Anti-h6 subtype avian influenza virus hemagglutinin protein monoclonal antibody 1h1 and application thereof

Abstract

The present application belongs to the field of biotechnology, and relates to anti-H6 subtype avian influenza virus hemagglutinin protein monoclonal antibody 1H1 and application thereof. The hybridoma cell line secreting hemagglutinin monoclonal antibody is obtained by using cell engineering and antibody engineering technology. The monoclonal antibody 1H1 against hemagglutinin protein is prepared by inducing ascites of the same strain of mice. The monoclonal antibody 1H1 is identified as IgG1, kappa type. The application of the antibody is realized by affinity purification and immunization technology. The present application has the advantages that the preparation method is simple and easy to operate. More importantly, the monoclonal antibody prepared by the method has multiple uses. The present application provides an effective tool for the auxiliary diagnosis of H6 subtype avian influenza virus infection in clinical samples, and can be applied to various detection technologies, clinical and experimental researches.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and relates to anti-H6 subtype avian influenza virus hemagglutinin protein monoclonal antibody 1H1 and its application. The application is obtained by using cell engineering and antibody engineering technology, and is a hybridoma cell line secreting hemagglutinin monoclonal antibody. The monoclonal antibody 1H1 against hemagglutinin protein is prepared by inducing ascites in mice of the same strain, and is identified as IgG1, κ type. The application of the antibody is realized by affinity purification and immunization technology. BACKGROUND

[0002] Avian influenza virus is a virus with high variability and potential cross-species transmission ability, which poses a significant threat to poultry farming and public health safety. As one of the important subtypes, H6 subtype avian influenza virus has attracted widespread attention due to its wide prevalence and potential zoonotic risk.

[0003] Currently, the diagnosis methods for H6 subtype avian influenza virus infection mainly include virus isolation and identification, serological methods and molecular biology methods such as reverse transcriptase polymerase chain reaction and real-time quantitative polymerase chain reaction. However, these methods require high technology and laboratory and are time-consuming. It is imperative to develop a rapid and sensitive H6 subtype virus detection product, which is conducive to promoting earlier and more extensive detection of H6 subtype avian influenza virus infection and controlling the spread. Although various detection technologies and vaccine development strategies have been proposed, there are still certain limitations in existing vaccines and treatment methods, especially in the case of rapid virus variation. Therefore, specific monoclonal antibodies become an important research direction, and detection methods based on monoclonal antibodies are widely used in virus detection. Therefore, the present application aims to describe a specific monoclonal antibody against H6 subtype avian influenza virus, which can be combined with immunofluorescence technology to detect H6 subtype avian influenza virus in samples. Through immunofluorescence technology, the fluorescence signal generated by the specific binding of viral antigens in the sample to the monoclonal antibody can be observed, thereby realizing rapid and sensitive detection of the virus.

[0004] Based on the above background, the present application selects H6 subtype avian influenza virus hemagglutinin protein as the target antigen, and uses fusion hybridoma technology to establish a hybridoma cell line stably secreting anti-H6 subtype avian influenza virus monoclonal antibody. The monoclonal antibodies are prepared, purified and identified in large quantities. The successful preparation of the monoclonal antibodies lays a material foundation for the establishment of a diagnostic method for H6 subtype avian influenza virus based on immunofluorescence technology. At the same time, it plays an important role in the research of disease pathogenesis, prognosis and efficacy determination, etc.

[0005] In addition, the monoclonal antibody can also be applied to other immunological techniques, such as immunoblotting, enzyme-linked immunosorbent assay, etc., to provide multiple choices for detection of the H6 subtype avian influenza virus and lay a material foundation for immunological technology-based diagnosis. SUMMARY

[0006] The purpose of the present application is to provide an anti-H6 subtype avian influenza virus hemagglutinin protein monoclonal antibody which can recognize the H6 subtype avian influenza virus.

[0007] The anti-H6 subtype avian influenza virus hemagglutinin protein monoclonal antibody 1H1 is of IgG1, κ type, can specifically bind to the H6 subtype avian influenza virus hemagglutinin protein antigen, and 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 (363bp)

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

[0011] CAGGTCCAACTGCAGCAGCCTGGGTCTGTGCTGGTGAGGCCTGGAGCTTCAGTGAAGCTGTCCTGCAA

[0012] GGCTTCTGGCTTCACCTTCACCAGCTCCTTGATACACTGGACAAAACAGAGGCCTGGACAAGGCCTTGA

[0013] GTGGATTGGAGAGATTCATCCTAATAGTGGTAATACTAACTACAATGAGAAGTTCAAGGGCAAGGCCACA

[0014] CTGACTGTAGACACATCCTCCAGCACAGCCTACGTGGATCTCAGCAGCCTGACATCTGAGGACTCTGCG

[0015] GTCTATTACTGTACAAGAGGGTATGATGGTTACTACGTACAAGCTATGGACTACTGGGGTCAAGGAACC

[0016] TCAGTCACCGTCTCCTCA

[0017] SEQ ID No. 2

[0018] Heavy chain: Amino acid sequence (121 AA)

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

[0020] QVQLQQPGSVLVRPGASVKLSCKASGFTFTSSLIHWTKQRPGQGLEWIGEIHPNSGNTNYNEKFKGKATLTVDTSSS

[0021] TAYVDLSSLTSEDSAVYYCTRGYDGYYVQAMDYWGQGTSVTVSS

[0022] SEQ ID No. 3

[0023] Light chain: DNA sequence (336 bp)

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

[0025] GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCA

[0026] GATCTAGTCAGAGCATTGTACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCC

[0027] AGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCA

[0028] GTGGATCAGGGACAGATTTCACACTCAAGATCAGTAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTG

[0029] CTTTCAAGGTTCACATGTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAASEQ ID No. 4

[0030] Light chain: Amino acid sequence (112 AA)

[0031] Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0032] DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTL

[0033] KISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIK

[0034] The second object of the present application provides a preparation method of a monoclonal antibody against H6 subtype avian influenza virus hemagglutinin protein, which is achieved by the following steps and technical solutions:

[0035] (1) Immunization of animals: 6-week-old BALB / C mice are selected, and the mice are immunized with purified H6N1 subtype avian influenza virus (A / chicken / Zhejiang / 1664 / 2017) hemagglutinin protein.

[0036] (2) Culture of mouse myeloma cells: mouse myeloma cells SP2 / 0 are cultured and kept in good growth state for cell fusion.

[0037] (3) Cell fusion: polyethylene glycol fusion method is used. BALB / C mouse peritoneal macrophages are used as feeder cells, and on the day before fusion, BALB / C mouse peritoneal macrophages are inoculated into 96-well culture plates, and the hypoxanthine-guanine-phosphoribosyl transferase culture medium containing 20% bovine serum is cultured for one day. The mice prepared in (1) are sacrificed, and the spleen lymphocytes are obtained. The mouse myeloma cells in (2) are collected. The above two cells are mixed and centrifuged, and then polyethylene glycol is used to mediate cell fusion. The fused cells are appropriately diluted and inoculated into the feeder cell culture plate, and cultured under appropriate conditions.

[0038] (4) Screening of hybridoma cells: the above culture is cultured in a hypoxanthine-phosphoribosyl transferase selective culture medium. When the cell colonies grow to an appropriate size, the cell culture supernatant is aspirated for antibody identification and screening of positive clones.

[0039] (5) Cloning of hybridoma cells: the hybridoma cells are cloned by limited dilution method, and the cells diluted to a certain density are inoculated into 96-well plates to make only one cell grow in each well. The wells forming cell colonies are taken for enzyme-linked immunosorbent assay to identify positive clones. The culture well with the highest antibody titer and single clone cell growth is selected, and limited dilution is carried out again, and the limited dilution is continuously carried out for more than 4 times, and the hybridoma cell strain stably expressing the anti-H6N1 subtype influenza virus monoclonal antibody is obtained by continuous passage for more than 20 generations. The cloned hybridoma cells are subjected to antibody identification and physicochemical property analysis.

[0040] (6) Induction of monoclonal antibody ascites: 8-week-old BALB / C healthy mice are selected, and 5 million positive hybridoma cells are inoculated into the abdomen of each mouse. About 10 days after inoculation of the cells, the abdomen of the mouse is significantly swollen, and the health status and abdominal signs of the mouse are closely observed. When the ascites is as much as possible, the ascites is collected and centrifuged, the antibody titer is determined, and the monoclonal antibody in the ascites is purified.

[0041] (7) Purification of monoclonal antibody: the monoclonal antibody in the ascites is purified by Protein G affinity purification method.

[0042] (8) The present application obtains a hybridoma cell line producing an anti-H6 subtype avian influenza virus monoclonal antibody, i.e. 1H1. The 1H1 hybridoma cell line is cloned for 4 times, and is continuously cultured for more than six months, and the antibody secretion is stable. The cell strain is stored in liquid nitrogen, and grows well after resuscitation, and the antibody secretion does not decline. The enzyme-linked immunosorbent indirect assay experiment shows that the culture supernatant titer of 1H1 is 1:128, and the ascites titer is 1:6400. The monoclonal antibody immunoglobulin subtype analysis shows that the antibody type produced by the hybridoma cell is IgG1.

[0043] The present application provides a hybridoma cell producing a monoclonal antibody, which is a mouse hybridoma cell line 1H1 obtained by fusion, screening, cloning, passage and repeated freezing and resuscitation of the spleen cells of immunized BALB / C mice and mouse myeloma cells SP2 / 0, and can stably secrete the monoclonal antibody 1H1 against the hemagglutinin protein of H6 subtype avian influenza virus.

[0044] Another object of the present application is to provide the application of the monoclonal antibody 1H1 in the preparation of a product for detecting H6 subtype avian influenza virus by fluorescence. The detection of H6 subtype avian influenza virus in body fluid, allantoic fluid or other environmental samples is realized by immunofluorescence detection.

[0045] The present application provides a monoclonal antibody 1H1 of hemagglutinin protein of H6 subtype avian influenza virus, and a method for detecting H6 subtype avian influenza virus by immunofluorescence technique. The present application has the advantages of simple preparation method, and more importantly, the monoclonal antibody prepared by the method can have multiple uses, providing an effective tool for the auxiliary diagnosis of H6 subtype avian influenza virus infection in clinical samples, and can be applied to various detection techniques and clinical and experimental researches.

[0046] Drawings

[0047] Figure 1 Analysis of immunoglobulin subtype of the monoclonal antibody 1H1.

[0048] Figure 2 Specificity of the immunofluorescence technique for detecting H6 subtype avian influenza virus. DETAILED DESCRIPTION

[0049] The present application will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not used to limit the scope of the present application.

[0050] Example 1. Preparation method of monoclonal antibody against hemagglutinin protein of H6 subtype avian influenza virus

[0051] (1) Immunization of mice: for the first immunization, the hemagglutinin protein of H6 subtype avian influenza virus was mixed with an equal volume of adjuvant uniformly, and the total volume was 600 microliters. Each BALB / C mouse was injected with 0.1 milliliter (containing 5 micrograms of hemagglutinin protein antigen of H6 subtype avian influenza virus) into the thigh muscle. On the 21st day, the same method was used for booster immunization. On the 35th day, a small amount of tail blood was collected for enzyme-linked immunosorbent assay, and the antibody titer reached 1:16000. Then, booster immunization was performed once through tail vein injection, and cell fusion was performed 3 days later.

[0052] (2) Culture of mouse myeloma cells SP2 / 0: the SP2 / 0 myeloma cell strain from BALB / C mice was cultured in a 10% bovine serum DMEM medium, and was cultured in a 37℃ incubator with 5% carbon dioxide saturation. The cells were subcultured one day before fusion to ensure that the cells were in the logarithmic growth phase at the time of fusion.

[0053] (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 in hypoxanthine-guanine-phosphoribosyl transferase medium containing 20% bovine serum for one day. Three days after the last boost, the spleen of the mouse was taken out and the spleen cells were isolated by pressure water injection method. After centrifugal washing twice, the cells were resuspended with culture medium. SP2 / 0 cells were collected, centrifuged, washed twice, and resuspended with culture medium as SP2 / 0 cells to be fused. One hundred million immune mouse spleen lymphocytes were mixed with 20 million mouse myeloma cells SP2 / 0, and the fusion was carried out under the action of polyethylene glycol. After mixing the two kinds of cells, they were washed once, centrifuged, and the supernatant was discarded. The cells were suspended by tapping the tube wall, and 0.9 ml of 37°C pre-warmed polyethylene glycol was added dropwise to the cell precipitate within 90 seconds. During this process, the centrifuge tube was gently shaken, but not blown, and then it was left for 1 minute. Then, 1 ml of serum-free DMEM was added within the first minute, 2 ml of serum-free DMEM was added within the second minute, 7 ml of serum-free DMEM was added within the third minute, and 40 ml of 37°C pre-warmed serum-free DMEM medium was gradually added within the next 1 minute. The cells were centrifuged at 1000 rpm for 10 minutes. Then, the medium was added and inoculated into 96-well culture plates with feeder cells. Generally, the cells for each fusion were plated into 2 plates, and then cultured in a cell incubator.

[0054] (4) Screening of hybridoma cells: The culture medium was changed every 4 days (containing hypoxanthine-guanine-phosphoribosyl transferase) once, and after 10 days, the culture medium containing hypoxanthine-phosphoribosyl transferase was used. The hybridoma cells after fusion were cultured in selective culture medium containing hypoxanthine-phosphoribosyl transferase for about two weeks. The culture supernatant was collected for enzyme-linked immunosorbent assay to screen positive clones. The positive hybridoma clones were screened by enzyme-linked immunosorbent assay indirect method. The main steps are as follows: ① H6 subtype hemagglutinin protein was diluted with 0.01 mol / L pH 9.6 carbonate buffer to a concentration of 20 ng / well, and 0.1 ml was added to each well of a 96-well enzyme-labeled plate and incubated at 4°C overnight; ② The plate was washed three times with 0.01 mol / L pH 7.4 phosphate buffer containing Tween 20; ③ The plate was blocked with 5% bovine serum albumin in 0.01 mol / L pH 7.4 phosphate buffer for 2 hours; ④ The plate was washed as above; ⑤ Hybridoma culture supernatant was added, 0.1 ml per well, and positive control (immune mouse serum), negative control (SP2 / 0 culture supernatant) and blank control were set up, and incubated at room temperature for 2 hours; ⑥ The plate was washed; ⑦ Horseradish peroxidase-labeled goat anti-mouse IgG was added at a dilution of 1:6000, 0.1 ml per well, and incubated at room temperature for 1 hour; ⑧ The plate was washed; ⑨ The substrate was added and incubated at room temperature for 5 minutes; ⑩ The reaction was terminated with 2 mol / L sulfuric acid; and 450 nm was used to measure the optical density value. The value was divided by the negative value, and ≥2.1 was considered positive.

[0055] (5) Cloning of hybridoma cells: The cloning of hybridoma cells was performed by limiting dilution method. The hybridoma cells which were positive for antibody detection were selected and proliferated. The cells were accurately counted and diluted to 10 cells per milliliter with complete DMEM medium. The cell suspension was inoculated into 96-well culture plates with feeder cells at 0.1 milliliter per well. After 10 days, the cell growth was observed and the antibody level in the supernatant was detected. Five wells with the highest antibody titer and single clone cell growth were selected for further limiting dilution. The limiting dilution was continuously performed for more than 4 times and the hybridoma cell strain stably expressing anti-H6 subtype influenza virus monoclonal antibody was obtained after 20 generations of continuous passage.

[0056] (6) Ascites induction: 8-week-old BALB / C healthy mice were selected and inoculated with 5 million positive hybridoma cells in the abdomen. About 7 days after inoculation, the mice showed obvious abdominal distension. The health status and abdominal signs of the mice were closely observed. The ascites was collected before the mice were close to death. The ascites was centrifuged, the antibody titer was determined, and the monoclonal antibody in the ascites was purified.

[0057] (7) Purification of monoclonal antibody: The affinity purification method (Protein G cross-linked Sepharose) was used to purify the monoclonal antibody in the ascites. ① The ascites was diluted 3 times with cold binding buffer and centrifuged at 10,000 rpm for 15 minutes at 4°C to remove the precipitate. ② The affinity purification column preloaded with Sepharose-Protein G was washed with 10 times the column bed volume of binding buffer. ③ The diluted ascites was loaded onto the column at a flow rate of 10 drops per minute. ④ The ascites eluted was loaded onto the column again. ⑤ The column was washed with 20 times the column bed volume of binding buffer until the absorbance of the eluate at 280 nm was less than 0.01. ⑥ The bound monoclonal antibody was eluted with elution buffer at a flow rate of 10 drops per minute. The eluate was collected in a collection tube pre-added with 0.1 milliliter of potassium phosphate buffer (pH 7.9). Each tube collected 0.5 milliliter of antibody-containing eluate, and more than 20 tubes were collected. ⑦ The absorbance of each tube of eluate was detected at 280 nm, and the eluate with an absorbance greater than 0.2 was collected. ⑧ The collected eluate was placed in a dialysis bag and dialyzed in 0.1 mol / L pH 7.4 phosphate buffer. The buffer was changed every 6 hours for 24 hours. ⑨ The dialyzed antibody solution was diluted and the protein content was measured at 280 nm. ⑩ The purified antibody was divided into small tubes and stored in a low-temperature refrigerator for use.

[0058] (8) Subtype identification of monoclonal antibody: The mouse monoclonal antibody immunoglobulin typing kit of Bio-Rad Company was used for analysis. The purified monoclonal antibody was appropriately diluted and detected. The operation was strictly performed according to the instructions of the kit. The test results showed that the monoclonal antibody secreted by 1H1 hybridoma cells was IgG1, κ type.

[0059] Results are shown in the following table Figure 1 .

[0060] Example 2. Qualitative detection of H6 subtype avian influenza virus using the monoclonal antibody

[0061] The H6 subtype avian influenza virus hemagglutinin protein monoclonal antibody prepared by the present application can be used for qualitative detection of H6 subtype avian influenza virus, and the identification method can be realized by the following method:

[0062] H6 subtype avian influenza virus immunofluorescence detection method:

[0063] (1) MDCK cells were seeded in a 48-well plate one day in advance at a density of 40,000 cells per well, and the cells were grown to 70% for standby;

[0064] (2) Take out the cell plate with cells plated, discard the culture supernatant, and wash once with phosphate buffered saline for standby;

[0065] (3) Determine the specificity of the immunofluorescence detection method for detecting H6 subtype avian influenza virus: dilute the virus with virus diluent, including H6N1 (A / chicken / Zhejiang / 1664 / 2017), H6N6 (A / chicken / Zhejiang / 727018 / 2014), H6N2

[0066] (A / chicken / Zhejiang / 727031 / 2014), H9N2 (A / chicken / Zhejiang / 221 / 2016), H10N7

[0067] (A / chicken / Zhejiang / 2CP8 / 2014), H7N9 (A / chicken / Zhejiang / ZJU01 / 2013), H1N1 (A / Michigan / 45 / 2015), H3N2 (A / Texas / 50 / 2012) and influenza B virus (B / Phuket / 3073 / 2013), the diluted virus solution infects the cells (multiplicity of infection is 0.5), and is cultured in a 37°C incubator containing 5% carbon dioxide for 2 hours;

[0068] (4) Take out the cell plate, discard the virus solution, wash the cells twice with phosphate buffered saline, and 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;

[0069] (5) Take out the cell plate, discard the culture supernatant, and wash the cells once with phosphate buffered saline;

[0070] (6) Add 4% paraformaldehyde to fix cells in each well of the cell plate, and fix at room temperature for 30 minutes, and wash with phosphate buffer for 3 times;

[0071] (7) Permeate cells with 0.5% Triton-X100, and permeate at room temperature for 30 minutes, and wash with phosphate buffer for 3 times;

[0072] (8) Block with phosphate buffer containing 3% bovine serum albumin, block at room temperature for 1 hour, and discard the bovine serum albumin solution;

[0073] (9) Dilute the monoclonal antibody to 10 micrograms per milliliter with phosphate buffer, and add 200 microliters to each well, and incubate at 4°C overnight, and wash with phosphate buffer for 3 times;

[0074] (10) Dilute the fluorescent secondary antibody to 5 micrograms per milliliter with 1% bovine serum albumin solution, and add 200 microliters to each well, and incubate in a 37°C incubator for 90 minutes in the dark, and wash with phosphate buffer for 3 times;

[0075] (11) Stain the cell nucleus with deoxyribonucleic acid fluorescent dye (4', 6-diamidino-2-phenylindole), and incubate at room temperature in the dark for 10 minutes, and wash with phosphate buffer for 3 times;

[0076] (12) Observe the experimental results under a fluorescence microscope, and green fluorescence indicates a positive result. The detection results show that the anti-H6 subtype avian influenza virus monoclonal antibody 1H1 developed based on the present research can detect H6 subtype avian influenza virus, and has good specificity.

[0077] The results are shown in the following table Figure 2 .

[0078] It should be understood that the present application is described in conjunction with the best embodiment, 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 appended claims of the present application.

Claims

1. A monoclonal antibody 1H1 against the hemagglutinin protein of the H6 subtype avian influenza virus. The monoclonal antibody is of IgG1 and κ subtype and can specifically bind to the hemagglutinin protein antigen of the H6 subtype avian influenza virus. 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.

2. Use of the monoclonal antibody 1H1 against the hemagglutinin protein of H6 subtype avian influenza virus according to claim 1 in the preparation of H6 subtype avian influenza virus detection products.

3. The use according to claim 2, characterized in that: The product detects H6 subtype avian influenza 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.

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