Hybridoma cell strain secreting monoclonal antibody for recognizing K subgroup avian leukosis virus and application of hybridoma cell strain

Abstract

The invention discloses a hybridoma cell strain capable of secreting a monoclonal antibody for recognizing K subgroup avian leukosis virus and application of the hybridoma cell strain, and belongs to the technical field of biology. The preservation number of the hybridoma cell strain provided by the invention is CGMCC (China General Microbiological Culture Collection Center) NO.23876. The monoclonal antibody generated by the hybridoma cell strain can recognize the antigen epitope (204-DWSRPQL-210) of the K subgroup avian leukosis virus gp85 envelope protein, the subtype of the antibody is IgG1, the monoclonal antibody can be used for developing a diagnostic reagent or medicine for the K subgroup avian leukosis, and a new substance is provided for clinical differential diagnosis and laboratory research of the K subgroup avian leukosis.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a hybridoma cell strain secreting a monoclonal antibody for recognizing K subgroup avian leukosis virus and an application thereof. Background Art

[0002] Avian leukosis (AL) is a contagious neoplastic disease of chickens caused by avian leukosis virus (ALV). Avian leukosis is widespread throughout the world and can cause immunosuppression, tumors, severe pathological damage and death in infected chickens, causing huge losses to the breeding industry every year. Currently, due to the high variability of ALV, there is a lack of effective vaccines or drugs. In clinical production, some countries control the disease by culling infected breeder chickens.

[0003] ALV has evolved into many subgroups with different pathogenic mechanisms. Eleven subgroups of ALV, ALV-A / B / C / D / E / F / G / H / I / J / K, have been identified, of which seven subgroups are from chickens. ALV-E is an endogenous virus that is usually not a target for clinical culling due to its weak pathogenicity, but it may interfere with the determination of other exogenous ALVs because its genome has been embedded in the host chicken during evolution. ALV-K is a new exogenous subgroup reported in recent years in local chickens in Asian countries, especially in China, which causes viremia, immunosuppression, and growth retardation in infected chickens; however, it is difficult to screen for ALV-K p27 antigen by ELISA because it replicates slowly in chicks. Therefore, a more specific and sensitive method is needed to detect ALV-K strains.

[0004] ALV is a single-stranded, positive-strand, linear RNA virus with a total length of approximately 7.2-7.8kb. In the ALV genome, the env gene is an important structural gene with a high mutation frequency, which contains the gp37 and gp85 genes. The gp37 gene encodes a transmembrane glycoprotein (TM) that is responsible for the transfer of ALV to host cells and is more conservative than the gp85 protein. The gp85 gene encodes a surface glycoprotein (SU) that determines the pathogenicity and virulence of different ALV subgroups. It is mainly responsible for mediating the binding process between the virus and the corresponding host cell membrane receptors, and can stimulate the body to produce specific antibodies. Usually, the gp85 gene is used to identify ALV subgroups based on the nucleotide homology of ALV, and to prepare neutralizing antibodies or cloned antibodies (MAbs) against ALV to establish specific detection methods.

[0005] At present, the K subgroup avian leukosis virus infection can be initially diagnosed through clinical autopsy and histopathological examination, and further diagnosis requires PCR, immunohistochemistry, indirect immunofluorescence and ELISA. The PCR method is easily interfered by many factors and may produce false positive and false negative results; the ELISA method currently has no finished kit for the specific detection of ALV-K on the market, and the immunohistochemistry and indirect immunofluorescence methods also require highly specific monoclonal antibodies.

[0006] In view of the above research status, it is imperative to prepare highly specific ALV-K virus monoclonal antibodies based on the isolated strains in my country to control and detect ALV-K infection in chicken flocks in my country. Summary of the invention

[0007] In view of the above-mentioned prior art, the object of the present invention is to provide a hybridoma cell line secreting monoclonal antibodies recognizing K subgroup avian leukosis virus and its application.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] The first aspect of the present invention provides a hybridoma cell line ALV-K-GW2012, which is classified and named as anti-ALV-K monoclonal cell line. It was deposited in the General Microbiology Center (CGMCC) of China Microorganism Culture Collection Administration on November 16, 2021, with the deposit number CGMCC No. 23876. The address of the deposit unit is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0010] The hybridoma cell line ALV-K-GW2012 of the present invention has the following characteristics:

[0011] (1) The number of chromosomes is 98, which is close to the sum of the chromosome numbers of the two parental cells;

[0012] (2) The cells have good cryopreservation stability. After the hybridoma cell line ALV-K-GW2012 stored in liquid nitrogen was revived, the cell viability was measured and the cell survival rate was above 96%;

[0013] (3) The secreted antibodies are stable. During the 60 cell passages, the titer of the secreted antibodies did not change much, showing a high consistency, and the antibody secretion stability was high.

[0014] (4) Produce monoclonal antibodies that specifically recognize K subgroup avian leukosis virus.

[0015] The second aspect of the present invention provides the use of the hybridoma cell line ALV-K-GW2012 in preparing a monoclonal antibody that recognizes the gp85 envelope protein of the K subgroup avian leukosis virus.

[0016] The third aspect of the present invention provides a monoclonal antibody 23H7, which is secreted and produced by the hybridoma cell line with the above-mentioned deposit number CGMCC No. 23876.

[0017] The subtype of the monoclonal antibody 23H7 of the present invention is IgG1; it can specifically recognize the gp85 envelope protein of the K subgroup avian leukosis virus. 204- DWSRPQL -210 The monoclonal antibody 23H7 of the present invention only reacts with ALV-K virus, but not with ALV-A, ALV-B, and ALV-J strains, and has strong specificity.

[0018] The fourth aspect of the present invention provides the use of the hybridoma cell line or monoclonal antibody in the preparation of a product for detecting ALV-K virus.

[0019] In the above application, preferably, the product can be an ELISA kit, a colloidal gold test strip or an indirect immunofluorescence assay (IFA) kit.

[0020] The fifth aspect of the present invention provides a kit for detecting ALV-K virus, which contains an effective amount of the above-mentioned monoclonal antibody 23H7.

[0021] Beneficial effects of the present invention:

[0022] According to the gene sequence of ALV-K-JS11C1 strain published in NCBI (GenBank: KF746200.1), the present invention intercepts a region of gp85 gene that has a wider representation (higher homology), a higher antigenic index, and expresses 103 amino acids as an immunogen, and obtains a hybridoma cell line after multiple screening and cloning. The hybridoma cell line can stably and efficiently secrete the monoclonal antibody 23H7, and can achieve large-scale batch production. The monoclonal antibody secreted by the hybridoma cell line screened by the present invention can specifically recognize K subgroup avian leukosis virus but not ALV-A / B / J, and the ascites antibody titer is high; the shortest linear epitope that can be recognized is ALVgp85 protein. 204- DWSRPQL -210 The 7 amino acids in the antigen epitope recognized by this monoclonal antibody are highly conserved in the published ALV-K strains, but very low in ALV-A / B / J / C / D / E strains. It is a good target for detecting the spread of ALV-K strains, which helps to improve the specificity and universality of detection technology and accelerate the purification of ALV-K. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 :Chromosome quantity analysis of hybridoma cells (ALV-K-GW2012).

[0024] Figure 2 : IFA mediated by monoclonal antibody 23H7 in DF-1 cells containing different ALV strains. Figure A, ALV-K-JS11C1 strain; Figure B, ALV-A-SDAU09C1 strain; Figure C, ALV-B-SDAU09C2 strain; Figure D, ALV-J-NX0101 strain.

[0025] Figure 3 : The titer determination results of monoclonal antibody 23H7.

[0026] Figure 4 : Subtype determination results of monoclonal antibody 23H7.

[0027] Figure 5 : Results of detecting ALV-K in chicken myocardial tissue using the monoclonal antibody 23H7 prepared by the hybridoma cell (ALV-K-GW2012) of the present invention (magnification 200 times); in the figure, A is a positive reaction between the monoclonal antibody 23H7 and the myocardial tissue of ALV-K infected chickens, and B is a negative reaction between the monoclonal antibody 23H7 and the myocardial tissue of normal chickens.

[0028] Figure 6 : Identification of secreted mAb 23H7 by immunoblotting.

[0029] Figure 7 :Analysis of linear antigen epitopes of monoclonal antibody 23H7 by immunoblotting with expressed protein fragments. Figure A, Strategy for expressing different protein fragments of gp85 protein antigen (red line indicates positive reaction in protein blot, black line indicates negative reaction). Figure B, Reaction results of 15 overlapping truncated protein fragments with prepared monoclonal antibody 23H7 in immunoblotting.

[0030] Figure 8 : ELISA of the shortest mAb antigen epitope using synthetic peptides; Figure A, strategy of synthetic peptides; Figure B, results of ELISA (mAb is ascites antibody; NC is negative mouse serum negative control); **, significant difference compared with the NC group (P<0.01).

[0031] Fig. 9 : Biochemical properties and spatial conformation prediction of monoclonal antibody 23H7 epitope; Figure A, predicted secondary structure and biological properties of monoclonal antibody 23H7 epitope; Figure B, predicted three-dimensional structure of monoclonal antibody 23H7 epitope on ALV-K gp85 protein. Figure C, predicted spatial conformation of monoclonal antibody 23H7 epitope on ALV-K gp85 protein. The gene sequence is derived from ALV-K-JS11C1 in Genbank (GenBank: KF746200.1). The model was constructed using Swiss model online prediction software.

[0032] Fig.10: Analysis of the variation of the antigen epitopes recognized by the monoclonal antibody (23H7) in different subgroups of ALV strains; Number of representative strains: 15 ALV-K strains; 8 ALV-A strains; 5 ALV-B strains; 6 ALV-J strains; 1 ALV-C strain; 1 ALV-D strain; 5 ALV-E strains. The ALV strains used in IFA are marked with a box. DETAILED DESCRIPTION

[0033] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0034] As mentioned above, ALV-K is a new exogenous subgroup reported in recent years, which can cause viremia, immunosuppression and growth retardation in infected chickens; currently there are relatively few reports on ALV-K detection.

[0035] Based on this, the present invention provides a hybridoma cell line that can stably secrete monoclonal antibodies that recognize K subgroup avian leukosis virus. The monoclonal antibodies secreted by the hybridoma cell line can be widely used for ALV-K differential diagnosis and ALV-Kgp85 protein recognition research, providing a material basis for the clinical differential diagnosis and laboratory research of K subgroup avian leukosis.

[0036] In the process of obtaining hybridoma cell lines, the screening of hybridomas after fusion is the most tedious and complicated. After the fused hybridomas are cultured in HAT medium for one week, the cell supernatant in each well plate is taken out and tested by ELISA to see whether it secretes specific antibodies and whether the antibody secretion ability is strong. If positive cells with strong secretion ability are detected, the positive cells need to be subcloned multiple times to obtain positive monoclonal hybridoma cell lines. In this process, due to cell variation and competition, strong positive cells are likely to be missed. Therefore, it is very difficult to obtain hybridoma cell lines that can continuously, stably and efficiently secrete monoclonal antibodies, and it is random and unpredictable.

[0037] The hybridoma cell line ALV-K-GW2012 screened by the present invention has the following characteristics:

[0038] (1) The results of the colchicine method for detecting chromosomes in the hybridoma cell (ALV-K-GW2012) showed that the number of chromosomes in the hybridoma cell (ALV-K-GW2012) was 98, which was close to the total number of chromosomes in the two parental cells.

[0039] (2) Take 10-week-old female Balb / C mice, treat them with liquid paraffin by intraperitoneal injection, and then inject the hybridoma cells (ALV-K-GW2012). After 14 days, it can be observed that the abdomen of the mice is obviously swollen, with tumors and a large amount of ascites. The ascites contains a large amount of monoclonal antibodies that recognize ALV-K, and the production of monoclonal antibodies is large.

[0040] (3) The cells have good cryopreservation stability. After the hybridoma cell line ALV-K-GW2012 stored in liquid nitrogen was revived, the cell viability was measured and the cell survival rate was above 96%;

[0041] (4) The secreted antibodies are stable. During the 60 cell passages, the titer of the secreted antibodies did not change much, showing a high consistency, and the antibody secretion was highly stable.

[0042] (5) IFA analysis results showed that the secreted antibody 23H7 obtained by the hybridoma cell line (ALV-K-GW2012) could only react with ALV-K virus but not with ALV-A / B / J strains.

[0043] (6) The hybridoma cell (ALV-K-GW2012) obtained monoclonal antibody 23H7, the antibody subtype of which was IgG1.

[0044] The subtype of monoclonal antibodies affects the purification, application range, sensitivity and stability of antibody detection methods. In the process of monoclonal antibody purification, the subtype of monoclonal antibodies determines what kind of purification method is used to obtain protein antibodies with higher specificity; in the use of monoclonal antibodies to establish corresponding identification detection methods, the subtype of monoclonal antibodies also determines which labeled secondary antibody is more specific, sensitive and stable when combined with the corresponding subtype monoclonal antibodies. For example, in the process of establishing indirect fluorescent immunoassay IFA, immunohistochemistry and immunoprotein blot analysis with high specificity using the monoclonal antibodies obtained from the hybridoma cells of the present invention, the sensitivity of the labeled anti-IgM or IgG secondary antibody will be higher and more stable; and for the ELISA method that does not require very high specificity, the subtype of monoclonal antibodies is not very demanding, and the key is that the titer of the antibody must be high enough.

[0045] The monoclonal antibody 23H7 obtained from the hybridoma cell (ALV-K-GW2012) of the present invention can specifically recognize the antigenic amino acid sequence corresponding to the base sequence from the 610th bp to the 630th bp on the ALV-K gp85 gene (GenBank: KF746200.1), that is, the antigenic region of the 204th aa-210aa; the antigenic epitope recognized by it is also different from the monoclonal antibodies Km3 and 30B9 secreted by the hybridoma cells reported in the prior art.

[0046] In summary, even if the same or similar acquisition methods are used, the biological characteristics of the hybridoma cell lines screened and the monoclonal antibodies secreted by them will be different, especially the recognition sites, stability, titer, application range, etc. of the secreted monoclonal antibodies are quite different. The hybridoma cell line (ALV-K-GW2012) with the deposit number of CGMCCNO.23876 of the present invention can secrete the monoclonal antibody 23H7 more efficiently and stably, and the titer of the monoclonal antibody 23H7 can reach 2 14 ; and the secreted monoclonal antibody 23H7 can specifically recognize the antigenic amino acid sequence corresponding to the base sequence from 610bp to 630bp on the ALV-K gp85 gene, that is, the antigenic region of 204aa-210aa.

[0047] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0048] The test materials used in the examples of the present invention are all conventional test materials in the art and can be purchased through commercial channels.

[0049] The methods used in the embodiments of the present invention that are not described in detail are all existing technologies in the art.

[0050] Example 1: Preparation of hybridoma cell line (ALV-K-GW2012)

[0051] (1) Based on the gene sequence of ALV-K-JS11C1 strain published in NCBI (GenBank: KF746200.1), a region of the gp85 gene with a wider representation (higher homology), a higher antigenic index, and expressing 103 amino acids was intercepted as an immunogen, and its amino acid sequence is shown in SEQ ID NO.1. The nucleotide sequence encoding the immunogen was inserted into the PET-32a vector to construct a recombinant expression vector; ALV-K gp85 recombinant protein was prepared and purified by conventional methods;

[0052] (2) The purified ALV-K gp85 recombinant protein was diluted to 1.0 mg / mL with sterile PBS solution and injected intraperitoneally into 6-week-old female Balb / C mice at a concentration of 0.2 mL per mouse for 4 immunizations.

[0053] The above recombinant protein was mixed with an equal volume of complete / incomplete Freund's adjuvant. The first immunization was performed at the 3rd and 5th weeks after the first immunization, and the protein dose used was the same as the first immunization. The adjuvant used for the first immunization was complete Freund's adjuvant, and the adjuvant for the booster immunization was changed to incomplete Freund's adjuvant. One week after the third immunization, blood was collected from the tail vein of the mice, serum was separated, and the antibody level of the mice was tested. Four weeks after the third immunization, a booster immunization was performed once, and 3 days later, the spleen cells of the immunized mice were taken for cell fusion with SP2 / 0.

[0054] (3) One day before fusion, spleen cells from healthy, unimmunized 6-week-old Balb / C mice were selected as feeder cells at a cell density of 1×10 6 pc / mL, added to 96-well cell culture plate, 100 μL / well, 37°C, 5% CO 2 Culture in an incubator.

[0055] The steps for preparing feeder cells are as follows:

[0056] One day in advance, spleen cell suspension was prepared from healthy 8-week-old Balb / c mice and plated in a 96-well cell plate as feeder cells. The method was as follows: the mice were killed by cervical dislocation and placed in a beaker containing 75% alcohol for 15 min; the mice were placed on their side on a sterile plate, and the fur and peritoneum on the left side of the mice were cut with sterile ophthalmic scissors and ophthalmic tweezers in an ultra-clean workbench. The scissors and tweezers were wiped with alcohol and sterilized with an alcohol lamp flame, and the spleen was gently clamped out with tweezers, taking care not to break it and removing the surrounding adhesion tissue, and then placed in 10 mL 4°C Rinse in a beaker of pre-cooled serum-free DMEM cell culture medium; place a 70μm cell sieve on top of a small beaker, cut the spleen on the cell sieve, and fully crush and grind it with a disposable 10mL sterile syringe plug, add a total of 10mL serum-free cell culture medium three times, and filter the spleen cell suspension into a small beaker; use a 5mL pipette to draw up the spleen cell suspension, centrifuge at 4℃, 1600rpm for 6min; discard the supernatant, add 10mL 4℃ pre-cooled red blood cell lysis buffer to resuspend the precipitate, and place the centrifuge tube at 4℃ for 10min to break the red blood cells. After the lysis is completed, centrifuge at 4℃, 1600rpm for 6min;

[0057] Discard the supernatant, add 10 mL of 4°C pre-cooled DMEM serum-free medium, resuspend and centrifuge at 4°C for washing; count in the same way as above. Discard the supernatant after full centrifugation, and resuspend the cell pellet with the required medium according to the counting results of the previous step so that the final cell density is 1×10 6 Splenocytes / mL; Plating: 100 μL of spleen cells per well was added to a 96-well cell culture plate and placed at 37°C with 5% CO 2 Culture the cells in a cell culture incubator. The next day, make sure that the cells are not contaminated and are transparent and evenly distributed.

[0058] According to the ratio of spleen cells: SP2 / 0 cells = 10:1, take an appropriate amount of SP2 / 0 cells and spleen cells and mix them thoroughly in a sterilized 50mL centrifuge tube, centrifuge at 1000rpm for 8min; discard the supernatant, use a pipette to absorb the residual liquid, and gently tap the bottom of the tube to make the cell pellet loose and uniform; place the centrifuge tube in a beaker filled with 37℃ warm water, use a pipette to absorb 1mL of 50% PEG preheated to 37℃, and add it dropwise on the cell pellet at a speed of 1mL / min, gently stir while adding, and let it stand for 1min after dropping; add 5mL of DMEM basal medium preheated to 37℃ at a speed of 1mL / min, then add 10mL of medium at a speed of 1mL / min, and finally add 15mL of medium evenly at a faster speed, and let it stand at 37℃ for 10min; centrifuge at 1000rpm for 5min, discard the supernatant; slowly add HAT medium preheated to 37℃, gently blow off the precipitate to suspend and mix it, the amount of HAT medium is 2×10 6 / mL spleen cells were added; the mixed cell suspension was added to a 96-well cell culture plate (100 μL of feeder layer cells had been laid in each well), 100 μL / well, and cultured in a 37°C, 5% CO2 incubator; medium change: half amount of HAT medium was replaced after 4 days, and HT medium was used after 10-13 days; then 20% DMEM medium was used according to cell proliferation; after 14 days of culture, the growth of hybridoma cells was frequently observed, recorded and tested in time, and when the hybridoma cells grew to more than 1 / 3 of the bottom area of ​​each well, the cell supernatant was aspirated for antibody detection.

[0059] (4) Screening, identification and cloning of positive hybridoma cells

[0060] The screening method is as follows:

[0061] (1) Preliminary screening of hybridoma cells by ELISA

[0062] The removed hybridoma cell supernatant was added to the ELISA plate coated with purified ALV-K gp85 recombinant protein, and the positive serum of immune Balb / C mice and SP2 / 0 cell culture supernatant were used as positive and negative controls, respectively. The ELISA method can be used for preliminary screening of a large number of fusion cells, and hybridoma cells that cannot target the ALV-K gp85 recombinant protein antigen will be discarded, reducing the workload of the IFA method during re-screening, saving time and cost, and improving screening efficiency. The specific method is as follows:

[0063] 1) Coating: Dilute the protein with coating solution to the detection concentration, add 100 μL to each well of the ELISA plate, and incubate at 4°C for about 12 hours. The next day, discard the coating solution and wash with PBST solution 4 times for a total of 2 minutes.

[0064] 2) Blocking: Add 200 μL of blocking solution (5% skim milk powder) to each well, incubate at 37°C for 1 h, and wash the plate in the same way;

[0065] 3) Add primary antibody: add the supernatant of hybridoma cells to be tested and positive serum of immune mice (mouse positive serum is diluted at a ratio of 1:10) 2 , 1:10 3 , 1:10 4 …1:10 8 After dilution, add the supernatant of SP2 / 0 cells to the reaction wells in turn, 100 μL per well, incubate at 37°C for 1 h, and wash the plate in the same way;

[0066] 4) Add secondary antibody: add 1:5000 diluted HRP-labeled goat anti-mouse IgG to each well, 100 μL per well, incubate at 37°C for 1 hour, and wash the plate in the same way;

[0067] 5) Add substrate: add 100 μL TMB single-component substrate colorimetric solution to each well and incubate at 37°C for 15 min;

[0068] 6) Stop color development: Add 50 μL of 2M H 2 SO 4 Stop solution to stop color development and gently shake to mix. 450nm Read the data at the time of immunization. Determine the antibody level and titer of mice after immunization; according to the sample OD 450nm / Negative OD 450nm >2.1 was used as the positive standard and screened again by IFA method.

[0069] (2) IFA screening of hybridoma cells

[0070] The hybridoma cell supernatant was used as the primary antibody and FITC-labeled goat anti-mouse IgG was used as the secondary antibody to react with DF-1 cells infected with ALV-K-JS11C1 virus.

[0071] 1) On the second day after DF-1 cells were passaged, when the cell density was about 75%, ALV-K-JS11C1 was inoculated. 1 mL of virus solution was taken to infect the cells. After 2 h, 4 mL of 2% culture medium was added for culture. After one passage, the cells were inoculated into a 96-well plate.

[0072] 2) Observe the growth status of the cells. When the cells are in good condition and the S / P value of the cell supernatant detected by the IDEXX ALV P27 antigen detection kit is greater than 1.5, take out the 24-well plate for IFA detection;

[0073] 3) Cell fixation: discard the upper layer of culture medium in the cell plate, wash once with PBS solution, 3 min / time, add fixative (acetone: anhydrous ethanol = 3:2) and fix at room temperature for 8 min, discard the fixative, and wash 3 times with PBS solution, 3 min / time;

[0074] 4) Dilute the selected serum with PBS solution at a multiple of 1:8, discard the PBS solution, add 200 μL of the diluted supernatant to each well, and incubate at 37°C for 1 hour;

[0075] 5) Discard the supernatant, wash with PBS solution 3 times, 3 min / time, add 200 μL 1:100 diluted FITC-labeled goat anti-mouse secondary antibody to each well, and incubate at 37°C in the dark for 1 h;

[0076] 6) Discard the secondary antibody, wash with PBS solution 4 times, 3 min / time, add 200 μL 50% glycerol to each well to cover the bottom cells, protect from light, and observe under a microscope. Under blue fluorescence excitation, observe the cytoplasm with green fluorescence under a microscope as positive cells.

[0077] After multiple cell fusions and screening, a hybridoma cell (ALV-K-GW2012) that can stably secrete monoclonal antibodies was obtained, named anti-ALV-K monoclonal cell line, and was deposited on November 16, 2021 at the General Microbiology Center of the China Culture Collection Administration, located at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the deposit number CGMCCNO.23876.

[0078] Example 2: Preparation of ascites antibodies using hybridoma cells (ALV-K-GW2012)

[0079] (1) Select healthy, unimmunized, 10-week-old female Balb / c mice and intraperitoneally inject liquid paraffin (0.5 mL / mouse) 1-2 weeks before inoculation of hybridoma cells;

[0080] (2) The adherent hybridoma cells (ALV-K-GW2012) were blown into a single cell suspension.

[0081] The tumor cells were washed 2-3 times with DMEM basal medium, and after low-speed centrifugation, the tumor cells were counted and diluted to 1×10 6 / mL. 0.2mL of cell suspension was slowly injected intraperitoneally into the treated mice in multiple directions;

[0082] (3) 10-14 days after the injection of hybridoma cells, observe the production of ascites in the mice. If the abdomen is obviously swollen and the skin feels tense when touched with fingers, the ascites can be slowly extracted with a 16-gauge needle syringe. Generally, the ascites can be collected 2-3 times in a row, with an interval of 2-3 days between each time.

[0083] (4) The collected ascites was centrifuged at 10,000 rpm for 10 min to remove cellular components and other precipitates, and the supernatant was collected. It was then filtered through a 0.45 μm filter membrane and purified by affinity chromatography. The purified antibody was collected and recorded as antibody 23H7.

[0084] Example 3: Investigation of the characteristics of hybridoma cells (ALV-K-GW2012):

[0085] The chromosomes in the nucleus of the hybridoma cell (ALV-K-GW2012) were identified by Giemsa staining under a microscope (10×100). The results showed that the number of chromosomes in the hybridoma cell (ALV-K-GW2012) was 98 ( Figure 1 ), which is close to the sum of the chromosome numbers of the two parental cells.

[0086] The hybridoma cell line (ALV-K-GW2012) obtained by screening in Example 1 was frozen and revived at 3, 6, 9, 12, 18 and 24 months, respectively, and passaged at least 3 times. The cell growth state was observed under a microscope and the antibody 23H7 secretion ability of the hybridoma cell line (ALV-K-GW2012) was determined by indirect ELISA.

[0087] The results showed that after cryopreservation and resuscitation, the cell survival rate of the hybridoma cell line (ALV-K-GW2012) of the present invention was above 96%, and the secretion capacity of the antibody 23H7 did not decrease for at least 24 months.

[0088] The hybridoma cell line (ALV-K-GW2012) obtained in Example 1 was subcultured, and the culture supernatant was collected at 1, 4, 8, 12, 16, 20, 24, 28, 32, 36, 42, 50, and 60 generations, respectively, and the stability of the cell-secreted antibody was analyzed by ELISA. The results showed that the titer of the secreted antibody did not change substantially after 60 cell subcultures, showing a high consistency.

[0089] Example 4: Specific detection of monoclonal antibody (23H7)

[0090] Indirect immunofluorescence (IFA) was used to detect the specificity of the ascites antibody 23H7 prepared in Example 2, and the steps were as follows:

[0091] DF-1 was passaged in 96-well cell culture plates. After the cells grew into a monolayer, DF-1 cells were infected with ALV-K-JS11C1 (GenBank: KF746200.1), ALV-A-SDAU09C1 (GenBank: HM452339), ALV-B-SDAU09C2 (GenBank: HM446005) and ALV-J-NX0101 (GenBank: AY897227), respectively. 5-7 days after infection, the cells were fixed with cold acetone-ethanol (6:4) for 5 min, washed once with PBS solution, dried in air, and stored at -20°C as detection antigens. When performing IFA, the ascites antibody to be detected was appropriately diluted and added dropwise to the 96-well cell culture plate infected with the virus. After incubation at 37°C for 30 minutes, it was washed 5 times with PBS solution, and then FITC-labeled goat anti-mouse secondary antibody was added. The cells were incubated for another 30 minutes, washed 5 times with PBS solution, and finally observed under a fluorescence microscope. The results are shown in Figure 2. Figure 2 shown.

[0092] Depend on Figure 2 It can be seen that the ascites antibody 23H7 prepared in Example 2 reacts positively with DF-1 cells infected with ALV-K, and reacts negatively with DF-1 cells infected with ALV-A / B / J, indicating that the ascites antibody 23H7 in Example 2 can specifically recognize ALV-K and has good specificity.

[0093] Example 5: Determination of the titer and subtype of monoclonal antibody 23H7

[0094] The indirect ELISA method was used to detect the titer of the ascites antibody prepared in Example 2. In addition to the ascites diluent, standard negative and positive serum, the ascites of SP2 / 0 cells should also be used as a control. The judgment criteria are: negative serum OD 450nm Value < 0.2, positive serum OD 450nm When the value is greater than 1.0 and S / N is greater than 2.1, the maximum dilution factor of the ascites antibody is the ELISA titer of the ascites. Figure 3 shown.

[0095] The monoclonal antibody subclass of the ascites antibody prepared in Example 2 was identified using a mouse antibody isotype ELISA kit. The results are as follows: Figure 4 shown.

[0096] Example 6: Detection of ALV-K in tissues

[0097] 1) Fixation: The size of tissue blocks should be limited to 0.5-1cm×0.5-0.2cm, and fixed with 1% paraformaldehyde for 24h;

[0098] 2) Dehydration: 30% sucrose dehydration for more than 48 hours;

[0099] 3) Sectioning: Use a cryostat to slice, and use an adhesive slicer to absorb the intact, wrinkle-free tissue slices. When the slices are half dry, soak them in a PBS solution for 10 minutes to remove OCT. If the slices are not stained immediately after freezing, they can be placed in a -80°C ultra-low temperature refrigerator. After taking them out, they can be half-dried at room temperature, fixed and soaked in a methanol solution pre-cooled in a -20°C refrigerator for 5 minutes, and then soaked in a PBS solution;

[0100] 4) Antigen repair: First, heat the sodium citrate solution to a boil, place the slices in a container filled with citrate buffer, and heat in a microwave oven to keep the liquid temperature in the container at medium-low heat (20 power) for 5 minutes. Open the door and let it air dry for 2 minutes. Repeat 3 times, remove the container, cool at room temperature for 1-2 hours, rinse with PBS 3 times, 5 minutes each time, and wipe off the surrounding moisture for the last time;

[0101] 5) Blocking: Block with 10% normal fetal bovine serum, incubate at room temperature at 37°C for 1 hour. Pour off the serum and spin dry;

[0102] 6) Add primary antibody: Add primary antibody 23H7 diluted in appropriate proportion, incubate at 37℃ for 1-2h or at 4℃ overnight. Rinse with PBS 3 times, 5min each time, and wipe off the surrounding water for the last time;

[0103] 7) Add secondary antibody: Add 1:500 diluted FITC-labeled goat anti-mouse IgG secondary antibody, incubate at 37°C for 1 hour, rinse with PBS 3 times, 5 minutes each time, and wipe off the surrounding water for the last time;

[0104] 8) Sealing: After adding anti-fluorescence quenching agent, seal the slides. Cells with green fluorescence under a fluorescence microscope are positive cells. Figure 5 shown.

[0105] Example 7: Antigen detection mediated by monoclonal antibody 23H7 by immunoblotting

[0106] The monoclonal antibody 23H7 prepared by hybridoma cells (ALV-K-GW2012) with the deposit number of CGMCCNO.23876 was used as the primary antibody, and HRP-labeled goat anti-mouse IgG was used as the secondary antibody. The method is as follows:

[0107] 1) After the gel tank is properly installed, check for leaks. If there is no leakage, add 12.5% ​​separation gel. After the separation gel solidifies, add 12.5% ​​concentrated gel. Insert a comb of appropriate size, and after solidification, slowly pour in the prepared electrophoresis buffer and equilibrate at 4°C overnight.

[0108] 2) Sample treatment: resuspend the bacterial precipitate with 20 μL PBS solution, add 1 / 5 of the final volume of 5×SDS loading buffer, and fully denature it in a boiling water bath for 10 minutes. Add protein marker, load 20 μL / well sample, turn on the power, perform electrophoresis at 150V and 90mA, and stop electrophoresis when the band reaches the bottom;

[0109] 3) After stopping the electrophoresis, cut the gel block into appropriate sizes and place the gel block in the rapid transfer buffer to keep it moist;

[0110] 4) Cut 6 filter papers and 1 PVDF membrane of the same size as the rubber block in advance, and activate the PVDF membrane by soaking it in methanol for 30 to 50 seconds. Then, place both the filter paper and the PVDF membrane in the rapid transfer solution.

[0111] 5) Use the transfer device to place two layers of sponge, three layers of filter paper, a rubber block, a PVDF membrane, three layers of filter paper, and two layers of sponge in order from the negative electrode to the positive electrode, clamp them and immerse them in the rapid transfer solution, turn on the power, transfer the membrane at 90V, 400mA for 30 minutes, and transfer is completed when there is no marker on the rubber block;

[0112] 6) After the transfer, the PVDF membrane was blocked with 5% skimmed milk powder and shaken at room temperature for 1 hour; after discarding the skimmed milk powder, the membrane was washed with PBST solution and shaken on a horizontal shaker for 10 minutes, and repeated 3 times; then the PVDF membrane was soaked with 23H7 monoclonal antibody prepared by hybridoma cells diluted 1:200 with PBST solution and placed at 4°C overnight for reaction; on the next day, the primary antibody 23H7 was discarded and the membrane was washed 3 times; finally, the membrane was placed in the HRP-labeled goat anti-mouse IgG secondary antibody diluted 1:5000 with 5% skimmed milk powder and shaken at room temperature for 1 hour; the secondary antibody was discarded and the membrane was washed 3 times; after discarding the eluent, a 1:1 prepared ECL colorimetric solution was added in a dark place, and the results were observed and saved on the instrument. The results are as follows Figure 6 shown.

[0113] Example 8: Identification of Antigen Epitopes Recognized by Monoclonal Antibody 23H7

[0114] In order to locate the antigen epitope recognized by the monoclonal antibody of ALV-K gp85 protein antigen, this study designed a series of overlapping truncated ALV-K gp85 fragments, amplified and cloned the truncated gp85 gene fragments into the pET-32a vector, and expressed them in Escherichia coli as a fusion protein containing a His tag. The purified protein product was used as the antigen, and the monoclonal antibody 23H7 was used as the first antibody. Then, the location of the antigen epitope was determined based on the binding reaction between the antigen and the antibody in the immunoblot. The results are shown in Figure 2. Figure 7 As shown, only gp85-3-4 and gp85-3-5 204- DWSRPQL -210To further verify the accuracy of the epitope, Figure 8 The seven peptides were selected and their reactions with the prepared monoclonal antibody 23H7 were analyzed by ELISA. The optical density values ​​at 450 nm were detected and statistically compared.

[0115] Example 9: Bioinformatic analysis of antigen epitope recognition by monoclonal antibody 23H7

[0116] Bioinformatics analysis software was used to predict the secondary structural domains (such as α-helix, β-pleated region / turn and random coil) and biochemical properties (such as hydrophilicity, surface accessibility and antigenic index) of the ALV-Kgp85 protein fragment containing the antigenic epitope. In addition, the partial gp85 protein amino acid sequence containing the antigenic epitope was sent to Swiss-Model (http: / / swissmodel.expasy.org / ) to construct a three-dimensional (3D) model online, which was also used to predict the spatial conformation of the antigenic epitope. Fig. 9 The prediction showed that the epitope of mAb 23H7 contained a turn and a β-fold, with a high antigenic index. The three-dimensional spatial structure model showed that the seven amino acids of the antigen epitope formed a curved linear spatial structure, which was exposed on the surface of ALV-K gp85 protein and was conducive to binding with mAb 23H7. These findings provide more evidence for a better understanding of the immune recognition between ALV-K gp85 protein and mAb 23H7.

[0117] The gp85 gene sequences of 41 representative ALV strains were obtained from GenBank, including 15 ALV-K strains, 8 ALV-A strains, 5 ALV-B strains, 6 ALV-J strains, 1 ALV-C strain, 1 ALV-D strain and 5 ALV-E strains. Fig.10 Using Megign software to compare the differences in antigenic epitopes, 7 antigenic epitopes of 15 ALV-K strains were highly conserved, including the ALV-K-JS11C1 strain used for immunofluorescence, while in ALV subgroup strains such as ALV-A-SDAU09C1, ALV-B-SDAU09C2 and ALV-J-NX0101, the conservation of 7 amino acids was very low (≤40%), which explains why the developed monoclonal antibody 23H7 can only recognize the ALV-K-JS11C1 strain, but not the ALV-A-SDAU09C1, ALV-B-SDAU09C2 and ALV-J-NX0101 strains in IFA.

[0118] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A hybridoma cell line ALV-K-GW2012, whose deposit number is CGMCC No.23876.

2. Use of the hybridoma cell line according to claim 1 in preparing a monoclonal antibody that recognizes the gp85 envelope protein of subgroup K avian leukosis virus.

3. A monoclonal antibody 23H7, characterized in that: It is secreted and produced by the hybridoma cell line with the deposit number CGMCC No. 23876 as described in claim 1.

4. The monoclonal antibody 23H7 according to claim 3, characterized in that: The antibody subclass of the monoclonal antibody 23H7 is IgG1.

5. The monoclonal antibody 23H7 according to claim 3, characterized in that: The monoclonal antibody 23H7 specifically recognizes the first 204- DWSRPQL -210 area.

6. Use of the hybridoma cell line ALV-K-GW2012 according to claim 1 or the monoclonal antibody 23H7 according to claim 3 in the preparation of a product for detecting ALV-K virus.

7. The use according to claim 6, characterized in that: The product is an ELISA kit, a colloidal gold test strip or an indirect immunofluorescence analysis kit.

8. A kit for detecting ALV-K virus, characterized in that: The kit contains an effective amount of the monoclonal antibody 23H7 according to claim 3.

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