A monoclonal antibody capable of recognizing novel duck reovirus σC protein, preparation method and application thereof

Abstract

The present invention discloses a monoclonal antibody capable of recognizing a novel duck reovirus NDRVσC protein, a preparation method and an application thereof, wherein the monoclonal antibody comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises a V sequence as shown in SEQ ID NOs: 3, 4, and 5. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the amino acid sequences shown in SEQ ID NOs: 6, 7, and 8. L CDR1, V L CDR2 and V L CDR3. The present invention also discloses a method for preparing a monoclonal antibody and a double-antibody sandwich ELISA method based on the antibody and the σC rabbit polyclonal antibody. This method can be used to quantify NDRV subunit vaccine antigens and determine animal immunization doses. Compared with the methods reported in the literature, this ELISA method is simpler to operate and has great potential for practical application. The present invention provides a new method for evaluating NDRV immune efficacy and controlling vaccine quality, which can ensure the upgrading of NDRV vaccines.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a monoclonal antibody capable of identifying a novel duck reovirus NDRV σC protein, a preparation method and an application thereof. Technical Background

[0002] Currently, there are three main types of avian reoviruses that harm poultry and waterfowl: chicken reovirus (CRV), genotype I duck reovirus (Muscovy duck reovirus (MDRV), and genotype II duck reovirus (Novel duck reovirus (NDRV)). NDRV poses a more serious threat. This is primarily due to two factors: First, NDRV has a broader host spectrum, infecting all species of ducklings and goslings. It can cause hemorrhagic necrotizing hepatitis in Muscovy ducks, Muscovy ducks, and geese, and severe splenic necrosis in Cherry Valley ducks and Shelducks. Second, NDRV carries a high morbidity and mortality rate, posing a significant threat to the poultry industry. Wu Hong et al. found that NDRV prevalence in Muscovy ducks and Cherry Valley ducks ranges from 20% to 60%, with a mortality rate of approximately 5% to 80%.

[0003] There is currently no safe and effective commercial vaccine for NDRV.

[0004] NDRV vaccines currently in the clinical trial approval phase primarily include attenuated live (MLV) vaccines. Compared to traditional MLV vaccines, subunit vaccines offer enhanced safety, stability, and embryo-free production, making them a promising vaccine format.

[0005] The insect cell-baculovirus expression system is a commonly used technology platform for developing subunit vaccines. To control costs, crude antigens harvested from baculovirus-infected insect cells are typically used for vaccine preparation. However, the baculovirus and insect cell proteins contained in the crude antigen significantly interfere with the precise quantification of NDRV antigenic proteins. Some studies have used recombinant Lactococcus lactis or Escherichia coli to express the NDRV σC protein and purify the protein via a His tag for antigen quantification. However, the protein purification process is complex, costly, and time-consuming, making it difficult to apply in large-scale vaccine production. Summary of the Invention

[0006] In view of at least one of the above technical problems, the present invention provides a monoclonal antibody capable of recognizing the novel duck reovirus NDRV σC protein, a preparation method and its application, and also provides a double-antibody sandwich ELISA method for NDRV σC protein, which can be used for the quantification of NDRV σC vaccine antigens, vaccine quality control and determination of animal immunization doses.

[0007] The method uses a monoclonal antibody capable of recognizing NDRV σC protein as a capture antibody to capture antigen components in a sample to be tested, and uses a polyclonal antibody against σC protein as a detection antibody to quantify the captured antigen.

[0008] The technical solutions provided by the present invention are as follows:

[0009] The present invention provides a monoclonal antibody, which comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises V H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes V L CDR1, V L CDR2 and V L CDR3; the V H The amino acid sequence of CDR1 is shown in SEQ ID NO: 3; H The amino acid sequence of CDR2 is shown in SEQ ID NO: 4;

[0010] The V H The amino acid sequence of CDR3 is shown in SEQ ID NO: 5;

[0011] The V L The amino acid sequence of CDR1 is shown in SEQ ID NO: 6;

[0012] The V L The amino acid sequence of CDR2 is shown in SEQ ID NO: 7;

[0013] The V L The amino acid sequence of CDR3 is shown in SEQ ID NO:8.

[0014] Furthermore, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 10; and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 12.

[0015] Furthermore, the monoclonal antibody includes all or part of the antibody heavy chain constant region and / or the antibody light chain constant region.

[0016] The present invention also provides a nucleic acid molecule, which encodes the monoclonal antibody.

[0017] Furthermore, the nucleic acid molecule has a nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody as shown in SEQ ID NO: 9 and a nucleotide sequence encoding the light chain variable region of the monoclonal antibody as shown in SEQ ID NO: 11.

[0018] The present invention also provides a vector / host cell, wherein the vector / host cell comprises the nucleic acid molecule.

[0019] The present invention also provides a method for preparing the monoclonal antibody, comprising:

[0020] The purified σC protein is used as an immunogen to immunize animals, and the monoclonal antibody is prepared by hybridoma technology.

[0021] Furthermore, the nucleotide sequence of the gene encoding the σC protein is shown in SEQ ID NO: 1. The amino acid sequence of the σC protein is shown in SEQ ID NO: 2.

[0022] The present invention also provides the use of the monoclonal antibody capable of recognizing the NDRV σC protein and the nucleic acid molecule in preparing a NDRV σC protein detection reagent.

[0023] The present invention also provides a double-antibody sandwich ELISA method for quantitative determination of NDRV σC protein, comprising: using the above-mentioned monoclonal antibody as a capture antibody to determine the concentration of the σC protein.

[0024] Furthermore, NDRV σC rabbit polyclonal antibody was used as the detection antibody.

[0025] Furthermore, the dilution ratio of the monoclonal antibody is 250 to 32,000 times, and the optimal dilution ratio is 1:32,000.

[0026] The method uses a monoclonal antibody capable of recognizing NDRV σC protein as a capture antibody to capture antigen components in a sample to be tested, and uses a polyclonal antibody against σC protein as a detection antibody to quantify the captured antigen.

[0027] Furthermore, the present invention provides a murine monoclonal antibody 5D6 specific for the NDRV σC protein as an antigen capture antibody. The antibody is a murine monoclonal antibody prepared by hybridoma technology using the expressed σC protein as an immunogen.

[0028] Furthermore, the present invention also provides a rabbit-derived polyclonal antibody specific for the NDRV σC protein as an antigen detection antibody. The antibody is a polyclonal antibody purified from the serum of inoculated New Zealand white rabbits using the expressed σC protein as an immunogen.

[0029] Furthermore, the present invention also provides an operating method for a double-antibody sandwich ELISA, comprising: diluting the capture antibody 5D6 at a preferred dilution of 1:32000, coating the ELISA plate, and coating at 4°C overnight; the blocking solution is PBST containing 5% skim milk, and the blocking is performed at 37°C for 1 h; the σC protein standard preferably has 10 concentrations of 0.5 μg / mL, 0.25 μg / mL, 0.125 μg / mL, 0.0625 μg / mL, 0.03125 μg / mL, 0.015625 μg / mL, 0.0078 μg / mL, 0.00390625 μg / mL, 0.001953125 μg / mL, and 0.0009765625, which are used for drawing a standard curve, and the incubation condition is 37°C for 1 h; the detection antibody is diluted at a preferred dilution of 1:3200, and incubated at 37°C for 1 h. h; horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG was diluted at a preferred dilution of 1:5000 and incubated at 37°C for 1 h; the substrate tetramethylbenzidine (TMB) was incubated at room temperature for 20 min, and then 2M sulfuric acid solution was added to terminate the reaction; at OD 450 Read the absorbance value.

[0030] Furthermore, the present invention also provides a product expressed by a recombinant baculovirus expressing σC protein in insect cells Sf9, which is used as a sample to be tested in a double-antibody sandwich ELISA.

[0031] Furthermore, the present invention also provides a method for quantifying σC protein in a baculovirus-insect cell expression product, which specifically comprises: diluting the capture antibody 5D6 at a ratio of 1:32000, coating the ELISA plate, and coating at 4°C overnight; blocking with PBST containing 5% skim milk, and incubating at 37°C for 1 h; drawing a standard curve with different concentrations of σC protein (0.5 μg / mL, 0.25 μg / mL, 0.125 μg / mL, 0.0625 μg / mL, 0.03125 μg / mL, 0.015625 μg / mL, 0.0078 μg / mL, 0.00390625μg / mL, 0.001953125μg / mL, 0.0009765625), and adding the baculovirus expression product at the same time, and incubating at 37°C for 1 h; diluting the detection antibody at a ratio of 1:1000, and incubating at 37°C for 1 h. h; HRP-labeled goat anti-rabbit IgG was diluted 1:5000 and incubated at 37°C for 1 h; TMB substrate was added and incubated at room temperature for 20 min; 2M sulfuric acid solution was added to terminate the reaction; at OD 450 The absorbance value was read at 400 nm; a standard curve was drawn based on the σC protein standard, and the σC protein content in the expression product to be tested was calculated based on the standard curve.

[0032] The main objectives of the present invention are achieved through the following technical solutions: expression and purification of NDRV σC protein; preparation and identification of mouse monoclonal antibodies and rabbit polyclonal antibodies specific for NDRV σC protein; determination of dilution multiples of capture antibodies and detection antibodies; establishment of a double-antibody sandwich ELISA method; and application of the double-antibody sandwich ELISA method to determine the content of NDRV σC protein in baculovirus-expressed products.

[0033] Beneficial effects: The monoclonal antibody provided by the present invention has high specificity and can be directly detected against NDRV σC protein, which is convenient, fast and highly accurate.

[0034] The existing technology purifies the σC protein expressed in different expression systems and tests the antigen content by measuring the protein concentration. This method is costly, complex to operate, and has a long cycle. It can only be performed under laboratory conditions and is difficult to apply in a large-scale production environment. The double-antibody sandwich ELISA method provided by the present invention can quantify the NDRV σC protein in the expression product. It has the advantages of high sensitivity, wide linear range, low cost, ease of use, and short operation time. It can be used for antigen quantification of NDRV protein subunit vaccines, providing a new method for evaluating NDRV immune efficacy and vaccine quality control, which can ensure the upgrading of NDRV vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Schematic diagram of the expression of NDRV σC protein in the embodiment of the present invention. A is the SDS-PAGE identification result of the recombinant σC protein induced by IPTG; B is the SDS-PAGE identification result of the purified σC protein.

[0036] Figure 2 Schematic diagram of the Western blotting reaction characteristics identification results of the σC monoclonal antibody and the σC protein in the embodiment of the present invention.

[0037] Figure 3 Schematic diagram of the results of IFA reaction characteristics identification between σC monoclonal antibody and σC protein in the examples of the present invention.

[0038] Figure 4 Schematic diagram of the ELISA reactivity characteristics identification results of the σC monoclonal antibody and the σC protein in the examples of the present invention.

[0039] Figure 5 Schematic diagram of the identification results of the purified σC rabbit polyclonal antibody in the examples of the present invention, wherein A is the SDS-PAGE identification result of the purified σC rabbit polyclonal antibody, and B is the Western blotting identification result of the purified σC rabbit polyclonal antibody.

[0040] Figure 6 Schematic diagram of a standard curve generated using a σC protein standard in an embodiment of the present invention.

[0041] Figure 7 Schematic diagram of the IFA results of recombinant baculovirus (rBac-σC) expressing NDRV σC protein in the present invention. A represents cells infected with rBac-σC, and B represents uninfected negative cells. DETAILED DESCRIPTION

[0042] The present invention will be further described below in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention.

[0043] Example 1: Expression and purification of NDRV σC protein

[0044] (1) Construction and identification of prokaryotic plasmids

[0045] The consensus sequence of the NDRV σC gene was analyzed and codon optimized using GenSmart™. The optimized sequence is SEQ ID NO: 1, and the amino acid sequence is SEQ ID NO: 2. SEQ ID NO: 1 was synthesized into the PET28a vector and transformed into competent Escherichia coli DH5a cells. After resistance screening, plasmids were extracted from cells positive for PCR identification. The recombinant plasmid was named PET28a-σC.

[0046] (2) Inducible expression of σC protein

[0047] The PET28a-σC recombinant plasmid was transformed into Escherichia coli BL21 competent cells and spread on LB agar plates with corresponding resistance. Positive monoclonal colonies were selected and inoculated into 5 mL LB liquid medium containing ampicillin resistance. The culture was shaken at 37°C and 220 rpm overnight. Subsequently, the bacterial solution was transferred to 20 mL LB liquid medium containing Amp at an inoculation ratio of 1:100 and continued to be shaken at 37°C and 220 rpm. When the OD of the bacterial solution reached 0. 600 When the value reached 0.6-0.8, IPTG (final concentrations of 0.5 μM and 1 mM) was added to induce expression. The bacterial cultures were collected at different time points (4 h and overnight culture) at 4°C and 37°C, respectively, and analyzed by SDS-PAGE.

[0048] (3) Detection of soluble expression of σB and σC proteins

[0049] A 20 mL bacterial culture, induced at 37°C with 1 μM IPTG for 4 hours, was centrifuged at 4°C and 12,000 rpm for 5 minutes. The supernatant was discarded, and the pellet was resuspended in 4 mL of PBS and sonicated. After centrifugation, the supernatant and pellet were collected separately, resuspended in 200 μL of PBS, and stored at -20°C until further use. Simultaneously, an empty vector containing pET-28a was induced under the same conditions as a blank control.

[0050] 4) Detection of expression products

[0051] A 100 μL sample was taken from each of the induced pET-28a, pET28a-σB, and pET28a-σC bacterial cultures and mixed with 25 μL of 5× SDS loading buffer. The cells were then denatured in a 100°C metal bath for 10 minutes. The treated samples were subjected to SDS-PAGE electrophoresis, and the proteins were transferred to a PVDF membrane. After blocking with 5% skim milk at room temperature for 2 hours, a mouse anti-His monoclonal antibody was used as the primary antibody at 4°C overnight. HRP-conjugated goat anti-mouse IgG was used as the secondary antibody for incubation at room temperature for 45 minutes. The color reaction was developed using an ECL colorimetric development kit.

[0052] (5) Protein purification and detection

[0053] The expressed σC protein was purified using a nickel column method. The entire purification process was completed by Jier Biochemical (Shanghai) Co., Ltd., and the purity of the purified protein reached or exceeded 90%. The results showed that E. coli transformed with the recombinant expression plasmid pET32a-σC successfully expressed the σC protein under the induction of IPTG ( Figure 1 A), the protein was expressed in the form of inclusion bodies; the σC protein was purified using a nickel column, and SDS-PAGE showed that the purified σC protein had a single band and high purity, with a molecular weight of approximately 38 kDa ( Figure 1 B). The results showed that the NDRV σC protein was successfully expressed in the E. coli expression system.

[0054] Example 2: Preparation and identification of NDRV σC protein-specific monoclonal antibodies

[0055] (1) Immunization of BALB / c mice

[0056] 1.1 Preparation of immunogen

[0057] The concentration of the purified prokaryotic σC protein was determined to be 1.04 mg / mL using a Bradford protein assay kit. The σC protein was mixed with an equal volume of adjuvant and homogenized in a homogenizer at 5500 rpm for 15 s, repeated eight times.

[0058] 1.2 Animal immunization

[0059] BALB / c mice approximately 6 weeks old were immunized according to the immunization schedule shown in Table 1. Blood was collected 2, 4, and 6 weeks after immunization, and serum was isolated and assayed for antibody levels.

[0060] Table 1 Immunization schedule for BALB / c mice

[0061]

[0062] (2) Preparation and identification of monoclonal antibodies

[0063] 2.1 Cell fusion

[0064] ① Three days before fusion, immunize mice with antigen pulses, remove their eyes, and collect blood for serum preparation. Centrifuge at 5000 rpm for 5 minutes, collect serum, and store at -20°C. After blood collection, sacrifice the mice by cervical dislocation and soak in 75% alcohol for 10 minutes.

[0065] ② Transfer to a biosafety cabinet. Secure the mouse on a foam board with the abdomen facing upward. Cut the skin to expose the peritoneum. Cut the peritoneum open, carefully remove the spleen, remove the fascia, place it on a cell strainer, and gently grind it.

[0066] Wash and collect the culture medium into a 50 mL centrifuge tube. Remove the six bottles of SP2 / 0 cells by pipetting and mixing with the spleen cells. Next, add culture medium to the mixture to a total volume of 30 mL. Centrifuge at 1000 rpm for 10 minutes to remove the supernatant. Gently tap the bottom of the tube to evenly disperse the cells. Next, preheat the tube in a 40°C water bath.

[0067] Pipette 1 mL of PEG solution, preheated to 37°C, and slowly add it dropwise to the fusion tube, swirling to mix thoroughly. The entire addition should be completed within 45-60 seconds. Within 90 seconds, add 30 mL of culture medium, preheated to 37°C, to terminate the fusion. Incubate the tube at 37°C for 10 minutes to allow the cell membrane to stabilize.

[0068] Centrifuge again at 1000 rpm for 10 min and discard the supernatant. After completing the above steps, add about 5 mL of HAT medium to the tube and suspend the cell pellet.

[0069] Add HAT medium to a total volume of 80 mL. Add the cell suspension to the 96-well plate containing feeder cells prepared 1 day earlier, adding 100 μL per well.

[0070] Place the cell plate in a 37°C, 6% CO2 incubator for static culture. On day 5, replace half of the medium in the well with HAT medium containing 15% FBS. On day 10, replace all of the medium in the well with HT medium containing 15% FBS. When the medium in the well turns yellow, aspirate it for analysis.

[0071] 2.2 Screening of positive hybridoma cells

[0072] When the cell supernatant in the 96-well plate turns yellow, aspirate it and perform ELISA. Sera from immune mice serve as a positive control, and serum from unimmunized mice serves as a negative control. A positive result is considered if the OD value of the cell supernatant compared to the OD value of the negative control is greater than 2.1.

[0073] 2.3 Subcloning of hybridoma cells

[0074] Before subcloning, prepare feeder cells and seed them into 96-well plates, adding 100 μL per well. Then, place the plates in an incubator and culture overnight.

[0075] Mix the cells in the positive wells by gently pipetting, aspirate 10 μL of the cell solution, mix with 0.2% trypan blue, and count the cells. Then, remove 100 cells and mix thoroughly with 10 mL of HT (15% FBS) medium.

[0076] Add 100 μL of hybridoma cell culture medium to each well of a 96-well plate. Finally, place the plate in a 37°C, 6% CO2 incubator for static culture. After 4–5 days of culture, observe the colonies in the wells and record them. When the culture medium turns yellow, assay for antibody levels.

[0077] Subcloning was performed three times in succession, and the obtained monoclonal cells were expanded and cultured.

[0078] 2.4 Sequencing of Hybridoma Cells

[0079] According to PrimeScript TM Total RNA was reverse transcribed into cDNA using reverse transcription primers according to the technical manual of the First Strand cDNA Synthesis Kit (TaKaRa, Cat # 2690A). V was then amplified according to the protocol of Biointron Biology Inc. H and V LThe antibody fragment was cloned into the TA / Blunt-Zero cloning vector. Clones were screened by PCR and the sequences of three positive clones were determined.

[0080] 2.5 Preparation of ascites

[0081] BALB / c female mice were selected as experimental subjects, and sterilized liquid paraffin was injected into their abdominal cavity. The injection volume for each female mouse was 0.5 mL.

[0082] After 10 days of waiting, the expanded hybridoma cells were centrifuged at 1000 rpm / min for 10 min, and then the cells were resuspended in fresh culture medium.

[0083] After counting the cells, they were diluted appropriately to ensure that each 0.2 mL of culture medium contained approximately 1 million cells. These cells were then injected intraperitoneally into mice.

[0084] After 7 days, when the abdomen of the mouse was observed to be swollen and the movement became slow, the ascites was collected.

[0085] The collected ascites was centrifuged at 4500 rpm / min for 5 minutes, and the supernatant was collected and filtered through a 0.22 μL filter. The processed ascites was packaged and stored at -20°C.

[0086] 2.6 Reactivity of monoclonal antibodies with NDRV

[0087] In order to evaluate the reaction characteristics between the prepared ascites and NDRV infected cells, Western blot and indirect immunofluorescence (IFA) identification methods were used. The results showed that 10 monoclonal antibodies (5D6, 2B4, 1C7, 5C3, 6B9, 6D11, 7B6, 7E10, 2G4, 3G5) against NDRV σC protein could react with NDRV σC protein, and a clear band around 34 kDa was visible ( Figure 2 ) In addition, 10 monoclonal antibodies against NDRV σC protein (5D6, 2B4, 1C7, 5C3, 6B9, 6D11, 7B6, 7E10, 2G4, 3G5) were able to react with NDRV WYC strain, and obvious fluorescence was observed ( Figure 3 ).

[0088] The above results show that the 11 σC monoclonal antibodies screened have both IFA and WB characteristics. The results showed that after DF-1 was infected with the NDRVWYC strain, the ascites secreted by the σC hybridoma cells was used as the primary antibody for IFA detection.

[0089] 2.7 Affinity determination of monoclonal antibodies for σC protein

[0090] The affinity of mouse ascites for σB and σC proteins was determined using an indirect ELISA. Ascites was diluted to 10 dilutions: 1:250, 1:500, 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, and 1:128000, with 100 μL / well added. Repeat wells were used. Negative mouse serum served as a control group, and the OD was measured. 450 The results showed that each monoclonal antibody had a high affinity with the protein ( Figure 4 ), the endpoint titers of the reactions were all greater than 40960, and some monoclonal antibodies had endpoint titers as high as 512000 (2B4, 5C3, 5D6, 7E10).

[0091] Example 3: Preparation of NDRV σC protein-specific polyclonal antibodies

[0092] 3.1 Preparation of rabbit polyclonal antibodies

[0093] New Zealand white rabbits were used for immunization. Before antigen injection, blood was collected for serum collection as a negative control. Rabbits were injected with antigen three times. For the first immunization, the purified σB and σC proteins were diluted to 600 μg / mL. 0.5 mL of the diluted protein was mixed with 0.5 mL of Freund's complete adjuvant and injected subcutaneously. For the second and third immunizations, incomplete Freund's adjuvant was used, and immunization conditions were the same as for the first immunization. Immunizations were repeated every two weeks.

[0094] 3.2 Purification of rabbit polyclonal antibodies

[0095] After complete immunization, serum was collected and rabbit polyclonal antibodies were purified using Protein A+G Agarose. The σB and σC antibodies in the antisera were verified by Western blotting. Antisera obtained after immunization and antisera collected before immunization (negative control) were diluted in antibody diluent (1:1000) and incubated with PVDF membranes overnight at 4°C. HRP-conjugated goat anti-rabbit IgG secondary antibody (1:1000) was added, incubated at room temperature for 1 hour, and visualized by ECL chemiluminescence. The results showed that rabbit polyclonal antibodies against NDRV σB and σC proteins were successfully prepared and purified, showing good reactivity with prokaryotic σB and σC proteins (Figure 5).

[0096] Example 4: Establishment of double antibody sandwich ELISA method

[0097] 1. Establishment of the double antibody sandwich ELISA method

[0098] A double-antibody sandwich ELISA method was established using σC monoclonal antibody 5D6 as the capture antibody, σC rabbit polyclonal antibody as the detection antibody, and σC protein as the protein standard. Array titration was used to optimize the parameters of the sandwich ELISA method. The specific steps are as follows:

[0099] Monoclonal antibodies were diluted with carbonate buffer in the following dilution ranges: 1: 250, 1: 500, 1: 1000, 1: 2000, 1: 4000, 1: 8000, 1: 16000, 1: 32000, 1: 64000, 1: 128000, and 1: 256000, at 100 μL / well. Coating was carried out overnight at 4°C.

[0100] Wash the coated ELISA plate three times with PBST buffer, 5 minutes per wash. Pat dry for the final wash. Add 100 μL / well of 5% skim milk and incubate at 37°C for 1 hour.

[0101] Wash the coated ELISA plate three times with PBST buffer, each wash lasting 5 minutes. Pat dry for the final wash.

[0102] Add σB and σC proteins at 0.08 μg / mL and 0.26 μg / mL, respectively, at 100 μL / well, and incubate at 37°C for 1 hour. Add PBST to another plate as a negative control. Wash the coated ELISA plate three times with PBST buffer, each wash lasting 5 minutes. Pat dry for the final wash.

[0103] σB and σC rabbit polyclonal antibodies were serially diluted in PBST (1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, and 1:12800) at 100 μL / well. Incubate at 37°C for 1 hour. Wash the coated ELISA plate three times with PBST buffer, each wash lasting 5 minutes. Pat dry after the final wash.

[0104] HRP-labeled goat anti-rabbit IgG antibody was diluted 1:5000 in PBST, 100 μL / well, and incubated at 37°C for 1 hour. The coated ELISA plate was washed five times with PBST buffer, each wash lasting 5 minutes.

[0105] TMB color development, 100 μL / well, room temperature for 20 min. 2 M sulfuric acid, stop the reaction, add 50 μL per well. Measure OD 450 value, calculate the P / N ratio.

[0106] The optimal antibody dilution ratio was found by the checkerboard method. It was found that the optimal dilution ratio of σC protein monoclonal antibody (capture antibody) was 1:32000, and the optimal dilution ratio of rabbit polyclonal antibody (detection antibody) was 1:3200 (Tables 2 and 3).

[0107] Table 2 OD values ​​of different σC capture antibodies and detection antibodies after pairing

[0108]

[0109] Table 3 P / N values ​​after pairing different σC capture antibodies and detection antibodies

[0110]

[0111] 2. Establishment of the Standard Curve

[0112] ELISA experiments were performed using optimized parameters, and a standard curve was established using different concentrations of σC protein (0.5 µg / mL, 0.25 µg / mL, 0.125 µg / mL, 0.0625 µg / mL, 0.03125 µg / mL, 0.015625 µg / mL, 0.0078 µg / mL, 0.00390625 µg / mL, 0.001953125 µg / mL, and 0.0009765625). The procedure was as described previously.

[0113] The results of the matrix titration show that the capture antibody (1:32000) paired with the detection antibody (1:3200) has the highest P / N value. The optimized ELISA experimental parameters are used to detect the σC standard, and the linear reactivity is good. The R 2 The detection limit was 3.125 ng, with high sensitivity (Figure 6).

[0114] Example 5: Preparation and quantitative analysis of σC protein expression products

[0115] To further validate the sandwich ELISA method for detecting σC protein in expression products, a recombinant baculovirus expressing NDRV σC protein was constructed and inoculated into insect cells sf9 to produce the σC protein expression product. The specific steps are as follows:

[0116] Based on the NDRV σC gene sequence published on GeneBank, after extensive alignment analysis, the most suitable antigen sequence was identified. The NDRV σC gene was then synthesized and incorporated into the pvL1393 vector. The recombinant transfer vector was co-transfected with the linearized genome of Autographa californica polyhedrosis virus (Spodoptera californica) into sf9 cells to rescue the recombinant baculovirus.

[0117] The recombinant baculovirus was inoculated into sf9 cells, and the expression of NDRV σC protein was identified by IFA method ( Figure 7 ).

[0118] The recombinant baculovirus was inoculated into suspension cultured sf9 cells at an MOI of 0.1. After culturing for 4 days, the culture product was harvested, the cells were lysed by ultrasonication, and the supernatant was collected after centrifugation.

[0119] The σC protein in the expression product was quantitatively analyzed using an established double-antibody sandwich ELISA method. The results showed that a recombinant baculovirus expressing the NDRV σC protein was successfully constructed. The σC protein expression product was prepared by inoculating sf9 cells with the recombinant baculovirus. The established double-antibody sandwich ELISA method could detect σC protein expression at a level of 2 μg / mL.

Claims

1. A monoclonal antibody that recognizes the novel duck reovirus NDRV σC protein, characterized in that: The monoclonal antibody comprises a heavy chain variable region and a light chain variable region; The heavy chain variable region includes V H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes V L CDR1, V L CDR2 and V L CDR3; The V H The amino acid sequence of CDR1 is shown in SEQ ID NO: 3; The V H The amino acid sequence of CDR2 is shown in SEQ ID NO: 4; The V H The amino acid sequence of CDR3 is shown in SEQ ID NO: 5; The V L The amino acid sequence of CDR1 is shown in SEQ ID NO: 6; The V L The amino acid sequence of CDR2 is shown in SEQ ID NO: 7; The V L The amino acid sequence of CDR3 is shown in SEQ ID NO:

8.

2. The monoclonal antibody according to claim 1, characterized in that The heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 10; the light chain variable region has the amino acid sequence shown in SEQ ID NO:

12.

3. The monoclonal antibody according to any one of claims 1 to 2, characterized in that The monoclonal antibody includes all or part of the antibody heavy chain constant region and / or the antibody light chain constant region.

4. A nucleic acid molecule, characterized in that The nucleic acid molecule is a nucleic acid molecule encoding the monoclonal antibody according to any one of claims 1 to 3.

5. The nucleic acid molecule according to claim 4, characterized in that The nucleic acid molecule has a nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody as shown in SEQ ID NO: 9 and a nucleotide sequence encoding the light chain variable region of the monoclonal antibody as shown in SEQ ID NO:

11.

6. A vector or host cell, characterized in that The vector or host cell comprises the nucleic acid molecule of claim 4 or 5.

7. Use of the monoclonal antibody according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4 or 5, the vector or host cell according to claim 6 in the preparation of a detection reagent for NDRV σC protein.

8. A double antibody sandwich ELISA method for quantification of NDRV σC protein for non-diagnostic purposes, characterized in that: The monoclonal antibody according to any one of claims 1 to 3 is used as a capture antibody to measure the concentration of σC protein.

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