Hybridoma cell secreting anti-pigeon IgG monoclonal antibody and application thereof

Abstract

The application discloses a hybridoma cell secreting anti-pigeon IgG monoclonal antibody and application, the monoclonal antibody prepared by the hybridoma cell line 2H5 is secreted, the monoclonal antibody can specifically recognize the pigeon immunoglobulin IgG as shown in sequence SEQ ID NO:6.The monoclonal antibody provided by the application can be specifically combined with the pigeon immunoglobulin IgG, and has high titer and good affinity, can be used for specifically detecting the pigeon immunoglobulin IgG, and is used for monitoring the antibody level in pigeon body.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a hybridoma cell that secretes anti-pigeon IgG monoclonal antibody and its application. Background Technology

[0002] Immunoglobulins (Ig) are globulins with antibody (Ab) activity or chemical structure, similar to antibody molecules. They are tetrapeptide chains composed of two identical light chains and two identical heavy chains linked by interchain disulfide bonds. Immunoglobulins are classified into five classes: immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), and immunoglobulin E (IgE). Immunoglobulin G (IgG) constitutes the largest proportion, accounting for approximately 70%-80% of the immunoglobulin content in pigeon serum. Following artificial immunization or natural infection, the antibodies produced in pigeons are primarily IgG, which is not only abundant but also has a long duration of action.

[0003] In recent years, the country has increasingly emphasized animal disease prevention and control, highlighting the growing importance of disease monitoring, spring and autumn immunization evaluation, and the creation of clean-up farms for breeding livestock and poultry. Studies have found that pigeons produce specific immunoglobulins when attacked by bacteria, viruses, and other infectious agents. Research has also revealed significant differences in the antigenicity of immunoglobulin IgG among different poultry species. However, existing research and applications of anti-immunoglobulin IgG antibodies primarily target common poultry such as chickens. Targeted research on the preparation and application of anti-pigeon immunoglobulin IgG monoclonal antibodies for smaller-scale farming is scarce. Furthermore, the development technology for secreting anti-pigeon immunoglobulin IgG monoclonal antibodies has significant shortcomings, and commercially available testing materials and application products are not yet on the market.

[0004] Therefore, a hybridoma cell that secretes anti-pigeon IgG monoclonal antibody and its application are urgently needed. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a hybridoma cell that secretes anti-pigeon IgG monoclonal antibodies and its applications.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0007] The first objective of this invention is to provide a hybridoma cell that secretes an anti-pigeon IgG monoclonal antibody, comprising the hybridoma cell line 2H5.

[0008] A second objective of the present invention is to provide a monoclonal antibody secreted by the hybridoma cell line 2H5 as described in claim 1, wherein the monoclonal antibody is capable of specifically recognizing pigeon immunoglobulin IgG as shown in sequence SEQ ID NO: 6.

[0009] Preferably, the monoclonal antibody includes a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2.

[0010] Preferably, both the heavy chain variable region and the light chain variable region are composed of a determining cluster complementary region and a frame region; the determining cluster complementary regions of both the heavy chain variable region and the light chain variable region are composed of CDR1, CDR2 and CDR3.

[0011] The amino acid sequence of CDR1 in the heavy chain variable region is shown as positions 26-33 of SEQ ID No. 1;

[0012] The amino acid sequence of CDR2 in the heavy chain variable region is shown as positions 51-58 of SEQ ID No. 1;

[0013] The amino acid sequence of CDR3 in the heavy chain variable region is shown as positions 97-110 of SEQ ID No. 1;

[0014] The amino acid sequence of CDR1 in the light chain variable region is shown as positions 27-32 of SEQ ID No. 2;

[0015] The amino acid sequence of CDR2 in the light chain variable region is shown as positions 49-53 of SEQ ID No. 2;

[0016] The amino acid sequence of CDR3 in the light chain variable region is shown as positions 89-97 of SEQ ID No. 2.

[0017] A third objective of the present invention is to provide a kit containing the aforementioned monoclonal antibody.

[0018] Preferably, the kit further contains a microplate, pigeon IgG, HRP-labeled anti-pigeon IgG monoclonal antibody, sample diluent, TMB substrate, and 2M H2SO4 stop solution.

[0019] A fourth objective of this invention is to provide the use of the monoclonal antibody as described above in the preparation of a reagent for monitoring antibody levels in pigeons.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The monoclonal antibody provided by this invention can specifically bind to pigeon immunoglobulin IgG, and has high titer and good affinity. It can be used to specifically detect pigeon immunoglobulin IgG and to monitor antibody levels in pigeons. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the SDS-PAGE results in Embodiment 1 of the present invention;

[0023] Figure 2 This is a schematic diagram of the Western blotting analysis results in Embodiment 1 of the present invention;

[0024] Figure 3 This is a schematic diagram of the SDS-PAGE results in Embodiment 2 of the present invention;

[0025] Figure 4 This is a schematic diagram of the Western Blotting results in Embodiment 2 of the present invention;

[0026] Figure 5 This is the standard curve of pigeon immunoglobulin (IgG) in Example 4 of the present invention. Detailed Implementation

[0027] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0028] Example 1: Expression, purification and identification of recombinant pigeon IgG protein.

[0029] Using RT-PCR, primers IgG-Fc-F and IgG-Fc-R were designed to amplify the Fc segment of the pigeon IgG gene (the gene sequence of IgG-Fc-F is shown in SEQ ID NO: 3, the gene sequence of IgG-Fc-R is shown in SEQ ID NO: 4, and the gene sequence of the pigeon IgG-Fc gene is shown in SEQ ID NO: 5). The cloned pigeon IgG-Fc gene was then inserted into the pcDNA3.1 vector containing a His tag, obtaining pigeon IgG-Fc-pcDNA3.1. The pigeon IgG-Fc-pcDNA3.1 plasmid was transformed into DH5α, and positive clones were selected. After amplification and culture of the selected positive clones, the recombinant plasmid pigeon IgG-Fc-pcDNA3.1 was extracted. Restriction digestion analysis with HindIII and BamHI showed that the digested fragments were of the expected size. Recombinant pigeon IgG-Fc-pcDNA3.1 with correct sequencing was transfected into 293 cells using PEI. Cells were cultured in serum-free medium (37℃, 8% CO2) for 6 days. The culture supernatant was collected and purified using a nickel column. Specifically, the cell culture medium was collected, cells were separated by centrifugation at 12000 rpm, and the supernatant was collected and filtered through a 0.45 μm filter. The filtrate was loaded onto a 5 mL Ni-NTA column, washed with 5 column volumes of 10 mmol / L imidazole solution, and then eluted with 300 mmol / L imidazole. The eluted protein solution was collected from each tube. SDS-PAGE was used to identify and quantify the protein purity, which reached 95% at a concentration of 5 mg / mL. The cell expression products were analyzed by SDS-PAGE, and the results are as follows: Figure 1 The results showed a specific expression band around 45kD. Western blotting analysis using Anti-6×His antibody yielded the following results: Figure 2 As shown, only one specific band indicates that the target protein has been obtained. This embodiment uses molecular biology methods to prepare a large amount of protein in one go as a subsequent immunogen, which is simpler to operate and has a higher efficiency in obtaining purified IgG protein.

[0030] Example 2: Preparation, purification and identification of pigeon IgG monoclonal antibody.

[0031] BALB / c mice were immunized subcutaneously with the purified pigeon IgG-Fc recombinant protein from Example 1. Each mouse received 50 μg of antigen per immunization, with an immunization volume of 100 μL. For the first immunization, pigeon IgG-Fc was mixed 1:1 with Freund's complete adjuvant; thereafter, pigeon IgG-Fc was mixed 1:1 with Freund's incomplete adjuvant, and immunizations were repeated every 14 days for a total of 4 immunizations. Finally, a booster immunization was administered intraperitoneally with 30 μg of adjuvant-free pigeon IgG-Fc. Three days later, spleen cells from the immunized mice were harvested, and mouse spleen cells and mouse myeloma cell line SP2 / 0 were fused using PEG-4000. Hybridoma cells were selected on 96-well cell culture plates using HAT selective medium, and cell lines producing anti-pigeon IgG-Fc protein (pigeon IgG monoclonal antibody) were identified by ELISA: hybridoma cell line 1D2, hybridoma cell line 1E6, hybridoma cell line 2G7, and hybridoma cell line 2H5. The obtained hybridoma cell lines were cultured in serum-free medium. The cell culture supernatant was collected, filtered through a 0.45 μm filter, and then 50% ammonium sulfate was added. The mixture was stirred at 4°C, allowed to stand for 1 hour, and the precipitate was collected at 12,000 rpm and redissolved in 50 mmol / L PBS. The crude purified antibody was loaded onto a Protein A-Sepharose affinity chromatography column at a flow rate of 1 mL / min using an AKTA purification system. The column was washed with 5 column volumes of binding buffer (50 mmol / L PBS, pH 7.0), and then eluted with 0.1 mol / L glycine-hydrochloric acid solution (pH 2.7) (each collection tube was pre-neutralized with 1 mol / L pH 9.0 Tris buffer) to obtain the target antibody. The isolated and purified monoclonal antibodies were then identified by SDS-PAGE and Western blotting and named 1D2 monoclonal antibody, 1E6 monoclonal antibody, 2G7 monoclonal antibody, and 2H5 monoclonal antibody, respectively. The SDS-PAGE results are shown below. Figure 3 As shown, the purified monoclonal antibody contained only a heavy chain at 50 kDa and a light chain at 25 kDa. Gray-scale analysis showed that the antibody purity reached 95%. 5 μg of recombinant pigeon IgG-Fc protein was loaded into each well, and the above monoclonal antibody was diluted 1:10000 for Western blotting. The results are shown below. Figure 4 As shown, the antibody specifically reacts with the recombinant protein.

[0032] Example 3: Pigeon immunoglobulin (IgG) pairing screening.

[0033] Pigeon IgG paired antibodies were screened using a double-antibody ELISA method. First, the pigeon IgG monoclonal antibodies obtained in Example 2 were coated onto ELISA plates at 5 μg / mL, and then HRP was labeled onto each plate. Different concentrations of recombinant pigeon IgG-Fc protein were added to the ELISA plates, and after incubation at 37°C for 1 h, the aforementioned HRP-labeled monoclonal antibodies were added, and after incubation at 37°C for 1 h, unbound labeled antibodies were washed away. Finally, chromogenic buffer was added for color development, and the reaction was terminated with 2 mol / L H₂SO₄. The OD₄₅₀ value was measured. The results are shown in Table 1. Ultimately, 1D₂ was selected as the capture antibody, and 2H₅ as the detection antibody.

[0034] Table 1: Pigeon IgG monoclonal antibody pairing results.

[0035]

[0036]

[0037] Example 4: Pigeon Immunoglobulin (IgG) Detection Kit (ELISA).

[0038] This kit uses a sandwich method. Specific anti-pigeon IgG antibody is coated onto a 96-well microplate. Pigeon IgG standards or samples are added to the wells, allowing the pigeon IgG protein in the standards or samples to bind to the anti-pigeon IgG antibody immobilized on the microplate. Then, HRP-labeled pigeon IgG antibody is added, followed by thorough washing and the addition of TMB substrate for color development. TMB is converted to blue under the catalysis of peroxidase, and then to yellow under acidic conditions. The color intensity is positively correlated with the amount of pigeon IgG protein in the sample. OD values ​​are measured at 450 nm using a microplate reader, and a standard curve is plotted. Figure 5 The sample concentration was calculated.

[0039] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A monoclonal antibody, characterized in that, The monoclonal antibody can specifically recognize pigeon immunoglobulin IgG as shown in SEQ ID NO:

6. The monoclonal antibody includes a heavy chain variable region and a light chain variable region. Both the heavy chain variable region and the light chain variable region are composed of a determinant complementary region and a framework region. The determinant complementary regions of the heavy chain variable region and the light chain variable region are composed of CDR1, CDR2 and CDR3. The amino acid sequence of CDR1 in the heavy chain variable region is shown as positions 26-33 of SEQ ID No. 1; The amino acid sequence of CDR2 in the heavy chain variable region is shown as positions 51-58 of SEQ ID No. 1; The amino acid sequence of CDR3 in the heavy chain variable region is shown as positions 97-110 of SEQ ID No. 1; The amino acid sequence of CDR1 in the light chain variable region is shown as positions 27-32 of SEQ ID No. 2; The amino acid sequence of CDR2 in the light chain variable region is shown as positions 49-53 of SEQ ID No. 2; The amino acid sequence of CDR3 in the light chain variable region is shown as positions 89-97 of SEQ ID No.

2.

2. The monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

2.

3. A reagent kit, characterized in that, The kit contains the monoclonal antibody as described in any one of claims 1-2.

4. The reagent kit according to claim 3, characterized in that, The kit also contains a microplate, pigeon IgG, sample diluent, TMB substrate, and 2M H2SO4 stop solution.

5. The use of a monoclonal antibody as described in any one of claims 1-2 in the preparation of a reagent for detecting IgG levels in pigeons.

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