Monoclonal antibody 1E1 against H10 subtype influenza virus hemagglutinin protein and its application

Abstract

The application belongs to the technical field of biology, and relates to an anti-H10 subtype influenza virus hemagglutinin protein monoclonal antibody and application thereof. The application is obtained by using cell engineering and antibody engineering technology, and is a hybridoma cell line secreting the anti-hemagglutinin protein monoclonal antibody. The anti-hemagglutinin protein monoclonal antibody 1E1 is prepared by inducing ascites of the same strain mouse, and is identified as IgG2a and kappa type. The application of the antibody is realized by affinity purification and immunization methods. The application can specifically recognize the hemagglutinin protein of the H10 subtype influenza virus. The heavy chain variable region amino acid sequence of the antibody is shown in SEQ ID No. 2, and the light chain variable region amino acid sequence is shown in SEQ ID No. 4. The application provides an effective tool for auxiliary diagnosis of H10 subtype influenza virus infection in clinical samples, and can be applied to various detection technologies and clinical and experimental researches.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to monoclonal antibodies against H10 subtype influenza virus hemagglutinin protein and applications thereof. The invention utilizes cell engineering and antibody engineering technologies to obtain a hybridoma cell line that secretes monoclonal antibodies against hemagglutinin protein, induces ascites in mice of the same strain, prepares monoclonal antibody 1E1 against hemagglutinin protein, identifies it as IgG2a, κ type, and then realizes application of the antibody through affinity purification, immunization methods and other technologies. Background Art

[0002] Influenza A viruses belong to the Orthomyxoviridae family and are classified into 18 HA and 11 NA subtypes based on the antigenic properties of their hemagglutinin (HA) and neuraminidase (NA) glycoproteins. Waterfowl are considered the natural reservoirs of influenza A viruses, and most influenza A virus subtypes have been identified in waterfowl. In recent years, H10 subtype influenza viruses, such as H10N1, H10N2, H10N3, H10N4, H10N5, H10N6, H10N7, H10N8, and H10N9, have been increasingly isolated from poultry. H10 subtype influenza viruses pose a potential pandemic threat to human health. Therefore, establishing rapid and accurate methods for detecting H10 subtype influenza viruses is crucial for influenza prevention and control.

[0003] Virus isolation in chicken embryos or MDCK cells is a current classic method for detecting influenza viruses. In recent years, molecular detection methods have also been greatly developed, and real-time quantitative polymerase chain reaction has been widely used in laboratory diagnosis of influenza virus infection. However, these methods are very demanding on technology and laboratory requirements and are time-consuming. Due to the development of monoclonal antibody technology, detection methods based on monoclonal antibodies are also widely used in virus detection. Therefore, the present invention aims to illustrate a specific monoclonal antibody for H10 subtype influenza virus, and combines it with immunofluorescence technology to detect H10 subtype influenza virus in samples. The above method has the advantages of being fast, sensitive and cheap, which will be conducive to earlier and more extensive discovery of H10 subtype influenza virus, effectively controlling viral spread.

[0004] In summary, the development of monoclonal antibodies against the H10 subtype influenza virus and the establishment of rapid and sensitive detection methods are of great significance for the prevention and control of the virus. Based on this background, this project selected the H10 subtype influenza virus hemagglutinin protein as the target antigen. Using fusion hybridoma technology, a hybridoma cell line stably secreting anti-hemagglutinin monoclonal antibodies was established. These monoclonal antibodies were then mass-produced, purified, and characterized. The successful acquisition of these monoclonal antibodies lays the material foundation for the development of a novel immunofluorescence-based diagnostic method for the H10 subtype influenza virus. It also plays a significant role in research into disease pathogenesis, prognosis, and therapeutic efficacy.

[0005] The present invention utilizes hybridoma cell technology. This technology fuses B lymphocytes from immunized mice with SP2 / 0 myeloma cells to establish a hybridoma cell line that secretes homogeneous antibodies. This technology, also known as monoclonal antibody technology, involves a series of methods, including animal immunization, cell culture, cell fusion, cell cloning culture, and immunoassays. Summary of the Invention

[0006] The present invention provides a monoclonal antibody against the hemagglutinin protein of influenza virus subtype H10, capable of recognizing influenza virus subtype H10. This monoclonal antibody, designated 1E1, is of IgG2a, κ subtype and specifically recognizes the hemagglutinin protein of influenza virus subtype H10. The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID No. 2, and the amino acid sequence of the light chain variable region is shown in SEQ ID No. 4.

[0007] SEQ ID No.1

[0008] Heavy chain:DNA sequence(375bp)

[0009] Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0010] CAGGTTACTCTGAAAGAGTCTGGCCCTGGGATATTGCAGCCCTCCCAGACCCTCAGTCTGACTTGTTCTTTCTCTGGG

[0011] TTTTCACTGAGCACTTCTGGTATGGGTGTGAGCTGGATTCGTCAGCCTTCAGGAAAGGGTCTGGAATGGCTGACACA

[0012] CATTTACTGGGATGATGACAAGCGCTATAACCCATCCCTGAAGAGCCGGCTCACAATCTCCAAGGATACCTCCAGA

[0013] AACCAGGTATTCCTCAAGATCACTAGTGTGGACACTGCAGATACTGCCACATACTACTGTGCTCGAAGCCCCCCTAC

[0014] GGACTACGGTAGTAGCTGGGGTGTTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCASEQ ID No.2

[0015] Heavy chain:Amino acid sequence(125AA)

[0016] Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0017] QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVSWIRQPSGKGLEWLTHIYWDDDKRYNPSLKSRLTISKDTSRNQVFLKITSVDT

[0018] ADTATYYCARSPPTDYGSSWGVMDYWGQGTSVTVSS

[0019] SEQ ID No.3

[0020] Light chain:DNA sequence(318bp)

[0021] Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0022] CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCGTCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAG

[0023] CTCGAGTATAACTTACATGCACTGGTACCAGCAGAAGTCAGGCACCTCCCCCAAAAGATGGATTTATGACACATCCA

[0024] AACTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATG

[0025] GAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTAACCCACTCACGTTCGGTGCTGGGACCAAGC

[0026] TGGAGCTGAAA

[0027] SEQ ID No.4

[0028] Light chain:Amino acid sequence(106AA)

[0029] Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0030] QIVLTQSPAIMSASPGEKVTMTCSASSSITYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQ

[0031] QWSSNPLTFGAGTKLELK

[0032] The second object of the present invention is to provide a method for preparing monoclonal antibodies against H10 subtype influenza virus hemagglutinin protein, which is achieved by the following steps and technical solutions:

[0033] (1) Animal immunization: 8-week-old BALB / C mice were selected and immunized with purified hemagglutinin protein of H10N7 subtype influenza virus (A / chicken / Zhejiang / 2CP8 / 2014).

[0034] (2) Culture of mouse myeloma cells: Mouse myeloma cells SP2 / 0 were cultured and maintained in a good growth state for cell fusion.

[0035] (3) Cell fusion: Polyethylene glycol-mediated cell fusion was used. The mice selected in step (1) were sacrificed and spleen lymphocytes were obtained. The SP2 / 0 cells from step (2) were collected, the two cells were mixed and centrifuged, and then the cells were fused using polyethylene glycol. The fused cells were appropriately diluted, inoculated into 96-well culture plates, and cultured under appropriate conditions.

[0036] (4) Screening of hybridoma cells: The above culture was cultured in a hypoxanthine-phosphoribosyltransferase selective medium. When the cell colonies grew to a suitable size, the cell culture supernatant was aspirated for antibody identification and positive clones were screened.

[0037] (5) Cloning of hybridoma cells: clone positive hybridoma cells by limiting dilution method, inoculate the cells diluted to a certain density into 96-well cell culture plates so that only one cell grows in each well. Take the supernatant from the wells where cell colonies are formed for enzyme-linked immunosorbent assay to screen and identify positive clones. Select the culture wells with the highest antibody titer and single clone cell growth, perform limiting dilution again, perform limiting dilution more than 3 times in succession, and continuously passage for more than 20 generations to obtain hybridoma cell lines that stably and efficiently express monoclonal antibodies against H10 subtype influenza virus. The cloned hybridoma cells are subjected to antibody identification and physical and chemical property analysis.

[0038] (6) Preparation of monoclonal antibody ascites: 10-week-old healthy BALB / C mice were selected and 5 million positive hybridoma cells were inoculated into each abdomen. About 10 days after the inoculation of cells, the abdomen of the mouse was significantly swollen. The health status and abdominal signs of the mouse were closely observed. When the ascites was as much as possible and the mouse was on the verge of death, the ascites was collected and centrifuged to determine the antibody titer and purify the monoclonal antibody in the ascites.

[0039] (7) Purification of monoclonal antibodies: Monoclonal antibodies were purified from mouse ascites using protein G agarose gel affinity purification.

[0040] (8) The present invention has obtained a hybridoma line, namely 1E1, which produces monoclonal antibodies against the hemagglutinin protein of influenza virus H10 subtype. The 1E1 hybridoma cell line was cloned three times and cultured continuously for 6 months, with stable antibody secretion. The cell line was frozen in liquid nitrogen and grew well after thawing. The titer of the 1E1 culture supernatant was 1:128 and the titer of the ascites fluid was 1:2048 as measured by indirect enzyme-linked immunosorbent assay. Monoclonal antibody immunoglobulin subtype analysis showed that the antibody produced by the hybridoma cells was IgG2a.

[0041] The present invention provides a hybridoma cell producing a monoclonal antibody, which is a mouse hybridoma cell line 1E1 obtained by fusing immune BALB / C mouse spleen cells and mouse myeloma cells SP2 / 0, screening, cloning and passage, and can stably secrete the monoclonal antibody 1E1 against the H10 subtype influenza virus hemagglutinin protein.

[0042] Another object of the present invention is to provide the use of the monoclonal antibody 1E1 in the preparation of a fluorescent detection product for H10 subtype influenza virus.

[0043] Detection of influenza virus H10 subtype in body fluids, allantoic fluid, or other environmental samples is achieved by immunofluorescence.

[0044] The present invention provides monoclonal antibody 1E1 against the hemagglutinin protein of the H10 subtype influenza virus, which serves as a probe for detecting H10 subtype influenza virus using immunofluorescence techniques. The present invention has the advantages of a simple and easy preparation method. More importantly, the monoclonal antibody prepared by this method can be used in a variety of applications, providing an effective tool for auxiliary diagnosis of H10 subtype influenza virus infection in clinical samples and can be widely applied to various detection technologies, as well as clinical and experimental research.

[0045] Figures in the specification

[0046] Figure 1 Immunoglobulin subtype analysis of monoclonal antibody 1E1.

[0047] Figure 2 The specificity of immunofluorescence technology for detecting H10 subtype influenza virus. DETAILED DESCRIPTION

[0048] The present invention will be further described below with reference to specific examples. It should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention.

[0049] Example 1. Preparation of Monoclonal Antibodies to H10 Subtype Influenza Virus Hemagglutinin Protein

[0050] (1) Immunization of mice: For the first immunization, H10 subtype influenza virus hemagglutinin protein and adjuvant were mixed evenly in a 1:1 ratio, with a total volume of 0.5 ml. 0.1 ml (containing 5 μg of H10 subtype influenza virus hemagglutinin protein antigen) was injected into the inner thigh muscle of each BALB / C mouse. On the 21st day, a booster immunization was performed in the same manner. On the 35th day, a small amount of tail blood was collected for enzyme-linked immunosorbent assay. The highest antibody titer reached 1:32,000. The mouse with the highest antibody titer was selected for a booster immunization via tail vein injection, and cell fusion was performed 3 days later.

[0051] (2) Culture and Passaging of Mouse Myeloma SP2 / 0 Cells: SP2 / 0 myeloma cells from BALB / C mice were cultured and passaged in DMEM medium containing 10% bovine serum in a 37°C incubator containing 5% carbon dioxide. Passaging is usually not performed the day before fusion to ensure that the cells enter the logarithmic growth phase at the time of fusion.

[0052] (3) Cell fusion: BALB / C mouse peritoneal macrophages were used as feeder cells. One day before fusion, BALB / c mouse peritoneal macrophages were inoculated into 96-well culture plates and cultured for one day in hypoxanthine-guanine-phosphoribosyltransferase medium containing 20% ​​bovine serum. Spleens were removed from mice 3 days after the last booster immunization. Splenic lymphocytes were isolated by pressure injection, washed by centrifugation, and resuspended in DMEM culture medium. SP2 / 0 cells were collected, centrifuged, washed, resuspended in DMEM culture medium, and counted. 300 million spleen lymphocytes from the immunized mice were mixed with 30 million mouse myeloma SP2 / 0 cells. The two cell types were mixed, centrifuged, and the supernatant discarded. The centrifuge tube was swirled to loosen the cell clumps. Polyethylene glycol pre-warmed at 37°C was slowly added to the fusion tube, gently shaking the tube during this process. The cells were aspirated into the fusion tube. After 90 seconds of quiescence, the cells were blown into the centrifuge tube. Then, following the principle of slowing down first and then increasing speed, 1 ml of DMEM medium was added within the first minute, 2 ml of DMEM within the second minute, and 7 ml of DMEM within the third minute. Over the next minute, 40 ml of DMEM pre-warmed at 37°C was gradually added. The tube was centrifuged at 800 rpm for 10 minutes. Hypoxanthine-guanine-phosphoribosyltransferase medium supplemented with 20% bovine serum was then added. The cells were then seeded into 96-well culture plates containing feeder cells using a glass pipette. Two plates were typically plated for each fusion. The cells were cultured in a 37°C incubator containing 5% carbon dioxide.

[0053] (4) Screening of hybridoma cells: half of the medium in 96-well plates is changed after 5 days (containing hypoxanthine-guanine-phosphoribosyl transferase) once, and the medium containing hypoxanthine-phosphoribosyl transferase is used after 10 days. The hybridoma cells after fusion are cultured in the selective medium containing hypoxanthine-phosphoribosyl transferase for about two weeks. When the cell colonies grow to an appropriate size (observed under 10x objective, the cell colony size is appropriate to fill one field), the cell culture supernatant is aspirated for enzyme-linked immunosorbent assay to screen positive clones. The positive hybridoma clones are screened by enzyme-linked immunosorbent assay indirect method. Main steps: ① H10 subtype hemagglutinin protein is diluted in 0.01 mol / L pH 9.6 carbonate buffer, then 0.1 mL is added to each well of 96-well enzyme-labeled plate, and the protein amount is 20 ng per well, 4°C overnight; ② the plate is washed 5 times with 0.01 mol / L pH 7.4 phosphate buffer (containing Tween 20); ③ blocked for 2 hours with 0.01 mol / L pH 7.4 phosphate buffer containing 5% bovine serum albumin; ④ washed 3 times; ⑤ add hybridoma culture supernatant, 0.1 mL per well, and set positive control (H10 subtype protein immunized mouse serum), negative control (SP2 / 0 culture supernatant) and blank control, and react at room temperature for 2 hours; ⑥ wash the plate 3 times; ⑦ add 1:10,000 diluted horseradish peroxidase-labeled goat anti-mouse IgG, 0.1 mL per well, and react at room temperature for 1 hour; ⑧ wash the plate 3 times; ⑨ add color developing solution, react at room temperature for 5 minutes; ⑩ terminate the reaction with 2 mol / L sulfuric acid; and measure the optical density value at 450 nm, and the value divided by the negative value is ≥2.1 to be positive.

[0054] (5) Cloning of hybridoma cells: the cloning of hybridoma cells is carried out according to the limited dilution method. The hybridoma cell holes with positive antibody detection are selected for appropriate proliferation, and the cells are accurately counted. The cell suspension diluted in complete DMEM medium to 10 cells per mL is inoculated into 96-well culture plates with feeder cells, 0.1 mL per well, and the cell growth is observed after 10 days, and the antibody level in the supernatant is detected. The culture hole with the highest antibody titer and single clone cell growth is selected for limited dilution again, and the limited dilution is continuously carried out for more than 3 times, and the hybridoma cell strain stably and highly expressing anti-H10 subtype influenza virus monoclonal antibody is obtained after continuous passage for more than 20 generations.

[0055] (6) Preparation of monoclonal antibody ascites: 10-week-old BALB / C healthy mice are selected, and each mouse is inoculated with 5 million positive hybridoma cells in phosphate buffered saline in the abdomen. The mouse abdomen is significantly swollen 10 days after inoculation of the cells, and the health status of the mouse and the abdominal signs are closely observed. The mouse ascites is collected when the ascites is as much as possible.

[0056] (7) Purification of the monoclonal antibody: The monoclonal antibody in the ascites was purified by affinity purification (protein G agar gel). ① The ascites was centrifuged at 10,000 rpm for 15 minutes at 4°C, and the precipitate was removed. The supernatant was mixed with 3 times the volume of binding buffer, and then centrifuged at 10,000 rpm for 15 minutes at 4°C to remove the precipitate. The precipitate was removed by centrifugation at 10,000 rpm for 15 minutes at 4°C. ② The affinity purification column preloaded with protein G agar gel was washed with 5 times the column bed volume of binding buffer. ③ The diluted ascites was loaded onto the column, and the flow rate was controlled at 10 drops per minute. ④ The ascites that had passed through the column was loaded onto the column again. ⑤ The purification column was washed with 5 times the column bed volume of binding buffer. ⑥ The bound monoclonal antibody was eluted with elution buffer, and the flow rate was controlled at 10 drops per minute. The eluate was collected in a collection tube preloaded with 0.1 mL of potassium phosphate buffer (pH 7.9), and 0.5 mL of the eluate containing the antibody was collected per tube. ⑦ The absorbance of each tube of eluate was measured at 280 nm, and the eluate with a protein content of greater than 0.1 mg / mL was collected. ⑧ The antibody eluate was added to an ultrafiltration centrifuge tube, and centrifuged at 10,000 rpm for 20 minutes at 4°C to a final volume of about 1 mL. 10 mL of 0.1 M phosphate buffer at pH 7.4 was added, and centrifuged at 10,000 rpm for 20 minutes at 8°C. The final concentration of the antibody was about 1 mL, and the concentrated antibody was collected in a collection tube. ⑨ The desalted antibody solution was diluted, and the protein content was measured at 280 nm. ⑩ The purified antibody was aliquoted into small tubes and stored in a low-temperature refrigerator for future use.

[0057] (8) Subtype identification of the monoclonal antibody: The mouse monoclonal antibody immunoglobulin typing kit from Bio-Rad was used for analysis. The purified monoclonal antibody was diluted appropriately and tested, and the operation was strictly in accordance with the instructions of the kit. The test results showed that the monoclonal antibody secreted by the 1E1 hybridoma cells was of the IgG2a, κ type.

[0058] Results are shown in the accompanying Figure 1 .

[0059] Example 2. Qualitative detection of H10 subtype influenza virus using the monoclonal antibody

[0060] The anti-H10 subtype influenza virus hemagglutinin protein monoclonal antibody prepared in the present application can be used for qualitative detection of H10 subtype influenza virus, and the identification method can be achieved by the following method:

[0061] H10 subtype influenza virus immunofluorescence detection method:

[0062] (1) MDCK cells were seeded in a 48-well plate at a density of 40,000 cells per well one day in advance, and the cells were grown to 70% confluence for use;

[0063] (2) Take out the cell plate with cells, discard the culture supernatant, and wash once with phosphate buffer solution for standby;

[0064] (3) Immunofluorescence detection method for detecting the specificity of H10 subtype influenza virus: dilute the virus with phosphate buffer solution, including H10N2 (A / duck / Zhejiang / 6D20 / 2013), H10N3 (A / chicken / Zhejiang / 8615 / 2016), H10N5 (A / Zhejiang / ZJU01 / 2023), H10N7 (A / chicken / Zhejiang / 2CP8 / 2014), H1N1 (A / Michigan / 45 / 2015), H2N8 (A / duck / Zhejiang / 6D10 / 2013), H3N2 (A / Texas / 50 / 2012), H4N6 (A / duck / Zhejiang / 409 / 2013), H5N1 (A / goose / Zhejiang / 97 / 2014), H6N1 (A / chicken / Zhejiang / 1664 / 2017), H7N9 (A / chicken / Zhejiang / ZJU01 / 2013) and H9N2 (A / chicken / Zhejiang / 221 / 2016) diluted virus solution to infect cells (multiplicity of infection is 0.5), and culture in a 37°C incubator containing 5% carbon dioxide for 2 hours;

[0065] (4) Take out the cell plate, discard the virus solution, and wash the cells twice with phosphate buffer solution, then add 200 microliters of virus culture solution to each well, and incubate in a 37°C incubator containing 5% carbon dioxide for 24 hours;

[0066] (5) Take out the cell plate, discard the culture supernatant, and wash the cells once with phosphate buffer solution;

[0067] (6) Add 4% paraformaldehyde to fix the cells in each well of the cell plate, and fix at room temperature for 30 minutes, then wash 3 times with phosphate buffer solution;

[0068] (7) Permeabilize the cells with 0.5% Triton-X100 at room temperature for 30 minutes, and wash 3 times with phosphate buffer solution;

[0069] (8) Block with 3% bovine serum albumin in phosphate buffer solution at room temperature for 1 hour, and discard the bovine serum albumin solution;

[0070] (9) Dilute the monoclonal antibody to 10 micrograms per milliliter with phosphate buffer solution, add 200 microliters to each well, and incubate at 4°C overnight, then wash 3 times with phosphate buffer solution;

[0071] (10) Dilute the fluorescent secondary antibody to 5 micrograms per milliliter with a 1% bovine serum albumin solution, add 200 microliters per well, and incubate in a 37°C incubator for 90 minutes in the dark, and wash 3 times with phosphate buffered saline;

[0072] (11) Stain the cell nucleus with a deoxyribonucleic acid fluorescent dye (4', 6-diamidino-2-phenylindole), incubate at room temperature for 10 minutes in the dark, and wash 3 times with phosphate buffered saline;

[0073] (12) Observe the experimental results under a fluorescence microscope, and green fluorescence indicates a positive result. The results show that the anti-H10 subtype influenza virus monoclonal antibody 1E1 developed in the present research has good specificity for detecting H10 subtype influenza virus.

[0074] It should be understood that the present application is described in conjunction with the preferred embodiments, however, those skilled in the art can make various modifications or changes to the present application after reading the above description of the present application, and these equivalent forms also fall within the scope defined by the claims attached hereto.

Claims

1. An anti-H10 subtype influenza virus hemagglutinin protein monoclonal antibody 1E1, the antibody subtype is IgG2a, κ type, can specifically bind to the H10 subtype influenza virus hemagglutinin protein antigen, the amino acid sequence of the antibody heavy chain variable region is shown in SEQ ID No. 2, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.

4.

2. Use of the monoclonal antibody 1E1 against the hemagglutinin protein of the H10 subtype influenza virus according to claim 1 in the preparation of a fluorescent detection product for H10 subtype influenza virus, wherein the H10 subtype influenza virus is one or more of H10N2, H10N3, H10N5 and H10N7, and the detection is a qualitative detection.

3. The use according to claim 2, characterized in that: The product detects H10 subtype influenza virus in samples through immunofluorescence technology.

4. The use according to claim 3, characterized in that: The sample is body fluid or allantoic fluid.

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