Anti-y series influenza b virus hemagglutinin protein monoclonal antibody 2h6 and its application in detection

Abstract

The application provides a monoclonal antibody 2H6 against hemagglutinin protein of Y-type influenza B virus and application thereof in detection. The heavy chain amino acid sequence of the monoclonal antibody 2H6 is shown in SEQ ID No. 2, and the light chain amino acid sequence is shown in SEQ ID No. 4. The application provides an effective tool for auxiliary diagnosis of influenza B virus infection in a clinical sample, and can be popularized and applied to various detection technologies and clinical and experimental researches.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and relates to preparation and application of anti-influenza B virus hemagglutinin protein monoclonal antibody. The application is obtained by using cell engineering and antibody engineering technology, and is a hybridoma cell line secreting anti-hemagglutinin protein monoclonal antibody. The anti-hemagglutinin protein monoclonal antibody 2H6 is prepared by inducing ascites of the same strain of mice, and is identified as IgG1, κ type. The application of the antibody is realized by affinity purification, immunization method and other technologies. BACKGROUND

[0002] At present, influenza A, B and C viruses can infect humans, among which influenza A and B viruses cause seasonal influenza outbreaks every year. Since 2000, two lineages of influenza B virus, B / Victoria (V lineage) and B / Yamagata (Y lineage), have emerged and have begun to circulate simultaneously or alternately in each influenza season. Generally, the clinical manifestations of influenza B virus are self-limiting diseases, mainly mild symptoms, but the elderly, infants and children, and some patients with chronic diseases are prone to influenza and progress to life-threatening severe acute respiratory disease. Studies have shown that influenza B virus is more likely to infect young people (≤65 years old) than influenza A virus. Although influenza A virus is considered the greatest public health problem, the high incidence and mortality of influenza B virus cannot be ignored, for example, influenza-related complications-myositis are more common in influenza B patients in children and young adults.

[0003] Although studies have shown that the antigenic drift rate of influenza B virus is slower than that of influenza A virus. Unlike influenza A virus, influenza B virus almost only infects humans, which limits the generation of new virus strains by genetic recombination of influenza B virus. However, during the 2012 / 2013 and 2017 / 2018 seasonal influenza epidemic seasons, a large number of influenza B virus infection cases were detected in most European countries, accounting for more than 50% of the total influenza outbreak cases. In addition, there are differences in epidemiology and transmission risk between different lineages of influenza B virus, and the clinical manifestations caused by Y-lineage influenza B virus are more severe than those of V-lineage.

[0004] Isolation of viruses in chicken embryos or MDCK cells is currently recognized as a 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 have high requirements for technology and laboratory and are time-consuming. Due to the development of monoclonal antibody technology, monoclonal antibody-based detection methods are also widely used in virus detection.

[0005] In summary, the development of Y series of influenza B virus monoclonal antibody, the establishment of rapid and sensitive detection method for the prevention and control of virus has important significance. Based on the above background, the hemagglutinin protein of Y series of influenza B virus is selected as the target antigen, the hybridoma cell line stably secreting the monoclonal antibody against the hemagglutinin protein is established by using the fusion hybridoma technology, and the monoclonal antibody is prepared, purified and identified in a large amount. The successful obtaining of the monoclonal antibody lays a material foundation for establishing a new type of Y series of influenza B virus diagnosis method based on immunological technology. Meanwhile, the monoclonal antibody plays an important role in the research on the pathogenesis, prognosis and curative effect determination of diseases.

[0006] The present application uses hybridoma cell technology. The technology fuses the B lymphocytes of immunized mice with myeloma cells SP2 / 0 to establish a hybridoma cell line secreting homogeneous antibodies, also known as monoclonal antibody technology. The technology involves a series of methods such as animal immunization, cell culture, cell fusion, cell cloning culture and immunoassay. SUMMARY

[0007] The purpose of the present application is to provide a Y series of influenza B virus hemagglutinin protein monoclonal antibody which can recognize Y series of influenza B virus. The monoclonal antibody subtype is IgG1, κ type, named 2H6, and can specifically recognize the hemagglutinin protein of Y series of influenza B 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.

[0008] SEQ ID No. 1

[0009] Heavy chain: DNA sequence (366bp)

[0010] Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0011] CAGATCCAGTTGGTGCAATCTGGACCTGAGCTGAAGAAACCTGGAGAGACAGTCAAGATCTCCTG

[0012] CAAGGCTTCTGGGTATACAATCACAGGCTATGGAATGAACTGGGTGAAGCAGGCTCCAGGAAAGGG

[0013] TTTAAAGTGGATGGGCTGGATAAACACCTACACTGGAGAGCCAACATATACTGATGACTTCAAGGGA

[0014] CGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAAAT

[0015] GAGGACATGGCTACATATTTCTGTGCAAGATCGGGAACTACGATACTAGATTACTATTCTATGGACTAC

[0016] TGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA

[0017] SEQ ID No. 2

[0018] Heavy chain: Amino acid sequence (122 AA)

[0019] Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0020] QIQLVQSGPELKKPGETVKISCKASGYTITGYGMNWVKQAPGKGLKWMGWINTYTGEPTYTDDFKGRF

[0021] AFSLETSASTAYLQINNLKNEDMATYFCARSGTTILDYYSMDYWGQGTSVTVSS

[0022] SEQ ID No. 3

[0023] Light chain: DNA sequence (321 bp)

[0024] Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0025] GACATTGTGATGACCCAGTCTCCCAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGTATCACCT

[0026] GCAAGGCCAGTCAGGATGTGAGTTCTGCTGTAGCCTGGTATCAACAGAAACCAGGACAATCTCCTAA

[0027] AGTACTGATTTACTCGGCATCCTACCGGTACACTGGAGTCCCTGATCGCTTCACTGGCAGTGAATCTG

[0028] GGACGGATTTCACTTTCACTATCAGCAGTGTGCAGTCTGAAGACCTGGCAGTTTATTACTGTCAGCA

[0029] ACATTATAGTATTCCTCCCACGTTCGGTGGTGGGACCAAGCTGGAGCTGAAA

[0030] SEQ ID No. 4

[0031] Light chain: Amino acid sequence (107 AA)

[0032] Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0033] DIVMTQSPKFMSTSVGDRVSITCKASQDVSSAVAWYQQKPGQSPKVLIYSASYRYTGVPDRFTGSESGTD

[0034] FTFTISSVQSEDLAVYYCQQHYSIPPTFGGGTKLELK

[0035] The second object of the present application provides a preparation method of a monoclonal antibody against hemagglutinin protein of Y-type influenza B virus, wherein the monoclonal antibody is produced by a hybridoma cell. The hybridoma cell producing the monoclonal antibody is a hybridoma cell line 2H6 obtained by fusion, screening, cloning and stable passage of spleen lymphocytes of an immunized BALB / C mouse and mouse myeloma cells SP2 / 0, and can stably secrete the monoclonal antibody 2H6 against hemagglutinin protein of Y-type influenza B virus.

[0036] The second object of the present application provides a preparation method of a monoclonal antibody against hemagglutinin protein of Y-type influenza B virus, wherein the monoclonal antibody is produced by a hybridoma cell. The hybridoma cell producing the monoclonal antibody is a hybridoma cell line 2H6 obtained by fusion, screening, cloning and stable passage of spleen lymphocytes of an immunized BALB / C mouse and mouse myeloma cells SP2 / 0, and can stably secrete the monoclonal antibody 2H6 against hemagglutinin protein of Y-type influenza B virus.

[0037] (1) Immunization of animals: 6-8 week old BALB / C mice are selected, and the mice are immunized with purified hemagglutinin protein of Y-type influenza B virus (B / Phuket / 3073 / 2013).

[0038] (2) Culture of mouse myeloma cells: mouse myeloma cells SP2 / 0 are cultured and kept in good growth state for cell fusion.

[0039] (3) Cell fusion: PEG-mediated cell fusion method was used. The selected mouse in step (1) was sacrificed to obtain spleen lymphocytes. The SP2 / 0 cells in step (2) were collected, and the two kinds of cells were mixed and centrifuged, and then PEG-mediated cell fusion was performed. The fused cells were diluted appropriately, inoculated into a 96-well culture plate, and cultured under appropriate conditions.

[0040] (4) Screening of hybridoma cells: the above culture was cultured in hypoxanthine-phosphoribosyl transferase selective medium. When the cell colonies grew to an appropriate size, the cell culture supernatant was aspirated for antibody identification, and positive clones were screened.

[0041] (5) Cloning of hybridoma cells: the positive hybridoma cells were cloned by limiting dilution method, and the cells diluted to a certain density were inoculated into a 96-well cell culture plate so that only one cell grew in each well. The supernatant of the well in which the cell colony was formed was taken for enzyme-linked immunosorbent assay to screen and identify positive clones. The culture well with the highest antibody titer and single clone cell growth was selected, and limiting dilution was performed again, and the limiting dilution was continuously performed for more than 4 times, and the hybridoma cell strain stably expressing the anti-Y series of influenza B virus monoclonal antibody was obtained by continuous passage for more than 20 generations. The cloned hybridoma cells were subjected to antibody identification and physicochemical property analysis.

[0042] (6) Preparation of monoclonal antibody ascites: 8-10-week-old BALB / C healthy mice were selected, and each mouse was inoculated with 5×10 6 positive hybridoma cells in PBS buffer. After 7-10 days of inoculation, the mouse abdomen was significantly swollen, and the health status of the mouse was 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, the antibody titer was determined, and the monoclonal antibody in the ascites was purified;

[0043] (7) Purification of monoclonal antibody: protein G agarose affinity purification method was used to purify the monoclonal antibody in the mouse ascites

[0044] (8) A hybridoma cell line producing anti-Y series of influenza B virus hemagglutinin protein monoclonal antibody, i.e. 2H6, was obtained. The 2H6 hybridoma cell line was cloned for 4 times, and the antibody secretion was stable after more than six months of continuous culture. The cell line was stored in liquid nitrogen, and grew well after resuscitation, and the antibody secretion did not decline. The enzyme-linked immunosorbent indirect method experiment showed that the culture supernatant titer of 2H6 was 1:128, and the ascites titer was 1:2048. The monoclonal antibody immunoglobulin subtype analysis showed that the antibody produced by the hybridoma cell was IgG1.

[0045] The present application provides a hybridoma cell for producing a monoclonal antibody, which is a mouse hybridoma cell line 2H6 obtained by fusion, screening, cloning and subculturing of spleen cells of immunized BALB / C mice and mouse myeloma cells SP2 / 0, and can stably secrete a monoclonal antibody 2H6 against hemagglutinin protein of Y-line influenza B virus.

[0046] Another object of the present application is to provide an application of the monoclonal antibody 2H6 in preparation of a detection product for Y-line influenza B virus.

[0047] The detection product is used for detection in a body fluid, allantoic fluid or other environmental sample containing the influenza B virus, and the detection is realized by an immunofluorescence detection method.

[0048] The present application has the advantage of providing a monoclonal antibody against hemagglutinin protein of Y-line influenza B virus, and the preparation method is simple and easy to operate, and more importantly, the monoclonal antibody prepared by the method can have various uses, such as qualitative diagnosis of influenza B virus samples in clinics and laboratories.

[0049] The present application provides a specific monoclonal antibody against Y-line influenza B virus, and the monoclonal antibody is combined with an immunofluorescence technology to detect Y-line influenza B virus in a sample. The above method has the advantages of rapidness, sensitivity and low cost, which can promote earlier and more extensive detection of Y-line influenza B virus and control the spread of the epidemic.

[0050] DRAWINGS

[0051] Figure 1 Analysis of immunoglobulin subtype of the monoclonal antibody 2H6.

[0052] Figure 2 Specificity of the immunofluorescence technology for detecting Y-line influenza B virus. DETAILED DESCRIPTION

[0053] The present application will be further described below in combination with specific examples. It should be understood that the examples are only used for illustrating the present application and are not used for limiting the scope of the present application.

[0054] Example 1. Preparation method of a monoclonal antibody against hemagglutinin protein of influenza B virus

[0055] (1) Mouse immunization: the first immunization, Y-type influenza B virus hemagglutinin protein was mixed with adjuvant 1:1 volume, the total volume was 0.5ml. Each BALB / C mouse 0.1ml (containing influenza B virus hemagglutinin protein antigen 100ug), intramuscular injection in the thigh. The same way to enhance immunity once on the 21st day. The antibody titer reached 1:256000 by ELISA test on the 35th day, and the mouse with the highest antibody titer was selected for intravenous injection of the tail vein for one time, and cell fusion was carried out 3 days later.

[0056] (2) Culture and passage of mouse myeloma cells SP2 / 0: the SP2 / 0 myeloma cell strain from BALB / C mice was cultured and passed with 10% bovine serum DMEM medium, and cultured in a 37°C incubator containing 5% carbon dioxide.

[0057] (3) Cell fusion: BALB / C mouse peritoneal macrophages were used as feeder cells, and the day before fusion, BALB / c mouse peritoneal macrophages were inoculated in 96-well culture plates, and cultured in hypoxanthine-guanine-phosphoribosyltransferase medium containing 20% bovine serum for one day. The spleen of the mouse immunized 3 days after the last boost was taken, and the spleen lymphocytes were separated by pressure water injection method. After centrifugal washing of the cells, they were resuspended with DMEM culture solution. SP2 / 0 cells were collected, centrifuged, washed and resuspended with DMEM culture solution, and counted. 2.5x10 8 immunized mouse spleen lymphocytes were mixed with 2.5x10 7 mouse myeloma cells SP2 / 0. The two cells were mixed, the supernatant was discarded after centrifugation, and the cell mass was loosened by gently rubbing the centrifuge tube with the palm. Slowly add 37°C pre-warmed polyethylene glycol to the fusion tube, gently shake the centrifuge tube, and suck the cells into the fusion tube. After 90 seconds, the cells were blown into the centrifuge tube, and then 1ml of DMEM culture medium was added within 1 minute, 2ml of DMEM culture medium was added within 2 minutes, 7ml of DMEM culture medium was added within 3 minutes, and 40ml of 37°C pre-warmed DMEM culture medium was gradually added within 1 minute. Centrifuge at 800rpm per minute for 10 minutes. Then add hypoxanthine-guanine-phosphoribosyltransferase medium containing 20% bovine serum, and inoculate into 96-well culture plates with feeder cells using a glass dropper. Generally, 2-4 plates of cells are fused each time, and cultured in a 37°C incubator containing 5% carbon dioxide.

[0058] (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. The main steps are as follows: ① 0.01 mol / L pH 9.6 carbonate buffer is used to dilute influenza B virus hemagglutinin protein, then 0.1 ml per well is added to 96-well enzyme-labeled plates, and the protein amount is 20 ng per well, and it is incubated at 4°C overnight; ② the plates are 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 (influenza B virus hemagglutinin protein immune mouse serum), negative control (SP2 / 0 culture supernatant) and blank control, and react at room temperature for 2 hours; ⑥ wash the plates 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 plates 3 times; ⑨ add color developing solution, react at room temperature for 5 minutes in the dark; ⑩ 2 mol / L sulfuric acid is used to terminate the reaction; and the optical density value is measured at 450 nm, and the value is divided by the negative value, and ≥2.1 is positive.

[0059] (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 with 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 again for limited dilution, and the limited dilution is continuously carried out for more than 4 times, and the hybridoma cell strain stably and highly expressing anti-Y series influenza B virus monoclonal antibody is obtained after continuous passage for more than 20 generations.

[0060] (6) Preparation of monoclonal antibody ascites: 8-10 week old BALB / C healthy mice are selected, and each mouse is inoculated with 5×10 6 positive hybridoma cells in PBS buffer. The mouse abdomen is significantly swollen 7-10 days after inoculation of the cells, and the health status of the mouse is closely observed. The mouse ascites is collected when the ascites is as much as possible.

[0061] (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 3-4 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 8-10 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 8-10 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 in each tube. ⑦ The absorbance of each tube of eluate was measured at 280 nm, and the eluate with a protein content of more 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 10-20 minutes at 4°C until the final volume of the antibody eluate was about 1 ml. 10 ml of 0.1 M phosphate buffer (pH 7.4) was added, and centrifuged at 10,000 rpm for 10-20 minutes at 8°C. The antibody was concentrated to a final volume of about 1 ml by centrifugation for the last time, and the concentrated antibody was taken up 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 in small tubes, and stored in a freezer for use.

[0062] (8) Subtype identification of the monoclonal antibody: The mouse monoclonal antibody immunoglobulin typing kit of Bio-Rad Company was used for analysis. The purified monoclonal antibody was diluted appropriately, and then subjected to detection. The operation was strictly performed according to the instructions of the kit. The test results showed that the monoclonal antibody secreted by the 2H6 hybridoma cell was of the IgG1, κ type.

[0063] The results are shown in the accompanying Figure 1 .

[0064] Example 2. Qualitative detection of influenza B virus using the monoclonal antibody

[0065] The anti-Y series influenza B virus hemagglutinin protein monoclonal antibody prepared in the present application can be used for qualitative detection of the Y series influenza B virus, and the identification method can be achieved by the following method:

[0066] Influenza B virus immunofluorescence detection method:

[0067] (1) MDCK cells were seeded in a 48-well plate at a density of 4 x 10 4 cells per well one day in advance, and the cells were grown to 70%, and then used;

[0068] (2) Take out the cell plate with cells, discard the culture supernatant, and wash once with phosphate buffer solution for standby;

[0069] (3) Determine the specificity of the influenza B virus immunofluorescence detection method for detecting Y series influenza B virus: dilute the virus with phosphate buffer solution, including B / Phuket / 3073 / 2013 (Y series), B / Shang Hai / 361 / 2002 (Y series), B / Florida / 4 / 2006 (Y series), B / Hubei-Wujiagang / 158 / 2009 (Y series), B / Massachusetts / 2 / 2012 (Y series), B / Malaysia / 2506 / 2004 (V series), B / Brisbane / 60 / 2008 (V series), B / Washington / 02 / 2019 (V series), A / Michigan / 45 / 2015 (Influenza A H1N1 virus) and A / Texas / 50 / 2012 (Influenza A H3N2 virus), and infect the cells after dilution (multiplicity of infection is 0.5), and culture in a 37°C incubator containing 5% carbon dioxide for 2 hours;

[0070] (4) Take out the cell plate, discard the virus solution, wash the cells twice with phosphate buffer solution, and then add 200 microliters of virus culture solution to each well, and culture in a 37°C incubator containing 5% carbon dioxide for 16 hours;

[0071] (5) Take out the cell plate, discard the culture supernatant, and wash the cells once with phosphate buffer solution;

[0072] (6) Add 4% paraformaldehyde to fix the cells in each well of the cell plate, and fix at room temperature for 30 minutes, and wash 3 times with phosphate buffer solution;

[0073] (7) Permeabilize the cells with 0.5% Triton-X100 at room temperature for 30 minutes, and wash 3 times with phosphate buffer solution;

[0074] (8) Block with 3% bovine serum albumin in phosphate buffer solution at room temperature for 1 hour, and discard the bovine serum albumin solution;

[0075] (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, and wash 3 times with phosphate buffer solution;

[0076] (10) Dilute the fluorescent secondary antibody to 5 micrograms per milliliter with 1% bovine serum albumin solution, add 200 microliters to each well, and incubate at 37°C in the dark for 90 minutes, and wash 3 times with phosphate buffer solution;

[0077] (11) Stain the cell nucleus with deoxyribonucleic acid fluorescent dye (4', 6-diamidino-2-phenylindole), incubate for 10 minutes at room temperature in the dark, and wash 3 times with phosphate buffer;

[0078] (12) Observe the experimental results under a fluorescence microscope, and green fluorescence is observed to be positive. The detection results show that the anti-Y type influenza B virus monoclonal antibody 2H6 developed based on the present research has good specificity for detecting the Y type influenza B virus.

[0079] It should be understood that the present application is described in connection with the preferred embodiments, but 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 to the present application.

Claims

1. An anti-Y strain influenza B virus hemagglutinin protein monoclonal antibody 2H6, the antibody subtype is IgG1, κ type, can specifically bind to the influenza B virus hemagglutinin protein antigen, the antibody heavy chain variable region amino acid sequence is shown in SEQ ID No. 2, and the light chain variable region amino acid sequence is shown in SEQ ID No.

4.

2. Use of the monoclonal antibody 2H6 against the hemagglutinin protein of Y-series influenza B virus according to claim 1 in the preparation of a Y-series influenza B virus detection product.

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

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

Images