Hybridoma cell strain and monoclonal antibody for detecting or identifying aMPV and application of hybridoma cell strain and monoclonal antibody

Abstract

The invention discloses a hybridoma cell strain and a monoclonal antibody for detecting or identifying aMPV and application of the hybridoma cell strain and the monoclonal antibody, and belongs to the field of veterinary biotechnology detection. The preservation number of the hybridoma cell strain for detecting or identifying the aMPV is CCTCC (China Center For Type Culture Collection) NO: C2025211. The monoclonal antibody is obtained by secreting a hybridoma cell strain or a passage cell strain thereof, and the passage cell strain of the hybridoma cell strain comprises a cell strain obtained by hybridoma cell passage for 30 times or within 30 times. The hybridoma cell strain and the monoclonal antibody can recognize different subtype aMPV strains, do not have cross reaction with avian reovirus, chicken egg drop syndrome virus and other common avian disease viruses, have good specificity and sensitivity, can be used for detecting aMPV exogenous viruses in avian virus live vaccines, and can be applied to detection of aMPV exogenous viruses in avian virus live vaccines. The kit can also be used for clinical identification, virus content determination and epidemiological investigation of aMPV.

Description

Technical Field

[0001] This invention belongs to the field of veterinary biotechnology detection, specifically relating to a hybridoma cell line and monoclonal antibody for detecting or identifying aMPV and their applications. Background Technology

[0002] Avian metapneumovirus disease (aMPV) is an infectious disease caused by avian metapneumovirus (aMPV), characterized by acute upper respiratory tract infection, decreased egg production, and reduced eggshell quality. aMPV can infect various poultry species, including chickens, ducks, and pigeons. In chickens, aMPV can cause swollen head syndrome, characterized by coughing, runny nose, foamy conjunctivitis, infraorbital sinus swelling, and swelling around the eyes and face. Recent studies have found that aMPV infection is severe in waterfowl and can cause a decrease in egg production and eggshell quality in laying ducks. Since its initial discovery in South Africa in 1978, aMPV has been reported in Asia, Africa, and Europe, becoming widespread globally and causing significant economic losses to the poultry industry. aMPV is a single-stranded, non-segmented, negative-sense RNA virus belonging to the Pneumoviridae family. Its genome size is 13.1–14.1 kb, and its genome structure is 5'-NPMF-M2-SH-GL-3'. Based on the different genes encoding the accessory glycoprotein G, aMPV is divided into subgroups A, B, C, and D. Epidemiological surveys show that subtypes A and B of aMPV are relatively common, subtype C of aMPV is mainly reported in the United States, France, and China, and subgroup D of aMPV is only reported in France.

[0003] Avian reticuloendotheliosis virus (REV) subgroup B is the dominant circulating subgroup in China, and most isolates in China belong to this subgroup. Given the serious harm and public health risks posed by aMPV to China's poultry industry, establishing a detection method for aMPV is of great significance for understanding its etiology, pathogenicity, and for the diagnosis and control of the virus. Indirect immunofluorescence (IFA) 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 antigen detection of avian reticuloendotheliosis virus (REV) and avian adenovirus group I (FadV-1). However, an IFA detection method for aMPV is currently lacking. Therefore, screening for monoclonal antibodies with high specificity and sensitivity to aMPV to achieve IFA detection of aMPV is particularly important in this field.

[0004] Chinese invention patent application CN103739679 A discloses an F protein polypeptide and its application. This antigenic polypeptide is synthesized using a prokaryotic expression system based on the F protein gene sequence. The amino acid sequence of the antigenic polypeptide is CTNAGSTAYYPNKDD. This antigenic polypeptide exhibits high conservation and strong antigenicity, and can be used to generate monoclonal antibodies against the avian metapneumovirus (aMPV) F protein. However, this technical solution has significant drawbacks in practical applications. The expression level of the eukaryotic expression system is relatively low, and the cost is relatively high, hindering widespread adoption. Furthermore, the technical solution does not validate the sensitivity and specificity of the method, and it is unclear whether it only detects a specific subtype of aMPV or a broad spectrum, thus failing to meet detection requirements.

[0005] Chinese invention patent application CN 120210132 A discloses a hybridoma cell line and monoclonal antibody for detecting or identifying MDV-1 and their applications. This application uses the pp38 protein of MDV-1 as an immunogen to screen for the hybridoma cell line MDV-1Mab-pp38-E2 (accession number CCTCC NO: C202501). The monoclonal antibody produced by this cell line can only recognize different serotype I Marek's disease virus strains in chickens, without cross-reacting with other common avian disease viruses such as serotype II Marek's disease virus, serotype III Marek's disease virus, and egg drop syndrome virus (EDSV). It exhibits good specificity and sensitivity and can be used for the detection of exogenous serotype I Marek's disease virus in avian viral live vaccines. It can also be used for the clinical identification, viral load determination, and epidemiological investigation of serotype I Marek's disease virus. However, MDV is an intracellular virus, which differs significantly from other extracellular viruses. Whether this method can be applied to extracellular infections caused by aMPV has not yet been reported. Furthermore, this method can only identify serotype I Marek's virus, and cannot identify serotypes II and III Marek's virus, so it is not broad-spectrum and has certain limitations. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing a hybridoma cell line for detecting or identifying aMPV, which specifically recognizes subtype B and subtype C of aMPV without cross-reacting with other common avian disease viruses such as avian reovirus (REO) and egg drop syndrome virus (EDSV), and has high sensitivity.

[0007] A second objective of this invention is to provide a monoclonal antibody.

[0008] A third objective of this invention is to provide the application of the hybridoma cell line and the monoclonal antibody.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is: a hybridoma cell line aMPV-Mab-F-E2 for detecting or identifying avian metapneumovirus (aMPV) in serum, with accession number CCTCC NO:C2025211.

[0010] A monoclonal antibody for detecting or identifying aMPV, secreted by the hybridoma cell line aMPV-Mab-F-E2 or its passaged cell line.

[0011] The hybridoma cell lines include those passaged 30 times or less.

[0012] The present invention also provides the application of the hybridoma cell line aMPV-Mab-F-E2 or monoclonal antibody in the preparation of products for detecting or identifying aMPV.

[0013] Preferably, the aMPV includes one or more of the B subtype strains and the C subtype strains.

[0014] Methods for detecting or identifying avian metapneumovirus in products include the following steps:

[0015] (1) The monoclonal antibody was used to incubate Vero cells inoculated with the sample to be tested for the first time, and the first incubation product was obtained after washing.

[0016] (2) The first incubation product was incubated a second time with FITC-labeled goat anti-mouse antibody, and the second incubation product was obtained after washing.

[0017] (3) The second incubation product is stained and observed at a wavelength of 490 nm. The results of the fluorescence staining are used to determine whether the sample contains avian metapneumovirus. If specific green fluorescence is observed, the sample does not contain avian metapneumovirus.

[0018] A kit for detecting or identifying aMPV, comprising the monoclonal antibody, a fluorescently labeled anti-mouse antibody, a diluent, and a washing solution.

[0019] Preferably, 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.

[0020] The present invention also provides the application of the hybridoma cell line aMPV-Mab-F-E2 described in the above technical solution, or the monoclonal antibody or the kit, in the detection or identification of aMPV-Mab-F-E2 F protein.

[0021] The present invention also provides the application of the hybridoma cell line aMPV-Mab-F-E2 described in the above technical solution, or the monoclonal antibody or the kit described therein, in detecting one or more of the purity, safety and illegal additives in avian viral live vaccines.

[0022] Preferably, the safety detection of the avian live virus vaccine includes detecting and / or monitoring the viral load of aMPV in the avian live virus vaccine.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows: the monoclonal antibody secreted by the hybridoma cell line aMPV-Mab-F-E2 can recognize different subtypes B and C of aMPV without cross-reacting with other common avian disease viruses such as avian reovirus (REO) and egg drop syndrome virus (EDSV). It has good specificity and sensitivity and can be used for the detection of exogenous aMPV in avian viral live vaccines. It can also be used for the clinical identification, viral content determination and epidemiological investigation of aMPV. Attached Figure Description

[0024] 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.

[0025] Figure 1 The image shows the SDS-PAGE results of low-level expression of aMPV-F recombinant protein; where M is the marker, 1 is before IPTG induction, and 2 is after IPTG induction.

[0026] Figure 2 The image shows the SDS-PAGE results of high-level expression of aMPV-F recombinant protein; where M is the marker, 1 is the supernatant after sonication, and 2 is the precipitate after sonication.

[0027] Figure 3 The image shows the SDS-PAGE results of purified aMPV-F recombinant protein; where M is the marker and 1-3 are the purified protein supernatant.

[0028] Figure 4 This is a graph showing the results of detecting subtype B aMPV B1 strain using the indirect immunofluorescence kit from Example 4;

[0029] Figure 5 This is a graph showing the results of detecting subtype B aMPV LN16 strain using the indirect immunofluorescence kit from Example 4;

[0030] Figure 6 The image shows the results of detecting subtype B aMPV RPVA0201 strain using the indirect immunofluorescence kit from Example 4.

[0031] Figure 7 The image shows the results of detecting subtype C aMPV GX strain using the indirect immunofluorescence kit from Example 4.

[0032] Figure 8 The image shows the results of detecting MDV-3FC126 strain using the indirect immunofluorescence kit from Example 4.

[0033] Figure 9 The image shows the results of detecting NDV Clone30 strain using the indirect immunofluorescence kit from Example 4.

[0034] Figure 10 The image shows the results of detecting the quail-adapted attenuated FPV strain using the indirect immunofluorescence kit from Example 4.

[0035] Figure 11 The image shows the results of detecting IBV H52 strain using the indirect immunofluorescence kit from Example 4.

[0036] Figure 12 The image shows the results of detecting IBDV BC6 / 85 strain using the indirect immunofluorescence kit from Example 4.

[0037] Figure 13 The image shows the detection results of EDSV K911 strain using the indirect immunofluorescence kit in Example 4.

[0038] Figure 14 This is a graph showing the detection results of ALV RAV-1 strain using the indirect immunofluorescence kit in Example 4;

[0039] Figure 15 The image shows the detection results of AIV (H9N2 subtype) AV1571 strain using the indirect immunofluorescence kit in Example 4.

[0040] Figure 16 This is a graph showing the detection results of FAdV CELO strain using the indirect immunofluorescence kit in Example 4.

[0041] Figure 17 The image shows the detection results of CIAV AV1550 strain using the indirect immunofluorescence kit in Example 4.

[0042] Figure 18 The image shows the detection results of ARV Reo S1133 strain using the indirect immunofluorescence kit in Example 4. Detailed Implementation

[0043] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings and specific embodiments. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. 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.

[0044] A hybridoma cell line, aMPV-Mab-F-E2, for detecting or identifying aMPV strains, is biologically classified as a hybridoma cell. Its biodeposit information is as follows: the biodeposit name is aMPV-Mab-F-E2, the biodeposit number is CCTCC NO: C2025211, the biodeposit date is July 10, 2025, and the biodepository center is the China Center for Type Culture Collection.

[0045] A monoclonal antibody for detecting or identifying aMPV, prepared by secretion from the hybridoma cell line aMPV-Mab-F-E2 or its passaged cell line.

[0046] As one implementation, the hybridoma cell line aMPV-Mab-F-E2 passaged cell line includes cell lines that have been passaged no more than 30 times.

[0047] As another embodiment, the hybridoma cell line aMPV-Mab-F-E2 passaged cell line includes a hybridoma cell line aMPV-Mab-F-E2 passaged 30 times.

[0048] The limited number of passages of the hybridoma cell line aMPV-Mab-F-E2 has the advantage of maintaining the purity and stability of monoclonal antibodies.

[0049] The F protein is highly conserved across all aMPV subtypes. This invention prepares the F protein based on the aMPV strain, uses the F protein as an immunogen to immunize mice, collects the spleen cell suspension from the immunized mice, fuses it with SP2 / 0 cells, and after culturing and screening, obtains a hybridoma cell line aMPV-Mab-F-E2. The monoclonal antibody secreted by this cell line can specifically recognize different B and C subtypes of aMPV without cross-reacting with other common avian diseases such as avian reovirus (REO) and egg drop syndrome virus (EDSV), demonstrating high sensitivity.

[0050] Given the aforementioned advantages of the hybridoma cell line aMPV-Mab-F-E2 and its passaged cell lines, the application of the hybridoma cell line aMPV-Mab-F-E2 or the monoclonal antibody described in this invention in the preparation of products for detecting or identifying aMPV also falls within the scope of protection of this invention.

[0051] The products used for detecting or identifying aMPV include reagents and / or kits. Preferably, the kit is an indirect immunofluorescence detection kit.

[0052] The method for detecting or identifying aMPV in products includes the following steps:

[0053] (1) Vero cells inoculated with the sample to be tested were incubated with a monoclonal antibody for the first time, and the first incubation product was obtained after washing; the monoclonal antibody is the monoclonal antibody secreted by the hybridoma cell line aMPV-Mab-F-E2 described in the above technical solution.

[0054] (2) The first incubation product was incubated a second time with FITC (fluorescein isothiocyanate) labeled goat anti-mouse antibody, and the second incubation product was obtained after washing.

[0055] (3) The second incubation product is stained and observed at a wavelength of 490 nm. The results of the fluorescence staining are used to determine whether the sample contains aMPV. If a specific green fluorescence appears, the sample contains aMPV. If no specific green fluorescence appears, the sample does not contain aMPV.

[0056] The Vero cells used to inoculate the sample to be tested are prepared by inoculating the sample into Vero cells (African green monkey kidney cells) for adsorption and culture. The adsorption time is 50–70 min. Preferably, the adsorption time is 60 min. The culture temperature is 37°C, and the culture time is 4–6 days; preferably, the culture time is 5 days.

[0057] The method described above for detecting or identifying aMPV can be used to detect aMPV without cross-reactivity with other viruses. It is convenient, efficient, highly operable, and has high specificity and sensitivity. Promoting and using the above technical solutions will help further ensure the purity and safety of avian viral live vaccines, thereby improving vaccine quality. It can also be used for clinical detection, viral load determination, and epidemiological investigation of aMPV.

[0058] The aMPV that can be detected and identified by this invention includes one or more of the following: subtype B aMPV strain B1, strain LN16, strain RPVA0201, and subtype C aMPV strain GX. Specifically, subtype B aMPV includes strains B1, LN16, and RPVA0201, and subtype C aMPV includes strain GX.

[0059] A kit for detecting or identifying aMPV, comprising the monoclonal antibody, a fluorescently labeled anti-mouse antibody, a diluent, and a washing solution.

[0060] The fluorescent label includes FITC (fluorescein isothiocyanate) labeling or CY3 (anthocyanin fluorescent dye) labeling. The anti-mouse antibody includes goat anti-mouse antibody or goat anti-mouse antibody, preferably goat anti-mouse antibody. The diluent and washing buffer each include phosphate buffer. The pH of the phosphate buffer is 7.2–7.4. Preferably, the pH of the phosphate buffer is 7.3. The concentration of the phosphate buffer is 9–11 mM. Preferably, the concentration of the phosphate buffer is 10 mM.

[0061] The kit for detecting or identifying aMPV also includes a chromogenic solution. The chromogenic solution comprises a citrate buffer containing 1 vol% solution A and 10 vol% solution B; solution A is a DMSO (dimethyl sulfoxide) solution with a TMB (3,3′,5,5′-tetramethylbenzidine) concentration of 1 wt%, and solution B comprises an aqueous H2O2 solution with a H2O2 concentration of 1 wt%.

[0062] As one embodiment, the present invention also provides the application of the kit for detecting or identifying aMPV in the detection or identification of aMPV F protein.

[0063] As another embodiment, the present invention also provides the application of the hybridoma cell line aMPV-Mab-F-E2, the monoclonal antibody, or the kit in detecting one or more of the purity, safety, and illegal additives in avian viral live vaccines.

[0064] The safety testing of avian viral live vaccines includes detecting and / or monitoring the viral load of aMPV in the avian viral live vaccine.

[0065] The avian live virus vaccines include fowlpox live vaccine, turkey herpesvirus live vaccine, Newcastle disease live vaccine, avian encephalomyelitis, fowlpox bivalent live vaccine, avian infectious bronchitis live vaccine, avian infectious laryngotracheitis recombinant fowlpox virus genetically engineered vaccine, or avian infectious bursal disease live vaccine.

[0066] The fowlpox live vaccine is a quail-attenuated live fowlpox vaccine strain. The turkey herpesvirus live vaccine is an HVT Fc-126 clone strain. The Newcastle disease live vaccine is a Newcastle disease live vaccine strain Clone30. As one embodiment, the avian encephalomyelitis and fowlpox bivalent live vaccine is an avian encephalomyelitis and fowlpox bivalent live vaccine strain YBF02 + a quail-attenuated live strain. The avian infectious bronchitis live vaccine is an avian infectious bronchitis live vaccine strain H120. The avian Newcastle disease live vaccine is a Newcastle disease live vaccine strain CS2. The avian infectious bursal disease live vaccine is an avian infectious bursal disease live vaccine strain B87.

[0067] When the safety of the avian live virus vaccine is tested using the hybridoma cell line aMPV-Mab-F-E2, the monoclonal antibody, or the kit described above, if no specific green fluorescence appears, it indicates that the avian live virus vaccine is not infected with aMPV and is safe.

[0068] Example 1

[0069] aMPV F protein sequence analysis

[0070] (1) Reference sequences of different aMPV strains were downloaded from NCBI (National Center for Biotechnology Information). Analysis revealed that the F protein showed high homology among different strains. A comprehensive secondary analysis and antigenicity analysis were performed on the F protein of the aMPV strain. The results showed that the 144-479aa region of the aMPV F protein was highly conserved and exhibited very low antigenicity, making it a potential candidate fragment for protein expression. After codon optimization, the gene was synthesized and cloned into the pET-32a vector. The fusion protein had a molecular weight of 56 kDa and incorporated Trx and 6×His tags. The sequence information is as follows, with underlined EcoRV and Xho I restriction sites: GATATC CTCGAG .

[0071] Example 2

[0072] Construction, expression, and purification of recombinant expression plasmids

[0073] 1. Construction of recombinant expression plasmids

[0074] The sequence in Example 1 and the purchased plasmid pET-32a were double-digested with restriction endonucleases EcoR V and Xho I, respectively. The purified and recovered fragments and the digestion products of the expression vector were ligated with a DNA Ligation Kit to obtain a recombinant expression plasmid, which was then transformed into competent cells (BL21).

[0075] 2. Low-level expression of aMPV F recombinant protein

[0076] (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 rpm until the OD of the culture medium reaches the target value. 600 The value was 0.6. IPTG (isopropyl-β-D-thiogalactoside) was added to the culture medium to induce induction. The final concentration of IPTG in the culture medium was 0.5 mM, the induction temperature was 37℃, the rotation speed was 200 rpm, and the induction time was 2 h.

[0077] (2) Take 1 mL of the induced bacterial culture, centrifuge at 12000 rpm for 1 min, discard the supernatant, and disperse the precipitate with 50 μL of 10 mM Tris-HCl solution (pH 8.0) (the amount of buffer added depends on the amount of bacterial cells). Add an equal volume of 2× loading buffer, incubate at 100℃ for 5 min, and then perform SDS-PAGE electrophoresis. The results are as follows: Figure 1 As shown, a specific target band of recombinant aMPVF protein was found at a size of 56 kDa, indicating that aMPVF protein expression was successful.

[0078] 3. High expression of aMPV F recombinant protein

[0079] (1) The transformed BL21 obtained in step 1 was identified by PCR, and the positive BL21 samples were 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℃ and 200 rpm until the OD of the culture medium was reached. 600The value was 0.6. IPTG was added to the culture medium to induce induction. The final concentration of IPTG in the culture medium was 0.5 mM, the induction temperature was 37℃, and the induction time was 3 h.

[0080] (2) Centrifuge the induced bacterial solution at 8000 rpm for 6 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 solution (pH 8.0) and then perform ultrasonic disruption. The ultrasonic disruption power is 500 W, and the ultrasonic disruption is performed 180 times, each time for 5 seconds, with a 5-second interval before the next ultrasonic disruption.

[0081] (3) Electrophoretic detection was performed on the products obtained from ultrasonic disruption. The specific procedure was as follows: 100 μL of the ultrasonically disrupted bacterial suspension was centrifuged at 12000 rpm 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 was detected in the precipitate, such as... Figure 2 As shown, the aMPV F protein of this recombinant bacterium is expressed as an inclusion body.

[0082] 4. Purification of aMPV F recombinant protein

[0083] The precipitate obtained in step 3 after ultrasonic disruption and centrifugation was resuspended in 20 mL of 10 mM Tris-HCl solution (pH 8.0) and allowed to stand for 10 min. The precipitate was then centrifuged at 12000 rpm for 10 min, and the supernatant was transferred to another tube for storage. The precipitate was resuspended in 20 mL of 10 mM Tris-HCl solution (pH 8.0) and allowed to stand for 10 min to obtain a bacterial suspension. The bacterial suspension was centrifuged at 12000 rpm for 10 min, and the supernatant was discarded to obtain the first precipitate. The resuspension and centrifugation steps were repeated once for the first precipitate to obtain the second precipitate. The second precipitate was first resuspended in a small amount of 10 mM Tris-HCl (pH 8.0) solution to disperse the protein. Then, 10 mL of 10 mM Tris-HCl (pH 8.0) solution containing 8 M urea was added to dissolve the protein. The mixture was centrifuged at 12000 rpm 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 aMPV F protein, such as... Figure 3 As shown. Using BSA (bovine serum albumin) as the standard, the purified protein concentration was estimated to be >0.5 mg / mL and the purity >85% by SDS-PAGE gel scanning analysis.

[0084] Example 3

[0085] Preparation of monoclonal antibodies

[0086] 1. Immunity in mice

[0087] The purified aMPV F 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 aMPV F 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 aMPV F 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 for a single intraperitoneal injection of 50 μg of immunogen (aMPV F protein).

[0088] 2. Cell fusion

[0089] Three days after intraperitoneal injection of the immunogenic shock, mouse spleens were aseptically harvested and prepared into a spleen cell suspension. An equal volume of the immunogenic 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, catalog number H0262).

[0090] 3. Cloning and screening of hybridoma cells

[0091] 3.1 The cultures from the 96-well plate described in step 2 were screened using an ELISA plate coated with aMPV F protein. The specific steps are as follows:

[0092] (1) Coating ELISA plates: Dilute the purified aMPV F 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 aMPV F protein to each well of the ELISA plate and incubate overnight at 4°C; then wash three times with PBST (PBS containing 0.05 vol% Tween-20).

[0093] (2) Blocking: Add 200 μL of PBS buffer 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 vol% Tween-20).

[0094] (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 (aMPV positive serum diluted 1000 times with PBS) as primary antibody, 100 μL / well, and incubate at 37℃ for 1 h.

[0095] (4) Washing: After incubating with the primary antibody, wash the ELISA plate (step (3)) with PBST (PBS containing 0.05 vol% Tween-20) for a total of 3 times.

[0096] (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.

[0097] (6) Washing: Wash the ELISA plate after incubation with secondary antibody in step (5) with PBST (PBS containing 0.05 vol% Tween-20) for a total of 3 times.

[0098] (7) Color development: Add 100 μL / well of color development solution (citric acid buffer containing 1 vol% solution A and 10 vol% solution B, solution A is DMSO solution with a TMB mass concentration of 1%; solution B is H2O2 aqueous solution with a H2O2 mass concentration of 0.1%), and the color development time is about 5 min.

[0099] (8) Add 50 μL of stop solution (containing 2M sulfuric acid) to each well to terminate the process.

[0100] (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 below.

[0101] 3.2 The cultures in the 96-well plate described in step 2 were screened by ELISA plates coated with TRS His tag protein. The specific steps were similar to those in 3.1, except that in step (1) when coating the ELISA plate, the purified aMPV F protein was replaced with TRS His tag protein; in step (3) when incubating the primary antibody, the positive control was aMPV positive serum collected from mice immunized with F recombinant protein. The results are shown in Table 1 below.

[0102] Table 1. Results of ELISA screening for hybridoma cell lines

[0103]

[0104] Note: A high absorbance value for the target protein and a low absorbance value for the tag protein indicate a high antibody titer secreted by the hybridoma cell line. As shown in Table 1, this example screened three ELISA-positive hybridoma cell lines (E2, H3, and G4) for further screening, with the E2 hybridoma cell line exhibiting the highest titer.

[0105] 4. Indirect immunofluorescence detection

[0106] 4.1 Preparation of positive virus plates: The viral solutions of different aMPV strains in Table 2 below were diluted to 100 TCID using DMEM culture medium containing 2% newborn calf serum. 50 / 0.1mL, seeded into a 96-well plate containing Vero cells, and after about 5 days, fix with cold methanol for 15 min. At the same time, set up a blank control of Vero cells for later use.

[0107] Table 2a Information on different MPV strains and control strains

[0108]

[0109]

[0110] Note: Subtype B1 strain is from: Yu Zekun, Jiang Chengyuan, Yuan Hongxing, et al. Construction and identification of infectious clones of subtype B avian metapneumovirus B1 isolate [J]. Journal of Animal Husbandry and Veterinary Medicine, 2025, 56(02):788-802;

[0111] The B subtype LN16 strain was obtained from: Feng Xiaoyan, Bao Yuanling, Yu Mengmeng, et al. Pathogenicity of B subtype avian metapneumovirus in commercial broilers [J]. Chinese Journal of Preventive Veterinary Medicine, 2022, 44(10):1034-1038;

[0112] Subtype B RPVA0201 strain: Wang You, Song Xinyu, Wang Haiwang, et al. Evaluation experiment on the protective efficacy of avian metapneumovirus inactivated vaccine against challenge [J]. China Animal Health, 2023, 25(04):115-116;

[0113] Subtype C GX strain is from: Sun Tong. Preparation of monoclonal antibody against N protein of avian type C metapneumovirus and isolation and identification of Guangxi GX strain [D]. Yangzhou University, 2023. DOI:10.27441 / d.cnki.gyzdu.2023.000637;

[0114] The remaining virus strains were obtained from the National Veterinary Microbiology Culture Collection Center. For details, please refer to the China Veterinary Drug Information Network, Microbial Culture Collection, and Microbial Culture Search.

[0115] 4.2 Fluorescent staining

[0116] (1) Fixation: After the positive virus plate is prepared, discard the cell culture medium in the 96-well plate, add about 250 μL PBS to each well, wash the cell surface once, discard as much PBS as possible, then add 100 μL cold methanol to each well, fix at room temperature for 15 min, discard the methanol, and air dry for 5 min.

[0117] (2) Add primary antibody (monoclonal antibody): After fixing the 96-well plate, wash the cell surface once with PBS (pH 7.2). Use the supernatant of the three positive hybridoma cell lines (H3, E2 and G4) obtained by ELISA screening in step 3 as primary antibody. Dilute the supernatant 10 times with PBS and add 50 μL to each well of the positive virus plate (washed with PBS once before use). Incubate at 37°C in the dark for 1 h.

[0118] (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.

[0119] (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.

[0120] (5) Washing: The method is the same as step (3).

[0121] (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 100x, the nucleus and cytoplasm of the infected cells were stained, the well was determined to be positive for aMPV 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 aMPV detection. As a result, a cell line with good reactivity to the whole virus, which was different from aMPV, was screened. This cell line expressed a monoclonal antibody against the aMPV F protein and was named aMPV Mab-F-E2.

[0122] 5. Identification of hybridoma cells

[0123] 5.1 Cultivation Characteristics

[0124] Hybridoma cell line aMPV Mab-F-E2 was cultured in DMEM medium containing 10% 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.

[0125] 5.2 Purity Test

[0126] 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 aMPV Mab-F-E2 was subjected to sterility testing, mycoplasma testing, and exogenous virus testing, and the results all met the requirements.

[0127] 5.3 Nucleic acid examination

[0128] The chromosome number of the hybridoma cell line aMPV Mab-F-E2, 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.

[0129] 5.4 Ascites titer determination

[0130] (1) Preparation of ascites fluid: Eight-week-old BALB / c rats were intraperitoneally injected with 0.5 mL of phenazine per rat. Seven days later, the rats were intraperitoneally injected with 10 mL of phenazine. 6 Each hybridoma cell line containing 0.5 mL of aMPV Mab-F-E2 was administered to each mouse. After 7 days, the mice were observed for abdominal distension and difficulty moving. Ascites fluid was collected, centrifuged at 3000 rpm for 10 minutes, and the supernatant was collected and stored at -40°C. If ascites fluid recurred after 2 days, it could be collected again. This ascites fluid was purified using Protein-A affinity to obtain mouse anti-aMPV monoclonal antibody.

[0131] (2) The ascites fluid that produces mouse anti-aMPV monoclonal antibody was serially diluted from a volume ratio of 1:100 to 1:16000, and the fluorescent antibody titer of the ascites fluid was determined to be 1:2000 according to the indirect immunofluorescence detection method in step 4.

[0132] Example 4

[0133] The indirect immunofluorescence kit for detecting aMPV included a monoclonal antibody against aMPV, a 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 phosphate-buffered saline (PBS) at pH 7.2. The monoclonal antibody against aMPV was produced from the hybridoma cell line aMPV Mab-F-E2 obtained in Example 3.

[0134] The steps and judgment criteria for detecting aMPV using this indirect immunofluorescence kit are as follows:

[0135] 1. Sample inoculation

[0136] 100 μL of the sample to be tested was seeded into a 96-well plate containing confluent Vero cells and cultured at 37°C for about 5 days.

[0137] 2. Fluorescent staining and result interpretation

[0138] (1) Fixation: After 1 hour, 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.

[0139] (2) Adding primary antibody: After air drying, wash the cell surface once with PBS (pH 7.2), then add 50 μL aMPV monoclonal antibody to each well and incubate at 37°C for 1 h. The added aMPV monoclonal antibody is diluted with PBS (pH 7.2) at a volume ratio of 1:100.

[0140] (3) Washing: Discard the aMPV monoclonal antibody, wash 5 times with PBS (pH 7.2), add 0.3 mL of washing buffer to each well each time, and gently shake to wash.

[0141] (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) at a volume ratio of 1:100.

[0142] (5) Washing: The method is the same as step (3).

[0143] (6) Observation and Judgment: Under an inverted fluorescence microscope with blue excitation light (wavelength 490nm), the cells showed intact cell morphology. When specific green fluorescence appeared in the field of view of the inoculation well, and the cell nucleus and cytoplasm of the infected cells were visible at 100x magnification, the well was judged to be positive for aMPV 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 judged to be negative for aMPV detection.

[0144] Example 5

[0145] Specific detection

[0146] Using the indirect immunofluorescence kit from Example 4, and following the established steps and judgment criteria, avian metapneumovirus (aMPV), turkey herpesvirus (MDV-3), Newcastle disease virus (NDV), fowlpox virus (FPV), infectious bronchitis virus (IBV), infectious bursal disease virus (IBDV), egg drop syndrome virus (EDSV), avian leukosis virus (ALV), and avian ororeovirus (ARV) were detected. The staining of virus-infected cells was observed to determine the specificity of the indirect immunofluorescence method.

[0147] Specific detection results show that the indirect immunofluorescence kit of Example 4 can specifically identify and detect different aMPV strains, as shown in the results below. Figures 4-7 As shown, all reaction results were positive, while those of MDV-3 ( Figure 8 ), NDV ( Figure 9 ), FPV ( Figure 10 ), IBV ( Figure 11 ), IBDV ( Figure 12 ), EDSV Figure 13 ), ALV( Figure 14 ), AIV Figure 15 ),FAdV( Figure 16 CIAV Figure 17 ), and ARV ( Figure 18 All reactions were negative, demonstrating that the established method has broad-spectrum applicability to all serotypes of aMPV and does not react with other viruses, exhibiting good specificity. This indirect immunofluorescence kit can be used for the specific detection of aMPV.

[0148] Example 6

[0149] Sensitivity detection

[0150] aMPV (RPVA0201 strain) was diluted to 100 TCID. 50 / 100μL, based on which serial dilutions were performed to 50TCID. 50 / 100μL, 10TCID 50 / 100μL, 5TCID 50 / 100μL, 2TCID 50 / 100μL, 1TCID 50 / 100μL. Samples at the above 5 dilutions were inoculated into Vero monolayers, 100μL / well, with 4 replicates per sample. Vero was also used as a negative control. Detection was performed using the indirect immunofluorescence kit from Example 4, following the established procedures and judgment criteria. The results showed that the aMPV detection results for each dose gradient were as follows: when the aMPV infection dose was 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 aMPV contamination using the indirect immunofluorescence kit of Example 4 is 1 TCID⁻¹. 50 .

[0151] Example 7

[0152] (1) Exogenous virus testing for avian viral live vaccines

[0153] Eight key poultry live vaccines produced by domestic manufacturers were selected (as shown in Table 3 below). Indirect immunofluorescence (IFA) detection of aMPV was performed using the indirect immunofluorescence kit in Example 4. Exogenous aMPV contamination was also tested in 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 an aMPV-infected group as a positive control. Specific test results are shown in Table 3 below.

[0154] Table 3 Results of Exogenous Virus Detection in Enterprise Vaccines

[0155]

[0156]

[0157] In summary, the anti-aMPV monoclonal antibody produced using the hybridoma cell line aMPV Mab-F-E2 of this invention can simultaneously recognize different strains of aMPV without cross-reacting with viruses such as avian reovirus (REO) and egg drop syndrome virus (EDSV). It has good sensitivity and specificity and can be used not only for the detection of exogenous aMPV in avian viral live vaccines (cell examination method), but also for the clinical detection and epidemiological investigation of aMPV.

[0158] 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 hybridoma cell line for detecting or identifying avian metapneumovirus, characterized in that, The accession number is CCTCC NO:C2025211.

2. A monoclonal antibody for detecting or identifying avian metapneumovirus, characterized in that, It is obtained from the hybridoma cell line of claim 1 or its passaged cell line.

3. A monoclonal antibody for detecting or identifying avian metapneumovirus according to claim 2, characterized in that, Hybridoma cell lines include those passaged 30 times or less.

4. The use of the hybridoma cell line for detecting or identifying avian metapneumovirus according to claim 1 or the monoclonal antibody for detecting or identifying avian metapneumovirus according to any one of claims 2 or 3 in the preparation of products for detecting or identifying avian metapneumovirus.

5. The method for detecting or identifying avian metapneumovirus in products according to claim 4, characterized in that, Includes the following steps: (1) The monoclonal antibody was used to incubate Vero cells inoculated with the sample to be tested for the first time, and the first incubation product was obtained after washing. (2) The first incubation product was incubated a second time with FITC-labeled goat anti-mouse antibody, and the second incubation product was obtained after washing. (3) The second incubation product is stained and observed at a wavelength of 490 nm. The results of the fluorescence staining are used to determine whether the sample contains avian metapneumovirus. If specific green fluorescence is observed, the sample does not contain avian metapneumovirus.

6. A kit for detecting or identifying avian metapneumovirus, characterized in that, Includes the monoclonal antibody as described in claim 2 or 3, the fluorescently labeled anti-mouse antibody, the diluent, and the washing solution.

7. The kit for detecting or identifying avian metapneumovirus according to claim 6, characterized in that, 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.

8. The kit for detecting or identifying avian metapneumovirus according to claim 6, characterized in that, Applications in detecting or identifying aMPV F protein, or in detecting the purity, safety, and illegal additives in avian viral live vaccines.

9. The application of the hybridoma cells according to claim 1 or the monoclonal antibody according to claim 2 in detecting the purity, safety, and illegal additives in avian viral live vaccines.

10. The application according to claim 9, characterized in that, Detecting the safety of avian live viral vaccines includes detecting and / or monitoring the viral load of aMPV in the avian live viral vaccine.

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