tested on eight negative sera from different sources under optimal experimental conditions. Each serum sample was performed in quadruplicate, and OD was measured. 450 After calculating the values, the mean (X), standard deviation (SD), and coefficient of variation (CV) of each serum OD value were calculated.

[0176] Table 9-1 GST-Fiber-ELISA Intergroup Repeatability Tests

[0177]

[0178] Table 9-2 GST-Fiber-ELISA Intergroup Repeatability Tests

[0179]

[0180]

[0181] Table 10-1 His-Fiber-ELISA Intergroup Repeatability Tests

[0182]

[0183] Table 10-2 His-Fiber-ELISA Intergroup Repeatability Tests

[0184]

[0185]

[0186] As shown in Tables 9 and 10, the inter-batch coefficient of variation for the blocking ELISA method established using GST-Fiber protein was 6.85%, which is less than 10%; the inter-batch coefficient of variation for the blocking ELISA method established using His-Fiber protein was 6.31%, which is less than 10%. This indicates that the established ELISA methods have good inter-batch reproducibility.

[0187] Example 8: Clinical application of the FAdV-11 Fiber protein blocking ELISA detection method

[0188] 1. Testing of clinical samples

[0189] Eighty identical clinical samples were tested using the established GST-Fiber-ELISA and His-Fiber-ELISA methods. The positive detection rate of the clinical samples was calculated based on the test results.

[0190] Table 11 Clinical Sample Testing

[0191]

[0192] As shown in Table 11, the results of the two blocking ELISA methods for detecting clinical samples showed a high degree of agreement, reaching 98.75%. The positive rates of both the GST-Fiber-ELISA and His-Fiber-ELISA methods for detecting clinical samples were 8.75%; the negative rates of the GST-Fiber-ELISA and His-Fiber-ELISA methods for detecting clinical samples were 90% and 91.25%, respectively.

[0193] 2. Sensitivity test for blocking ELISA

[0194] Clinically positive serum for FAdV-11 was diluted with PBS at volume ratios of 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, and 1:1280 before being subjected to blocking ELISA detection. The results were based on the detected OD... 450 The PI value is calculated to determine the maximum dilution at which it can be detected.

[0195] Table 12 Sensitivity Tests

[0196]

[0197] Table 12 shows that when serum was diluted 320 times, the PI value of GST-Fiber-ELISA was 26.07% (greater than the positive cutoff value of 24.17%), and the PI value of His-Fiber-ELISA was 25.88% (greater than the positive cutoff value of 23.10%). Both ELISA methods yielded positive results. Therefore, the established GST-Fiber-ELISA and His-Fiber-ELISA detection methods have high sensitivity.

[0198] Example 9: Establishment and Comparison of GST-Fiber-ELISA and His-Fiber-ELISA

[0199] This invention successfully established a GST-Fiber-ELISA detection method using GST-Fiber protein expressed and purified via an *E. coli* expression system, and a His-Fiber-ELISA detection method using His-Fiber protein expressed and purified via a mammalian cell expression system. The optimal experimental conditions for the GST-Fiber-ELISA method were determined through optimization: GST-Fiber protein coating concentration of 0.5 μg / mL, coating at 4℃ for 12 h; blocking buffer of 1% BSA, blocking at 37℃ for 1 h; serum dilution of 8-fold, incubation at 37℃ for 2 h; and anti-His-Fiber serum dilution of 2 × 10⁻⁶. 5 The optimal experimental conditions for the His-Fiber-ELISA method were determined as follows: His-Fiber protein coating concentration of 1 μg / mL, coating at 4℃ for 12 h; blocking buffer of 5% skim milk powder, blocking at 37℃ for 1 h; serum dilution of 8-fold, incubation at 37℃ for 1 h; and Anti-GST-Fiber serum dilution of 2 × 10⁻⁶. 5The enzyme-labeled secondary antibody was diluted 5000 times and incubated at 37℃ for 1 hour. The TMB color development time was 10 minutes. The optimal protein coating concentrations of GST-Fiber-ELISA and His-Fiber-ELISA methods are lower than the optimal coating concentration of ELISA-I established in existing literature 1 (Wang Jing. Identification of avian adenovirus group I monoclonal antibody epitopes and establishment of ELISA antibody detection method [D]. Chinese Academy of Agricultural Sciences, 2020), and comparable to the optimal coating concentration of the competitive ELISA established in existing literature 2 (Wang Weikang. The role of spike protein in serum type 4 avian adenovirus infection and its application in diagnosis [D]. Yangzhou University, 2022).

[0200] The established GST-Fiber-ELISA and His-Fiber-ELISA methods showed no cross-reactivity with positive sera from common avian diseases such as FAdV-4, FAdV-8b, AIVH9, AIVH5, AIVH7, IBDV, IBV, and NDV, indicating good specificity. Both GST-Fiber-ELISA and His-Fiber-ELISA exhibited high sensitivity (1:320) and good reproducibility, with coefficients of variation less than 10% both between and within groups, making them suitable for large-scale clinical detection of FAdV-11 infection. The results of testing 80 clinical serum samples showed that the concordance between GST-Fiber-ELISA and His-Fiber-ELISA was 98.75%, with a positive concordance rate of 100%. This concordance was higher than that of the indirect ELISA for detecting FAdV-8b specific antibodies established in existing literature 3 (Lu Hao. Research on diagnostic technology, chimeric vaccine and infection mechanism of avian adenovirus based on spike protein [D]. Yangzhou University, 2022).

[0201] This invention establishes GST-Fiber-ELISA and His-Fiber-ELISA by coating with the optimal concentration of purified protein. There is no significant difference in specificity, sensitivity, and stability, and both can be used for clinical testing. Compared to the cost of mammalian cell culture, protein expression, and purification, the E. coli expression system for expressing and purifying GST-Fiber protein is less expensive, requires less sophisticated experimental equipment and conditions, and has a shorter cycle time. Therefore, both the established GST-Fiber-ELISA and His-Fiber-ELISA can be used for the detection of FAdV-11, but GST-Fiber-ELISA has a lower clinical application cost and is easier to promote.

[0202] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A recombinant FAdV-11 Fiber protein, characterized in that, The amino acid sequence of the recombinant protein is shown in SEQ ID NO.

1.

2. A gene encoding the FAdV-11 Fiber recombinant protein as described in claim 1, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID NO.2 or SEQ ID NO.

3.

3. A recombinant vector containing the gene as described in claim 2.

4. The recombinant vector according to claim 3, characterized in that, The recombinant vector used is either pGEX-4T-1 or pCAGGS.

5. A method for preparing the FAdV-11 Fiber recombinant protein as described in claim 1, characterized in that, When prokaryotic cell expression is used, the method includes: (1) Homology comparison analysis was performed using the FAdV-11 Fiber gene sequence already available in the GenBank database. One strain was selected and codon optimization was performed to suit the Escherichia coli Rosetta expression system. EcoRI and Xhol sequences were introduced at the 5' and 3' ends, respectively. Gene synthesis was performed to obtain the target sequence shown in SEQ ID NO.

2. The target sequence was cloned into the pGEX-4T-1 plasmid vector with the GST tag to construct the prokaryotic expression vector pGEX-GST-Fiber. (2) The pGEX-GST-Fiber expression vector was transformed into Rosetta(DE3) competent cells and cultured at 37℃ in an inverted state. Single colonies were picked and inoculated into LB liquid medium and cultured until the OD600 of the bacterial culture was 0.6-0.

8. The bacterial culture was mixed with glycerol and stored at -80℃ to obtain Rosetta(DE3) strain containing the pGEX-GST-Fiber expression vector. (3) Rosetta (DE3) strain was inoculated into LB liquid medium and activated at 37°C. The activated strain was then inoculated into LB liquid medium and cultured until the OD600 of the bacterial culture was 0.6-0.

8. IPTG was added and the culture was induced at 20°C. The induced bacterial culture was centrifuged at 8000 r / min at 4°C, the supernatant was discarded, and the bacterial cells were collected. Sterile PBS was added, and the cells were repeatedly resuspended by pipetting. The cells were centrifuged at 8000 r / min at 4°C, the supernatant was discarded, and the operation was repeated to resuspend the bacterial cells with PBS. Lysozyme was added and the culture was lysed on ice. The cells were then sonicated. The lysate was centrifuged at 10000 r / min at 4°C, and the supernatant and precipitate were collected to obtain the protein sample. The protein sample was mixed with protein loading buffer at a volume ratio of 1:

4. When using eukaryotic cell expression, this method includes: (1) Based on the FAdV-11 Fiber gene sequence in the GenBank database, codon optimization was performed to suit the mammalian cell expression system. EcoRI sequence, kozak sequence and IL-10 signal peptide were introduced sequentially at the 5' end, and 6×His and Xhol sequences were added at the 3' end. Gene synthesis was performed to obtain the target sequence as shown in SEQ ID NO.

3. The target sequence was ligated into the pCAGGS plasmid vector to construct the eukaryotic expression vector pCAGGS-His-Fiber. The nucleotide sequence of the kozak is shown in SEQ ID NO.4; the amino acid sequence of the IL-10 signal peptide is shown in SEQ ID NO.5; (2) Transform the pCAGGS-His-Fiber expression vector into DH5α competent cells, incubate at 37℃ in an inverted state, pick single colonies, inoculate them into LB liquid medium, and incubate until the OD600 of the bacterial culture is 0.6-0.

8. Mix the bacterial culture with glycerol and store at -80℃ to obtain DH5α bacteria containing the pCAGGS-His-Fiber expression vector. Inoculate the DH5α bacteria into LB liquid medium and incubate at 37℃ to activate them. Inoculate the activated bacterial culture into LB liquid medium and incubate at 37℃ and 220 r / min. Collect the bacterial culture, centrifuge at 8000 r / min, discard the supernatant, and collect the bacterial cells. Repeat the steps until all bacterial cells are collected. Extract the pCAGGS-His-Fiber expression vector and transfect it into 293F cells.

6. The preparation method according to claim 5, characterized in that, When prokaryotic cell expression is used, in steps (2) and (3), the LB liquid medium contains 50 μg / mL Amp. + .

7. The preparation method according to claim 5, characterized in that, When prokaryotic cells are used for expression, in step (3), the final concentration of IPTG is 0.8 mM; the disruption is performed in groups of 5 seconds followed by a 5-second pause, for a total of 20 groups; the final concentration of lysozyme is 1 mg / mL.

8. The preparation method according to claim 5, characterized in that, When using eukaryotic cells for expression, the transfection conditions include: a PEI to pCAGGS-His-Fiber expression vector mass ratio of 4:1, and a transfection time of 120 h.

9. The preparation method according to claim 5, characterized in that, When eukaryotic cells are used for expression, the method for purifying the obtained FAdV-11 Fiber recombinant protein is as follows: (1) Sample processing: The collected culture medium was centrifuged at 4℃ and 10000r / min, and the supernatant was filtered to remove cell debris from the culture medium to obtain the sample; (2) Peristaltic pump cleaning: Extend the pipe at the inlet end below the liquid surface, add sterile ddH2O to submerge the pipe; turn on the switch, adjust the flow rate to 2mL / min, quickly clean the flow pipe, and remove air bubbles in the pipe at the same time; (3) Preparation of chromatography column: His Trap TM The Excel pre-packed column is connected to one end of the peristaltic pump pipeline. The flow rate is adjusted to 1 mL / min, allowing ddH2O to slowly pass through the pre-packed column to wash the matrix in the pre-packed column. Then, the inlet end is connected to the sample loading volumetric flask of the binding buffer, allowing the binding buffer to pass through the pre-packed column to equilibrate the matrix in the column. The volume of ddH2O is 5 times that of the pre-packed column. (4) Sample loading: Add the sample obtained in step (1) into the sample loading volumetric flask, and let the culture medium slowly pass through the pre-packed column. When the His-Fiber protein flows through the chromatography column, it binds with nickel in the matrix; collect the flow liquid with a sterile collection bottle. (5) Elution: The proteins bound in the matrix were eluted in a gradient using elution buffers containing different concentrations of imidazole through a pre-packed column. At the same time, the protein samples eluted from the outlet were collected in sterile centrifuge tubes. The His-Fiber protein in the recovered imidazole elution buffer was determined by SDS-PAGE. (6) Cleaning: The matrix in the column is cleaned sequentially with binding buffer, sterile ddH2O and 20% ethanol.

10. The use of the FAdV-11 Fiber recombinant protein as described in claim 1 or the recombinant vector as described in claim 4 in the preparation of a reagent for detecting FAdV-11 virus.

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