Pigeon type I paramyxovirus HN protein monoclonal antibody as well as preparation and application thereof

Abstract

The invention discloses a pigeon I-type paramyxovirus HN protein monoclonal antibody as well as preparation and application thereof. The monoclonal antibody is secreted by a monoclonal antibody hybridoma positive cell strain 4H3 secreting pigeon I-type paramyxovirus HN protein. The hybridoma positive cell strain 4H3 is preserved in Institute of Microbiology, Chinese Academy of Sciences on August 3, 2023, the address is No.3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number of the preservation center is CGMCC No.45648. The invention solves the problem of lack of HN protein specific monoclonal antibody in the clinical detection and basic research process of pigeon type I paramyxovirus at present. According to the present invention, the monoclonal antibody secreted by the 4H3 can specifically react with the pigeon source Newcastle disease virus and the chicken source Newcastle disease virus, and does not react with the clinical common vaccine strain Lasota, and the antibody 4H3 has advantages of high titer, easy mass production, and wide application prospect.

Description

Technical Field

[0001] This invention relates to a hybridoma cell line for a monoclonal antibody, specifically to a monoclonal antibody against pigeon type I paramyxovirus HN protein and its preparation and application. Background Technology

[0002] Pigeon Newcastle disease is an acute, septicemic infectious disease caused by pigeon paramyxovirus type I (PPMV-1). It can cause neurological symptoms and gastrointestinal symptoms such as enteritis and diarrhea in pigeons. Unlike Newcastle disease virus (NDV), PPMV-1 hosts are typically limited to pigeons and wild birds. In recent years, pigeon Newcastle disease has broken out in meat pigeons and racing pigeons in many provinces of my country, causing significant harm to the pigeon farming industry. Pigeon paramyxovirus type I is an enveloped virus, with a viral diameter of approximately 200–300 nm. Its genome is a single-stranded negative-sense RNA without segmentation, with a genome length of 15192 nt, similar to NDV. The genome of pigeon paramyxovirus type I includes a 55-nt leader sequence at the 3' end and a 114-nt trailer sequence at the 5' end. The middle coding region encodes six essential proteins, including: nucleocapsid protein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase protein (HN), and large protein (L). Among them, the NP structural protein is involved in viral genome replication and, together with the L protein, P protein, and viral genome RNA, constitutes the viral nucleocapsid; the HN protein and F protein are two glycoproteins on the viral surface. The HN protein plays several important roles in the life cycle of type I pigeon paramyxovirus, including: binding to sialic acid receptors on the cell surface, promoting viral adhesion to host cells; enhancing the fusion activity of the F protein, thereby promoting viral invasion; and possessing neuraminidase activity, cleaving sialic acid from the sugar side chain, playing a role in the release of progeny virus particles from the infected cell surface. Furthermore, the HN protein is one of the main protective antigens of pigeon Newcastle disease virus, playing a crucial role in the virus's pathogenicity.

[0003] NDV has only one serotype, but there are significant differences between their genomes. Currently, NDV is divided into two major categories: Class I and Class II. Among them, Class II NDV strains of genotypes II, IV, and VII have all been isolated from pigeons, but the predominant strain in pigeon flocks is genotype VI, which includes subtypes such as VIa-VIk. Reference 1 (Pei Yu et al., Genomic sequence and pathogenicity analysis of 4 pigeon Newcastle disease virus strains [J], Acta Virologica Sinica, 2022, 38(2):402-413) indicates that the VIb subtype is the predominant strain isolated from pigeon flocks in my country. Currently, genotype VII Newcastle disease is mainly prevalent in chicken flocks in my country. Because the commonly used Lasota vaccine strain does not match the genotype and antigenicity of genotype VII Newcastle disease virus, the Lasota vaccine strain has a weak ability to clear the virus and a poor immune effect. With the development and promotion of genotype VII inactivated Newcastle disease vaccine, reference 2 (Liu Xiufan, Development of genotype VII novel vaccine and progress in the prevention and control of Newcastle disease in my country [J], Veterinary Guide, 2020(19): 4-5) shows that genotype VII inactivated Newcastle disease vaccine plays an important role in promoting the prevention and control of Newcastle disease in poultry. However, since there is currently no commercially available vaccine for pigeon Newcastle disease, the chicken vaccines such as Lasota used cannot provide effective protection against pigeon Newcastle disease, and the spread of pigeon Newcastle disease in pigeon flocks will cause significant harm to the pigeon industry. Summary of the Invention

[0004] The purpose of this invention is to provide a monoclonal antibody against the HN protein of pigeon type I paramyxovirus, its preparation and application, which solves the problem of the lack of HN protein-specific monoclonal antibodies in the current clinical detection and basic research of pigeon type I paramyxovirus.

[0005] To achieve the above objectives, this invention provides a monoclonal antibody hybridoma-positive cell line 4H3 that secretes pigeon type I paramyxovirus HN protein. This hybridoma-positive cell line 4H3 was deposited on August 3, 2023, at the Institute of Microbiology, Chinese Academy of Sciences, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 45648. The heavy chain subclass of the hybridoma-positive cell line 4H3 is IgG2a, and the light chain subclass is κ chain.

[0006] The present invention also provides a monoclonal antibody against pigeon type I paramyxovirus HN protein, wherein the monoclonal antibody is secreted by the monoclonal antibody hybridoma positive cell line 4H3 that secretes pigeon type I paramyxovirus HN protein as described above; wherein, the amino acid sequence shown in SEQ ID NO.1 is selected as the immunogen of pigeon type I paramyxovirus HN protein.

[0007] Preferably, the antigenic epitope of the monoclonal antibody is located on the pigeon type I paramyxovirus HN protein.

[0008] Preferably, the immunogen of the pigeon type I paramyxovirus HN protein is prepared by the following method:

[0009] (1) The amino acid sequence of pigeon type I paramyxovirus HN protein with optimized codons was synthesized into pET30a expression vector through EcoRI / XhoI restriction site gene, transformed into Escherichia coli BL21 expression strain, and single clones were picked from the transformed plate into LB liquid medium containing kanamycin resistance and activated by culturing at 37°C.

[0010] (2) Inoculate the activated bacterial culture into LB liquid medium containing kanamycin resistance and incubate at 37°C;

[0011] (3) The cultured bacterial culture was transferred to LB liquid medium containing kanamycin resistance, and cultured at 37°C until the optical density (OD) was 0.6-0.8 and the concentration of isopropyl-β-D-thiogalactoside (IPTG) was 0.5 mM. The culture was then induced overnight at 16°C.

[0012] (4) Centrifuge the overnight induced bacterial cells and discard the supernatant; then sonicate to break down the bacteria; purify the protein after sonication to obtain the immunogen of the pigeon type I paramyxovirus HN protein.

[0013] Preferably, in step (4), the bacterial collection is carried out at 6000 rpm and centrifugation for 8 min; the ultrasonic disruption is performed by blowing the bacterial cells with a 20-30 mL, 10 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) solution at pH 8.0, followed by ultrasonic disruption.

[0014] Preferably, in step (4), the ultrasonic breaking power is 500W, the number of times is 180, the time for each ultrasonic breaking is 5s, and the interval between adjacent breaking is 5s.

[0015] Preferably, in steps (1) to (3), the 37°C incubation is carried out at 200 rpm.

[0016] This invention provides an application of a monoclonal antibody against pigeon type I paramyxovirus HN protein as described above in the detection of Newcastle disease virus.

[0017] Preferably, the Newcastle disease virus comprises pigeon Newcastle disease virus and / or chicken Newcastle disease virus.

[0018] More preferably, the pigeon-derived Newcastle disease virus is PG / SX / 01 and / or PG / JX / 812.

[0019] More preferably, the Newcastle disease virus of chicken origin is CK / SX / 01 and / or CK / HuN / 905.

[0020] This invention discloses a monoclonal antibody against the HN protein of pigeon type I paramyxovirus, its preparation, and its application. This invention solves the problem of the lack of HN protein-specific monoclonal antibodies in the current clinical detection and basic research of pigeon type I paramyxovirus, and has the following advantages:

[0021] 1. In this invention, the relatively conserved region 198-390aa of the HN protein of pigeon type I paramyxovirus is selected as the immunogen. The antibody 4H3 obtained against this polypeptide can recognize pigeon type I paramyxovirus more broadly.

[0022] 2. The antibody 4H3 of the present invention, which is a pigeon Newcastle disease virus HN protein, can specifically bind to the pigeon Newcastle disease virus HN protein and has good reactivity in both Western blot and immunofluorescence detection. The results show that antibody 4H3 can specifically react with Class II pigeon Newcastle disease virus (PG / SX / 01 and PG / JX / 812) and chicken Newcastle disease virus (CK / SX / 01 and CK / HuN / 905), but does not react with the Lasota vaccine strain currently used in clinical practice.

[0023] 3. The antibody 4H3 of the pigeon Newcastle disease virus HN protein of the present invention can be used for immunoblotting and immunofluorescence detection, and can be widely used in the basic research field of pigeon type I paramyxovirus. It has the advantages of high potency and easy mass production, and has a wide range of application prospects. Attached Figure Description

[0024] Figure 1 This is a detailed analysis diagram of the PG1-HN protein sequence according to the present invention.

[0025] Figure 2 This is a diagram (I) showing the secondary structure analysis of the PG1-HN protein according to the present invention.

[0026] Figure 3 This is diagram (II) showing the secondary structure analysis of the PG1-HN protein according to the present invention.

[0027] Figure 4 This is a gel image of the purified PG1-HN(198-390aa) immunoprotein of the present invention.

[0028] Figure 5 This is a Western blot image of the monoclonal antibody 4H3 of this invention.

[0029] Figure 6 This is an immunofluorescence detection image of the monoclonal antibody 4H3 of this invention. Detailed Implementation

[0030] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] A monoclonal antibody against pigeon type I paramyxovirus HN protein is disclosed. The monoclonal antibody is obtained from a hybridoma-positive cell line 4H3 that secretes pigeon type I paramyxovirus HN protein. This hybridoma-positive cell line 4H3 was deposited on August 3, 2023, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China (CGMCC No. 45648). Amino acids 198–390 of the pigeon type I paramyxovirus HN protein were selected as the immunogen, and the sequence of amino acids 198–390 is shown in SEQ ID NO. 1. The sequence of the gene encoding amino acids 198–390 is shown in SEQ ID NO. 2.

[0032] The following experimental examples provide a detailed description of the hybridoma-positive cell line 4H3 and the pigeon type I paramyxovirus HN protein monoclonal antibody provided by this invention.

[0033] Experimental Example 1

[0034] In 2021, the Poultry Disease Prevention and Control Laboratory of the College of Veterinary Medicine, Shanxi Agricultural University, isolated three strains of pigeon type I paramyxovirus (Newcastle disease virus) from pigeons in Shanxi Province during routine monitoring of pathogens carried by domestic meat pigeons and racing pigeons. These three strains of pigeon Newcastle disease virus were purified on SPF chicken embryos using a limiting dilution method. After three rounds of purification, the purified viruses were frozen at -80°C. Sequence alignment and secondary structure analysis of the HN protein of the three strains of pigeon Newcastle disease virus revealed that the 198-390 region of the HN protein is relatively conserved in pigeon Newcastle disease, and this region has low hydrophobicity, high antigenicity, and surface accessibility, making it suitable as an immunogen for preparing monoclonal antibodies against the HN protein.

[0035] like Figure 1 The figure shown is a detailed analysis of the PG1-HN protein sequence. Figure 1 As can be seen, the amino acid sequences of the HN protein from three pigeon-derived Newcastle disease viruses were analyzed using Geneious software to observe their sequence homology. The black areas represent regions with identical sequences, while the gray areas represent regions with different amino acids. It is evident that the amino acid sequence in the 198-390aa region is the most conserved, and this region was selected as the antigenic epitope for the construction of the expression plasmid.

[0036] like Figure 2The diagram shown is an analysis of the secondary structure of the PG1-HN protein (I). (From...) Figure 2 As can be seen, using Geneious software to align and analyze amino acid sequences, TM Prediction predicts membrane proteins, with out representing the extramembrane region, tm the supramembrane region, in the intramembrane region, and alternating regions representing transmembrane regions; Hydrophobicity predicts hydrophobicity, with red bars representing hydrophobic amino acids and blue bars representing hydrophilic amino acids. It is evident that the 198-390 amino acid region has low hydrophobicity, which is beneficial for synthesizing the correct polypeptide.

[0037] like Figure 3 The diagram shown is a secondary structure analysis of the PG1-HN protein (II). (From...) Figure 3 As can be seen, analysis using Protean software in DNASTAR (top right shows the protein's secondary domains), Alpha (α-helix), Beta (β-sheet), Turn (β-turn), Coil (random coil), Hydrophobicity (predicted hydrophobicity), Antigenicity (predicted antigenicity), and Surface Probability (predicted antigen surface accessibility, predicting antigen exposure), along with a comprehensive analysis of the HN protein amino acid sequence, revealed that the 198-390 aa region possesses high antigenicity and surface accessibility, capable of evoking a strong immune response. Therefore, the 198-390 aa region of the HN protein was selected as the immunogen.

[0038] Example 2: Preparation of PG1-HN (198-390aa) immunogen

[0039] 1. A method for preparing a PG1-HN(198-390aa) immunogen, the method comprising:

[0040] (1) The codon-optimized PG1-HN (198-390aa) was cloned into the pET30a expression vector through the EcoRI / XhoI restriction site and transformed into Escherichia coli BL21 expression strain. Single clones were picked from the transformed plate and transferred to 1.5 mL of LB liquid medium containing kanamycin resistance (1 g tryptone, 1 g NaCl, 0.5 g yeast extract, and distilled water to a final volume of 100 mL followed by autoclaving; all of the above were purchased from Beijing Solarbio Co., Ltd.) and cultured at 37 °C and 200 rpm for activation (Shanghai Zhicheng Analytical Instruments Manufacturing Co., Ltd.).

[0041] (2) Add 50 μl of activated bacterial culture to 5 mL of LB liquid medium containing kanamycin resistance and incubate at 37°C and 200 rpm.

[0042] (3) Transfer the cultured bacterial culture to 200 mL of LB liquid medium containing kanamycin resistance, and culture at 37°C and 200 rpm until OD = 0.6-0.8. Induce overnight with IPTG (0.5 mM) at 16°C.

[0043] (4) Harvest the bacterial culture after overnight induction: Centrifuge at 6000 rpm for 8 min and discard the supernatant; then sonicate to break the bacteria (EppendORF; catalog number: 5804R): Bacterial cells are dispersed with 20-30 mL of 10 mM Tris-HCl (pH 8.0) solution and sonicated (500 W, 180 times, 5 s each time, 5 s interval; Ningbo Xinzhi Biotechnology Co., Ltd.); The protein after sonication is purified by nickel column purification (Beyotime Biotechnology Co., Ltd.) to obtain purified PG1-HN (198-390aa) immunoprotein.

[0044] like Figure 4 The image shown is an SDS-PAGE gel image of purified PG1-HN (198-390aa) immunoprotein. Figure 4 The target band was detected at approximately 33 kDa, and the band was single. Compared with the BSA control, the purity and concentration of the purified PG1-HN (198-390 aa) immunoprotein were both good.

[0045] (5) Six SPF-grade female BALB / c mice were subcutaneously immunized with purified PG1-HN (198-390aa) immunoprotein at a dose of 60 μg protein / mouse. Two booster immunizations were performed two weeks after the initial immunization (the booster immunization dose was 30 μg protein / mouse). After the two booster immunizations, blood was collected to measure the serum titer, which was greater than 25600. Three days before the fusion of mouse spleen cells with SP2 / 0 myeloma cells, the mice were intraperitoneally boosted with 50 μg of immunogen (purified PG1-HN 198-390aa immunoprotein) to obtain immunized BALB / c mice.

[0046] 2. Preparation and screening of hybridoma cell lines containing monoclonal antibodies against pigeon type I paramyxovirus HN protein

[0047] 1) Preparation of hybridoma cell lines

[0048] (1) Gently blow the healthy SP2 / 0 cells (myeloma cells) off the culture flask wall and aspirate them into a 50mL centrifuge tube.

[0049] (2) After euthanizing the immunized BALB / c mice, soak them in 75% alcohol for 5 min. Pour 5 ml of serum-free IMDM medium (Gibco; catalog number: 12440053) into a petri dish. Place the spleen of the immunized BALB / c mouse on a cell sieve and gently crush the spleen with the inner core of a syringe. Aspirate the crushed cells into a centrifuge tube containing SP2 / 0 cells and centrifuge at 1500 r / min for 5 min to obtain the centrifuged cells.

[0050] (3) Remove the thymus from the immunized BALB / c mice with scissors and tweezers, crush it, transfer the crushed thymocytes to a 15mL centrifuge tube, add 2mL of HAT (Sigma; catalog number: H0262-10VL) and 1mL of HT (Sigma; catalog number: H0137-10VL) to obtain thymocytes, and place them in an incubator for later use.

[0051] (4) Discard the supernatant of the centrifuged cells from step (2), gently and carefully mix the cells with serum-free IMDM, centrifuge (1500 rad / min, 5 min), discard the supernatant, tap the bottom of the centrifuge tube to fully suspend the cells, place the centrifuge tube in 37°C warm water, slowly add 1 mL of PEG (Sigma; catalog number: P7181) within 1 minute, let it stand in warm water for 1 min, then slowly add 10 mL of serum-free IMDM, centrifuge at 1000 r / min, discard the supernatant after 5 min, add 10 mL of serum, carefully mix the cells, and pour into the thymocytes obtained in step (3). Add sterilized semi-solid HAT hybridoma cell selection medium (IMDM complete medium containing 15% serum, Gibco, catalog number: 12440053; 1 ml HAT, Sigma, catalog number: H0262-10VL; 2.3% methylcellulose, Sigma, catalog number: M0262-100G) to a final volume of 50 mL, mix thoroughly, and obtain hybridoma cell solution.

[0052] (5) Pour 50 mL of hybridoma cell solution evenly into 30 cell culture dishes, each containing 6 mL of hybridoma cell solution. Place all cell culture dishes containing hybridoma cell solution into a humidified box and then into an incubator for culture.

[0053] 2) Screening of positive hybridoma cell lines

[0054] (1) Discard all the supernatant of monoclonal cells in the 96-well culture plate, add 200 μL of IMDM medium (containing HT) with 20% fetal bovine serum to each well for the first screening.

[0055] (2) The purified PG1-HN(198-390aa) protein was used to coat the ELISA plate, and the positive clones were screened a second time using the ELISA method. The selected clones were coated with PG1-HN(198-390aa) protein and screened a second time using the ELISA method, resulting in 7 positive hybridoma cell lines:

[0056] ① Dilute “PG1-HN(198-390aa) protein” with sodium carbonate-sodium bicarbonate buffer (pH 9.6) as the coating solution. The final concentration of PG1-HN(198-390aa) protein is 2 μg / mL. 100 μL / well, 4℃, overnight, followed by washing 3 times with washing buffer.

[0057] ② Seal with blocking solution (2% skim milk powder), 200 μL / well, incubate at 37℃ for 2 h, then wash 3 times with washing solution.

[0058] ③ Add cell culture supernatant or negative control (SP2 / 0 culture supernatant) or blank control (PBS) or positive control (positive serum PBS diluted 1000 times) to each well in sequence, 100 μL / well, incubate at 37℃ for 1 h, and then wash 3 times with washing buffer.

[0059] ④ Add PBS to dilute the secondary antibody 20,000 times, 100 μL / well, incubate at 37°C for 1 h, and wash 3 times with washing buffer after taking it out.

[0060] ⑤ Add 100 μL of colorimetric reagent per well, and the color development time is about 10 min.

[0061] ⑥ Add 50 μL of stop solution to each well to terminate the process.

[0062] ⑦ Measure the absorbance using a microplate reader (Bio-Tek, USA) with dual wavelengths (450, 630), record and save the data, and screen for positive clones.

[0063] (3) Third screening of positive clones

[0064] ELISA plates were coated with PG1-HN (198-390aa) protein and HIS synthetic peptide respectively. The ELISA method was used to screen 7 positive hybridoma cell lines for the third time. The operation steps were as described in step (2) above, and 6 positive hybridoma cell lines were obtained, one of which was the hybridoma positive cell line 4H3.

[0065] 3) Cloning of pigeon type I paramyxovirus HN protein monoclonal antibody hybridoma cells

[0066] Subcloning of the above-obtained positive hybridoma cell line 4H3 is performed using a method comprising:

[0067] First, gently pipette the cells in the culture wells containing the positive hybridoma cell line 4H3 to mix them well, then count the cells. Dilute the cells with DMEM (Gibco; catalog number: 11995065) containing 20% ​​FBS to approximately 5 cells per mL, mix well, and add 200 μL to each well of a 96-well plate. Incubate in a cell culture incubator containing 5% CO2. Observe the cell condition in the 96-well plates daily until cell clumps appear, and label the single-cell wells. When the cells have grown to about half their maximum size, perform positive hybridoma cell selection. Repeat the above subcloning method three times.

[0068] 3. Preparation of ascites fluid containing monoclonal antibody against pigeon type I paramyxovirus HN protein

[0069] A method for large-scale preparation of the above-mentioned monoclonal antibody 4H3, the method comprising:

[0070] (1) Eight-week-old BALB / c mice were selected, and each mouse was injected intraperitoneally with 0.4 mL of Freund's incomplete adjuvant. Three days after injection, healthy 4H3 hybridoma cells were washed with sterile PBS, resuspended, and diluted to [a specific concentration]. Take 500 μL (i.e.) 10 5 Hybridoma cells were injected into the peritoneal cavity of mice.

[0071] (2) 7-10 days after the injection of hybridoma cells, observe the mouse abdomen every day. When the mouse abdomen is obviously distended, collect the mouse ascites with a sterile syringe, incubate overnight at 4°C, centrifuge at 4000r / m for 10min, collect the ascites supernatant, and store it in a -70°C refrigerator for later use.

[0072] The aforementioned pigeon type I paramyxovirus HN protein monoclonal antibody hybridoma-positive cell line 4H3 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 45648, and deposited on August 3, 2023.

[0073] Example 3: Subclass identification and titer determination of monoclonal antibodies against pigeon type I paramyxovirus HN protein

[0074] 1. Subclass identification of HN protein monoclonal antibodies

[0075] This subclass identification method includes:

[0076] (1) Dilute the subclass coated antibody (GoatAnti-Mouse Ig, Human ads-UNLB) with coating buffer to a final concentration of 2 μg / mL, 100 μl / well, incubate overnight at 4°C, and then wash 3 times with washing buffer PBST (PBS buffer containing 0.05% Tween).

[0077] (2) Block with blocking solution (2% skim milk), 200 μL / well, incubate at 37°C for 2 h, and then wash 3 times with washing solution PBST (PBS buffer containing 0.05% Tween).

[0078] (3) Add 100 μl of cell culture supernatant of positive clone of 4H3 hybridoma cell in Example 1 or negative control (SP2 / 0 culture supernatant) to each well, incubate at 37°C for 1 h, and then wash 3 times with washing buffer PBST (PBS buffer containing 0.05% Tween).

[0079] (4) Dilute each type of subtype of secondary antibody with PBS to 100 μl / well and add to the corresponding well. Incubate at 37°C for 1 h. After incubation, wash three times with washing buffer.

[0080] (5) Add 100 μL of color development solution (1% solution A + 10% solution B, where solution A: 1% TMB dissolved in DMSO; solution B: 0.1% CH4N2O·H2O2 dissolved in citrate buffer) per well, and the color development time is about 10 min.

[0081] (6) Add 50 μL of stop solution (0.5 M sulfuric acid) to each well to terminate the process.

[0082] (7) The absorbance was measured using a microplate reader (Bio-Tek, USA) at dual wavelengths (450, 630). The results of the 4H3 antibody subclass identification are shown in Table 1 below:

[0083] Table 14 H3 Antibody Subclass Identification Results

[0084]

[0085]

[0086] Based on the results in Table 1, the heavy chain subclass of 4H3 is determined to be IgG2a, and the light chain subclass is κ chain.

[0087] 2. Valence determination

[0088] The PG1-HN (198-390 aa) protein was coated onto an ELISA plate and incubated overnight at 4°C. After washing the plate three times with PBST, 200 μL of 5% skim milk was added for blocking, and the plate was incubated at 37°C for 1 h. The supernatant of the hybridoma-positive cell line 4H3 from Example 1, or the prepared ascites fluid (mouse ascites fluid from the large-scale preparation method of monoclonal antibody 4H3 in Example 1), was added at dilutions of 200, 400, 800, 1600, 3200, 6400, 12800, 25600, 51200, 102400, 204800, and 409600 times as primary antibodies, and incubated at 37°C for 1 h. After washing three times with PBST, 100 μL of HRP-labeled goat anti-mouse secondary antibody diluted 1:5000 was added to each well, and the plate was incubated at 37°C for 1 h. After washing with PBST, 100 μL of TMB chromogenic buffer was added to each well for color development. After 15 min of development, 50 μL of 2M H2SO4 was added to each well to stop the reaction. The OD values ​​were read using a microplate reader (Bio-Tek, USA). 450 The final ELISA titer for 4H3 hybridoma cell supernatant was determined to be 1:6400, and the ELISA titer for ascites was determined to be 1:204800.

[0089] Example 4: Western blot detection of monoclonal antibody against pigeon type I paramyxovirus HN protein

[0090] Pigeon Newcastle disease virus PG / SX / 01, PG / JX / 812, chicken Newcastle disease virus CK / SX / 01, CK / HuN / 905, duck Newcastle disease virus DK / GZ / 664, DK / GX / 463, and the classic Newcastle disease vaccine strain Lasota were inoculated into DF1 cells at MOI=1. Twelve hours post-infection, cells were lysed using NP-40 lysis buffer (Beyotime Biotechnology Co., Ltd.), centrifuged at 12,000 rpm for 10 min at 4°C, and the supernatant was mixed with 5×SDS-PAGE loading buffer (Beijing Solarbio Co., Ltd.) and heated for denaturation for 10 min. After cooling, SDS-PAGE electrophoresis was performed, and the cells were transferred to nitrocellulose membranes (NC membranes) (Thermo Fisher Scientific, USA). After transfer, the membranes were blocked with 5% skim milk (Beijing Solarbio Co., Ltd.) and incubated at 37°C for 1 h. Purified 4H3 monoclonal antibody (1:2000 dilution) was added as the primary antibody. For the internal control (Wuhan Sanying Biotechnology Co., Ltd.), β-actin was diluted 1:5000 as the primary antibody. The membrane was incubated at 37°C on a shaker for 1 hour. The membrane was washed three times with PBST solution (Beijing Solarbio Co., Ltd.) for 10 minutes each time. After washing, horseradish peroxidase-labeled goat anti-mouse secondary antibody (Abcam, UK) diluted 1:5000 was added, and the membrane was incubated at 37°C for 1 hour. After washing three times as described above, the membrane was scanned using a chemiluminescence imaging system (Bio-Rad Laboratories, USA).

[0091] like Figure 5 The image shows a Western blot detection of monoclonal antibody 4H3. The first row shows the detection of HN, and the second row shows the detection of the internal control β-actin. The control group is a blank control. PG / SX / 01 and PG / JX / 812 are pigeon Newcastle disease virus, CK / SX / 01 and CK / HuN / 905 are chicken Newcastle disease virus, DK / GZ / 664 and DK / GX / 463 are duck Newcastle disease virus, and Lasota is the classic Newcastle disease vaccine strain. Figure 5 It is evident that 4H3 can specifically react with Class II pigeon Newcastle disease virus (PG / SX / 01 and PG / JX / 812) and chicken Newcastle disease virus (CK / SX / 01 and CK / HuN / 905), but shows poor reactivity with Class I chicken and duck Newcastle disease virus, and does not react with the Lasota vaccine strain.

[0092] Example 5: Application of monoclonal antibody against pigeon type I paramyxovirus HN protein in immunofluorescence detection.

[0093] Pigeon Newcastle disease virus PG / SX / 01, PG / JX / 812, chicken Newcastle disease virus CK / SX / 01, CK / HuN / 905, duck Newcastle disease virus DK / GZ / 664, DK / GX / 463, vaccine strain Lasota, avian influenza virus AIV / SX / 4302, and chicken infectious bronchitis virus IBV / SX / 06 were infected with DF1 cells for 12 hours. The cells were then fixed with 4% paraformaldehyde (Lanjieke Technology Co., Ltd.) for 20 minutes. The fixative was discarded, and the cells were washed three times with PBS for 5 minutes each time. The cells were permeabilized with 0.5% Triton X-100 (Beijing Solarbio Co., Ltd.) (dissolved in PBS) for 10 minutes, followed by three washes with PBS for 5 minutes each time. Finally, the cells were blocked with 5% BSA (Beijing Solarbio Co., Ltd.) (500 μL per well) for 1 hour. The prepared monoclonal antibody was diluted 1:300 with 4H3 as the primary antibody and incubated at room temperature for 1 hour. It was then washed three times with PBS as described above. The secondary antibody was a 1:700 dilution of FITC-labeled goat anti-mouse fluorescent secondary antibody (Abcam, UK), incubated in the dark for 1 hour. After washing with PBS as described above, the results were observed and recorded under a fluorescence microscope (Leica, Germany).

[0094] like Figure 6The image shows the immunofluorescence detection pattern of monoclonal antibody 4H3, where A represents pigeon Newcastle disease virus PG / SX / 01; B represents pigeon Newcastle disease virus PG / JX / 812; C represents chicken Newcastle disease virus CK / SX / 01; D represents chicken Newcastle disease virus CK / HuN / 905; E represents duck Newcastle disease virus DK / GZ / 664; F represents duck Newcastle disease virus DK / GX / 463; G represents the classic Newcastle disease vaccine strain Lasota; H represents chicken infectious bronchitis virus IBV / SX / 06; I represents avian influenza virus AIV / SX / 4302; and J represents the negative control. Figure 6 It is evident that 4H3 can specifically react with Class II pigeon Newcastle disease virus (PG / SX / 01 and PG / JX / 812) and chicken Newcastle disease virus (CK / SX / 01 and CK / HuN / 905), but shows weak reactivity with Class I duck Newcastle disease virus, and does not react with Lasota vaccine strain, avian influenza virus AIV / SX / 4302, or chicken infectious bronchitis virus IBV / SX / 06.

[0095] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A monoclonal antibody-positive hybridoma cell line 4H3 that secretes pigeon type I paramyxovirus HN protein, characterized in that, The hybridoma-positive cell line 4H3 was deposited on August 3, 2023, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China. The accession number is CGMCC No. 45648. The heavy chain subclass of the hybridoma-positive cell line 4H3 is IgG2a, and the light chain subclass is κ chain.

2. A monoclonal antibody against the HN protein of pigeon type I paramyxovirus, characterized in that, The monoclonal antibody is obtained from the monoclonal antibody hybridoma positive cell line 4H3 that secretes pigeon type I paramyxovirus HN protein as described in claim 1. Among them, the amino acid sequence shown in SEQ ID NO.1 was selected as the immunogen of the pigeon type I paramyxovirus HN protein.

3. The pigeon type I paramyxovirus HN protein monoclonal antibody according to claim 1, characterized in that, The antigenic epitope of the monoclonal antibody is located on the HN protein of pigeon type I paramyxovirus.

4. The pigeon type I paramyxovirus HN protein monoclonal antibody according to claim 1, characterized in that, The immunogen of the pigeon type I paramyxovirus HN protein was prepared by the following method: (1) The amino acid sequence of pigeon type I paramyxovirus HN protein with optimized codons was synthesized into pET30a expression vector through EcoRI / XhoI restriction site gene, transformed into Escherichia coli BL21 expression strain, and single clones were picked from the transformed plate into LB liquid medium containing kanamycin resistance and activated by culturing at 37°C. (2) Inoculate the activated bacterial culture into LB liquid medium containing kanamycin resistance and incubate at 37°C; (3) The cultured bacterial culture was transferred to LB liquid medium containing kanamycin resistance, and cultured at 37°C until the optical density (OD) was 0.6-0.8, the concentration of isopropyl-β-D-thiogalactoside was 0.5mM, and induced overnight at 16°C; (4) Centrifuge the overnight induced bacterial cells and discard the supernatant; then sonicate to break down the bacteria; purify the protein after sonication to obtain the immunogen of the pigeon type I paramyxovirus HN protein.

5. The pigeon type I paramyxovirus HN protein monoclonal antibody according to claim 4, characterized in that, In step (4), the bacterial collection is carried out at 6000 rpm and centrifugation for 8 min; the ultrasonic disruption is performed by blowing the bacterial cells with a 20-30 mL, 10 mM tris(hydroxymethyl)aminomethane hydrochloride solution at pH 8.0 and then performing ultrasonic disruption; the ultrasonic disruption power is 500 W, the number of times is 180, the duration of each ultrasonic disruption is 5 s, and the interval between adjacent disruptions is 5 s.

6. The pigeon type I paramyxovirus HN protein monoclonal antibody according to claim 4, characterized in that, In steps (1) to (3), the 37°C incubation is carried out at 200 rpm.

7. The application of a monoclonal antibody against pigeon type I paramyxovirus HN protein as described in any one of claims 1-6 in the field of detecting Newcastle disease virus.

8. The application according to claim 7, characterized in that, The Newcastle disease virus includes pigeon-derived Newcastle disease virus and / or chicken-derived Newcastle disease virus.

9. The application according to claim 8, characterized in that, The Newcastle disease virus from pigeons is PG / SX / 01 and / or PG / JX / 812.

10. The application according to claim 8, characterized in that, The Newcastle disease virus from chickens is CK / SX / 01 and / or CK / HuN / 905.

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