Chicken infectious anemia virus VP1 recombinant protein, monoclonal antibody, blocking ELISA kit and preparation method thereof

Abstract

The invention belongs to the technical field of biological medicines, and discloses a chicken infectious anemia virus VP1 recombinant protein, a monoclonal antibody, a blocking ELISA kit and a preparation method of the blocking ELISA kit, pCold-His-TF-vp1 and pCold-His-TF-vp1 are used as prokaryotic expression vectors to establish chicken infectious anemia virus related recombinant proteins His-VP1 and StrepII-VP1, the expression of VP1 can be helped, and the recombinant proteins His-VP1 and StrepII-VP1 can be used for preparing the chicken infectious anemia virus related recombinant proteins His-VP1 and StrepII-VP1. Compared with the prior art, the method has the advantages that the method is simple and convenient to operate, good immunoreaction after immunization is facilitated, the monoclonal antibody with the correct epitope is generated, further, the monoclonal antibody with the correct epitope and the appropriate affinity can serve as a foundation for developing a blocking ELISA kit, and therefore a foundation can be laid for research on detection or diagnosis of the chicken infectious anemia virus.

Description

Technical Field

[0001] The present invention discloses a chicken infectious anemia virus VP1 recombinant protein, a monoclonal antibody, a blocking ELISA kit and a preparation method thereof, and specifically relates to a His-VP1 and StrepII-VP1 recombinant protein expressing chicken infectious anemia virus, a monoclonal antibody based on the recombinant protein, a blocking ELISA kit and a preparation method thereof, and belongs to the technical field of biomedicine. Background Art

[0002] Chicken Infectious Anemia Virus (CIAV) is a pathogen that causes severe immunosuppression in chicks under 3 weeks of age and old chickens. Existing studies have found that highly pathogenic strains of CIAV can also cause obvious clinical symptoms in 10-week-old adult chickens. The key pathological changes caused by CIAV infection mainly include damage to bone marrow hematopoietic tissue and atrophy of immune organs (especially thymus and bursa of Fabricius), leading to immune suppression and aplastic anemia. This immunosuppression makes chickens more susceptible to infection by other pathogens, such as infectious bursal disease virus (IBDV), Marek's disease virus (MDV) or reticuloendotheliosis virus (REV), increasing the risk of mixed infection and vaccine immunization failure, resulting in poor feed conversion rate, threatening the health and welfare of chickens, and having a huge impact on the global poultry industry, resulting in serious economic losses in the poultry industry.

[0003] In 1979, CIAV was first discovered in contaminated vaccines by researchers such as Yuasa in Japan. The disease caused by the virus is mainly manifested as anemia and immunosuppression in chicks, which has attracted widespread attention from the scientific community. CIAV has only one serotype and is the only member of the genus Gyrovirus in the family Anelloviridae. It is a non-enveloped single-stranded circular DNA virus. The diameter of the virus particle is approximately between 29.1nm and 26.5nm. The capsid of CIAV is composed of 12 pentagonal trumpet-shaped units, which show a distinct protruding morphology under an electron microscope. CIAV infection was first reported in China in 1996. To date, the virus can be detected in chicken flocks in most parts of China, resulting in significant economic losses to the poultry industry. Sun et al. collected 573 clinical samples from 197 broiler farms in 17 provinces in China from 2020 to 2021, of which 375 samples tested positive for CIAV (65.4%). The positive rate of CIAV detection in different regions was different, 46.67% in northern China and 81.25% in southern China, indicating that the virus is common in my country. CIAV can be transmitted horizontally and vertically in chickens. The fecal-oral route has been shown to be the main route of horizontal transmission of CIAV. The feces of infected chickens are the main source of horizontal transmission of the virus in chickens. In addition, the feathers of positive chickens can also be used as potential sources of infection to spread the virus. The most important way of transmission of CIAV is vertical transmission through eggs. Hoop et al. confirmed that the semen of hens infected with CIAV and CIAV-positive roosters is a source of vertical transmission, which can be sustained for up to 6 weeks. Studies have shown that even if hens infected with CIAV produce an immune response and form antibodies, the possibility of vertical transmission still exists. Offspring of hens with high titers of neutralizing antibodies may not show clinical symptoms due to the protection of maternal antibodies, but CIAV DNA can still be detected in these offspring. Since CIAV is vertically transmitted and present in many SPF chicken flocks, another way CIAV is transmitted is through contaminated vaccines. CIAV is injected into chickens together with live vaccines, where it recovers virulence and causes viral infection. Therefore, it is necessary to take certain quality control measures to monitor vaccines and control the further spread of CIAV.

[0004] The main mode of transmission of CIAV is vertical transmission. At present, the only effective means of control is to combine poultry biosafety measures with vaccination. The commercial vaccine for CIAV is mainly a live attenuated vaccine, but the necessity of CIAV vaccination has not been widely recognized because the vaccine cost is high and the live vaccine has a certain risk of re-strengthening, and there is a possibility of introducing vaccine strains. Therefore, regular monitoring of the antibody level of CIAV in chicken flocks, timely elimination of positive chickens and harmless treatment, and isolation of surrounding chicken flocks are measures taken by most chicken farms to prevent the further spread of the disease. Among the many detection methods in various countries, the ELISA detection method is widely used in clinical detection because of its sensitivity, specificity and ease of operation. This method is suitable for diagnosis and serological detection of the disease in grassroots veterinary departments and chicken farms. Many scholars have established a variety of methods for detecting CIAV serum antibodies, but none of them have been commercialized. Currently, there are four common CIAV commercial ELISA kits, all of which are imported kits: X-OVO's FlockscreenELISA, Zoetis's ProFLOK Chicken Anemia Virus (CAV) Antibody Test Kit, BioChek's CK126 CAV and IDEXX's FlockCheck CAV ELISA. The first three are indirect ELISAs, and IDEXX's is a blocking ELISA. Therefore, the development of a chicken infectious anemia virus serum antibody detection kit with independent intellectual property rights in my country and its industrial production are of great significance for the purification of chicken infectious anemia in my country.

[0005] As the only structural protein of CIAV, VP1 protein plays a key role in the infection and replication of the virus, and is the only protein with virus neutralizing epitopes. Therefore, VP1 protein is an important protein for understanding the pathogenesis, separation characteristics, epidemiology and development of diagnostic reagents of CIAV. In the prior art, Chinese patent CN116813717A discloses a coating antigen, ELISA kit and application thereof for detecting chicken infectious anemia virus antibodies based on virus-like particles, wherein the recombinant VP1 protein of chicken infectious anemia virus-like particles is used as the coating antigen, wherein the recombinant VP1 protein is obtained by connecting the VP1 gene to the Pgex-4T-1 backbone vector through designed amplification primers, and then expressing and purifying it by Escherichia coli BL21 (DE3). Since the virus-like particles have an external structure similar to the original virus, they are more suitable for the development of serological diagnostic methods and can efficiently detect CIAV antibodies in chickens in farms. The patent CN116813717A is an indirect ELISA, and the detection method involves an additional step, using another antibody, namely a secondary antibody that specifically binds to the antigen-antibody complex, a rabbit anti-chicken IgG antibody labeled with horseradish peroxidase, so it is very easy to produce cross-reactions, which may enhance non-specific binding and background signals. Blocking ELISA reduces the interference of non-specific background signals, and the enzyme-labeled detection antibody does not bind to the sample serum antibody, which has a lower background signal. Summary of the invention

[0006] The purpose of the present invention is to provide a chicken infectious anemia virus VP1 recombinant protein, a monoclonal antibody, a blocking ELISA kit and a preparation method thereof. The recombinant proteins His-VP1 and StrepII-VP1 related to chicken infectious anemia virus are established by using pCold-His-TF-vp1 and pCold-His-TF-vp1 as prokaryotic expression vectors, which can help the expression of VP1, help to produce a better immune response after immunization, and produce monoclonal antibodies with correct antigen epitopes. Furthermore, the monoclonal antibodies with correct epitopes and appropriate affinity can be used as the basis for developing a blocking ELISA kit, thereby laying a foundation for research on detecting or diagnosing chicken infectious anemia virus.

[0007] The present invention includes the following technical solutions:

[0008] (1) A chicken infectious anemia virus VP1 recombinant protein, comprising a His-VP1 recombinant protein and a StrepII-VP1 recombinant protein,

[0009] The His-VP1 recombinant protein is obtained by inducing expression of the prokaryotic expression vector pCold-His-TF-vp1, and the sequence of the His-VP1 recombinant protein is shown in SEQ ID NO: 1;

[0010] The StrepII-VP1 recombinant protein is obtained by inducing expression of the prokaryotic expression vector pET-TwinstrepII-SUMO-vp1. The sequence of the StrepII-VP1 recombinant protein is shown in SEQ ID NO:2.

[0011] Wherein, SEQ ID NO: 1 is:

[0012]

[0013] SEQ ID NO: 2 is:

[0014]

[0015] Furthermore, the sequences of amplification primers for constructing the prokaryotic expression vector pCold-His-TF-vp1 are shown in SEQ ID NO: 3 and SEQ ID NO: 4; the sequences of amplification primers for constructing pET-TwinstrepII-SUMO-vp1 are shown in SEQ ID NO: 5 and SEQ ID NO: 6.

[0016] Among them, SEQ ID NO: 3 (upstream primer) is 5'-ATGGAGCTCGGTACCCTCGAGatggcaagacgag-3'; SEQ ID NO: 4 (downstream primer) is 5'-AGCAGAGATTACCTATCTAGAtcagggctgcgtc-3'; SEQ ID NO: 5 (upstream primer) is 5'-GATCCGTCGACGAATTCatggcaagacg-3'; SEQ ID NO: 6 (downstream primer) is 5'-AGCCGGATCTCACTCGAGtcagggctg-3'.

[0017] (ii) A method for preparing the above-mentioned chicken infectious anemia virus VP1 recombinant protein, wherein the vp1 gene is constructed into the expression vectors pCold-His-TF and pET-TwinstrepII-SUMO, respectively, to obtain the prokaryotic expression vectors pCold-His-TF-vp1 and the prokaryotic expression vectors pET-TwinstrepII-SUMO-vp1, and after induced expression, the His-VP1 recombinant protein and the StrepII-VP1 recombinant protein are obtained.

[0018] Furthermore, the sequences of amplification primers for constructing the expression vector pET-TwinstrepII-SUMO are shown in SEQ ID NO:7 and SEQ ID NO:8.

[0019] SEQ ID NO: 7 (upstream primer) is 5'-AGAAGGAGATATACATATGAGCGCCTGGAG-3'; SEQ ID NO: 8 (downstream primer) is 5'-ACATGCTAGCCATATGTTTTTCGAACTG-3'.

[0020] Preferably, the prokaryotic expression vector pCold-His-TF-vp1 and the prokaryotic expression vector pET-TwinstrepII-SUMO-vp1 are respectively transformed into Escherichia coli competent cells, such as Transetta competent cells, for induced expression.

[0021] When inducing expression, the bacterial solution of the prokaryotic expression vector pCold-His-TF-vp1 is spread on the LBA solid culture medium; the bacterial solution of the prokaryotic expression vector pET-TwinstrepII-SUMO-vp1 is spread on the LBK solid culture medium.

[0022] (III) A method for preparing monoclonal antibodies against chicken infectious anemia virus, using the above-mentioned His-VP1 recombinant protein through immune reaction, cell fusion and screening to obtain positive hybridoma cell lines 4C11 and 5C11.

[0023] (IV) An anti-chicken infectious anemia virus monoclonal antibody prepared by the above preparation method.

[0024] BALB / c mice were immunized, spleen cells from the mice were fused with myeloma SP2 / 0 cells, and hybridoma cells that stably secreted anti-VP1 antibodies were obtained by multiple subcloning.

[0025] (V) A blocking ELISA kit for detecting antibodies to chicken infectious anemia virus, comprising the above-mentioned His-VP1 recombinant protein and anti-chicken infectious anemia virus monoclonal antibody, in addition to antigen coating stock solution, serum antibody diluent, stop solution and other related reagents.

[0026] Furthermore, in the blocking ELISA kit, the His-VP1 recombinant protein is the coating antigen, and the anti-chicken infectious anemia virus monoclonal antibody is the detection antibody.

[0027] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0028] (1) Based on the VP1 gene sequence of CIAV, the present invention designs a plasmid with pET-TwinstrepII-SUMO or pCold-His-TF as an expression vector. The plasmid is induced to express through the E. coli expression system to obtain two recombinant proteins of His-VP1 and StrepII-VP1. Among them, the plasmid with different solubilizing tags can better help VP1 to express in the supernatant, which helps to produce a better immune response after immunizing mice and produce monoclonal antibodies with correct antigenic epitopes. Secondly, the E. coli expression system has many advantages in expressing heterologous recombinant proteins, including high cost-effectiveness, time saving, easy production, and easier confirmation of the antigenic characteristics of recombinant proteins. Etc. Therefore, monoclonal antibodies with correct epitopes and appropriate affinity are also the basis for developing blocking ELISA kits.

[0029] (2) The present invention provides His-VP1 recombinant protein and StrepII-VP1 recombinant protein expressed in prokaryotes, which can be used to develop diagnostic kits and establish CIAV serum antibody blocking ELISA detection kits. In the ELISA kit, StrepII-VP1 recombinant protein is used as coating antigen, and the positive hybridoma cell line 4C11 monoclonal antibody immunolabeled with His-VP1 recombinant protein is used as detection antibody, thereby realizing the localization of chicken infectious anemia virus ELISA detection kit and reducing the high cost of using imported ELISA kits for detection. It can not only be used for early diagnosis of CIAV infection, but also suitable for evaluating the effect of preventive vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the cloning and restriction digestion of the target gene vp1.

[0031] Figure 2 PCR identification of recombinant prokaryotic expression plasmid.

[0032] Figure 3 This is the induced expression and solubility analysis of 1His-VP1 and StrepII-VP1 recombinant proteins.

[0033] Figure 4 Western Blot identification of recombinant VP1 protein.

[0034] Figure 5 For the purification of His-VP1 and StrepII-VP1 recombinant proteins.

[0035] Figure 6 For the purification of monoclonal antibodies of 5C11 and 4C11 strains.

[0036] Figure 7 is the affinity of the monoclonal antibody.

[0037] Figure 8 Schematic diagram of the structure of full-length VP1 and its truncated protein.

[0038] Fig. 9 Inducible expression of VP1 truncated protein.

[0039] Fig.10 is the recognition range of the antibody.

[0040] Fig.11 Cross-reactivity of antibodies.

[0041] Fig.12 Western blot was used to detect the VP1 protein expressed in MDCC-MSB1 cells infected with CIAV.

[0042] Fig.13 IFA was used to detect the VP1 protein expressed in CIAV-infected MDCC-MSB1 cells. DETAILED DESCRIPTION

[0043] The invention objectives, technical solutions and beneficial effects of the present invention are further described in detail below.

[0044] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the claimed invention, and unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs.

[0045] Test Materials:

[0046] (1) Virus strains, bacterial strains, plasmids, proteins, cells, experimental animals, serum and antibodies

[0047] CIAV Cux-1 strain was obtained from the Institute of Animal Husbandry and Veterinary Medicine, Beijing Academy of Agricultural and Forestry Sciences; DH5α and Transetta (DE3) competent cells were obtained from Beijing Quanshijin Biotechnology Co., Ltd. and Tiangen Biochemical Technology (Beijing) Co., Ltd.; prokaryotic expression plasmid pCold-His-TF was obtained from the College of Veterinary Medicine, China Agricultural University; prokaryotic expression plasmid pET-His-SUMO was obtained from Shanghai Biyuntian Biotechnology Co., Ltd.; plasmid pcDNA3.1-HA-TwinstrepII was obtained from the College of Veterinary Medicine, China Agricultural University; prokaryotic expression of avian infectious bursal disease virus VP2 protein (His-IBDV-VP2) and avian reticuloendotheliosis virus P30 protein His-REV-P30, Mycoplasma synoviae P50 protein (His-MS-P50) and Mycoplasma synoviae P60 protein (His-MS-P60) were obtained from the College of Veterinary Medicine, China Agricultural University; MDCC-MSB1 cells and myeloma cells SP2 / 0 were obtained from the College of Veterinary Medicine, China Agricultural University; BALB / c mice were obtained from the Institute of Laboratory Animals, Chinese Academy of Medical Sciences; positive sera of chicken infectious anemia virus (CIAV), Newcastle disease virus (NDV), avian reovirus (ARV), reticuloendotheliosis virus (REV) and infectious bursal disease virus (IBDV) and negative sera of SPF chicken were obtained from the College of Veterinary Medicine, China Agricultural University. The monoclonal antibody 4C11 strain was labeled with HRP (HRP-4C11) by Beijing Bioson Biotechnology Co., Ltd.

[0048] (2) Main reagents

[0049] DNA polymerase: Nanjing Novozyme Biotechnology Co., Ltd.; Agarose: Beijing Qingke Biotechnology Co., Ltd.; Agarose gel DNA recovery kit: Guangzhou Meiji Biotechnology Co., Ltd.; Homologous recombination enzyme: Nanjing Novozyme Biotechnology Co., Ltd.; Restriction endonuclease: TaKaRa; 2×M5 HiPer Taq PCR mix: Beijing Polymer Biotechnology Co., Ltd.; Goldview nucleic acid dye: Beijing Dingguo Changsheng Biotechnology Co., Ltd.; Low endotoxin plasmid mini-preparation kit: Guangzhou Meiji Biotechnology Co., Ltd.; 6×DNA loading buffer: Beijing Bomade Gene Technology Co., Ltd.; PTG, kanamycin, ampicillin: Amresco; SDS-PAGE gel kit: Genefist; Coomassie brilliant blue fast staining solution: Affinibody; PVDF membrane: Millipore; Protein Marker: Nanjing Novozyme Biotechnology Co., Ltd.; His-tag purification beads: QIAGEN; Strep-Tactin XT4Flow: IBA Lifesciences; Protease inhibitor cocktail: MCE; 6× protein loading buffer: Beijing Jumei Biotechnology Co., Ltd.; His-Tag Mouse mAb, Strep-Tag Mouse mAb: Abmart; Freund's complete adjuvant and Freund's incomplete adjuvant: Sigma; Fetal bovine serum, high-glucose DMEM powder: Gibco; Polyethylene glycol (PEG4000): MERCK; DNA Marker: Zhongke Ruitai; Mouse Monoclonal Antibody Isotyping Reagents kit: Sigma; dialysis bag: Millipore; HT and HAT powders: Sigma; 96-well single-strip detachable ELISA plate, cell culture plate: Costar; HRP-labeled goat anti-chicken IgG: Beijing Biosun Biotechnology Co., Ltd.; HRP-labeled goat anti-mouse IgG: Beijing Dingguo Changsheng Biotechnology Co., Ltd.; HRP-labeled horse anti-goat IgG, FITC-labeled goat anti-chicken IgG, FITC-labeled goat anti-mouse IgG: Beijing Dingguo Changsheng Biotechnology Co., Ltd.; DAPI solution (10 μg / mL): Beijing Solebow Technology Co., Ltd.; anti-fluorescence quenching sealing fluid: Shanghai Biotech Biotechnology Co., Ltd.; BSA: Beijing Weidayouer Trading Co., Ltd.; FlockCheck CAV ELISA kit is from IDEXX.

[0050] (3) Main instruments:

[0051] Fully automatic digital gel image analysis system: Shanghai Tianneng Technology Co., Ltd.; UV spectrophotometer: magnetic stirrer; Eppendorf: IKA; ultrasonic cell disruptor: Ningbo Xinzhi Biotechnology Co., Ltd.; nucleic acid and protein electrophoresis instrument: Bio-Rad; PCR gene amplifier: Guangzhou Langji Scientific Instrument Co., Ltd.; inverted fluorescence microscope: Olympus; microplate reader: TECAN; microscope: Chongguang Instrument Co., Ltd.; carbon dioxide constant temperature cell culture incubator: Thermo Fisher Scientific.

[0052] (IV) Preparation of reagents

[0053] 1% agarose gel: Add 0.5 g of agar powder to 50 mL of 1×TAE solution, heat in a microwave for 2 minutes, remove and cool, then add 1:10000 nucleic acid dye, mix well and pour into a gel plate.

[0054] LB liquid medium: 1% tryptone, 0.5% yeast extract and 1% NaCl dissolved in ddH2O and sterilized by autoclaving.

[0055] Ampicillin stock solution: Take 1.0 g of ampicillin powder, dissolve it in 10 mL of ddH2O, filter it with a 0.22 μm filter, and store it at -20°C.

[0056] Kanamycin stock solution: Take 1.0 g of kanamycin powder, dissolve it in 10 mL of ddH2O, filter it with a 0.22 μm filter, and store it at -20°C.

[0057] LBA / LBK liquid culture medium: Add ampicillin stock solution or kanamycin stock solution to the autoclaved LB liquid culture medium at a ratio of 1:1000, mix well and set aside.

[0058] LBA / LBK solid medium: Add 1.5% agar powder to LB liquid medium, sterilize under high pressure, add ampicillin stock solution or kanamycin stock solution at a ratio of 1:1000 when the temperature is not too hot, mix well and pour into a disposable sterilized plate, set aside after solidification.

[0059] 1M IPTG stock solution: Dissolve 2.38 g of IPTG powder in 10 mL of ddH2O, filter with a 0.22 μm filter, dispense into 1.5 mL centrifuge tubes, and store at -20°C.

[0060] 50% glycerol: Mix glycerol and ddH2O in a ratio of 1:1, sterilize by autoclave, and store at 4°C.

[0061] PBS buffer: weigh 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4 and 0.24 g KH2PO4, dissolve in 1 L ddH2O, and store at room temperature.

[0062] PBST: Add Tween-20 to PBS at a ratio of 1:2000 and mix thoroughly on a magnetic stirrer.

[0063] Lysis Buffer: 50 mM NaH2PO4 (or NaH2PO4·2H2O), 300 mM NaCl, 10 mM imidazole, dissolved in 500 mL ddH2O, pH adjusted to 8.0, stored at 4°C.

[0064] Wash Buffer: 50 mM NaH2PO4 (or NaH2PO4·2H2O), 1 M NaCl, 30 mM imidazole, dissolved in 200 mL ddH2O, pH adjusted to 8.0, stored at 4°C.

[0065] Elution Buffer: Add 100mM, 150mM, 200mM, 250mM, 300mM, and 350mM imidazole to Lysis Buffer to prepare Elution Buffer containing different imidazole concentrations. Adjust the pH to 8.0 and store at 4°C.

[0066] 10×Buffer W: 1M Tris-HCl, 1.5M NaCl, 10mM EDTA·2Na·2H2O, dissolved in 50mL ddH2O, pH adjusted to 8.0, stored at 4°C.

[0067] Buffer E: Add 5 mM desthiobiotin to 40 mL 1×Buffer W, adjust the pH to 8.0, and store at 4°C.

[0068] 5×SDS-PAGE electrophoresis buffer: Dissolve 15.1 g Tris, 94 g glycine and 5 g SDS in 1 L ddH2O and stir to mix.

[0069] 10× transfer solution: 302.8g Tris, 1441.3g glycine, dissolved in 10L ddH2O, stir and mix. When using, take 200mL 10× transfer solution, add 1800mL ddH2O, then add 500mL anhydrous methanol, stir and mix thoroughly.

[0070] Antigen coating solution: Weigh 0.4 g NaHCO3 and 0.73 g Na2CO3, dissolve in 250 mL ddH2O, stir and mix, and store at 4°C.

[0071] Cell freezing solution: DMSO: fetal bovine serum = 1:9, prepared and used immediately.

[0072] Red blood cell lysis solution: Weigh 0.83 g NH4Cl and 0.1214 g Tris, dissolve in 100 mL ddH2O, sterilize by high pressure, and store at 4°C.

[0073] 50% PEG4000: sterilize 10 g of PEG4000 at 115°C, add 10 mL of preheated DMEM, and store at -20°C after aliquoting.

[0074] HAT and HT selection medium: Add 5 mL of DMEM to HAT or HT lyophilized powder, store at -20°C, and add to the culture medium at a ratio of 1:100 when using.

[0075] Saturated ammonium sulfate solution: Heat and dissolve 425 g NH4SO4 in 500 mL ddH2O, cool naturally, adjust the pH to 7.4 with concentrated ammonia water, and store at room temperature.

[0076] Antigen coating solution: Weigh 0.4 g NaHCO3 and 0.73 g Na2CO3, dissolve in 250 mL ddH2O, stir and mix, and store at 4°C.

[0077] 1% BSA: weigh 0.5 g BSA and dissolve it in 50 mL PBST and store at 4°C.

[0078] Serum antibody diluent (self-prepared): Na2HPO4 5.8g, NaH2PO4 0.593g, NaCl 9.0g, BSA 10g, sucrose 100g, add 900mL ddH2O, dilute to 1L after fully dissolving, store at 2-8℃ for later use.

[0079] Stop solution (2M H2SO4): Measure 44.4 mL of concentrated sulfuric acid, add it to 355.6 mL of ddH2O, mix thoroughly with a glass rod, and store at room temperature.

[0080] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

[0081] Example 1: Construction of prokaryotic expression vector

[0082] Design synthetic PCR primers: Design primers with TwinstrepII sequence and homology arms of pET-His-SUMO expression vector according to the sequence of plasmid pcDNA3.1-HA-TwinstrepII. Design primers with homology arms of pET-TwinstrepII-SUMO and pCold-His-TF expression vector according to the vp1 gene sequence of CIAV Cux-1 (NC_001427.1) in GenBank. The amplification primers are shown in Table 1.1 below.

[0083] Table 1.1 Amplification primers

[0084]

[0085]

[0086] Amplification of TwinstrepII and CIAV vp1 genes: Using the plasmid pcDNA3.1-HA-TwinstrepII stored in the laboratory and the total DNA extracted from the strain CIAV Cux-1 as templates, the target gene was amplified using the corresponding primers. The reaction system is shown in Table 1.2 below.

[0087] Table 1.2 Reaction system

[0088]

[0089] Mix the above-prepared system evenly, centrifuge it momentarily and put it into the PCR instrument. The reaction program is shown in Table 1.3 below.

[0090] Table 1.3 Reaction procedure

[0091] program time 95℃ pre-denaturation 2min 95℃ denaturation 15s Annealing at 58°C 15s 72℃ extension 10-15s / kb, 32 cycles Final extension at 72°C 5min

[0092] After the PCR program is completed, add 10μL 6×DNA loading buffer to the reaction system, mix well and add to the prepared 1% agarose nucleic acid gel channel, start electrophoresis at a voltage of 150V, stop electrophoresis when bromophenol blue reaches the 2 / 3 position of the gel, put the agarose gel into a gel imager to view the results, cut out the gel block of the target band, purify and recover it using a DNA gel recovery and purification kit, and store the recovered fragment in a -20℃ refrigerator.

[0093] Enzyme digestion and recovery of pET-His-SUMO vector: The pET-His-SUMO empty vector plasmid was digested with NdeI restriction endonuclease. The restriction sites were located on both sides of the His tag. The enzyme digestion reaction system is shown in Table 1.4 below.

[0094] Table 1.4 Enzyme digestion reaction system

[0095]

[0096]

[0097] After mixing the above system, place it in a 37°C incubator for enzyme digestion for 3 hours. After the reaction is completed, perform 1% agarose nucleic acid gel electrophoresis, cut the gel to recover and purify the target band, and store the recovered fragment in a -20°C refrigerator.

[0098] Ligation and transformation: homologous recombination was performed between the target gene TwinstrepII and the pET-His-SUMO linearized plasmid digested with enzymes. The reaction system is shown in Table 1.5 below.

[0099] Table 1.5 Homologous recombination reaction system

[0100] Element volume 2×Clone Express Mix 5μL Target gene 3μL Linearized plasmid 2μL

[0101] After mixing the reaction system, put it into the PCR instrument and react at 50°C for 15 minutes.

[0102] Add the connected homologous recombination reaction product to 50μL DH5α competent cells, mix gently and place on ice for 30min, open the water bath in advance, heat shock at 42℃ for 45s, and let it stand on ice for 2min. Open the clean bench with LB culture medium in advance, add the mixture to 500μL LB culture medium, and shake at 37℃ for 1h. After the rejuvenation is completed, centrifuge the bacterial solution at 2000rpm for 3min, discard 450μL supernatant, mix the remaining liquid with the precipitate, spread it on LBK solid culture medium, and culture it in a 37℃ incubator overnight.

[0103] Pick and screen positive clones: Use tweezers to pick up several monoclonal colonies with a 10μL pipette tip, place them in a centrifuge tube containing 500μL LBK liquid culture medium, and culture them in a 37℃ shaker for 3-5h. When the bacterial solution becomes turbid, take 1μL for bacterial solution PCR identification. The reaction system is shown in Table 1.6 below. The positive bacterial solution will be sent to Beijing Bomed Gene Technology Co., Ltd. for sequencing. The correctly sequenced bacterial solution will be added to the new LBK liquid culture medium at a ratio of 1:50, and cultured in a 37℃ shaker for 8h. The bacterial solution will be mixed with an equal volume of 50% glycerol, preserved and frozen in a -80℃ refrigerator. After the remaining bacterial solution is continued to be cultured in a 37℃ shaker for a total of 12h, the plasmid will be extracted using a low endotoxin plasmid mini-extraction kit, the DNA concentration will be determined using a spectrophotometer, and stored in a -20℃ refrigerator.

[0104] Table 1.6 Colony PCR reaction system

[0105]

[0106] Enzyme digestion and recovery of pET-TwinstrepII-SUMO and pCold-His-TF vectors: The pET-TwinstrepII-SUMO empty vector plasmid was digested with EcoRI and XhoI restriction endonucleases. The enzyme digestion reaction system is shown in Table 1.7 below.

[0107] Table 1.7 Enzyme digestion reaction system

[0108] Element volume EcoRI 1μL XOt 1μL Cutsmart 5μL Plasmids 2μg <![CDATA[ddH2O]]> Up to 50 μL

[0109] The pCold-His-TF empty vector plasmid was digested with XhoI and XbaI restriction endonucleases. The digestion reaction system is shown in Table 1.8 below.

[0110] Table 1.8 Enzyme digestion reaction system

[0111] Element volume XOt 1μL Xgf 1μL Cutsmart 5μL Plasmids 2μg <![CDATA[ddH2O]]> Up to 50 μL

[0112] After mixing the above system, place it in a 37°C incubator for enzyme digestion for 3 hours. After the reaction is completed, perform 1% agarose nucleic acid gel electrophoresis, cut the gel to recover and purify the target band, and store the recovered fragment in a -20°C refrigerator.

[0113] According to the above steps, the total DNA extracted from the strain CIAV Cux-1 was used as a template, and the 1350bp vp1 gene was amplified using specific primers. Then, the vp1 gene and the linearized fragments of the pCold-His-TF and pET-TwinstrepII-SUMO vectors after restriction digestion were homologously recombined and connected, and then transformed into DH5α competent cells. Monoclonal colonies were picked for colony PCR verification and positive monoclonal colonies were sequenced. The results showed that the prokaryotic expression vectors pCold-His-TF-vp1 and pET-TwinstrepII-SUMO-vp1 with different tags were successfully constructed. For details, see Figure 1 and Figure 2 shown.

[0114] Figure 1 In the figure, A is the cloning of vp1 gene, 1 is DNA Marker and 2 is vp1; B is the restriction digestion of pCold-His-TF empty vector, 1 is DNA Marker and 2 is the linearized empty vector of pCold-His-TF; C is the restriction digestion of pET-TwinstrepII-SUMO empty vector, 1 is DNA Marker and 2 is the linearized empty vector of pET-TwinstrepII-SUMO.

[0115] Figure 2 In the figure, A is the PCR identification of the prokaryotic expression vector pCold-His-TF-vp1, in which 1 is a DNA Marker, 2 is a negative control, and 3 to 5 are monoclonal colonies of pCold-His-TF-vp1; B is the PCR identification of the prokaryotic expression vector pET-TwinstrepII-SUMO-vp1, in which 1 is a DNA Marker, 2 is a negative control, and 3 to 5 are monoclonal colonies of pET-TwinstrepII-SUMO-vp1.

[0116] Example 2: Inducible expression, solubility analysis and purification of His-VP1 and StrepII-VP1 recombinant proteins

[0117] The constructed expression vectors pCold-His-TF-vp1 and pET-TwinstrepII-SUMO-vp1 were transformed into Transetta competent cells, and monoclonal colonies were picked for bacterial liquid PCR identification. After the positive monoclonal colonies were propagated in large quantities, IPTG was used to induce expression at low temperature overnight. The bacteria were collected for ultrasonic disruption, and the supernatant and precipitate were collected separately after centrifugation. SDS-PAGE was used to identify the protein expression, and it was also determined whether the protein was expressed in the supernatant or inclusion body. His and Strep monoclonal antibodies were used as primary antibodies, and Western Blot was performed to identify the induced expressed proteins. The specific steps are as follows:

[0118] (1) Take out the Transetta competent cells from the -80℃ refrigerator and place them on ice for fusion. Take out the pET-TwinstrepII-SUMO-vp1 and pCold-His-TF-vp1 plasmids from the -20℃ refrigerator. When the competent cells melt and become cloudy, take 1μL of the plasmid and transform it into 50μL of competent cells. Let it stand on ice for 30 minutes. The remaining transformation steps are the same as 2.3.1.4. Finally, spread the bacterial solution of the pET-TwinstrepII-SUMO-vp1 plasmid on the LBK solid medium, and spread the bacterial solution of the pCold-His-TF-vp1 plasmid on the LBA solid medium.

[0119] (2) Pick positive monoclonal colonies and perform colony PCR identification. The steps are the same as 2.3.1.5. Keep the positive monoclonal colonies with correct colony PCR results.

[0120] (3) Take 500 μL of the correct bacterial culture identified by PCR and add it to 50 mL of LBA / K liquid culture medium. Shake the culture medium at 37°C and 220 rpm overnight. Prepare 1 L of LB liquid culture medium and sterilize it under high pressure. After the sterilization is completed and the temperature drops to room temperature, add ampicillin storage solution or kanamycin storage solution at a ratio of 1:1000. Shake well and place in a 4°C refrigerator. Take out the 50 mL of LBA / K liquid culture medium that has been cultured overnight, add it to 1 L of LBA / K liquid culture medium in a clean bench, and shake it at 37°C and 220 rpm for 3-5 hours. Wait until the OD of the bacterial culture reaches 90%. 600 When the value was 0.5-0.6, IPTG was added at a ratio of 1:1000 in a clean bench, and then the bacterial solution was placed in a 16°C shaker at 150 rpm to induce expression for 20-24 hours.

[0121] (4) After induction, the bacterial solution was centrifuged at 4°C and 6000 rpm for 5 min to collect the bacterial cells. The collected bacterial cells were resuspended and washed once with an appropriate amount of PBS, and centrifuged again at 4°C and 6000 rpm for 5 min to collect the bacterial precipitate.

[0122] (5) Resuspend the bacterial precipitates of pCold-His-TF-vp1 plasmid and pET-TwinstrepII-SUMO-vp1 plasmid in 40 mL His Lysis Buffer and Strep 1×Buffer W respectively in 50 mL centrifuge tubes. Place the 50 mL centrifuge tube in an ice bucket and put it into an ultrasonic disruptor. Raise the ultrasonic probe 2-3 cm below the liquid surface without contacting the surrounding tube wall. Set the ultrasonic condition to 47% power, ultrasonicate for 2 seconds, pause for 3 seconds, and perform ultrasonic disruption for a total of 15 minutes. Centrifuge the bacterial solution after ultrasonication at 10,000 rpm at 4°C for 40 minutes, and collect the supernatant and precipitate respectively. Take part of the supernatant and precipitate respectively in 1.5 mL EP tubes, add 6× protein loading buffer, mix well, place in boiling water for 10 minutes, let stand on ice for 2 minutes, centrifuge and vortex, and store in a -80°C refrigerator.

[0123] (6) SDS-PAGE electrophoresis

[0124] ① Clean the 1.5mm glass plate and let it dry naturally, then assemble and place it in the glue dispensing device.

[0125] ② Add the separation solution A, solution B, 10% APS and TEMED in the SDS-PAGE gel kit to a 50mL centrifuge tube, vortex and mix thoroughly, add to the glass plate, take 1mL of isopropanol and add to the glass plate to press the gel. After the separation gel is completely solidified, pour out the upper isopropanol and control the liquid, add the concentrated gel A, solution B, 10% APS and TEMED to a 50mL centrifuge tube, vortex and mix thoroughly, add to the glass plate, fill the glass plate until it overflows slightly, gently put in the pre-washed comb, and after the concentrated gel is solidified, gently remove the glass plate and assemble it into the electrophoresis tank.

[0126] ③ Gently pull out the comb and fill the electrophoresis tank with electrophoresis solution. Add the protein marker and sample into the wells in sequence, and add 1× loading buffer into the blank wells to ensure that the bands are neat during electrophoresis.

[0127] ④Fill up the outer liquid of the electrophoresis tank so that the inner liquid and the outer liquid are basically at the same height. Place the entire electrophoresis tank in a foam box, fill it with ice cubes all around, adjust the voltage to 150V and start electrophoresis. When the bromophenol blue runs to the bottom of the gel plate, end the electrophoresis.

[0128] ⑤Take out the glass plate, gently pry open the two glass plates, take out the protein gel, put the protein gel into Coomassie Brilliant Blue Rapid Staining Solution, stain for 20 minutes, and check the results in the gel imaging system.

[0129] (7) Western Blot Identification of Recombinant Protein

[0130] ①Reload the protein sample onto a new protein gel for SDS-PAGE.

[0131] ② Pour transfer solution into the transfer box, put in sponge and filter paper, cut a PVDF membrane of appropriate size according to the size of the protein gel, activate the PVDF membrane in anhydrous methanol for 30 seconds, and then put it into the transfer box. The protein gel is also placed in the transfer box.

[0132] ③ Place the black side of the transfer splint at the bottom, and place the sponge, filter paper, protein glue, PVDF membrane, filter paper, and sponge in order, then cover with the white side, and clamp the transfer clamp. Be careful not to generate bubbles during the whole process. You can use a syringe to absorb the transfer liquid and gently drip it on the protein glue and membrane to keep it moist, and use a glass rod to gently press the sponge to drive out bubbles before covering the white side. Place the transfer clamp in the transfer tank, with the positive and negative poles corresponding, fill the surrounding area with transfer liquid, put in an ice bag, cover the lid, and connect the power supply. 90V transfer for 70 minutes.

[0133] ④ Blocking: After the transfer is completed, take out the PVDF membrane, place it in 5% skim milk, and block it at 4°C overnight.

[0134] ⑤ Washing: Place the PVDF membrane in PBST and wash on a shaker, changing the solution every 5-10 minutes for a total of 4-6 times.

[0135] ⑥ Incubate with primary antibody: Place the PVDF membrane in a ziplock bag containing diluted His-Tag Mouse mAb or Strep-Tag Mouse mAb and incubate at room temperature for 2 hours.

[0136] ⑦Washing: Repeat step ⑤.

[0137] ⑧ Incubate with secondary antibody: dilute HRP secondary antibody at a ratio of 1:10000 in 5% skim milk, place the PVDF membrane in it, and incubate at room temperature for 35 minutes.

[0138] ⑨Washing: Repeat step ⑤.

[0139] ⑩ Color development and exposure: Turn on the chemiluminescence instrument in advance and wait for the chemiluminescence instrument to cool down to -30℃. Mix the appropriate amount of color development solution A and solution B in a 1:1 ratio, use absorbent paper to dry the water on the surface of the PVDF membrane, place it on PE gloves, add the mixed color development solution dropwise under light-proof conditions, so that the color development solution covers the PVDF membrane, and color development for 2 minutes in a light-proof condition. Put the PVDF membrane into the chemiluminescence instrument for automatic exposure and save the image.

[0140] The results showed that the E. coli transformed with the expression vectors pCold-His-TF-vp1 and pET-TwinstrepII-SUMO-vp1 were successfully induced to express after adding IPTG at low temperature overnight. The supernatant and inclusion bodies were expressed with molecular weights of 105 kDa and 67 kDa, which were consistent with the theoretical values. Figure 3 Western Blot showed that compared with before induction, both His-VP1 and StrepII-VP1 had bands at the target molecular weight, as shown in Figure 4 shown.

[0141] Figure 3 In the figure, A is the induced expression and solubility analysis of His-VP1 recombinant protein; B is the induced expression and solubility analysis of StrepII-VP1 recombinant protein. Figure 4 In the figure, A is the Western Blot identification of His-VP1 recombinant protein; B is the Western Blot identification of StrepII-VP1 recombinant protein.

[0142] Since both His-VP1 and SterpII-VP1 recombinant proteins were expressed significantly in the supernatant, the recombinant proteins in the supernatant were further purified. The specific steps of His-VP1 recombinant protein purification can be summarized as follows:

[0143] (1) Filter the supernatant after ultrasonic high-speed centrifugation through a 0.45 μm filter.

[0144] (2) Pipette 2 mL of Ni-NTA filler into a 12 mL empty affinity chromatography column, place on ice for 5 min, and open the bottom stopper to allow the ethanol to flow out. Add 5 mL of ddH2O to resuspend the filler, place the column on ice for 5 min, open the bottom stopper to allow the liquid to flow out, and repeat twice. Add 10 mL of Lysis Buffer solution to equilibrate the chromatography column, resuspend the filler, place on ice for 5 min, and open the bottom stopper to allow the liquid to flow out.

[0145] (3) Add a small amount of protein supernatant to resuspend the Ni-NTA filler, pour the mixture back into the 50 mL centrifuge tube containing a large amount of protein supernatant, and place the centrifuge tube on a rocking table and incubate at 4°C for 3-5 h.

[0146] (4) Pour the protein solution into a 12 mL empty affinity chromatography column. The flow-through solution flows out under natural gravity, leaving Ni-NTA filler in the affinity chromatography column. Pour the flow-through solution into the affinity chromatography column again, repeat this step once, and collect the flow-through solution.

[0147] (5) Add 10 mL of Wash Buffer solution, and collect the wash buffer solution, which will flow out under natural gravity.

[0148] (6) Add 1 mL of Elution Buffer containing 100 mM imidazole to resuspend the filler, let stand on ice for 5 min, collect in a 1.5 mL EP tube, add 1 mL of Elution Buffer containing 150 mM imidazole, repeat the above steps, collect in a new 1.5 mL EP tube, then add 1 mL of Elution Buffer containing 200 mM, 250 mM, 300 mM and 350 mM imidazole in sequence, repeat the above steps, and collect the eluate.

[0149] (7) Take a small amount of penetration buffer, washing buffer and eluent containing different concentrations of imidazole, add 6× protein loading buffer, mix well and boil for 10 minutes to prepare protein samples for subsequent SDS-PAGE and Western Blot identification. After SDS-PAGE identification, the eluents with target protein and relatively pure are mixed together, and protease inhibitors are added at a ratio of 1:100 and stored in a -80℃ refrigerator.

[0150] The specific steps for purification of StrepII-VP1 recombinant protein can be summarized as follows:

[0151] (1) Filter the supernatant after ultrasonic high-speed centrifugation through a 0.45 μm filter.

[0152] (2) Pipette 1 mL of Strep-Tactin filler into a 12 mL empty affinity chromatography column, place on ice for 5 min, and open the bottom stopper to allow the ethanol to flow out. Add 5 mL of 1× Buffer W solution to equilibrate the column, resuspend the filler, and place on ice for 5 min. Open the bottom stopper to allow the liquid to flow out, and repeat this step three times.

[0153] (3) Add a small amount of protein supernatant to resuspend the Strep-Tactin filler, pour the mixture back into the 50 mL centrifuge tube containing a large amount of protein supernatant, and place the centrifuge tube on a rocking table and incubate at 4°C for 3-5 hours.

[0154] (4) Pour the protein solution into a 12 mL empty affinity chromatography column. The permeate will flow out under natural gravity, leaving Strep-Tactin filler in the affinity chromatography column. Pour the permeate into the affinity chromatography column again, repeat this step once, and collect the permeate.

[0155] (5) Add 5 mL of 1× Buffer W solution, and allow the wash solution to flow out under natural gravity. Collect the wash solution and repeat this step five times.

[0156] (6) Add 0.5 mL of Buffer E solution to resuspend the filler, let it stand on ice for 5 min, collect it in a 1.5 mL EP tube, add 0.5 mL of Buffer E solution again, repeat the above steps, collect it in a new 1.5 mL EP tube, repeat the above steps seven times, and collect the eluate.

[0157] (7) Take a small amount of penetration buffer, washing buffer and seven elutions, add 6× protein loading buffer, mix well and boil for 10 minutes to prepare protein samples for subsequent SDS-PAGE and Western Blot identification. After SDS-PAGE identification, the relatively pure elutions containing the target protein are mixed together, and protease inhibitors are added at a ratio of 1:100 and stored in a -80℃ refrigerator.

[0158] The results showed that the His-VP1 recombinant protein could elute the target protein at 100mM-350mM imidazole concentration, with the highest protein concentration at 150mM and 200mM imidazole. BSA quantification showed that the protein concentration was about 0.5mg / mL. After purification of the StrepII-VP1 recombinant protein, many impurities were observed, which were caused by the breakage of the recombinant protein during and after induced expression. The protein after four inductions was concentrated using an ultrafiltration tube with a cutoff size of 50kDa. After concentration, the impurities still existed but were reduced. The concentration of the StrepII-VP1 protein was about 0.05mg / mL. Figure 5 shown.

[0159] Figure 5 In the figure, A is the purification and concentration determination of His-VP1; B is the purification of StrepII-VP1; C is the concentration determination of StrepII-VP1.

[0160] Example 3: Monoclonal Antibodies

[0161] The purified His-VP1 recombinant protein is subjected to immune reaction, cell fusion and screening to obtain the positive hybridoma cell line 4C11, i.e., the monoclonal antibody. The specific steps are as follows:

[0162] (I) Immunization of mice

[0163] The purified His-VP1 recombinant protein was used to immunize 6-8 week old BALB / c female mice. For the first immunization, the protein was mixed with an equal volume of Freund's complete adjuvant and emulsified. When the milky white liquid droplets were dropped on the water surface and did not diffuse or disappear within 30 minutes, it indicated that the emulsification was complete. The emulsified protein was used to immunize mice. Each mouse was immunized with 50 μg of protein, and the total volume did not exceed 200 μL. Multiple injections were made into the back of the neck. Two weeks after the first immunization, the second immunization was carried out. The protein was mixed with an equal volume of Freund's incomplete adjuvant and emulsified. Each mouse was immunized with 100 μg of protein and multiple injections were made into the back of the neck. The third immunization was carried out four weeks after the second immunization. The immunization dose and method were the same as the second immunization. Ten days after the third immunization, the mice were tail-cut and blood was collected. After standing at room temperature for 2-3 hours, the blood was centrifuged at 3000 rpm for 10 minutes. The upper serum was collected and temporarily stored at 4°C to determine the mouse serum antibody titer.

[0164] The details are as follows:

[0165] (1) Antigen coating: Dilute StrepII-VP1 recombinant protein to 1 μg / mL with antigen coating solution, add 100 μL of diluted protein coating solution to each well of a 96-well ELISA plate, and coat in a 4°C refrigerator for 12-14 h.

[0166] (2) Washing: Shake off the liquid in the wells, place the 96-well ELISA plate in an ELISA plate washer, wash three times with PBST, and pat dry the remaining liquid in the wells on absorbent paper.

[0167] (3) Blocking: Prepare 5% skim milk, add 300 μL to each well, and block in a 37°C incubator for 2 h.

[0168] (4) Washing: Same as (2). After this step, the ELISA plate can be placed in a -80°C refrigerator for long-term storage.

[0169] (5) Incubation with primary antibody: Dilute mouse serum with PBS from 1:1000 to 1:12800, add 100 μL of the diluted serum to each well of the ELISA plate, set up duplicate and negative controls, and incubate in a 37°C incubator for 1 h.

[0170] (6) Washing: Same as (2).

[0171] (7) Incubation with secondary antibody: dilute goat anti-mouse IgG secondary antibody with PBST at a ratio of 1:10000, add 100 μL per well of the ELISA plate, and incubate in a 37°C incubator for 35 min.

[0172] (8) Washing: Same as (2).

[0173] (9) Color development: Add 100 μL of TMB color development solution to each well and develop the color for 10-15 min in the dark.

[0174] (10) Termination: When the negative wells turn slightly blue, add 50 μL 2M H2SO4 to each well to terminate the reaction.

[0175] (11) Reading: Place the ELISA plate into the ELISA reader and read the OD 450 Absorbance value. Positive / negative cutoff value = negative sample OD 450 Mean + 2.58 × SD.

[0176] The test results showed that the serum antibody titer of mice immunized with His-VP1 recombinant protein reached 1:12800, as shown in Table 3.1 below, which met the requirements for cell fusion. Mouse No. 4 with the highest titer was selected for intraperitoneal booster immunization.

[0177] Table 3.1 Determination of serum antibody titers of mice immunized with His-VP1 (mean value = 0.0834; standard deviation = 0.0070; critical value = 0.1015)

[0178] Serum dilution Unimmunized mice Mouse No. 1 Mouse No. 2 Mouse No. 3 Mouse No. 4 1:1000 0.1010 2.3500 2.2830 2.0300 2.3500 1:2000 0.0850 2.1280 2.0145 1.7020 2.1200 1:4000 0.0800 1.7990 1.6250 1.2680 1.8170 1:8000 0.0770 1.2760 1.0665 0.7870 1.4230 1:16000 0.0810 0.7530 0.6575 0.3870 0.8800 1:32000 0.0790 0.3200 0.3450 0.1920 0.5220 1:64000 0.0810 0.1850 0.1920 0.1340 0.3320 1:128000 0.0830 0.1530 0.1560 0.1190 0.2450

[0179] (II) Cell fusion

[0180] Prepare to revive SP2 / 0 cells two weeks before cell fusion. Open the 37℃ water bath in advance, take the SP2 / 0 cells out of the liquid nitrogen, place them in the water bath, and shake them continuously to accelerate their melting. After the cells are thawed, take them into the clean bench. Add the cells to a 15mL centrifuge tube containing 2mL DMEM, gently blow and mix, centrifuge at 1000rpm for 3min, and discard the supernatant. Add 1mL10% DMEM to resuspend the cells, aspirate the SP2 / 0 cells and add them to a 10cm cell culture dish, then add 4-8mL10% DMEM, gently shake the culture dish using the eight-character method to evenly distribute the cells, and place it in a cell culture incubator.

[0181] After 24 hours of recovery, change the cell medium, discard the original culture medium, rinse gently with PBS twice, add 8-10mL10% DMEM, and put it into the cell culture incubator. Observe the cell status every day, and change the medium if the culture medium turns slightly yellow. Subculture after the cells grow to 80% of the culture dish. First discard the old culture medium, rinse the cell surface with an appropriate amount of PBS, discard PBS, add 3mL DMEM to blow the cells and transfer them to a 15mL centrifuge tube, centrifuge at 1000rpm for 2min, discard the supernatant, resuspend the cells with 2mL10% DMEM, add the cell suspension to two 10cm culture dishes, 1mL per dish, and add 9mL10% DMEM to each dish. Gently shake the culture dish using the eight-character method to evenly distribute the cells and put it into the cell culture incubator. Prepare four dishes of SP2 / 0 cells in the logarithmic growth phase for cell fusion.

[0182] Three days before fusion, mice with the highest serum antibody titer were boosted with an intraperitoneal injection of 100 μg of protein.

[0183] Light the alcohol lamp, collect blood from the eyeballs of the mice used for fusion, let stand at room temperature, centrifuge at 3000rpm for 10min, collect the upper serum, and store at -20℃. After blood collection from the eyeballs, dislocate the cervical vertebrae of the mice and kill them. Put them in a small bucket filled with 75% alcohol. During this period, use tweezers to wash the mice against their fur to ensure that the skin of the mice is completely soaked with alcohol. Take out the mice, fix the limbs of the mice on a clean dissection board with a needle, with the abdomen facing up, and use sterile scissors and tweezers to cut the skin of the mice to fully expose the peritoneum. The skin on both sides of the mouse abdomen can be fixed with a needle. Use another set of new sterile scissors and tweezers to open the peritoneum, remove the spleen completely, and put it in a cell culture dish on ice with 8mL of pre-cooled DMEM.

[0184] Place the culture dish containing the spleen in the clean bench, and transfer the spleen to another dish containing DMEM for washing. Place the autoclaved cell grinding sieve in a 10 cm sterilized culture dish, soak the sieve with 5 mL of DMEM, place the spleen on the sieve, cut it into pieces with scissors, and then grind it thoroughly with a 5 mL syringe handle. Pipette the ground cell suspension into a 50 mL centrifuge tube, centrifuge it at 2000 rpm for 10 minutes, and discard the supernatant. Resuspend the cells with 10 mL of red blood cell lysis buffer, let it stand for 10-15 minutes, add 10 mL of DMEM to stop the suspension after it becomes clear, centrifuge it at 2000 rpm for 10 minutes, and discard the supernatant.

[0185] Resuspend spleen cells and SP2 / 0 cells with DMEM, mix them in the same centrifuge tube at a volume ratio of 1:1, centrifuge at 2000rpm for 10min, and discard the supernatant. Place the centrifuge tube containing the mixed cells in a preheated beaker filled with distilled water. Take 1mL PEG4000, add the cell pellet dropwise within 1min, stir while adding, and continue stirring with a pipette tip for 1min after adding. Take 1mL DMEM, add dropwise within 1min, stir while adding, and then add 7mL DMEM within 3min. Centrifuge at 2000rpm for 10min, and discard the supernatant. Add preheated 44mL 20% DMEM to resuspend the cells, and spread the cells to a 96-well plate with 100μL per well. Do not add cell solution to the last column of each plate and place it in a cell culture incubator.

[0186] 12h after cell fusion, add 100μL of 20% DMEM containing HAT to each well. Half-change the medium with 20% DMEM containing HAT on days 3, 4, 5, 7, 9, and 11 after fusion. Half-change the medium with 20% DMEM containing HT on day 14 after fusion. Use 20% DMEM culture medium on day 15 and thereafter after fusion.

[0187] (III) Preparation of mouse thymocyte supernatant

[0188] Eyeballs of 6-week-old mice were collected and serum was collected as negative control. The mice were killed by cervical dislocation and soaked in 75% alcohol. The mouse limbs were fixed on a clean dissection board with needles, with the abdomen facing up, and the skin of the mouse was cut open with sterile scissors and forceps to expose the peritoneum. The peritoneum was cut open with another set of sterile scissors and forceps, and the heart was clamped with forceps. The thymus was removed and placed in a cell culture dish on ice with 8 mL of pre-cooled DMEM.

[0189] Take the plate to the clean bench, rinse the thymus with DMEM, transfer the rinsed thymus to a sterile filter, and grind it thoroughly with a 5mL syringe handle. The ground cell suspension is aspirated into a 50mL centrifuge tube, centrifuged at 2000rpm for 10min in a horizontal centrifuge, and the supernatant is discarded. Resuspend the cells with 10mL of red blood cell lysis buffer, let stand for 10-15min, add 10mL of DMEM after the suspension becomes clear, centrifuge at 2000rpm for 10min in a horizontal centrifuge, and discard the supernatant. Resuspend the cells with 8mL of 20% DMEM, dispense into 4 10cm plates, 2mL per plate, add 8mL of 20% DMEM, gently shake the culture plate with the eight-character method to evenly distribute the cells, and put it into a cell culture incubator. After 3-4 days, collect the thymic cell supernatant, filter it with a 0.45μm filter, and dispense it, and store it at -20℃.

[0190] (IV) Screening and subcloning of positive hybridoma cells

[0191] When the cell mass in the 96-well plate grows to about 30%, the first ELISA screening can be performed, and the steps are the same as 2.3.4.2. StrepII-VP1 recombinant protein is the coating antigen, the cell supernatant is the primary antibody, the positive control is the serum of the fusion-positive mouse, and the negative control is the supernatant of SP2 / 0 cells. Repeat the test two days later, and sub-gram and expand the hybridoma cell wells that are positive in both tests.

[0192] Discard the old culture medium in the wells to be subcloned, blow off the cells with 100μL of 10% DMEM containing thymocyte supernatant, and transfer to a 1.5mL EP tube. Take 10μL of cell suspension to a 1.5mL EP tube, take out the clean bench, add 10μL of trypan blue dye solution and mix evenly, take 10μL and add it to the cell counting plate for counting. Dilute the cell concentration to 150 cells / 10mL by limiting dilution method, mix thoroughly and inoculate 100μL per well in a 96-well plate, and freeze the remaining cells in liquid nitrogen after expansion. The next day, count the number of cells inoculated in each well of the 96-well plate, mark the wells with only one cell in each well, and perform ELISA screening when the cell cluster of the well grows to about 30%. The positive cell wells will continue to be subcloned. Hybridoma cells that are positive for three consecutive subclones are considered to be able to stably secrete positive antibodies, and they are expanded and frozen in large quantities.

[0193] Finally, two positive hybridoma cell lines that could stably secrete anti-VP1 antibodies were screened out and named 4C11 and 5C11. The two positive hybridoma cells 4C11 and 5C11 were expanded and cultured and cryopreserved for seed preservation.

[0194] (V) Large-scale preparation and purification of monoclonal antibodies

[0195] Resuscitate the frozen positive hybridoma cells and expand them in large quantities. Five multiparous BALB / c female mice were intraperitoneally injected with 500 μL of liquid paraffin each. After 14 days, positive hybridoma cells were intraperitoneally injected. In the clean bench, the hybridoma cells in the 10 cm flat dish were blown off and transferred to a 15 mL centrifuge tube. Centrifuge at 1000 rpm for 3 min, the supernatant was discarded, and the cells were resuspended in DMEM. Each mouse was injected with 5×10 5 ~1×10 6 The total injection volume for each mouse should not exceed 500 μL. After the injection, gently massage the mouse abdomen.

[0196] After injection, the mice were observed every day. When the abdominal cavity of the mice was obviously distended and the spirit was poor, the ascites was extracted from the abdominal cavity of the mice with a syringe. The extraction was continued every day until the mice died. The extracted ascites was centrifuged at 2000rpm for 10 minutes at room temperature, and the yellow supernatant was aspirated. Then, it was centrifuged at 6000rpm for 5 minutes at room temperature to avoid the upper fat layer, and the yellow clear liquid was aspirated. The collected ascites was stored in a -20℃ refrigerator.

[0197] Mouse ascites was purified using the n-octanoic acid-saturated ammonium sulfate method. All the ascites of the same monoclonal antibody were dissolved and mixed together, centrifuged at 4℃5000rpm for 20min, and the supernatant was taken. The ascites was transferred to a small beaker with a built-in magnetic stirrer, placed in an ice box, and the ice box was placed on a magnetic stirrer, while ensuring that the magnetic stirrer could be used normally. Turn on the magnetic stirrer to keep the magnetic stirrer working at a low speed and stability. Slowly add 2 times the volume of 0.06M acetate solution to the ascites, adjust the pH of the mixture solution to 4.8 with 1M hydrochloric acid, add n-octanoic acid dropwise, add 400μL n-octanoic acid for every 10mL of ascites, stir at room temperature for 30min, and stand at 4℃ for 2h. Transfer the contents of the beaker to a 50mL centrifuge tube, centrifuge at 4℃10000rpm for 30min, discard the precipitate, and adjust the pH of the supernatant to 7.2 with 1M NaOH. Measure the volume of the supernatant and transfer it to a 100mL beaker, place a magnetic stirrer inside, place it in an ice box, place the ice box on the magnetic stirrer, and ensure that the magnetic stirrer can be used normally. Turn on the magnetic stirrer and keep the magnetic stirrer working at a low speed and stability. Slowly add saturated ammonium sulfate solution to the ascites to make its saturation 50%, stir at 4℃ for 30min, and let stand at 4℃ for 2-4h. Transfer the contents of the beaker to a 50mL centrifuge tube, centrifuge at 4℃12000rpm for 30min, discard the supernatant, dissolve the precipitate in PBS, and use an amount of PBS equal to the volume of purified ascites. Transfer the product to a dialysis bag and dialyze at 4℃ for 48-60h. During this period, replace the PBS external solution twice a day, and use SDS-PAGE to identify the purity of the purified antibody.

[0198] The results showed that the two purified antibodies 4C11 and 5C11 had obvious bands at 50 kDa and 25 kDa, which were the heavy chain and light chain of the antibody, respectively. Figure 6 shown.

[0199] Example 4: Identification of monoclonal antibodies

[0200] (I) Identification of monoclonal antibody subtypes

[0201] According to the instructions of the Mouse Monoclonal Antibody Isotyping Reagents kit of Sigma Company, the subtype identification of the two VP1 monoclonal antibodies prepared in Example 3 was performed. 1 μg / mL StrepII-VP1 recombinant protein was used as the coating antigen, the positive hybridoma cell supernatant to be identified was the primary antibody, the goat anti-mouse IgA, IgM, IgG1, IgG2a, IgG2b, and IgG3 in the kit were the secondary antibodies, and the HRP-labeled horse anti-goat IgG was the tertiary antibody. The steps were the same as the determination of the mouse serum antibody titer in Example 3.

[0202] The results showed that the subtype of the 5C11 strain was IgG2b, and the subtype of the 4C11 strain was IgG2a, see Table 4.1 below.

[0203] Table 4.1 Identification of monoclonal antibody subtypes

[0204] Antibody subtype 5C11 strain 4C11 strain IgM 0.1130 0.1110 IgA 0.1130 0.0900 IgG1 0.1160 0.0810 IgG2a 0.1110 0.9410 IgG2b 2.8790 0.1760 IgG3 0.1140 0.0760

[0205] (II) Affinity determination of monoclonal antibodies

[0206] 1 μg / mL StrepII-VP1 recombinant protein was used as the coating antigen, the diluted monoclonal antibody was used as the primary antibody, and the HRP-labeled goat anti-mouse IgG was used as the secondary antibody. The steps were the same as 2.3.4.2. 450 The absorbance value is the ordinate and the monoclonal antibody concentration is the abscissa. The Michaelis-Menten equation is used to draw the curve in GraphPad Prism and obtain the affinity dissociation constant (K d value).

[0207] The affinity dissociation constants (K) of monoclonal antibodies of 5C11 and 4C11 strains d ) are 9.361×10 -11 and 2.806×10 -9 ,like Figure 7 As shown ( Figure 7 In the figure, A is the affinity test of 5C11 monoclonal antibody; B is the affinity test of 4C11 monoclonal antibody). It is generally believed that high affinity antibody K d The value is 10 -7 Up to 10 -9 , a very high affinity antibody K d The value is 10 -10 Up to 10 -12 , so the 5C11 strain antibody has a very high affinity, and the 4C11 strain antibody has a high affinity.

[0208] (III) Analysis of monoclonal antibody epitope recognition region

[0209] The CIAV vp1 gene was truncated to different lengths, and primers with homology arms of the expression vector pET-TwinstrepII-SUMO were designed for each truncation. The primer sequences are shown in Table 4.2 below. Using the expression plasmid pET-TwinstrepII-SUMO-vp1 as the amplification template, PCR was performed to truncate the genes of different lengths, and the expression vector pET-TwinstrepII-SUMO was double-digested and homologous recombination was performed with the truncated fragments. The recombinant plasmids of different truncations of vp1 were transformed into E. coli DH5α competent cells, and positive monoclonal colonies were picked and sequenced. The colonies with correct sequencing were amplified and preserved, and the plasmids were extracted, and then transformed into E. coli Transetta competent cells again for protein induction expression. The bacterial precipitate after IPTG-induced expression was collected, resuspended with an appropriate amount of PBS, boiled for 10 minutes, and placed on ice for 2 minutes to prepare protein samples. SDS-PAGE electrophoresis was performed to determine the expression of the truncated protein, and Western Blot was used to identify the linear epitope interval recognized by the monoclonal antibody.

[0210] Table 4.2 Primers for truncated fragments of vp1

[0211] Primer name Primer sequence (5'-3') Δ1: 1-261aa upstream primer AGATTGGTGGATCCGTCGACGAATTCatggcaagacga Δ1: 1-261aa downstream primer TGTTAGCAGCCGGATCTCACTCGAGtcaaatgccccc Δ2: 128-450aa upstream primer ATTGGTGGATCCGTCGACGAATTCcccatcgccggt Δ2: 128-450aa downstream primer TGTTAGCAGCCGGATCTCACTCGAGtcagggctgcgt Δ3: 261-450aa upstream primer ATTGGTGGATCCGTCGACGAATTCattgcttatgcg Δ3: 261-450aa downstream primer TGTTAGCAGCCGGATCTCACTCGAGtcagggctgcgt Δ4: 358-450aa upstream primer ATTGGTGGATCCGTCGACGAATTCccgctcggcacg Δ4: 358-450aa downstream primer TGTTAGCAGCCGGATCTCACTCGAGtcagggctgcgt

[0212] The results showed that both the full-length and truncated proteins carried TwinstrepII tags at the N-terminus. The schematic diagram and nomenclature of the truncated proteins are as follows: Figure 8 As shown; SDS-PAGE identification results showed that the truncated proteins with TwinstrepII tags were successfully constructed and expressed, and the molecular weight of each truncated protein was consistent with the theoretical results, as shown in Fig. 9 As shown; Western Blot results showed that the 5C11 strain antibody recognized amino acids 128-261, such as Fig.10 As shown in Figure A, the 4C11 strain antibody recognizes amino acids 358-450. Fig.10 As shown in B, the interval diagram is as follows Fig.10 As shown in C.

[0213] Fig.10 In the figure, A is the antigen epitope recognition interval of 5C11 monoclonal antibody; B is the antigen epitope recognition interval of 4C11 monoclonal antibody; C is a schematic diagram of the monoclonal antibody antigen epitope recognition interval.

[0214] (IV) Determination of monoclonal antibody cross-reactivity

[0215] The prokaryotic expressed chicken infectious anemia virus VP1 protein (His-CIAV-VP1), avian infectious bursal disease virus VP2 protein (His-IBDV-VP2), avian reticuloendotheliosis virus P30 protein (His-REV-P30), galliformis Mycoplasma P50 protein (His-MS-P50) and galliformis Mycoplasma P60 protein (His-MS-P60) were used as antigens, purified ascites of 5C11 and 4C11 strains and His-Tag Mouse mAb were used as primary antibodies, and HRP-labeled goat anti-mouse IgG was used as secondary antibody. The cross-reactivity of monoclonal antibodies was identified by Western Blot.

[0216] The results showed that both monoclonal antibodies only recognized VP1 protein and had good reactivity, but did not recognize His-IBDV-VP2, His-REV-P30, His-MS-P50 and His-MS-P60. Fig.11 shown.

[0217] (V) Identification of VP1 protein after virus infection by monoclonal antibodies

[0218] MDCC-MSB1 cells in the logarithmic growth phase were plated into a 12-well plate at a density of 60% and inoculated with CIAV Cux-1 strain. A non-inoculated MOCK control was set up. After 72 hours, the cells were collected and transferred to a 1.5 mL EP tube, centrifuged at 1000 rpm for 5 min at room temperature, the supernatant was discarded, and the cells were resuspended in an appropriate amount of PBS and washed two to three times. After each wash, the cells were centrifuged at 1000 rpm for 5 min at room temperature and the supernatant was discarded.

[0219] Collect total protein for Western Blot identification: resuspend the cell pellets of the inoculated group and MOCK with 100 μL of cell lysis buffer (with protease inhibitors added at a ratio of 1:100), lyse on ice for 30 min, centrifuge at 12500 rpm at 4°C for 12 min, collect the supernatant, add 6× protein loading buffer, boil for 10 min, and perform Western Blot identification.

[0220] Indirect immunofluorescence assay (IFA): The cell pellets of the inoculated group and MOCK were resuspended in PBS, and the cell suspension was applied to a cationic anti-shedding slide and dried naturally. 4% paraformaldehyde fixative was added to the slide and incubated at room temperature for 15-20 minutes. Pre-cooled PBS was used for washing three times. 0.2% Triton X-100 permeabilization solution was added and incubated on ice for 15-20 minutes. Pre-cooled PBS was used for washing three times. 1% BSA solution was added and blocked at 37°C for 1 hour. Pre-cooled PBS was used for washing three times. 5C11 monoclonal antibody, 4C11 monoclonal antibody, IgG2a isotype control, IgG2b isotype control, chicken CIAV positive serum (PC) or chicken CIAV negative serum (NC) were added to the slides of the inoculated group cells and incubated at 37°C for 1 hour. 5C11 monoclonal antibody or 4C11 monoclonal antibody was added to the slides of the MOCK group cells and incubated at 37°C for 1 hour. Wash four times with pre-cooled PBST, 5 minutes each time. Add FITC-labeled goat anti-mouse IgG or FITC-labeled goat anti-chicken IgG at an appropriate dilution ratio to the slide and incubate at 37°C for 30 minutes. Wash four times with pre-cooled PBST, 5 minutes each time. Add DAPI solution, incubate at room temperature for 10 minutes, and wash twice with pre-cooled PBST. Add anti-fluorescence quenching sealing solution to the slide, gently cover with a coverslip, and observe the results under a fluorescence microscope.

[0221] The results showed that both monoclonal antibodies could recognize the natural CIAV VP1 protein, such as Fig.12 and Fig.13 shown.

[0222] Fig.12 In the figure, A shows the recognition of VP1 protein expressed in CIAV-infected MDCC-MSB1 cells by 5C11 strain; B shows the recognition of VP1 protein expressed in CIAV-infected MDCC-MSB1 cells by 4C11 strain.

[0223] Example 5: ELISA test kit

[0224] A blocking ELISA method for detecting CIAV serum antibodies was established using purified His-VP1 recombinant protein as coating antigen and HRP-4C11 as detection antibody. The ELISA operation steps are shown in Table 5.1.

[0225] Table 5.1 Blocking ELISA operation steps

[0226]

[0227] (1) Determination of antigen coating concentration and serum dilution multiple

[0228] The concentration of His-VP1 recombinant protein was diluted to 12μg / mL, 6μg / mL, 3μg / mL, and 1.5μg / mL with antigen coating solution and coated at 4℃ overnight; CIAV negative and positive sera were diluted 1:2, 1:4, 1:8, and 1:16 with serum antibody diluent. The optimal antigen coating concentration and serum dilution multiple were determined by chessboard titration. Blocking rate PI = (sample OD 450 - Negative control OD 450 ) / negative control OD 450 . Select a larger PI value, a smaller amount of antigen, and a negative serum OD 450 The value is close to 1.0 and the positive serum OD 450 The antigen coating concentration and serum dilution factor should be as low as possible.

[0229] As shown in Table 5.2, the coating antigen concentration is 12 μg / mL and the PI value is the highest when the serum is diluted 1:8. However, under this condition, the amount of antigen used is large and the positive OD 450nm After comprehensive consideration, the optimal concentration of the coating antigen CIAV VP1 protein was finally determined to be 6 μg / mL, and the serum was diluted 1:4, which was the optimal antigen coating concentration and serum dilution multiple.

[0230] Table 5.1 Optimization of the optimal coating concentration of antigen VP1 protein and the optimal dilution multiple of the test serum

[0231]

[0232]

[0233] (2) Determination of antigen coating conditions

[0234] According to the determined antigen coating concentration and serum dilution multiple, different coating temperatures and times were set to determine the optimal coating conditions. Four coating conditions were set: 4°C overnight, 37°C 1h, 37°C 2h, and 37°C 3h. The remaining steps were performed according to Table 5.1 and the optimized conditions.

[0235] The results of ELISA test are shown in Table 5.3 below, which show that the PI value is the largest when coated at 37°C for 3 hours, and the coating effect is the best.

[0236] Table 5.3 Optimization results of coating conditions

[0237] Coating conditions 4℃ overnight 37℃1h 37℃2h 37℃3h <![CDATA[P(OD 450nm )]]> 0.2215 0.3460 0.3715 0.3245 <![CDATA[N(OD 450nm )]]> 0.4240 0.5535 0.5890 0.6725 PI(%) 47.76％ 37.49％ 36.93％ 51.75％

[0238] (3) Determination of blocking solution

[0239] Select commonly used ELISA blocking reagents, dilute skim milk, BSA and FBS with PBST respectively as blocking solutions, block at 37℃ for 2h, and perform the remaining steps according to Table 5.1 and the optimized conditions.

[0240] Specifically, 2% FBS, 1% BSA, 5% skim milk and 5% skim milk + 1% BSA were selected as blocking solutions, and ELISA detection was performed using the above optimized conditions. The results are shown in Table 5.4 below, which show that the PI value is the largest when 1% BSA is used as the blocking solution, and the blocking effect is the best.

[0241] Table 5.4 Optimization results of blocking solution

[0242]

[0243] (4) Determination of closure conditions

[0244] Four blocking conditions were set at 4°C overnight, 37°C for 1h, 37°C for 2h and 37°C for 3h respectively. The remaining steps were carried out according to Table 5.1 and the optimized conditions.

[0245] The results of ELISA test are shown in Table 5.5 below, which show that the PI value is the largest and the blocking effect is the best when blocked overnight at 4°C.

[0246] Table 5.5 Optimization results of closed conditions

[0247] Closure conditions 4℃ overnight 37℃1h 37℃2h 37℃3h <![CDATA[P(OD 450nm )]]> 0.3980 0.3900 0.3125 0.3020 <![CDATA[N(OD 450nm )]]> 0.9285 0.7450 0.6925 0.6950 PI(%) 57.14％ 47.65％ 54.87％ 56.55％

[0248] (5) Determination of serum antibody dilution

[0249] Four conditions were set: 1% BSA, 5% skim milk, PBST and self-prepared serum antibody diluent. The remaining steps were performed according to Table 5.1 and the optimized conditions.

[0250] The results of ELISA testing are shown in Table 5.6 below, which show that the PI value is the largest and the ELISA effect is the best when the self-prepared solution is used as the serum antibody diluent.

[0251] Table 5.6 Optimization results of serum antibody dilution

[0252]

[0253] (6) Determination of serum incubation time

[0254] Set the incubation time at 37°C to 30 min, 45 min, 1 h and 2 h. Perform the remaining steps according to Table 5.1 and the optimized conditions.

[0255] The results of ELISA test are shown in Table 5.7 below, which show that the PI value is the largest when incubated at 37°C for 45 minutes, and the ELISA effect is the best.

[0256] Table 5.7 Optimization results of serum incubation time

[0257]

[0258]

[0259] (7) Determination of the dilution multiple of enzyme-labeled monoclonal antibodies

[0260] Set eight dilution multiples of HRP-4C11 at 1:100, 1:300, 1:500, 1:700, 1:900, 1:1100, 1:1300 and 1:1500, and perform the remaining steps according to Table 5.1 and the optimized conditions.

[0261] The results of ELISA testing are shown in Table 5.8 below, which show that when HRP-4C11 is diluted at 1:300, the PI value is the largest and the ELISA effect is the best.

[0262] Table 5.8 Optimization results of enzyme-labeled antibody dilution

[0263]

[0264] (8) Determination of enzyme-labeled monoclonal antibody incubation time

[0265] Set the incubation time at 37°C to 30 min, 45 min, 1 h and 2 h. Perform the remaining steps according to Table 5.1 and the optimized conditions.

[0266] The results of ELISA test are shown in Table 5.9 below, which show that the PI value is the largest when incubated at 37°C for 45 minutes, and the ELISA effect is the best.

[0267] Table 5.9 Optimization results of enzyme-labeled antibody incubation time

[0268]

[0269] (9) Determination of color development time

[0270] Set four time periods of 5min, 10min, 15min and 20min for color development at room temperature and away from light. Perform the remaining steps according to Table 3-1 and the optimized conditions.

[0271] The results of ELISA test are shown in Table 5.10 below. The results show that the PI value is the largest and the ELISA effect is the best when the color is developed for 15 minutes at room temperature away from light.

[0272] Table 5.10 Optimization results of color development time

[0273] Color development time 5min 10min 15min 20min <![CDATA[P(OD 450nm )]]> 0.1845 0.2610 0.2830 0.3700 <![CDATA[N(OD 450nm )]]> 0.3700 0.6730 0.7470 0.8920 PI(%) 50.14％ 61.22％ 62.12％ 58.52％

[0274] (10) Determination of critical value

[0275] According to the optimal reaction conditions determined above, 32 CIAV negative sera were tested to establish the positive and negative critical values ​​of blocking ELISA. Three replicates were set for each negative serum, and positive and negative controls were set to measure OD 450 The cut-off value = mean value of negative serum PI + 2.58 × standard deviation (SD).

[0276] According to the OD of serum samples 450nm The blocking rate PI was calculated based on the value, as shown in Table 5.11 below. The average blocking rate of 32 negative sera was calculated to be 33.29%, with a standard deviation of 6.70%. The critical value = PI average + 2.58 × standard deviation (SD), that is, the critical value is 50.57%. Therefore, when the sample blocking rate PI ≥ 50.57% is judged as positive, and when it is < 50.57%, it is judged as negative.

[0277] Table 5.11 Determination of positive and negative critical values ​​(negative control OD450 nm =2.1690)

[0278]

[0279] (11) Cross-reactivity evaluation

[0280] According to the established blocking ELISA method, positive sera of Newcastle disease virus (NDV), avian reovirus (ARV), reticuloendotheliosis virus (REV) and infectious bursal disease virus (IBDV) were tested. At the same time, controls of positive and negative sera were set up, and the PI value of each sample was calculated (see Table 5.12 below), and the cross-reactivity was evaluated.

[0281] The results showed that the blocking rates were all less than 50.57%. According to the blocking rate PI, it was negative when it was less than 50.57%. It was found that the antigen-coated plate had no nonspecific reaction to the positive sera of REV, ARV, IBDV and NDV, indicating that the blocking ELISA kit had no cross-reaction with the positive sera of these viruses and had good specificity.

[0282] Table 5.12 Evaluation of cross-reactivity of blocking ELISA

[0283]

[0284] (12) Sensitivity evaluation

[0285] The CIAV-positive serum was diluted at 1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, and 1:128, for a total of 8 samples, and the blocking ELISA test was performed to evaluate the sensitivity.

[0286] The results are shown in Table 5.13 below, which show that when the dilution of CIAV-positive serum was 1:8, the PI value was 55.51%, and the sensitivity of the established blocking ELISA for detecting CIAV-positive serum was 1:8.

[0287] Table 5.13 Evaluation of sensitivity of blocking ELISA

[0288] Serum dilution multiple 1:2 1:4 1:8 1:16 1:32 1:64 Negative control <![CDATA[OD 450nm Mean]]> 0.5837 0.7503 0.9520 1.1470 1.5513 1.7637 2.1400 PI(%) 72.73％ 64.94％ 55.51％ 46.40％ 27.51％ 17.59％

[0289] (13) Repeatability test

[0290] The same batch of coated ELISA plates were used to test one positive serum and one negative serum, with 48 replicates for each serum, and the coefficient of variation was calculated, CV = (standard deviation / mean) × 100%, to analyze the intra-batch repeatability. Three different batches of coated ELISA plates were used to test one positive serum and one negative serum, with 16 replicates for each batch, and the coefficient of variation was calculated to analyze the inter-batch repeatability of the blocking ELISA.

[0291] The results are shown in Table 5.14 below. The coefficient of variation of intra-batch repetitions was between 4.12% and 6.39%, all less than 10%; the coefficient of variation of inter-batch repetitions was between 6.15% and 10.66%, all less than 15%, indicating that the kit has good repeatability and can be used for the detection of clinical samples.

[0292] Table 5.14 Repeatability analysis of competitive ELISA method

[0293] sample Intra-batch coefficient of variation (%) Coefficient of variation between batches (%) Negative serum 4.12％ 6.15％ Positive serum 6.39％ 10.66％

[0294] (14) Comparison rate with IDEXX commercial test kit

[0295] A total of 267 clinical serum samples were tested simultaneously using the established blocking ELISA method and the commercial IDEXX CIAV ELISA kit, and the test results of the two methods were compared and analyzed.

[0296] The statistical results are shown in Table 5.15 below. The concordance rate between the established blocking ELISA method and the IDEXX commercial kit for positive samples is 71.49%, the concordance rate for negative samples is 97.44%, the overall concordance rate is 75.28%, and the Kappa value is 0.4102.

[0297] Table 3-2 Clinical sample testing

[0298]

[0299] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any simple modification or equivalent change made to the above embodiment based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A chicken infectious anemia virus VP1 recombinant protein, characterized in that: Including His-VP1 recombinant protein and StrepII-VP1 recombinant protein, The His-VP1 recombinant protein is obtained by inducing expression of the prokaryotic expression vector pCold-His-TF-vp1, and the sequence of the His-VP1 recombinant protein is shown in SEQ ID NO: 1; The StrepII-VP1 recombinant protein is obtained by inducing expression of the prokaryotic expression vector pET-TwinstrepII-SUMO-vp1. The sequence of the StrepII-VP1 recombinant protein is shown in SEQ ID NO:

2.

2. The VP1 recombinant protein according to claim 1, characterized in that: The sequences of amplification primers for constructing the prokaryotic expression vector pCold-His-TF-vp1 are shown in SEQ ID NO: 3 and SEQ ID NO: 4; the sequences of amplification primers for constructing pET-TwinstrepII-SUMO-vp1 are shown in SEQ ID NO: 5 and SEQ ID NO:

6.

3. A method for preparing the recombinant protein of chicken infectious anemia virus VP1 according to any one of claims 1 or 2, characterized in that: The vp1 gene was constructed into expression vectors pCold-His-TF and pET-TwinstrepII-SUMO, respectively, to obtain prokaryotic expression vectors pCold-His-TF-vp1 and prokaryotic expression vectors pET-TwinstrepII-SUMO-vp1. After induced expression, His-VP1 recombinant protein and StrepII-VP1 recombinant protein were obtained.

4. The preparation method according to claim 3, characterized in that: The sequences of the amplification primers for constructing the expression vector pET-TwinstrepII-SUMO are shown in SEQ ID NO: 7 and SEQ ID NO:

8.

5. The preparation method according to claim 3, characterized in that: The prokaryotic expression vector pCold-His-TF-vp1 and the prokaryotic expression vector pET-TwinstrepII-SUMO-vp1 were respectively transformed into competent Escherichia coli cells for induced expression.

6. The preparation method according to claim 3, characterized in that: When inducing expression, the bacterial solution of the prokaryotic expression vector pCold-His-TF-vp1 is spread on the LBA solid culture medium; the bacterial solution of the prokaryotic expression vector pET-TwinstrepII-SUMO-vp1 is spread on the LBK solid culture medium.

7. A method for preparing an anti-chicken infectious anemia virus monoclonal antibody, characterized in that: The His-VP1 recombinant protein according to claim 1 or 2 is used to obtain positive hybridoma cell lines 4C11 and 5C11 through immune reaction, cell fusion and screening.

8. An anti-chicken infectious anemia virus monoclonal antibody prepared according to the preparation method of claim 7.

9. A blocking ELISA kit for detecting antibodies to chicken infectious anemia virus, characterized in that: The invention comprises the His-VP1 recombinant protein as described in claim 1 or 2, and the anti-chicken infectious anemia virus monoclonal antibody as described in claim 8.

10. The blocking ELISA kit according to claim 9, characterized in that: The His-VP1 recombinant protein is a coating antigen, and the anti-chicken infectious anemia virus monoclonal antibody is a detection antibody.

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