CN110452885B - Hybridoma cell 4F6 strain secreting monoclonal antibody against newcastle disease virus NP protein - Google Patents
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Abstract
The invention discloses a hybridoma cell 4F6 strain secreting a monoclonal antibody against Newcastle disease virus NP protein, belonging to the technical field of biology. The invention screens out a hybridoma cell 4F6 strain from the established hybridoma cell bank secreting monoclonal antibody against Newcastle Disease Virus (NDV), the biological performance is very excellent, and the reaction titer of the mouse ascites monoclonal antibody prepared by the hybridoma cell to NDV is as high as 10 9 The reaction titer to NP protein is as high as 10 10 And the reactivity to a plurality of NDV subtype strains such as genes II, III, IV, VI, VII and IX and the like and chicken, duck, goose and pigeon-sourced NDV strains is strong, the range of the anti-NDV strains is wide, and the application prospect is good.
Description
Technical Field
The invention discloses a hybridoma cell 4F6 strain secreting a monoclonal antibody against Newcastle disease virus NP protein, belonging to the technical field of biology.
Background
Newcastle Disease (ND) is an acute, virulent and highly contagious infectious disease caused by poultry infected by a virulent strain of Newcastle Disease Virus (NDV), and is one of animal epidemics of a type of animal epidemic disease that is preferentially prevented in the national middle and long term animal epidemic prevention program (2012-2020) of our country.
In recent years, the disease presents new epidemic characteristics, such as atypical newcastle disease, NDV new gene (subtype) appearance, host range expansion, virulence enhancement and the like, so that the newcastle disease prevention and control are complicated. Currently, typical newcastle disease is well controlled in production practice, but local areas are seriously polluted by virus, and epidemic situations are continuously prevalent. In recent years, the disease presents new epidemic characteristics, such as atypical newcastle disease, NDV new gene (subtype) appearance, host range expansion, virulence enhancement and the like, so that the newcastle disease prevention and control are complicated. The results of molecular epidemiological studies show that the currently popular NDV in China is mainly of gene VII type (chicken, duck and goose group) (Pan Tung, et al. Biological characteristics of different host-derived isolates of Newcastle disease [ J ]. Jiangsu agricultural bulletin 2011,27 (6): 1325-1329.). Therefore, basic and long-term monitoring and early warning of Newcastle disease antigens/antibodies on the poultry flock are necessary.
The existing ND monitoring means has certain limitation on scientific evaluation of ND occurrence risk and vaccine immune effect. ND hemagglutination and hemagglutination inhibition tests are often influenced by various factors and are easy to cause deviation; the RT-PCR method has good specificity and sensitivity, but has complex operation and needs special instruments. The immunological detection technology (such as ELISA and immune colloidal gold test paper) has the advantages of strong specificity, high sensitivity, good stability and the like, and is suitable for detecting large-scale samples. The screened monoclonal antibody with high quality (strong specificity, high sensitivity and good stability) is a necessary component for developing an ND immunological detection kit.
NDV is avian paramyxovirus type I of paramyxovirus of Paramyxoviridae, the genome is nonsegmented single-stranded negative-strand RNA, the genome structural pattern is 3'-NP-P-M-F-HN-L-5', and six structural proteins are sequentially coded: nucleocapsid Protein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin-Neuraminidase protein (HN), and macromolecular protein (Large protein, L). The NP protein is the most abundant protein in NDV particles, and it forms the nucleocapsid structure of the core helix with the RNA of the genome, protecting the RNA from degradation by nuclease activity. At the same time, NP is the only viral polypeptide that cannot be removed from the nucleocapsid, indicating that the NP protein is important for NDV assembly.
At present, there areHybridoma cell lines secreting NDV monoclonal antibodies have been reported, although some cell lines secrete monoclonal antibodies directed against the NP protein. For example, jass et al immunized mice with purified NP recombinant protein (from Duck/JS/10 low virulent strain of Class I), fused, screened and subcloned to finally obtain 2 hybridoma cell lines stably secreting monoclonal antibodies, which were named as 1H3 and 3F5, respectively. The 2 hybridoma cells were inoculated into abdominal cavities of mice to prepare ascites. The titer of the 2 monoclonal antibody ascites is 1 by 3.5 multiplied by 10 respectively according to ELISA determination 7 And 1 5 (Zhanyue, et al. Class I Newcastle disease virus Duck/JS/10 low virulent strain NP recombinant protein expression and preparation of broad-spectrum NDV monoclonal antibody [ J]Chinese veterinary science 2014 (6): 611-616). Liuwenli and the like use NDV F48E9 strain pET32a-NP recombinant protein to immunize a BALB/c mouse, and 2 hybridoma cell strains which stably secrete anti-NP recombinant protein monoclonal antibodies are obtained by adopting a hybridoma cell technology and indirect ELISA screening, and are respectively named as 1G3 and 4B12. The ascites antibody titer was 1.56 × 10 by indirect ELISA assay 5 、1:5.12×10 5 (Liu Wen Li, et al. Newcastle disease Virus F 48 E 9 Preparation and identification of monoclonal antibody against recombinant NP protein [ J ]]The university of northeast agriculture proceedings 2011,42 (9): 131-135.). The recombinant cell strains A1 and B2 which can stably secrete anti-recombinant protein MAb are obtained from 2 strains of the recombinant cell strains A1 and B2 by ELISA detection 12 The potency of induced ascites is 10 7 And 10 6 Preparation of (Yandili, etc. Duck source Newcastle disease virus nucleocapsid protein monoclonal antibody [ J]Chinese preventive veterinary bulletin 2013 (08): 666-668). From the existing literature, the immunogens are all NP proteins expressed by recombination, and the source strains of the proteins (such as Duck/JS/10 low-virulent strain and NDV F48E9 strain of the Class I Newcastle disease virus) are not currently circulating VII strains; in addition, the titers of monoclonal antibodies against the NP protein are not the same, and the titers of response to different NDV strains are not reported to be complete.
Monoclonal antibodies are highly homogeneous antibodies produced by a single B cell clone directed against only a particular epitope (i.e., a recognized epitope) (Touchi, et alAnalysis [ J]Bio-industrial technology, 2012 (6): 78-86). An epitope of a monoclonal antibody (i.e., the amino acid sequence of the antibody that binds to the antigen) is specific. For example, the epitope recognized by the monoclonal antibody 18C6 of the NP protein of the Newcastle disease virus prepared by Liupexin is positioned between the 473 rd and 481 th amino acids of the NP protein, and the polypeptide sequence of the epitope is 473 PPPTPGASQ 481 (ii) a The epitope recognized by the monoclonal antibody 24F9 is positioned between 470-478 th amino acids of NP protein, and the polypeptide sequence is 470 HPEPPPTPG 478 . Therefore, the analysis of the epitope recognized by the monoclonal antibody has important significance in the aspects of subsequent newcastle disease virus protein function research, diagnosis and detection and the like.
Disclosure of Invention
Technical problem
The invention aims to provide a hybridoma cell strain secreting the anti-newcastle disease virus NP protein monoclonal antibody, and the mouse ascites monoclonal antibody prepared by the hybridoma cell strain has high reaction titer and wide range of anti-NDV strains.
The technical proposal is that,
In order to achieve the purpose, the method is realized by the following technical scheme:
a hybridoma cell 4F6 strain secreting monoclonal antibody against NP protein of Newcastle disease virus, wherein the hybridoma cell 4F6 strain is deposited in China center for type culture Collection in 2019, 5, 9 days, and the address is as follows: wuhan university in Wuhan, china, the preservation number of the preservation center is CCTCC NO: c2019102, classification and naming: a monoclonal antibody hybridoma cell strain 4F6 secreting NP protein of the anti-newcastle disease virus. The epitope recognized by the monoclonal antibody of the NP protein of the Newcastle disease virus prepared by the hybridoma cell 4F6 strain is the amino acid from 140 th to 148 th of the NP protein of the Newcastle disease virus, and the polypeptide sequence is SNGTPFVTA.
The hybridoma cell 4F6 strain secreting the anti-newcastle disease virus NP protein monoclonal antibody can be applied to preparation of a newcastle disease virus NP protein monoclonal antibody diagnostic detection reagent.
The hybridoma cell 4F6 strain can be used for preparing a Newcastle disease virus NP protein monoclonal antibody. The Newcastle disease virus NP protein monoclonal antibody can be used for preparing a Newcastle disease virus NP protein monoclonal antibody diagnostic detection reagent.
Advantageous effects
The invention uses purified VII type Newcastle disease virus antigen immune BALB/c mouse to prepare immune spleen cell, and fuses with SP2/0 myeloma cell line, and uses prokaryotic expression NP protein and purified Newcastle disease virus to make double screening to obtain a monoclonal antibody hybridoma cell strain which can react with prokaryotic expression NP protein and Newcastle disease virus, and its name is 4F6, and said cell strain is very excellent in biological property, and the reaction titer of mouse ascites monoclonal antibody prepared by using it to NDV is up to 10 9 The reaction titer to NP protein is as high as 10 10 (the titer is obviously higher than the parameter values reported by the existing data), and the reactivity to a plurality of NDV subtype strains such as genes II, III, IV, VI, VII, IX and the like and chicken, duck, goose and pigeon-sourced NDV strains is strong, the range of the anti-NDV strains is wide, and the application prospect is good. In addition, the epitope recognized by the 4F6 monoclonal antibody screened by a peptide scanning method is at the 140 th to 148 th amino acids of the NP protein of the Newcastle disease virus, the polypeptide sequence of the epitope is SNGTPFVTA, and the epitope is not reported yet.
The invention has the following characteristics and advantages:
1. the BALB/C mouse immunogen used in the invention is prepared from a virulent strain of gene VII in which NDV is currently circulating. The immunogen of the screened NP protein monoclonal antibody is the recombined NP protein, and the source strain of the protein (such as Duck/JS/10 low virulent strain and NDV F48E9 strain of the Class I Newcastle disease virus) is not the current VII strain, and the immune antigen is different.
2. The invention screens out a hybridoma cell 4F6 strain from a established hybridoma cell bank in which 78 strains secrete anti-NDV monoclonal antibodies, has very excellent biological performance, and the reaction titer of the induced BALB/C mouse ascites monoclonal antibodies to NDV is as high as 10 9 The reaction titer to NP protein is as high as 10 10 And the reactivity to a plurality of NDV subtype strains such as genes II, III, IV, VI, VII and IX and the like and chicken, duck, goose and pigeon-sourced NDV strains is strong, and the range of anti-NDV strains is wide.
3. The epitope recognized by the Newcastle disease virus NP protein monoclonal antibody (4F 6) is at amino acids 140-148 of the Newcastle disease virus NP protein, the polypeptide sequence of the epitope is SNGTPFVTA, and the epitope is not reported yet.
Biological preservation
The hybridoma cell 4F6 strain was deposited in the chinese type culture collection on 2019, 5/9, at address: the preservation number of the preservation center of Wuhan university in Wuhan, china is CCTCC NO: c2019102, classification and naming: a monoclonal antibody hybridoma cell strain 4F6 secreting NP protein of the anti-newcastle disease virus.
Detailed Description
Establishment of monoclonal antibody hybridoma cell strain
Preparation of NDV antigen
Inoculating a currently popular VII type NDV virulent strain (purchased from Nanjing Tianbang Biotechnology Co., ltd.) into 9-day-old SPF chick embryos (purchased from Beijing Meiliyangtong laboratory animal technology Co., ltd.), collecting allantoic fluid after 48h, centrifuging at 8000r/min for 30min, taking supernatant, purifying by adopting PEG precipitation and discontinuous sucrose density gradient methods, determining the virus concentration by adopting a BCA protein quantitative detection kit (product of Semmerfell science Co., ltd.), and storing at-70 ℃ for later use.
2. Animal immunization
Female BALB/C mice (purchased from the university of Yangzhou, comparative medicine laboratory center) at the age of 8 weeks were immunized with purified NDV immunizing antigen by intraperitoneal injection at 50. Mu.g/mouse. The antigen was emulsified with an equal volume of Freund's complete adjuvant (Sigma) and then immunized 2 times every 14 days with Freund's incomplete adjuvant (Sigma) such as floral antigen. Collecting blood after 3 rd immunization at 6d tail-broken part, measuring serum antibody titer by indirect ELISA method, selecting antibody titer > 10 6 3d, re-boosting with purified NDV antigen before fusion.
3. Cell fusion
Mixing well SP2/0 myeloma cells (purchased from ATCC cell bank) and immunized BALB/C mouse spleen cells at a ratio of 1 4000 (Sigma Co., ltd.) 1mL of the medium was gently shaken while adding the medium, 15mL of fetal calf serum-free RPMI-1640 medium preheated to 37 ℃ was added to 90 seconds, the mixture was left standing at room temperature for 10min, centrifuged at 1000r/min for 10min, the supernatant was discarded, resuspended in 10% Fetal Calf Serum (FCS) (Seimer Feishell technology Co., ltd.) and HAT (Sigma Co., ltd.) containing RPMI-1640 medium, and the resulting suspension was dispensed onto feeder cells-containing 96-well plates and subjected to 5% CO addition 2 Culturing in an incubator. After 3d, the cells were supplemented with RPMI-1640 medium containing HAT (Sigma) and 10% FCS (Sammer Field technologies), after 5d, the cells were cultured in RPMI-1640 medium containing HT (Sigma) and 10% FCS (Sammer Field technologies), after 10d, the cells were cultured in RPMI-1640 medium containing 10% FCS (Sammer Field technologies), and when the fused cells grew to 1/5 of the bottom area of the well of the 96-well plate, the supernatant was collected and subjected to antibody detection.
4. Screening of hybridoma cell lines
Determining the coating concentration of the purified NDV antigen according to a square matrix method, diluting the purified NDV antigen by using a carbonate buffer solution with 0.05mol/LpH 9.6 of a coating solution in a multiple ratio, coating an ELISA plate with the amount of the NDV antigen diluted to 800 times, coating 100 mu L/hole, standing at 4 ℃ for coating overnight, washing 3 times by PBST (Poly-N-phenylenebenzobisthiazole), 5min each time, and finally beating to dry; sealing each well with PBST containing 10% calf serum at 200 μ L/well, standing at 37 deg.C for 2h, washing with PBST for 3 times, each for 5min, and drying at last; the cell supernatant 12d after fusion, 1 diluted immune mouse positive serum and 1 diluted mouse negative serum 1000 were added into the corresponding wells, 100 μ L/well, acted at 37 ℃ for 1h, pbst washed 3 times, 5min each time, and finally patted dry; adding Horse Radish Peroxidase (HRP) labeled goat anti-mouse IgG (Shanghai Biyuntian biotechnology, co., ltd.) diluted at 1:5000 at a concentration of 100. Mu.L/well, standing at 37 ℃ for 1h, washing with PBST for 3 times, each for 5min, and drying by beating for the last time; adding TMB substrate, 100 μ L/hole, and developing in dark at room temperature for 10min; the reaction was stopped by adding 50. Mu.L of 2mol/L sulfuric acid per well. OD determination by enzyme-linked immunosorbent assay 450nm Values were zeroed for blank control, P is the value of each well, N is the OD of the negative reference serum 450nm The value is that when the OD450nm value of the negative reference serum is less than or equal to 0.1, the OD of the positive reference serum 450nm Value and negative reference bloodClear OD 450nm The ratio of the values is more than or equal to 2.1, namely the detection holes with the P/N more than or equal to 2.1 are judged to be positive under the premise that negative and positive controls are established, the detection is carried out once after 2-3 days, and the hybridoma cells with positive detection results of the two times are cloned.
5. Cloning of hybridoma cells
First, viable cells in positive wells were stained and counted with trypan blue, diluted to 100 cells/15 mL of medium with 10% FCS (Saimeishiell technologies) in RPMI-1640 medium, and the diluted cell suspension was added to a 96-well cell culture plate, which was 0.15mL/well, 37 ℃ and 5% CO2, and cultured in an incubator for 4 to 5 days, then formation of cloned cells was observed under a microscope, only single cloned wells were recorded, and cell supernatants were collected at 8 to 9 days to immediately perform ELISA detection. Selecting positive monoclonal cell, cloning for more than 3 times until all cell wells have positive supernatant detection and each well detects OD 450nm The values are closer. And (3) performing expanded culture on the cloned NDV specific monoclonal antibody hybridoma cell strain, and freezing and storing. After 20 times of cell fusion, screening, cloning and identification, a 78-strain hybridoma cell bank stably secreting the NDV specific monoclonal antibody is established.
6. Expression and purification of newcastle disease virus NP protein
According to NP gene sequence information in a current popular VII type NDV virulent strain (purchased from Nanjing Tianbang biological technology Co., ltd.), a nucleic acid sequence (synthesized by general biological System (Anhui) Co., ltd.) optimized by an escherichia coli codon is artificially synthesized, and Nhe I enzyme cutting sites and Hind III enzyme cutting sites are respectively introduced into two ends of the sequence. Then, the artificially synthesized sequence and prokaryotic expression plasmid pET28a (+) (product of Beijing Sorbao technology, inc.) are subjected to enzyme digestion of Nhe I and Hind III respectively, after being identified by agarose gel electrophoresis, fragments are respectively recovered by using a gel recovery kit (product of Tiangen Biochemical technology, inc.), and are incubated for 16h at 4 ℃ under the connection effect of T4 ligase (product of Beijing technology, inc.) and are transformed into E.coli Rosetta competence (product of Beijing technology, inc.), so that expressible expression plasmid pET28a (+) (product of Beijing Sorbao technology, inc.) is obtainedA strain of newcastle disease virus NP protein. The strain was cultured to OD 600nm When the value is about 1.0, IPTG (product of Baoriji medical science (Beijing) Co., ltd.) with the final concentration of 1mmol/L is added, induced expression is carried out for 6h at 37 ℃, thalli are collected and purified by a Ni-NTA affinity chromatography column (product of GE Co., ltd.), purified recombinant protein is collected, the concentration is 1.25mg/mL through a BCA protein quantitative detection kit (product of Saimeifeishilai technology Co., ltd.) and the recombinant protein is stored for standby at-70 ℃.
7. Screening of Newcastle disease virus NP protein monoclonal antibody hybridoma cell strain
Determining the coating concentration of the purified NP protein according to a matrix method, performing multiple dilution on the purified NP protein by using a carbonate buffer solution with a coating solution of 0.05mol/LpH 9.6, diluting to 0.1 mu g/hole, coating overnight at 4 ℃, washing for 3 times by PBST (PBST), washing for 5min each time, and finally beating to dry; sealing each well with PBST containing 10% calf serum at 200 μ L/well, standing at 37 deg.C for 2h, washing with PBST for 3 times, each for 5min, and drying at last; adding 78 cell supernatants (1 diluted by 1000) stably secreting NDV specific monoclonal antibodies into corresponding holes, performing 1h at 37 ℃, washing 3 times by PBST, each time for 5min, and finally drying by beating; adding Horse Radish Peroxidase (HRP) labeled goat anti-mouse IgG (Shanghai Biyuntian biotechnology, co., ltd.) diluted at 1:5000 at a concentration of 100. Mu.L/well, standing at 37 ℃ for 1h, washing with PBST for 3 times, each for 5min, and drying by beating for the last time; adding TMB substrate, 100 μ L/hole, and developing in dark at room temperature for 10min; the reaction was stopped by adding 50. Mu.L of 2mol/L sulfuric acid per well. OD determination by enzyme-linked immunosorbent assay 450nm Value, OD is selected 450nm The NDV specific monoclonal antibody hybridoma cell strain corresponding to the highest value. The results of the above experiments repeated 3 times show that the OD of the hybridoma cell line 4F6 monoclonal antibody 450nm The value is highest.
8. Preparation of ascites
The sterilized liquid paraffin was injected intraperitoneally with 0.5 mL/mouse of BALB/C (purchased from the center of comparative medicine laboratory of Yangzhou university) at an age of 8-10 weeks, and after 7 days, hybridoma cell lines were injected into the abdominal cavity of each mouse at an amount of 0.2mL (containing 2X 10 cells) 6 ～5×10 6 Individual hybridoma cell), ascites of the mouse is collected after 7 to 10 days, and the mouse is centrifuged at 3000r/minCollecting supernatant after 10min, subpackaging, and storing at-20 deg.C for use.
Biological Properties of monoclonal antibody 4F6
1. Chromosome analysis of hybridoma cell lines
The hybridoma cells were chromosome-counted by giemsa staining. Respectively taking SP2/0 myeloma cells and positive hybridoma cells for culture, growing to a logarithmic phase, adding colchicine into a cell bottle to enable the final concentration to be 0.1 mu g/ml, and then putting the cell bottle into a cell culture box for continuous culture for 4-5 h. The cells were blown up and mixed evenly with 5mL of 0.075mol/LKCI hypotonic solution pre-warmed at 37 ℃, placed in a 37 ℃ incubator for 30min, added with L mL of a newly prepared fixative (methanol: glacial acetic acid: 3) while being added dropwise and mixed evenly, and centrifuged at 1000r/min for 10min. Discarding the supernatant, collecting cell precipitate, blowing up the cell with 5mL of stationary liquid, performing at 37 deg.C for 30min, centrifuging at 1000r/min for 10min, and repeating the above steps once. The cell sediment is suspended and mixed evenly by using the lmL stationary liquid, 1 drop of the suspension is absorbed by a dropper, is dripped on a pre-frozen glass slide, is laid on the glass slide, and is dried naturally. Staining with newly prepared Giemsa staining solution for 10min, washing with tap water, air drying, and observing under microscope. The number of chromosomes of the hybridoma cell line is 94, the number of chromosomes of myeloma cells is 54-64, and the number of chromosomes of mouse spleen cells is 40, so that the obtained hybridoma cell line is proved to be the result of fusion of the two cells.
2. Specificity identification of monoclonal antibodies
Experiments were performed in 24-well cell culture plates. Respectively inoculating a current epidemic VII type NDV virulent strain, an infectious bursal disease virus strain and an H9N2 subtype avian influenza virus strain (the strains are purchased from Nanjing Tianbang Biotechnology Co., ltd.) to primary Chick Embryo Fibroblast (CEF) cells, after culturing for 72H of pathological change, sucking cell culture fluid, washing for 2 times by using serum-free culture fluid, then adding-20 ℃ precooled absolute ethyl alcohol 1 mL/hole into the cell culture hole, fixing for 30min at 4 ℃, washing for 3 times by using PBS (phosphate buffer solution), and beating to dry; adding culture supernatant of hybridoma cell 4F6, incubating at 200 μ L/well for 1h at 37 deg.C, washing with PBS for 3 times, and patting to dry; a200-fold dilution of FITC-labeled goat anti-mouse IgG antibody (product of WU Han Dynasty, dr. Bioengineering, ltd.) was added thereto at 200. Mu.L/well, incubated at 37 ℃ for 1h, washed 5 times with PBS, and observed under a fluorescence microscope. Under a fluorescence microscope, the monoclonal antibody can only react with CEF cells infected by the Newcastle disease virus WJ10001 strain to generate fluorescence, but does not have fluorescence with CEF cells infected by other pathogens, and the specificity of the monoclonal antibody to NDV is proved.
3. Determination of monoclonal antibody type
The subclass of the monoclonal antibody secreted by the hybridoma cell 4F6 strain was determined using a monoclonal antibody subclass identification kit (seimer feishell technologies), and the result showed that the monoclonal antibody subclass was IgM κ.
4. Stability assay for monoclonal antibodies
Continuously culturing and subculturing the obtained hybridoma cell 4F6 strain for 50 times, freezing and storing by liquid nitrogen and recovering, and continuously detecting the antibody titer in the culture supernatant of the hybridoma cell by using an indirect ELISA method to be 10 5 The hybridoma cell strain 4F6 is proved to be capable of continuously and stably secreting the anti-NDV monoclonal antibody.
5. Potency assay for monoclonal antibodies
The indirect ELISA method using VII type NDV strain (purchased from Nanjing Tianbang Biotech Co., ltd.) as coating antigen (see the detailed description of the invention, the establishment of monoclonal antibody hybridoma cell strain, 4. Screening of hybridoma cell strain section contents) was used to determine the titer of hybridoma cell culture supernatant and mouse ascites, and the results showed that the ELISA titer (i.e., reaction titer) of hybridoma cell 4F6 culture supernatant was 10 5 Ascites reaction titer of 10 9 。
The titer of hybridoma culture supernatant and mouse ascites was determined by indirect ELISA method using NP protein as envelope antigen (see the detailed description of the invention, section (I) establishment of monoclonal antibody hybridoma cell line, 7. Screening of Newcastle disease virus NP protein monoclonal antibody hybridoma cell line), and the results showed that the titer of hybridoma 4F6 culture supernatant was 10 6 Ascites reaction titer is 10 10 。
For gene II type NDV strain (LaSota strain, purchased from China institute of veterinary medicine), gene III type NDV strain (Mu)ktesfar strain purchased from chinese veterinary medicine institute), gene IV type NDV strain (Herts 33 strain purchased from chinese veterinary medicine institute), gene VI type NDV strain (purchased from south beijing tianbang biotechnology limited), gene VII type NDV strain (purchased from south beijing tianbang biotechnology limited), gene IX type NDV strain (F48E 9 strain purchased from chinese veterinary medicine institute), and chicken, duck, goose, pigeon-derived NDV strain (purchased from south beijing tianbang biotechnology limited) were subjected to antigen purification and concentration measurement, respectively (the specific method refers to the "detailed embodiment" of the present invention, (one) establishment of monoclonal antibody hybridoma cell strain, preparation of 1.ndv antigen "chapter contents"), and the amount of NDV antigen diluted to 800 times with coating solution (0.05 mol/LpH 9.6 carbonate buffer solution) was coated on ELISA plate, 100 μ L/well, coated at 4 ℃, PBST was washed 3 times, 5min each time; blocking each well with PBST containing 10% calf serum at 200 μ L/well, standing at 37 deg.C for 2h, washing with PBST for 5min each time; respectively adding the monoclonal antibody cell supernatant and ascites with different dilution times and the negative serum diluted by 1 1000 into corresponding holes, performing 1h at the temperature of 37 ℃ in each hole, and washing for 3 times for 5min by PBST (basic particle beam diffraction); adding Horse Radish Peroxidase (HRP) labeled goat anti-mouse IgG (Shanghai Biyuntian biotechnology, co., ltd.) diluted at 1; adding TMB substrate, 100 μ L/hole, and developing in dark at room temperature for 10min; the reaction was stopped by adding 50. Mu.L of 2mol/L sulfuric acid per well. OD determination by enzyme-linked immunosorbent assay 450nm Values were zeroed for blank control, P is the value of each well, N is the OD of the negative reference serum 450nm Value, OD of negative reference serum 450nm Value less than or equal to 0.1, OD of positive reference serum 450nm Value and OD of negative reference serum 450nm The ratio of the values is more than or equal to 2.1, namely the detection holes with the P/N more than or equal to 2.1 are judged to be positive on the premise that negative and positive controls are established, and the highest dilution multiple of the positive holes of the monoclonal antibody is taken as the reaction titer. The result shows that the reaction titer of the culture supernatant of the hybridoma cell 4F6 to NDV strains with different genotypes and NDV strains with different animal sources reaches 10 5 Ascites reaction titer reaches 10 9 。
6. Recognition epitope screening of monoclonal antibodies
The recognition epitope of the prepared monoclonal antibody is screened by a peptide scanning method, 15 peptides are synthesized according to the amino acid sequence of the truncated NP protein for screening the antigen epitope, the step of each synthetic peptide is 5 peptides, and the synthetic peptides and the former synthetic peptide and the latter synthetic peptide have 5 amino acid repeats respectively. 67 continuous overlapping short peptides were synthesized, which were coated with 100 ng/well of an ELISA plate, and the prepared 4F6 ascites (1: 10000) was used as a primary antibody and HRP-IgG was used as a secondary antibody, and the detection was performed by an indirect ELISA method (see the present invention, "detailed description of embodiment (one) establishment of monoclonal antibody hybridoma cell line, 7. Screening of Newcastle disease Virus NP protein monoclonal antibody hybridoma cell line" ("section contents"), based on the detected OD 450nm The recognition epitope of the monoclonal antibody is preliminarily identified. The result shows that the epitope recognized by the 4F6 monoclonal antibody is at the 140 th to 148 th amino acids of the NP protein, the polypeptide sequence is SNGTPFVTA, and the epitope is not reported yet.
The Newcastle disease virus NP protein monoclonal antibody hybridoma cell strain 4F6 has very excellent biological performance, and the reaction titer of the mouse ascites monoclonal antibody prepared by the hybridoma cell strain on NDV is as high as 10 9 The reaction titer to NP protein is as high as 10 10 (the titer is obviously higher than the parameter values reported by the existing data), and the reactivity to a plurality of NDV subtype strains such as genes II, III, IV, VI, VII, IX and the like and chicken, duck, goose and pigeon-sourced NDV strains is strong, the range of the anti-NDV strains is wide, and the application prospect is good. In addition, the epitope recognized by the 4F6 monoclonal antibody screened by a peptide scanning method is at the 140 th to 148 th amino acids of the NP protein of the Newcastle disease virus, the polypeptide sequence of the epitope is SNGTPFVTA, and the epitope is not reported yet.
Claims (4)
1. A hybridoma cell 4F6 strain secreting monoclonal antibody against NP protein of Newcastle disease virus, wherein the hybridoma cell 4F6 strain is deposited in China center for type culture Collection in 2019, 5, 9 and the address: the preservation number of the preservation center of Wuhan university in Wuhan, china is CCTCC NO: c2019102, classification and naming: the monoclonal antibody hybridoma cell strain 4F6 for secreting anti-Newcastle disease virus NP protein, the epitope recognized by the Newcastle disease virus NP protein monoclonal antibody prepared by the hybridoma cell 4F6 strain is 140-148 th amino acid of the Newcastle disease virus NP protein, and the polypeptide sequence is SNGTPFVTA.
2. The use of the hybridoma cell 4F6 secreting monoclonal antibody against NP protein of Newcastle disease virus of claim 1 for the preparation of diagnostic reagents for the detection of NP protein of Newcastle disease virus.
3. A monoclonal antibody against NP protein of Newcastle disease virus prepared from the hybridoma cell strain 4F6 according to claim 1.
4. The reagent for diagnosing and detecting the NP protein monoclonal antibody of Newcastle disease virus prepared by the NP protein monoclonal antibody of Newcastle disease virus of claim 3.
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