Monoclonal antibody for resisting duck adenovirus 3 type Fiber2 protein and application of monoclonal antibody for detecting or identifying duck adenovirus 3 type

Abstract

The invention belongs to the technical field of veterinary biotechnology detection, and particularly relates to a monoclonal antibody for resisting duck adenovirus 3 type Fiber2 protein and application of the monoclonal antibody to detection or identification of duck adenovirus 3 type. The duck adenovirus 3 type Fiber2 protein is taken as an immunogen to screen a hybridoma cell strain with good reactivity to different strain whole viruses of the duck adenovirus 3 type, an antibody generated by the hybridoma cell strain is analyzed, the monoclonal antibody resisting the duck adenovirus 3 type Fiber2 protein is obtained, and the sequence information is shown in a table 3 in the specification. The monoclonal antibody provided by the invention can identify different strains of the duck adenovirus 3, does not generate cross reaction with other common poultry disease viruses such as fowl adenovirus, novel duck reovirus and the like, has good specificity and sensitivity, can be used for detecting the exogenous virus of the duck adenovirus 3 in poultry virus live vaccines, and has good application prospects. And the kit can also be used for clinical identification, virus content determination and epidemiological investigation of the duck adenovirus type 3.

Description

Technical Field

[0001] This invention belongs to the field of veterinary biotechnology detection technology, specifically relating to a monoclonal antibody against duck adenovirus type 3 Fiber2 protein and its application in detecting or identifying duck adenovirus type 3. Background Technology

[0002] Duck adenovirus serotype 3 (DAdV-3) is a newly emerging duck-derived adenovirus in my country in recent years. It primarily infects ducks aged 10-40 days, with a morbidity rate between 35% and 75%. Currently, DAdV-3 has become one of the important pathogens threatening duck farming, especially Muscovy duck farming, causing serious economic losses to the waterfowl industry. Given the serious harm and public health risks posed by DAdV-3 to the poultry industry, establishing detection methods for DAdV-3 is of great significance for understanding its etiology, pathogenicity, and for the diagnosis and control of the virus.

[0003] Indirect immunofluorescence (IFA) is an immunological technique that uses fluorescently labeled antibodies to detect antigens. It is rapid, simple, sensitive, specific, and inexpensive, and has been included in the *Veterinary Pharmacopoeia of the People's Republic of China* (Volume III) for the detection of avian reticuloendotheliosis virus (REV) and avian adenovirus group I (FAdV-1). However, an IFA detection method for DAdV-3 is currently lacking. Therefore, screening for monoclonal antibodies with high specificity and sensitivity to DAdV-3 to achieve IFA detection of DAdV-3 is of particular importance in this field. Summary of the Invention

[0004] The purpose of this invention is to provide a monoclonal antibody that specifically detects duck adenovirus type 3, does not cross-react with other common avian viruses such as avian adenovirus (FAdV) and novel duck reovirus (NDRV), and has high detection sensitivity.

[0005] This invention provides a monoclonal antibody against duck adenovirus type 3 Fiber2 protein, wherein the light chain complementarity-determining region CDR1 of the monoclonal antibody includes the amino acid sequence shown in SEQ ID NO:2, the light chain complementarity-determining region CDR2 includes the amino acid sequence KVS, and the light chain complementarity-determining region CDR3 includes the amino acid sequence shown in SEQ ID NO:3. The heavy chain complementarity-determining region (CDR1) of the monoclonal antibody includes the amino acid sequence shown in SEQ ID NO:5, the heavy chain complementarity-determining region (CDR2) includes the amino acid sequence shown in SEQ ID NO:6, and the heavy chain complementarity-determining region (CDR3) includes the amino acid sequence shown in SEQ ID NO:7.

[0006] Preferably, the light chain variable region of the monoclonal antibody includes an amino acid sequence as shown in SEQ ID NO:1, or has at least 75% identity with the sequence shown in SEQ ID NO:1 and has an amino acid sequence capable of binding to duck adenovirus type 3. The heavy chain variable region of the monoclonal antibody includes an amino acid sequence as shown in SEQ ID NO:4, or an amino acid sequence that has at least 75% identity with the sequence shown in SEQ ID NO:4 and has the ability to bind to duck adenovirus type 3.

[0007] This invention provides a nucleic acid molecule that encodes the monoclonal antibody described in the above-mentioned technical solution.

[0008] Preferably, the sequence of the nucleic acid molecule encoding the variable region of the light chain of the monoclonal antibody includes the nucleotide sequence shown in SEQ ID NO:8; the sequence of the nucleic acid molecule encoding the variable region of the heavy chain of the monoclonal antibody includes the nucleotide sequence shown in SEQ ID NO:11.

[0009] This invention provides a biological material that expresses the monoclonal antibody described in the above technical solution; the biological material includes a carrier or cells.

[0010] This invention provides the application of the monoclonal antibody described in the above technical solution, or the monoclonal antibody encoded by the nucleic acid molecule described in the above technical solution, or the monoclonal antibody expressed by the biological material described in the above technical solution, in one or more of the following: (1) Prepare products for detecting or identifying duck adenovirus type 3 Fiber2 protein; (2) Prepare products for the detection or identification of duck adenovirus type 3.

[0011] The present invention provides a kit comprising the monoclonal antibody described in the above technical solution.

[0012] Preferably, the kit further includes one or more of the following: fluorescently labeled anti-mouse antibody, diluent, washing solution, and positive control; The diluent and washing solution each comprise a phosphate buffer; the phosphate buffer has a pH of 7.2-7.4 and a concentration of 9-11 mM. The positive control is serum collected from mice immunized with DAdV-3 Fiber2 recombinant protein or DAdV-3 positive serum.

[0013] This invention provides the application of the monoclonal antibody described in the above-mentioned technical solution, or the monoclonal antibody encoded by the nucleic acid molecule described in the above-mentioned technical solution, or the monoclonal antibody expressed by the biological material described in the above-mentioned technical solution, or the kit described in the above-mentioned technical solution, in detecting the safety of avian virus live vaccines; The safety testing of avian live virus vaccines includes detecting and / or monitoring whether the avian live virus vaccine is contaminated with duck adenovirus type 3, or the concentration of duck adenovirus type 3 in the avian live virus vaccine.

[0014] This invention provides a method for detecting the safety of avian viral live vaccines, comprising the following steps: The sample to be tested was inoculated into a culture plate containing chicken liver cancer cells and cultured. The culture medium was discarded and the cells were fixed to obtain the test sample. The test sample is incubated with a monoclonal antibody, washed, and then the incubated test cells are obtained; the monoclonal antibody is the monoclonal antibody described in the above technical solution, or the monoclonal antibody encoded by the nucleic acid molecule described in the above technical solution, or the monoclonal antibody expressed by the biological material described in the above technical solution. The incubated test cells were then incubated a second time using fluorescently labeled anti-mouse antibodies, washed, and observed under a fluorescent inverted microscope. When specific fluorescence appeared in the cell detection wells and the cell nuclei and cytoplasm of the infected cells were visible at 200-400x magnification, the duck adenovirus type 3 test was considered positive. When no specific fluorescence appeared in the cell detection wells and the field of view was dark, it indicated that the cells were not infected, and the duck adenovirus type 3 test was considered negative.

[0015] Beneficial effects: This invention uses the Fiber2 protein of duck adenovirus type 3 as an immunogen to screen hybridoma cell lines that exhibit good reactivity to different strains of duck adenovirus type 3. The antibodies produced by these hybridoma cell lines are then analyzed to obtain a monoclonal antibody against the Fiber2 protein of duck adenovirus type 3. The sequence information is shown in Table 3 of the specification. The monoclonal antibody provided by this invention can recognize different strains of duck adenovirus type 3 without cross-reactivity with other common avian disease viruses such as avian adenovirus (FAdV) and novel duck reovirus (NDRV). It has good specificity and sensitivity and can be used for the detection of exogenous duck adenovirus type 3 in avian viral live vaccines. It can also be used for the clinical identification, viral load determination, and epidemiological investigation of duck adenovirus type 3. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1The image shows the SDS-PAGE results of low-level expression of DAdV-3-Fiber2 recombinant protein; where M is the marker, 1 is after IPTG induction, and 2 is before IPTG induction. Figure 2 The image shows the SDS-PAGE results of high-level expression of DAdV-3-Fiber2 recombinant protein; where M is the marker, 1 is the supernatant after sonication, and 2 is the precipitate after sonication. Figure 3 The image shows the SDS-PAGE results of purified DAdV-3-Fiber2 recombinant protein; where M is the marker and 1-3 are the purified protein supernatant. Figure 4 The image shows the results of detecting DAdV-3 GD24 strain using the indirect immunofluorescence kit from Example 4. Figure 5 The image shows the results of detecting DAdV-3 GDMM strain using the indirect immunofluorescence kit from Example 4. Figure 6 The image shows the results of detecting HVT FC126 strain using the indirect immunofluorescence kit from Example 4. Figure 7 The image shows the results of detecting NDV Clone30 strain using the indirect immunofluorescence kit from Example 4. Figure 8 The image shows the results of detecting the POX quail-adapted attenuated strain using the indirect immunofluorescence kit from Example 4. Figure 9 The results of detecting IBV H120 strain using the indirect immunofluorescence kit from Example 4 are shown in the figure. Figure 10 The image shows the results of detecting IBDV B87 strain using the indirect immunofluorescence kit from Example 4. Figure 11 The image shows the results of detecting EDSV K911 strain using the indirect immunofluorescence kit from Example 4. Figure 12 The image shows the results of detecting ALV RAV-1 strain using the indirect immunofluorescence kit from Example 4. Figure 13 The image shows the results of detecting ALV RAV-2 strain using the indirect immunofluorescence kit from Example 4. Figure 14 The image shows the results of detecting AIV (H9N2 subtype) AV1571 strain using the indirect immunofluorescence kit from Example 4. Figure 15 The image shows the results of detecting FAdV GY strain using the indirect immunofluorescence kit from Example 4. Figure 16The image shows the results of detecting CIAV AV1550 strain using the indirect immunofluorescence kit from Example 4. Figure 17 The image shows the results of detecting ILTV ILT / 13 strain using the indirect immunofluorescence kit from Example 4. Figure 18 The image shows the results of detecting ARV Reo S1133 strain using the indirect immunofluorescence kit from Example 4. Figure 19 The image shows the results of detecting NDRV GX2022 strain using the indirect immunofluorescence kit from Example 4. Figure 20 The image shows the results of detecting DPV attenuated strains in chicken embryos using the indirect immunofluorescence kit from Example 4. Figure 21 The image shows the results of detecting MDPV PF7 strain using the indirect immunofluorescence kit from Example 4. Detailed Implementation

[0018] This invention provides a monoclonal antibody against duck adenovirus type 3 Fiber2 protein. The light chain complementarity-determining region (CDR1) of the monoclonal antibody includes the amino acid sequence shown in SEQ ID NO:2, the light chain complementarity-determining region (CDR2) includes the amino acid sequence KVS, and the light chain complementarity-determining region (CDR3) includes the amino acid sequence shown in SEQ ID NO:3. The heavy chain complementarity-determining region (CDR1) of the monoclonal antibody includes the amino acid sequence shown in SEQ ID NO:5, the heavy chain complementarity-determining region (CDR2) includes the amino acid sequence shown in SEQ ID NO:6, and the heavy chain complementarity-determining region (CDR3) includes the amino acid sequence shown in SEQ ID NO:7.

[0019] As one embodiment, the light chain variable region of the monoclonal antibody of the present invention includes an amino acid sequence as shown in SEQ ID NO:1, or an amino acid sequence having at least 75% identity with the sequence shown in SEQ ID NO:1 and having the ability to bind to duck adenovirus type 3.

[0020] As one embodiment, the heavy chain variable region of the monoclonal antibody of the present invention includes an amino acid sequence as shown in SEQ ID NO:4, or an amino acid sequence that has at least 75% identity with the sequence shown in SEQ ID NO:4 and has the ability to bind to duck adenovirus type 3.

[0021] This invention provides a nucleic acid molecule that encodes the monoclonal antibody described in the above-mentioned technical solution.

[0022] In one embodiment, the sequence encoding the variable region of the light chain of the monoclonal antibody by the nucleic acid molecule of the present invention comprises the nucleotide sequence shown in SEQ ID NO:8. In another embodiment, the sequence encoding the complementarity-determining region (CDR1) of the light chain of the monoclonal antibody by the nucleic acid molecule of the present invention comprises the nucleotide sequence shown in SEQ ID NO:9. In yet another embodiment, the sequence encoding the complementarity-determining region (CDR2) of the light chain of the monoclonal antibody by the nucleic acid molecule of the present invention comprises the nucleotide sequence AAAGTTTCC. In yet another embodiment, the sequence encoding the complementarity-determining region (CDR3) of the light chain of the monoclonal antibody by the nucleic acid molecule of the present invention comprises the nucleotide sequence shown in SEQ ID NO:10.

[0023] In one embodiment, the sequence of the nucleic acid molecule encoding the variable region of the heavy chain of a monoclonal antibody comprises the nucleotide sequence shown in SEQ ID NO:11. In another embodiment, the sequence of the nucleic acid molecule encoding the complementarity-determining region (CDR1) of the heavy chain of a monoclonal antibody comprises the nucleotide sequence shown in SEQ ID NO:12. In another embodiment, the sequence of the nucleic acid molecule encoding the complementarity-determining region (CDR2) of the heavy chain of a monoclonal antibody comprises the nucleotide sequence shown in SEQ ID NO:13. In yet another embodiment, the sequence of the nucleic acid molecule encoding the complementarity-determining region (CDR3) of the heavy chain of a monoclonal antibody comprises the nucleotide sequence shown in SEQ ID NO:14.

[0024] This invention provides a biological material that expresses the monoclonal antibody described in the above technical solution; the biological material includes a carrier or cells.

[0025] In one embodiment, the vector of the present invention includes a base vector and a nucleic acid molecule as described in the above-described technical solution inserted into the base vector. In one embodiment, the base vector of the present invention includes pET30a. In one embodiment, the cell of the present invention is a hybridoma cell.

[0026] The present invention provides the application of the monoclonal antibody described in the above technical solution or the monoclonal antibody generated by the nucleic acid molecule described in the above technical solution or the monoclonal antibody expressed by the biological material described in the above technical solution in one or more of the following: (1) preparing a product for detecting or identifying duck adenovirus type 3 Fiber2 protein; (2) preparing a product for detecting or identifying duck adenovirus type 3.

[0027] In one embodiment, the product of the present invention includes a reagent kit. In another embodiment, the reagent kit of the present invention is an indirect immunofluorescence reagent kit.

[0028] The present invention provides a kit comprising the monoclonal antibody described in the above technical solution.

[0029] As one embodiment, the kit of the present invention further includes one or more of the following: fluorescently labeled anti-mouse antibody, diluent, washing solution, and positive control.

[0030] In one embodiment, the fluorescently labeled anti-mouse antibody of the present invention is FITC-labeled goat anti-mouse IgG. When observed under a fluorescence inverted microscope with blue excitation light (wavelength 490 nm), the FITC-labeled goat anti-mouse IgG of the present invention exhibits specific green fluorescence.

[0031] In one embodiment, the diluent and washing solution of the present invention each comprise a phosphate buffer; the pH value of the phosphate buffer is 7.2-7.4, and the concentration is 9-11 mM; in another embodiment, the diluent and washing solution of the present invention each comprise a phosphate buffer; the pH value of the phosphate buffer is 7.2, and the concentration is 10 mM.

[0032] In one embodiment, the positive control of the present invention is serum collected from mice immunized with DAdV-3 Fiber2 recombinant protein or DAdV-3 positive serum. In another embodiment, the nucleotide sequence of the DAdV-3 Fiber2 recombinant protein of the present invention is shown in SEQ ID NO:15.

[0033] This invention provides the application of the monoclonal antibody described in the above-mentioned technical solution, or the monoclonal antibody encoded by the nucleic acid molecule described in the above-mentioned technical solution, or the monoclonal antibody expressed by the biological material described in the above-mentioned technical solution, or the kit described in the above-mentioned technical solution, in detecting the safety of avian viral live vaccines; the detection of the safety of avian viral live vaccines includes detecting and / or monitoring whether the avian viral live vaccine is contaminated with duck adenovirus type 3, or the concentration of duck adenovirus type 3 in the avian viral live vaccine.

[0034] This invention provides a method for detecting the safety of avian viral live vaccines, comprising the following steps: The sample to be tested was inoculated into a culture plate containing chicken liver cancer cells and cultured. The culture medium was discarded and the cells were fixed to obtain the test sample. The test sample is incubated with a monoclonal antibody, washed, and then the incubated test cells are obtained; the monoclonal antibody is the monoclonal antibody described in the above technical solution, or the monoclonal antibody encoded by the nucleic acid molecule described in the above technical solution, or the monoclonal antibody expressed by the biological material described in the above technical solution. The incubated test cells were then incubated a second time using fluorescently labeled anti-mouse antibodies, washed, and observed under a fluorescent inverted microscope. When specific fluorescence appeared in the cell detection wells and the cell nuclei and cytoplasm of the infected cells were visible at 200-400x magnification, the duck adenovirus type 3 test was considered positive. When no specific fluorescence appeared in the cell detection wells and the field of view was dark, it indicated that the cells were not infected, and the duck adenovirus type 3 test was considered negative.

[0035] In this invention, the sample to be tested is inoculated into a culture plate containing chicken liver cancer cells, the culture medium is discarded, and the cells are fixed to obtain the test sample.

[0036] In one embodiment, the culture temperature of the present invention is 36~38℃; in another embodiment, the culture temperature of the present invention is 37℃. In one embodiment, the culture time of the present invention is 4~6 days; in another embodiment, the culture time of the present invention is 5 days. In one embodiment, the present invention uses a culture plate containing chicken liver cancer cells without inoculating the test sample as a negative control, and a culture plate containing chicken liver cancer cells containing serum collected from mice immunized with DAdV-3 Fiber2 recombinant protein or DAdV-3 positive serum as a positive control.

[0037] After culturing, the cultured cells are fixed to obtain a test sample. As one embodiment, the fixation described in this invention utilizes methanol.

[0038] After obtaining the test sample, the present invention uses a monoclonal antibody to perform a first incubation on the test sample, followed by washing, to obtain incubated test cells; the monoclonal antibody is the monoclonal antibody described in the above technical solution, or the monoclonal antibody encoded by the nucleic acid molecule described in the above technical solution, or the monoclonal antibody expressed by the biological material described in the above technical solution.

[0039] In one embodiment, the first incubation temperature of the present invention is 36~38℃; in another embodiment, the first incubation temperature of the present invention is 37℃. In one embodiment, the first incubation time of the present invention is 0.5~2h; in another embodiment, the first incubation time of the present invention is 1h. In one embodiment, the monoclonal antibody of the present invention is used after being diluted 10~1000 times with a diluent; in another embodiment, the monoclonal antibody of the present invention is used after being diluted 100 times with a diluent.

[0040] In one embodiment, the washing process of the present invention is performed 2 to 5 times. In another embodiment, the diluent and washing solution of the present invention each comprise a phosphate buffer; the pH value of the phosphate buffer is 7.2 to 7.4, and the concentration is 9 to 11 mM. In yet another embodiment, the diluent and washing solution of the present invention each comprise a phosphate buffer; the pH value of the phosphate buffer is 7.2, and the concentration is 10 mM.

[0041] After obtaining the incubated test cells, the present invention uses fluorescently labeled anti-mouse antibodies to perform a second incubation on the incubated test cells, followed by washing and observation under a fluorescence inverted microscope. When specific fluorescence appears in the cell detection wells and is magnified to 200-400 times, and the nuclei and cytoplasm of the infected cells are stained, the duck adenovirus type 3 test is determined to be positive. When no specific fluorescence appears in the cell detection wells and the field of view is dark, it proves that the cells are not infected, and the duck adenovirus type 3 test is determined to be negative.

[0042] In one embodiment, the second incubation temperature of the present invention is 36~38℃; in another embodiment, the second incubation temperature of the present invention is 37℃. In one embodiment, the second incubation time of the present invention is 0.5~2h; in another embodiment, the second incubation time of the present invention is 1h. In one embodiment, the fluorescently labeled anti-mouse antibody of the present invention is diluted 10~1000 times with a diluent before use; in another embodiment, the fluorescently labeled anti-mouse antibody of the present invention is diluted 100 times with a diluent before use. In one embodiment, the fluorescently labeled anti-mouse antibody of the present invention is FITC-labeled goat anti-mouse IgG. In one embodiment, the number of washes in the present invention is 2~5 times. In one embodiment, the diluent and washing solution of the present invention each include phosphate buffer; the pH value of the phosphate buffer is 7.2~7.4, and the concentration is 9~11mM; in another embodiment, the diluent and washing solution of the present invention each include phosphate buffer; the pH value of the phosphate buffer is 7.2, and the concentration is 10mM.

[0043]

[0044] This invention provides the application of the recombinant epitope of duck adenovirus type 3 Fiber2 protein described in the above-mentioned technical solution in the preparation of antibodies against duck adenovirus type 3 Fiber2 protein. This invention optimizes the encoding gene of duck adenovirus type 3 Fiber2 protein, enabling its high-level expression in *E. coli*, and can be used to prepare antibodies with high sensitivity and specificity for detecting duck adenovirus type 3.

[0045] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a monoclonal antibody against duck adenovirus type 3 Fiber2 protein and its application in detecting or identifying duck adenovirus type 3, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0046] Example 1 DAdV-3 Fiber2 protein sequence analysis 1. Reference sequences of different DAdV-3 strains were downloaded from NCBI. Analysis revealed that the Fiber2 protein showed high homology among the different strains. Comprehensive secondary analysis and antigenicity analysis were performed on the Fiber2 sequence of the DAdV-3 TZ193 strain. The results showed that the DAdV-3 Fiber2 protein sequence was highly conserved with extremely low antigenicity differences, making it a potential candidate fragment for protein expression.

[0047] 2. Based on protein sequence secondary structure analysis and antigenicity, using the Fiber2 sequence of DAdV-3 TZ193 strain (Genbank accession number: MT934842.1) as a basis, the full-length gene sequence of the protein (1440 bp) was selected, and after codon optimization, the sequence was synthesized to obtain the DAdV-3 Fiber2 protein recombinant epitope, which was then added to the 5' and 3' ends. EcoR V (5'-GATATC-3') and Xho The I (5'-CTCGAG-3') restriction enzyme site is used for vector cloning. The sequence was synthesized at Nanjing Platinum Biotech Co., Ltd., and the specific sequence is: 5'- GATATC CTCGAG -3' (SEQ ID NO:16); where the underscore is an extra one. EcoR V and Xho I restriction site.

[0048] Example 2 Construction, expression, and purification of recombinant expression plasmids 1. Construction of recombinant expression plasmids Using restriction endonucleases EcoR V and Xhoo I performed double enzyme digestion on the SEQ ID NO:12 sequence and plasmid pET30a in Example 1, and then ligated the purified and recovered fragment and the enzyme digestion product of the expression vector with a DNA Ligation Kit to obtain the recombinant expression plasmid, which was then transformed into competent cells (BL21).

[0049] 2. Low-level expression of DAdV-3 Fiber2 recombinant protein (1) Select the transformed BL21 clones that were positive by PCR in step 1 and put them into 1.5 mL of LB liquid medium containing 50 μg / mL kanamycin resistance. Incubate at 37 °C and 200 r / min until the OD of the culture medium reaches the target value. 600 The value was 0.6~0.8. IPTG was added to the culture medium to induce induction. The final concentration of IPTG in the culture medium was 0.5mM. The induction temperature was 16℃, the rotation speed was 200r / min, and the induction time was 12h.

[0050] (2) Take 1 mL of the induced bacterial culture, centrifuge at 12000 r / min for 2 min, discard the supernatant, and disperse the precipitate with 50 μL of 10 mM Tris-HCl (pH 8.0) solution (the amount of buffer added depends on the amount of bacterial cells). Add 2× loading buffer of the same volume as the buffer, and keep at 100℃ for 5 min before performing SDS-PAGE electrophoresis. The results showed a specific target band of recombinant DAdV-3 Fiber2 protein at a size of approximately 65 KD. Figure 1 This indicates that DAdV-3 Fiber2 protein expression was successful.

[0051] 3. High expression of DAdV-3 Fiber2 recombinant protein (1) The transformed BL21 obtained in step 1 was identified by PCR, and the BL21 that was identified as positive was cultured. The cultured bacterial solution was transferred to 250 mL of LB liquid medium containing 50 μg / mL kanamycin resistance at a volume ratio of 1:50, and cultured with shaking at 37 °C and 200 r / min until the OD of the culture medium was reached.600 The value was 0.6~0.8. IPTG was added to the culture medium to induce induction. The final concentration of IPTG in the culture medium was 0.5mM, the induction temperature was 16℃, and the induction time was 12h.

[0052] (2) Centrifuge the induced bacterial solution for 6 minutes at a speed of 8000 r / min, discard the supernatant to obtain bacterial cells; perform ultrasonic disruption on the obtained bacterial cells. The specific process is as follows: blow the obtained bacterial cells with 30 mL of 10 mM Tris-HCl (pH value of 8.0) solution and then perform ultrasonic disruption. The ultrasonic disruption power is 500 W, and ultrasonic disruption is performed 180 times, each time for 5 seconds, with a 5-second interval before the next ultrasonic disruption.

[0053] (3) Electrophoretic detection was performed on the product obtained by ultrasonic disruption. The specific procedure was as follows: 100 μL of the ultrasonically disrupted bacterial suspension was centrifuged at 12000 r / min for 10 min. After centrifugation, 50 μL of supernatant and the resulting precipitate were retained. The precipitate was dispersed with 50 μL of 10 mM Tris-HCl solution (pH 8.0). The supernatant and the solution obtained after dispersing the precipitate were respectively subjected to SDS-PAGE detection. The results showed that a large amount of the target protein ( Figure 2 This indicates that the DAdV-3Fiber2 protein of the recombinant bacteria is expressed in a soluble form.

[0054] 4. Purification of DAdV-3 Fiber2 recombinant protein The supernatant obtained after ultrasonic disruption and centrifugation in step 3 was added to 5-10 mL of 10 mM Tris-HCl solution (pH 8.0) containing 8 M urea to dissolve the protein. The mixture was centrifuged at 12000 r / min for 10 min, and the supernatant was collected. 50 μL of the sample was subjected to SDS-PAGE electrophoresis. The results showed a large amount of purified DAdV-3 Fiber2 protein (…). Figure 3 Using BSA (bovine serum albumin) as a standard, the purified protein concentration was estimated to be >0.5 mg / mL and the purity >85% by SDS-PAGE gel scanning analysis.

[0055] Example 3 Preparation of monoclonal antibodies 1. Immunity in mice The purified DAdV-3 Fiber2 protein from Example 2 was emulsified with Freund's complete adjuvant at a 1:1 volume ratio. Four SPF-grade BALB / c female mice were subcutaneously injected with 60 μg of DAdV-3 Fiber2 protein per mouse (this was the primary immunization). Booster immunizations were administered subcutaneously at 2, 4, and 6 weeks after the primary immunization, with a dose of 30 μg of DAdV-3 Fiber2 protein per mouse. The intervals between the primary and first booster immunizations were 14 days, as were between the first and second booster immunizations and the second and third booster immunizations. Ten days after the third booster immunization, blood was collected from the orbital sinus, and serum titers were measured using indirect ELISA. Mice with high serum titers (ELISA antibody titer of 1:12800) were selected and given a single intraperitoneal injection of 50 μg of the immunogen (DAdV-3 Fiber2 protein) as a pulse immunization.

[0056] 2. Cell fusion Three days after intraperitoneal injection of immunization shock, mouse spleens were aseptically harvested and prepared into a spleen cell suspension. An equal volume of the immunized spleen cell suspension was mixed with SP2 / 0 cells, and cell fusion was performed using the standard 50% PEG method. The resulting fused cells were then placed in five 96-well plates and selectively cultured in HAT medium (purchased from Sigma).

[0057] 3. Cloning and screening of hybridoma cells 3.1 The cultures from the 96-well plate described in step 2 were screened using an ELISA plate coated with DAdV-3 Fiber2 protein. The specific steps are as follows: (1) Coating ELISA plates: Dilute the purified DAdV-3 Fiber2 protein from Example 2 with sodium carbonate-sodium bicarbonate buffer at pH 9.6 to a final concentration of 2 μg / mL. Add 100 μL of diluted DAdV-3 Fiber2 protein to each well of the ELISA plate and incubate overnight at 4°C; then wash three times with PBST (PBS containing 0.05% Tween-20).

[0058] (2) Blocking: Add 200 μL of PBS containing 2% milk to each well of the coated ELISA plate, incubate at 37°C for 2 h, and then wash 3 times with PBST (PBS containing 0.05% Tween-20).

[0059] (3) Incubation of primary antibody: After blocking, add the hybridoma cell culture supernatant obtained after cell fusion in step 2, negative control (SP2 / 0 culture supernatant), blank control (PBS), and positive control (DAdV-3 positive serum diluted 1000 times with PBS) as primary antibody, all at 100 μL / well, and incubate at 37℃ for 1 h.

[0060] (4) Washing: After incubating with the primary antibody, wash the ELISA plate in step (3) with PBST (PBS containing 0.05% Tween-20) for a total of 3 times.

[0061] (5) Incubation of secondary antibody: After washing, add 100 μL of goat anti-mouse IgG / HRP diluted 20,000 times with PBS to each ELISA plate as secondary antibody, and incubate at 37°C for 1 h.

[0062] (6) Washing: Wash the ELISA plate after incubation with PBST (PBS containing 0.05% Tween-20) in step (5) a total of 3 times.

[0063] (7) Color development: Add 100 μL / well of color development solution (citric acid buffer containing 1% solution A and 10% solution B; solution A: TMB is prepared to a mass concentration of 1% with DMSO; solution B: H2O2 aqueous solution with a mass concentration of 0.1%) and the color development time is about 5 min.

[0064] (8) Add 50 μL of stop solution (containing 2M sulfuric acid) to each well to terminate the process.

[0065] (9) Reading: The absorbance was measured at two wavelengths (450nm, 630nm), and the data was recorded and saved. The results are shown in Table 1.

[0066] 3.2 The cultures in the 96-well plate described in step 2 were screened by ELISA plates coated with His tag protein. The specific steps were similar to those in 3.1, except that in step (1) when coating the ELISA plate, His tag protein was used to replace the purified DAdV-3Fiber2 protein; in step (3) when incubating the primary antibody, the positive control was serum collected from mice immunized with DAdV-3 Fiber2 recombinant protein or DAdV-3 positive serum. The results are shown in Table 1.

[0067] Table 1. ELISA screening results of hybridoma cell lines

[0068] Note: A high absorbance value for the target protein and a low absorbance value for the tag protein indicate a high titer of the hybridoma cell line.

[0069] As shown in Table 1, this embodiment screened out three ELISA-positive hybridoma cell lines, namely #1, #7 and #18, for further screening. Among them, hybridoma cell line #1 had the highest absorbance titer.

[0070] 4. Indirect immunofluorescence detection 4.1 Preparation of positive virus plates: The viral solutions of different DAdV-3 strains in Table 2 were diluted to 100 TCID using DMEM culture medium containing 2% newborn calf serum. 50 / 0.1mL was inoculated into a 96-well plate containing chicken liver cancer cells (LMH). After about 5 days, the cells were fixed with cold methanol for 15 minutes. At the same time, LMH cells without virus inoculation were set up as a control for later use.

[0071] Table 2 Information on different DAdV-3 strains and control strains

[0072] Note: Duck adenovirus type 3 (DAdV-3) GDMM strain was obtained from: Xinjin S, Xinyu Z, Haiwei S, et al. Isolation and pathogenic characterization of duck adenovirus 3 mutant circulating in China[J].Poultry Science,2022,101(1):101564-101564; Novel duck reovirus (NDRV) GX2022 strain was obtained from: Kong Dongni, Deng Yong, Chen Mengjiao, et al. Isolation, identification and cell adaptation of novel duck reovirus[J]. Progress in Animal Medicine,2025,46(05):48-52.

[0073] 4.2 Fluorescent staining (1) Fixation: After the positive virus plate is prepared, discard the cell culture medium in the 96-well plate, add about 250 μL of PBS to each well, wash the cell surface once, discard as much PBS as possible, then add 100 μL of cold methanol to each well, fix at room temperature for 10-15 min, discard the methanol, and air dry for 2-5 min.

[0074] (2) Add primary antibody (monoclonal antibody): After fixing the 96-well plate, wash the cell surface once with PBS (pH 7.2). Divide the supernatant of the three positive hybridoma cell lines (1#, 7# and 18#) obtained by ELISA screening in step 3 into primary antibodies. Dilute the supernatant 10 times with PBS and add it to the positive virus plate (washed once with PBS before use), 50 μL per well, and incubate at 37°C in the dark for 1 h.

[0075] (3) Washing: After adding primary antibody to the 96-well plate, discard the monoclonal antibody in the wells, wash 5 times with PBS, adding 0.3 mL of washing buffer to each well each time, and gently shake to wash.

[0076] (4) Fluorescent secondary antibody staining: Discard as much washing buffer as possible, add 50 μL of fluorescently labeled goat anti-mouse IgG diluted with PBS to each well, and incubate at 37°C in the dark for 1 h. The volume ratio of fluorescently labeled goat anti-mouse IgG diluted with PBS is 1:100~1:200.

[0077] (5) Washing: The method is the same as step (3).

[0078] (6) Observation and result determination: Under a fluorescence inverted microscope with blue excitation light (wavelength 490nm), the cells were observed to have complete cell morphology. When specific green fluorescence appeared in the field of view of the inoculation well, and when magnified to 200-400 times, the nucleus and cytoplasm of the infected cells were stained, the well was determined to be positive for DAdV-3 detection. When no specific green fluorescence appeared in the inoculation well, the field of view was dark, indicating that the cells were not infected, and the well was determined to be negative for DAdV-3 detection. The results showed that the IFA antibody titer of cell line number 18# was higher than that of cell line number 1#, and cell line number 7# had no IFA antibody titer. In this example, a cell line with good reactivity to the whole virus of a different strain from DAdV-3 was screened. This cell line expressed a monoclonal antibody against DAdV-3 Fiber2 protein and was named DAdV-3-Mab-Fiber2-18#.

[0079] 5. Identification of hybridoma cells 5.1 Cultivation Characteristics Hybridoma cell line DAdV-3-Mab-Fiber2-18# was cultured in DMEM medium containing 10%~15% fetal bovine serum at 37°C in a 5% CO2 incubator. The cell morphology of the hybridoma cell line was examined under a microscope. The cells should be uniform in morphology, which indicates that the cells are in good condition.

[0080] 5.2 Purity Test According to the methods in the appendix of the current Chinese Veterinary Pharmacopoeia (edited by the Chinese Veterinary Pharmacopoeia Committee, Veterinary Pharmacopoeia of the People's Republic of China, 2020 edition, China Agriculture Press, 2020, hereinafter referred to as the Chinese Veterinary Pharmacopoeia), the hybridoma cell line DAdV-3-Mab-Fiber2-18# was subjected to sterility testing, mycoplasma testing, and exogenous virus testing, and the results all met the requirements.

[0081] 5.3 Nucleic acid examination The chromosome number of the hybridoma cell line DAdV-3-Mab-Fiber2-18#, which had been cultured for 24 hours, was examined using the colchicine method, and the chromosome characteristics were observed to be consistent with the staining characteristics of hybridoma cells.

[0082] 5.4 Ascites titer determination (1) Preparation of ascites: 8-10 week old BALB / c mice were intraperitoneally injected with 0.5 mL of phenazine per mouse. 7-10 days later, the mice were intraperitoneally injected with 10 mL of phenazine. 6 ~10 7 0.5 mL of DAdV-3-Mab-Fiber2-18# hybridoma cells per mouse was administered. After 7-10 days, the mice were observed. When the abdomen became significantly distended and the mice exhibited difficulty in movement, ascites fluid was collected. The fluid was centrifuged at 3000 rpm for 10 minutes, and the supernatant was collected and stored at -40°C. If ascites fluid recurred after 2-3 days, it could be collected again. This ascites fluid was purified using Protein-A affinity to obtain mouse anti-DAdV-3 monoclonal antibody.

[0083] (2) The ascites fluid that produces mouse anti-DAdV-3 monoclonal antibody was serially diluted from a volume ratio of 1:100 to 1:3200, and the fluorescent antibody titer of the ascites fluid was determined to be 1:1600 according to the indirect immunofluorescence detection method in step 4.

[0084] 6. Identification of the sequence of the anti-DAdV-3 Fiber2 protein monoclonal antibody The structure of the hybridoma cell line DAdV-3-Mab-Fiber2-18# was identified by Shanghai Nearshore Technology Co., Ltd. The specific sequence information is shown in Table 3.

[0085] Table 3 Sequence information of monoclonal antibodies against DAdV-3 Fiber2 protein

[0086] Example 4 1. An indirect immunofluorescence kit for detecting DAdV-3, comprising the anti-DAdV-3 Fiber2 protein monoclonal antibody obtained in Example 3, commercially available FITC-labeled goat anti-mouse antibody (purchased from Sigma, catalog number F2057), sample diluent, and washing buffer. Both the diluent and washing buffer were 10 mM pH 7.2 phosphate-buffered saline (PBS); the anti-DAdV-3 Fiber2 protein monoclonal antibody was generated from the hybridoma cell line DAdV-3-Mab-Fiber-18# obtained in Example 3.

[0087] 2. The steps and judgment criteria for detecting DAdV-3 using the indirect immunofluorescence kit from step 1 are as follows: 2.1 Sample inoculation 100 μL of the sample to be tested was seeded into a 96-well plate confluent with LMH cells and cultured at 37°C for about 5 days.

[0088] 2.2 Fluorescent staining and result interpretation (1) Fixation: 1 h after inoculation, discard the cell culture medium in the 96-well cell plate, add about 0.3 mL of PBS (pH 7.2) to each well and gently wash the cell surface once. Discard as much PBS as possible, then add 0.2 mL of cold methanol to each well, fix at room temperature for 15 min, discard the methanol, and air dry for 5 min.

[0089] (2) Adding primary antibody: After air drying, wash the cell surface once with PBS (pH 7.2), and then add 50 μL of anti-DAdV-3 Fiber2 protein monoclonal antibody to each well and incubate at 37°C for 1 h. The added anti-DAdV-3 Fiber2 protein monoclonal antibody is diluted with PBS (pH 7.2~7.4) at a volume ratio of 1:100.

[0090] 2.3 Washing: Discard the anti-DAdV-3 Fiber2 protein monoclonal antibody, wash 5 times with PBS (pH 7.2), adding 0.3 mL of washing buffer to each well each time, and gently shake to wash.

[0091] 2.4 Fluorescent secondary antibody staining: Discard as much washing buffer as possible, add 50 μL of FITC-labeled goat anti-mouse IgG to each well, and incubate at 37°C for 1 h. Dilute the added FITC-labeled goat anti-mouse IgG with PBS (pH 7.2-7.4) at a volume ratio of 1:100.

[0092] 2.5 Washing: Same as step 2.3.

[0093] 2.6 Observation and Judgment: Under an inverted fluorescence microscope with blue excitation light (wavelength 490nm), the cells exhibited intact cellular morphology. When specific green fluorescence appeared in the field of view of the inoculated well, and at magnification of 200-400 times, the nucleus and cytoplasm of the infected cells were visible and stained, the well was considered positive for DAdV-3 detection. If no specific green fluorescence appeared in the inoculated well, and the field of view was dark, it indicated that the cells were not infected, and the well was considered negative for DAdV-3 detection.

[0094] Example 5 Specific detection Using the indirect immunofluorescence kit from Example 4, and following the established steps and judgment criteria, different DAdV-3, turkey herpesvirus (HVT), Newcastle disease virus (NDV), fowlpox virus (POX), infectious bronchitis virus (IBV), infectious bursal disease virus (IBDV), egg drop syndrome virus (EDSV), avian leukosis virus (ALV), avian influenza virus (AIV, H9N2 subtype), avian adenovirus (FAdV), infectious anemia virus (CIAV), infectious laryngotracheitis virus (ILTV), avian reovirus (ARV), novel duck reovirus (NDRV), duck plague virus (DPV), and Muscovy duck parvovirus (MDPV) were detected. The staining of infected cells was observed to determine the specificity of the indirect immunofluorescence method.

[0095] Specific detection results showed that the indirect immunofluorescence kit of Example 4 could specifically identify and detect different DAdV-3 strains ( Figures 4-5 The reaction results were all positive, and were also positive for other common avian viruses such as HVT (Hydrovirus Vulcanii). Figure 6 ), NDV ( Figure 7 ), POX ( Figure 8 ), IBV ( Figure 9 ), IBDV ( Figure 10 ), EDSV ( Figure 11 ), ALV ( Figures 12-13 ), AIV Figure 14 ),FAdV ( Figure 15 CIAV Figure 16 ), ILTV ( Figure 17 ), ARV ( Figure 18 ), NDRV ( Figure 19 ), DPV ( Figure 20 ), MDPV ( Figure 21 All reactions were negative, demonstrating that the established method has good specificity, and this indirect immunofluorescence kit can be used for the specific detection of DAdV-3.

[0096] Example 6 Sensitivity detection Dilute DAdV-3 (GD24 strain) to 100 TCID 50 / 100μL, diluted to 20 TCID based on this. 50 / 100μL, 10 TCID 50 / 100μL, 5 TCID 50 / 100μL, 2 TCID 50 / 100μL, 1 TCID 50 / 100μL. The five dilutions were seeded into confluent LMH cells at 100μL / well, with four replicates per sample. Uninoculated LMH cells served as a negative control. Detection was performed using the indirect immunofluorescence kit from Example 4, following the established procedures and criteria. Results showed that DAdV-3 detection was positive at all dose gradients, with a DAdV-3 infection dose greater than or equal to 1 TCID. 50 At that time, the virus test result was positive. It can be seen that the limit of detection for DAdV-3 contamination using the indirect immunofluorescence kit of Example 4 is 1 TCID. 50 .

[0097] Example 7 Exogenous virus testing for avian viral live vaccines Eight key poultry live vaccines produced by domestic manufacturers were selected (Table 4). Indirect immunofluorescence (IFA) detection of DAdV-3 was performed using the indirect immunofluorescence kit in Example 4. Serological testing for DAdV-3 contamination was conducted on the selected poultry live vaccines according to the 2020 edition of the Chinese Veterinary Pharmacopoeia, Part III. A PBS group was set as a negative control, and a DAdV-3-infected group as a positive control. Specific test results are shown in Table 4.

[0098] Table 4. Results of Exogenous Virus Detection in Enterprise Vaccines

[0099] As can be seen from Table 4, the results of serological testing and the detection using the indirect immunofluorescence kit in Example 4 are consistent. All selected poultry live vaccines are free of DAdV-3 contamination. The negative control is negative for DAdV-3, and the positive control is positive for DAdV-3.

[0100] As can be seen from the above, the monoclonal antibody against duck adenovirus type 3 Fiber2 protein provided by the present invention has good specificity and sensitivity against DAdV-3. It can be used for the detection of exogenous DAdV-3 virus in avian viral live vaccines, and can also be used for the clinical identification, viral content determination and epidemiological investigation of DAdV-3.

[0101] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A monoclonal antibody against duck adenovirus type 3 Fiber2 protein, characterized in that, The light chain complementarity-determining region (CDR1) of the monoclonal antibody includes the amino acid sequence shown in SEQ ID NO:2, the light chain complementarity-determining region (CDR2) includes the amino acid sequence KVS, and the light chain complementarity-determining region (CDR3) includes the amino acid sequence shown in SEQ ID NO:

3. The heavy chain complementarity-determining region (CDR1) of the monoclonal antibody includes the amino acid sequence shown in SEQ ID NO:5, the heavy chain complementarity-determining region (CDR2) includes the amino acid sequence shown in SEQ ID NO:6, and the heavy chain complementarity-determining region (CDR3) includes the amino acid sequence shown in SEQ ID NO:

7.

2. The monoclonal antibody according to claim 1, characterized in that, The light chain variable region of the monoclonal antibody includes an amino acid sequence as shown in SEQ ID NO:1, or an amino acid sequence that has at least 75% identity with the sequence shown in SEQ ID NO:1 and has the ability to bind to duck adenovirus type 3. The heavy chain variable region of the monoclonal antibody includes an amino acid sequence as shown in SEQ ID NO:4, or an amino acid sequence that has at least 75% identity with the sequence shown in SEQ ID NO:4 and has the ability to bind to duck adenovirus type 3.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the monoclonal antibody as described in claim 1 or 2.

4. The nucleic acid molecule according to claim 3, characterized in that, The sequence of the nucleic acid molecule encoding the variable region of the light chain of the monoclonal antibody includes the nucleotide sequence shown in SEQ ID NO:8; the sequence of the nucleic acid molecule encoding the variable region of the heavy chain of the monoclonal antibody includes the nucleotide sequence shown in SEQ ID NO:

11.

5. A biomaterial, characterized in that, The biological material is a biological material expressing the monoclonal antibody as described in claim 1 or 2; the biological material includes a carrier or cells.

6. The use of the monoclonal antibody of claim 1 or 2, or the monoclonal antibody encoded by the nucleic acid molecule of claim 3 or 4, or the monoclonal antibody expressed by the biological material of claim 5, in one or more of the following: (1) Prepare products for detecting or identifying duck adenovirus type 3 Fiber2 protein; (2) Prepare products for the detection or identification of duck adenovirus type 3.

7. A reagent kit, characterized in that, The kit includes the monoclonal antibody as described in claim 1 or 2.

8. The reagent kit according to claim 7, characterized in that, The kit also includes one or more of the following: fluorescently labeled anti-mouse antibody, diluent, washing solution, and positive control; The diluent and washing solution each comprise a phosphate buffer; the phosphate buffer has a pH of 7.2-7.4 and a concentration of 9-11 mM. The positive control is serum collected from mice immunized with DAdV-3 Fiber2 recombinant protein or DAdV-3 positive serum.

9. The use of the monoclonal antibody of claim 1 or 2, or the monoclonal antibody encoded by the nucleic acid molecule of claim 3 or 4, or the monoclonal antibody expressed by the biological material of claim 5, or the kit of claim 7 or 8, in detecting the safety of avian viral live vaccines; The safety testing of avian live virus vaccines includes detecting and / or monitoring whether the avian live virus vaccine is contaminated with duck adenovirus type 3, or the concentration of duck adenovirus type 3 in the avian live virus vaccine.

10. A method for detecting the safety of a live avian virus vaccine, characterized in that, Includes the following steps: The sample to be tested was inoculated into a culture plate containing chicken liver cancer cells and cultured. The culture medium was discarded and the cells were fixed to obtain the test sample. The test sample is incubated with a monoclonal antibody, washed, and the incubated test cells are obtained; the monoclonal antibody is the monoclonal antibody according to claim 1 or 2, or the monoclonal antibody encoded by the nucleic acid molecule according to claim 3 or 4, or the monoclonal antibody expressed by the biological material according to claim 5. The incubated test cells were then incubated a second time using fluorescently labeled anti-mouse antibodies, washed, and observed under a fluorescent inverted microscope. When specific fluorescence appeared in the cell detection wells and the cell nuclei and cytoplasm of the infected cells were visible at 200-400x magnification, the duck adenovirus type 3 test was considered positive. When no specific fluorescence appeared in the cell detection wells and the field of view was dark, it indicated that the cells were not infected, and the duck adenovirus type 3 test was considered negative.

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