Pig IL-15 monoclonal antibody as well as preparation method and application thereof

Abstract

The invention provides a porcine IL-15 monoclonal antibody as well as a preparation method and application thereof, and relates to the technical field of immunological detection. The porcine IL-15 monoclonal antibody is prepared from a hybridoma cell strain with the preservation number of CCTCC NO: C2024127, a double-antibody sandwich ELISA detection method for quantitatively detecting the content of porcine IL-15 in a biological sample is established through the antibody, the method has high sensitivity, specificity and accuracy, an important tool is provided for relevant research of porcine IL-15, and the porcine IL-15 monoclonal antibody can be applied to quantitative detection of porcine IL-15. Good application prospects are realized.

Description

Technical Field

[0001] The present invention relates to the technical field of immunological detection, and in particular to a porcine IL-15 monoclonal antibody and a preparation method and application thereof. Background Art

[0002] Interleukin-15 (IL-15) is a pleiotropic cytokine discovered by Grabstein in 1994 when testing the culture supernatant of the simian kidney epithelial cell line CV-1 / EBNA. It is produced by a variety of cells such as stromal cells, monocytes, macrophages, dendritic cells, epithelial cells, etc. IL-15 mRNA exists in various immune or non-immune cells and tissues, but its protein is mainly confined to monocytes, macrophages and dendritic cells. IL-15 is a 14 to 15 ku member of the 4α-helical bundle cytokine family and signals by binding to the IL-15Rα / IL-2Rβ / γc heterotrimeric receptor, where IL-15 receptor α (IL-15Rα, CD215) is the specific receptor for IL-15, IL-2Rβ (CD122, also known as IL-15Rβ) is shared with IL-2, and γc (CD132) is shared with IL-4, IL-7, IL-9, and IL-21.

[0003] IL-15 is a pleiotropic cytokine that can promote the proliferation of natural killer cells (NK cells), NK-T, γδT and memory CD8 T cells, and plays a key role in maintaining the survival, homeostatic proliferation and renewal of CD8 T cells. In addition, IL-15 is a hematopoietic factor for the generation of NK cells, which can replace the bone marrow environment to promote NK cell differentiation and maturation, regulate NK cell activation and maintain NK cell homeostasis. IL-15 is an important immune molecule for studying the interaction between pathogenic microorganisms and hosts and evaluating the status of cellular immunity.

[0004] However, the prior art lacks detection methods related to porcine IL-15. Summary of the invention

[0005] In view of this, the present invention provides a porcine IL-15 monoclonal antibody and a preparation method and application thereof. The present invention uses a hybridoma cell line with a deposit number of CCTCC NO: C2024127 to prepare a porcine IL-15 monoclonal antibody, and establishes a double antibody sandwich ELISA detection method for quantitatively detecting the content of porcine IL-15 in biological samples through the antibody. The method has high sensitivity, specificity and accuracy, provides an important tool for porcine IL-15 related research, and has a good application prospect.

[0006] The porcine IL-15 specific monoclonal antibody of the present invention is prepared by using a hybridoma cell line with a deposit number of CCTCC NO: C2024127.

[0007] The method for preparing the porcine IL-15 monoclonal antibody of the present invention comprises the following steps:

[0008] S21, extract nucleic acid from porcine inguinal lymph node tissue, reverse transcribe cDNA and perform PCR amplification. The porcine IL-15 primers are:

[0009] ePig IL-15U: 5′-CTAGAATTCGCCACCATGAGAATTTTAAAAC-3′

[0010] ePorcine IL-15L: 5′-GACCTCGAGTCAAGAAGGGTTGATG-3′;

[0011] The PCR product, pET28α(+) and pcDNA3.1(+) were digested with restriction endonucleases, the digested PCR product was connected with the plasmid to construct the recombinant plasmids pET-28α-porcine IL-15 and pcDNA3.1-porcine IL-15, the pET-28α-porcine IL-15 recombinant plasmid was transformed into Escherichia coli Rosetta (DE3) competent cells, and the pcDNA3.1-porcine IL-15 recombinant plasmid was transfected into PK-15 cells;

[0012] S22, recombinant porcine IL-15 protein, using purified porcine IL-15 protein to boost immunization after mouse immunization, preparing spleen cell suspension and fusing spleen cells with SP2 / 0 myeloma cells, screening hybridoma cell lines that stably secrete porcine IL-15-specific monoclonal antibodies (deposited in China Center for Type Culture Collection, deposit address is Wuhan University, Wuhan, China, deposit number is CCTCC NO: C2024127, deposit date is October 31, 2024, classified and named as hybridoma cell line Hybridoma cell line Pocine-IL-15-2C10), preparing ascites, purifying antibodies using the ammonium sulfate method, and completing the preparation of porcine IL-15 monoclonal antibodies. Preferably, the restriction endonuclease is EcoR I and XOt I.

[0013] Preferably, after the recombinant plasmid is constructed, the recombinant plasmid is digested with enzymes and sequenced.

[0014] Preferably, the pcDNA3.1-porcine IL-15 recombinant plasmid is transfected by liposome transfection.

[0015] Preferably, SDS-PAGE and Western Blot analysis are used to demonstrate the expression of porcine IL-15 protein in E. coli.

[0016] Preferably, after screening out hybridoma cell lines that stably secrete porcine IL-15-specific monoclonal antibodies, at least one of subcloning and antibody titer determination, monoclonal antibody class / subclass identification, monoclonal antibody identification, and specificity detection is performed. More preferably, Western Blot is used to confirm the expression of recombinant porcine IL-15 protein and the specificity of the monoclonal antibody, and the intracellular fluorescent signal is observed by fluorescence microscopy to verify the expression of porcine IL-15 in PK-15 cells and the binding ability of the monoclonal antibody; cross-reactivity detection is used to ensure the specificity of the monoclonal antibody to porcine IL-15 and avoid nonspecific binding with other cytokines.

[0017] The present invention also provides a double antibody sandwich ELISA detection method for quantitatively detecting the content of porcine IL-15 in a biological sample, and the method is performed using the porcine IL-15 monoclonal antibody described above.

[0018] Preferably, the biological sample is at least one of serum, tissue fluid and cell culture.

[0019] The method for establishing the pig IL-15 double antibody sandwich ELISA detection of the present invention comprises the following steps:

[0020] Preferably, the diabody comprises a capture antibody that can specifically bind to porcine IL-15 and an enzyme-labeled antibody.

[0021] Preferably, the capture antibody is the monoclonal antibody 2D10, and the enzyme-labeled antibody is the monoclonal antibody 2C10.

[0022] The establishment and evaluation method of the porcine IL-15 double antibody sandwich ELISA detection of the present invention comprises the following steps: S11, affinity determination of monoclonal antibodies;

[0023] S12, performing a monoclonal antibody pairing test;

[0024] S13, determination of antibody coating concentration;

[0025] S14. Determination of blocking solution;

[0026] S15. Determination of dilution multiple of enzyme-labeled monoclonal antibody;

[0027] S16, determining the minimum detectable concentration of porcine IL-15;

[0028] S17, evaluate the specificity of the double antibody sandwich ELISA method;

[0029] S18. Evaluate the repeatability of the double antibody sandwich ELISA method.

[0030] Preferably, the steps of the monoclonal antibody pairing test are as follows: labeling the purified monoclonal antibody, selecting an unlabeled antibody as a capture antibody, coating an ELISA plate, incubating, washing the ELISA plate, adding a blocking solution for blocking, adding recombinant porcine IL-15 protein, setting up a negative control, incubating, washing the plate, adding different combinations of HRP-labeled antibodies, incubating, adding a color developing solution for color development, adding a stop solution to stop the reaction, and 450 nm The best antibody combination was selected by calculating the P / N value.

[0031] Preferably, the antibody coating concentration is the concentration at which the P / N value is the highest. In some specific embodiments of the present invention, the antibody coating concentration is 5 μg / mL.

[0032] Preferably, the blocking solution is determined as follows: at a specific antibody coating concentration, after blocking with different blocking solutions, ELISA is performed and the P / N value is calculated. The best blocking solution is the one with the largest P / N value. In some specific embodiments of the present invention, the blocking solution is 2% BSA-PBST.

[0033] Preferably, the dilution multiple of the enzyme-labeled monoclonal antibody is determined by performing sandwich ELISA on enzyme-labeled antibodies of different dilution multiples, calculating the P / N value, and the maximum P / N value is the optimal dilution multiple. In some specific embodiments of the present invention, the dilution multiple of the enzyme-labeled antibody is 1:2000.

[0034] In addition, in addition to the above-mentioned porcine IL-15 double antibody sandwich ELISA detection method, the porcine IL-15 monoclonal antibody of the present invention can also be used in Western blot, immunofluorescence, immunohistochemistry, flow cytometry and other methods.

[0035] Compared with the prior art, the beneficial technical effects of the present invention are:

[0036] The porcine IL-15 double antibody sandwich ELISA detection method established by the present invention can detect a minimum of 31.25 ng / mL of porcine IL-15 protein, and has no cross-reaction with other cytokines or proteins (IL-1, IL-2, IL-4, IL-6, IL-10, IL-12, Gzms-B, IFN-γ, TNF-α). The mean values ​​of the coefficients of variation of the intra-batch and inter-batch repeatability experiments of the method are 2.56% and 3.39%, respectively.

[0037] The porcine IL-15 double antibody sandwich ELISA detection method of the present invention can quantitatively detect porcine IL-15 in samples such as serum, tissue fluid and cell culture, has high sensitivity, specificity and accuracy, provides an important tool for porcine IL-15 related research, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The present invention will be further described below in conjunction with the accompanying drawings.

[0039] Figure 1 The figure shows the identification of the recombinant plasmid pET-28α-porcine IL-15 and the expression of the porcine IL-15 protein. A is the amplification of the porcine IL-15 gene and the PCR and restriction enzyme digestion identification of the recombinant plasmid. M is a 5000bp DNA ladder. 1, 2 and 5 are the PCR products of the empty plasmid pET28α(+), the recombinant plasmid pET28α-porcine IL-15 and the porcine lymphoid tissue, respectively. 3 and 4 are the EcoR I-Xho I double enzyme digestion products of pET28α-porcine IL-15 and pET28α(+), respectively. B is the SDS-PAGE analysis of the induced expression of the recombinant protein. M is a 10-250ku protein molecular weight standard. 1 and 2 are the porcine IL-15 protein expression bacteria before and after induction, respectively. C is the Western blot analysis of the porcine IL-15 recombinant protein. Blot identification, M is the 8-180 ku protein molecular weight standard, 1 is the reaction result of the induced product of the empty plasmid pET-28α(+) transformed E. coli with the His tag antibody, and 2 is the reaction result of the expressed porcine IL-15 protein with the His tag antibody;

[0040] Figure 2 Identification of recombinant plasmid pcDNA3.1-porcine IL-15 and expression of porcine IL-15 in PK-15, wherein A is identification of pcDNA3.1-porcine IL-15, M is 5000bp DNA ladder, 1 and 2 are PCR products of empty plasmid pcDNA3.1(+) and recombinant plasmid pcDNA3.1-porcine IL-15, 3 and 4 are EcoRI-Xho I double restriction products of pcDNA3.1-porcine IL-15 and pcDNA3.1(+), respectively; B is Western blotting identification, M is 8-180ku protein molecular weight standard, 1 and 2 are the reaction results of cells transfected with pcDNA3.1-porcine IL-15 and porcine IL-15 antiserum and porcine IL-15 antibody negative serum, 3 is the reaction result of cells transfected with empty plasmid pcDNA3.1(+) and porcine IL-15 antiserum, C is IFA identification (100×);

[0041] Figure 3 is the titer of the monoclonal antibody, where A is the cell culture supernatant and B is the ascites;

[0042] Figure 4 The Western Blotting results of monoclonal antibodies, where M is a 10-250 ku protein molecular weight standard; 1-9 are monoclonal antibodies 1E8, 1F4, 2D3, 2C10, 2F3, 2D10, 3F4, 3D4 and D5, 10 is positive mouse serum, 11 and 12 are negative mouse serum and SP2 / 0 cell supernatant, respectively;

[0043] Figure 5 The results of indirect immunofluorescence identification (100×) of monoclonal antibodies, where AG are monoclonal antibodies 1E8, 1F4, 2D3, 2C10, 2F3, 2D10, and 3D5, H is the positive mouse serum of porcine IL-15 antibody, and I is the negative mouse serum;

[0044] Figure 6 It is the result of monoclonal antibody specificity test;

[0045] Figure 7 is the affinity curve of the monoclonal antibody;

[0046] Figure 8 are the P / N values ​​of different antibody working concentrations and blocking solutions; A, B and C are the coating concentration of capture antibody 2D10, the dilution of blocking solution and enzyme-labeled antibody 2C10, respectively;

[0047] Fig. 9 The linear range of the standard curve and the sensitivity analysis result of sandwich ELISA; A is the linear range of the standard curve, and B is the sensitivity analysis result;

[0048] Fig.10 It is the specificity test result of sandwich ELISA;

[0049] Fig.11 The quantitative detection results of IL-15, where A is Western blotting; B is the gray value analysis of the reaction band, where 1-16 are DCs isolated from different pig lymph node samples. DETAILED DESCRIPTION

[0050] The present invention provides a method for establishing a pig IL-15 double antibody sandwich ELISA test. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they all fall within the scope of protection of the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0051] In the present invention, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B may represent: A exists alone, A and B exist at the same time, and B exists alone. A and B may be singular or plural.

[0052] In the present invention, "at least one" means one or more, and "more than one" means two or more.

[0053] Many terms are used in the present invention. Unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art.

[0054] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0055] The embodiments of the present invention involve SP2 / 0 myeloma cells, Escherichia coli (E. coli) Rosetta (DE3) competent cells and PK-15 cells. These E. coli are commercially available products and therefore do not require additional preservation evidence and genetic resource registration information, plasmids pET28α(+), pcDNA3.1(+).

[0056] Restriction endonucleases (EcoR I and Xho I) were purchased from Bao Biotechnology (Dalian) Co., Ltd.; gel recovery kit and plasmid extraction kit were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; T4 DNA Ligase was purchased from Thermo Scientific Co., Ltd.; Freund's complete adjuvant and Freund's incomplete adjuvant were products of Sigma; Dulbecco's Modified Eagle Medium was purchased from GIBCO; fluorescein isothiocyanate (FITC)-labeled goat anti-mouse IgG and His-tag protein purification kit were purchased from Beijing Solarbio Technology Co., Ltd. (Solarbio); mouse monoclonal antibody Ig class / subclass / identification ELISA kit and horseradish peroxidase (HRP)-labeled goat anti-mouse IgG were purchased from Suzhou Biolong Technology Co., Ltd.; multicolor prestained protein marker and ECL chemical exposure color development kit were purchased from Shanghai Yazyme Biotechnology Co., Ltd.

[0057] SPF BALB / c mice were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

[0058] Example 1 Preparation, Identification, and Specificity Detection of Swine IL-15 Monoclonal Antibodies

[0059] 1.1 Gene cloning, protein expression and identification of porcine IL-15

[0060] The nucleic acid was extracted from the porcine inguinal lymph node tissue, and PCR was performed after reverse transcription of cDNA (annealing temperature was 55°C, and the amplified fragment size was 498 bp).

[0061] The porcine IL-15 primers are:

[0062] ePig IL-15U: 5′-CTAGAATTCGCCACCATGAGAATTTTAAAAC-3′

[0063] ePorcine IL-15L: 5′-GACCTCGAGTCAAGAAGGGTTGATG-3′.

[0064] The purified PCR product, plasmid pET28α(+) and pcDNA3.1(+) were digested with EcoR I and Xho I restriction endonucleases, respectively. The digested target gene was ligated with different vectors at 16°C for 16 h using T4 DNA ligase to construct pET-28α-porcine IL-15 recombinant plasmid and pcDNA3.1-porcine IL-15 recombinant plasmid, and the recombinant plasmids were identified by restriction digestion and sequencing (completed by Sangon Biotechnology Co., Ltd.).

[0065] The pET-28α-porcine IL-15 recombinant plasmid was transformed into Escherichia coli Rosetta (DE3) competent cells and induced to activate. The bacterial liquid was spread on a kanamycin-resistant LB (Kana+ / LB) plate; a positive single colony was picked and inoculated into a Kana+ / LB liquid culture medium, and cultured with shaking at 37°C until the logarithmic growth phase. The culture was induced with 0.25 mmol / L IPTG at 32°C for 6 h, the bacteria were collected and ultrasonically lysed, the protein expression was analyzed by SDS-PAGE, and the expressed protein was identified by Western blot.

[0066] Western blot process: transfer the proteins in the gel to the PVDF membrane; block the membrane at 4°C overnight; wash the membrane, add 1:5000 diluted anti-His tag mouse monoclonal antibody, incubate at room temperature for 2 hours; wash the membrane, add 1:5000 diluted goat anti-mouse IgG-HRP, incubate at room temperature for 1.5 hours; wash the membrane, add CEL substrate colorimetric solution, and observe the reaction between the antibody and the expressed protein.

[0067] The pcDNA3.1-porcine IL-15 recombinant plasmid was transfected into PK-15 cells by liposome transfection. 48 hours after transfection, the expression of porcine IL-15 was detected by indirect immunofluorescence assay (IFA). The specific method was as follows: PK-15 cells were fixed with pre-cooled anhydrous methanol, and then reacted with porcine IL-15 mouse polyclonal antibody (1:200) and FITC-labeled goat anti-mouse IgG (1:300 dilution) in sequence, and the green fluorescence in the cells was observed under a fluorescence microscope to identify the expression of porcine IL-15.

[0068] The results showed that the present invention used RT-PCR to amplify the porcine IL-15 gene (498 bp) from the piglet lymph node. Figure 1 A). The constructed porcine IL-15 recombinant expression plasmids pET-28α-porcine IL-15 and pcDNA3.1-porcine IL-15 were identified by PCR and could amplify the target fragment with the expected size. After double digestion with restriction endonucleases EcoR I-Xho I, a DNA fragment with the same size as the target gene could be released, while the empty plasmid control plasmids pET28α(+) and pcDNA3.1(+) did not release the target band ( Figure 1 A and Figure 2 A).

[0069] The sequencing results showed that the cloned target gene sequence was consistent with the reference sequence. This indicated that the pET-28α-porcine IL-15 and pcDNA3.1-porcine IL-15 recombinant plasmids were successfully constructed. Escherichia coli Rosetta (DE3) transformed with the recombinant plasmid pET-28α-porcine IL-15 was induced by IPTG at 32°C for 6h. SDS-PAGE showed that the target recombinant protein ( Figure 1 B).

[0070] Western blot analysis showed that after the expressed protein reacted with the anti-His tag antibody, a reaction band with the expected size appeared in the corresponding lane ( Figure 1 C). It shows that porcine IL-15 protein is expressed.

[0071] PK-15 cells transfected with recombinant plasmid pcDNA3.1-swine IL-15 were collected and identified by Western blot. After reacting with the pig IL-15 antiserum, a reaction band of the expected size appeared in the corresponding lane, while no reaction band appeared in the corresponding lane of the negative control ( Figure 2 B).

[0072] IFA identification showed that specific green fluorescence appeared in PK-15 cells transfected with pcDNA3.1-porcine IL-15 in response to porcine IL-15 antiserum, but no green fluorescence appeared after reaction with porcine IL-15 antibody negative serum ( Figure 2 C). This indicates that porcine IL-15 is expressed in PK-15 cells.

[0073] 1.2 Screening of hybridoma cell lines that stably secrete porcine IL-15-specific monoclonal antibodies and identification of monoclonal antibody classes / subclasses

[0074] The expressed recombinant porcine IL-15 protein was purified using a His tag protein purification kit, and the protein was subcutaneously inoculated into 6-8w SPF mice (50μg / mouse) for 3 consecutive times with an interval of 14d. The mice with the highest antibody level were selected and injected with 80μg porcine IL-15 protein intraperitoneally 3d before cell fusion for booster immunization. The spleen of the booster immunized mice was aseptically removed to prepare a spleen cell suspension; the spleen cells were fused with SP2 / 0 myeloma cells under the action of PEG4000. On the 9th day after fusion, the culture supernatant of the cell clone well was collected, and the porcine IL-15-specific antibody was detected by ELISA to screen the hybridoma cell lines that stably secreted porcine IL-15-specific monoclonal antibodies.

[0075] During ELISA testing, the ELISA plate was coated with 1 μg / mL of porcine IL-15 recombinant protein, and an antigen control was set up for the induced expression product of E. coli transformed with an empty plasmid to exclude nonspecific reactions. The dilution factor of goat anti-mouse IgG-HRP was 1:10,000.

[0076] The positive hybridoma cells screened were subcloned 3 to 5 times continuously to obtain a hybridoma cell line that stably secreted porcine IL-15-specific monoclonal antibody (the deposit number is CCTCC NO: C2024127). 2 ~10 4 After multiple dilution, ELISA was used to detect the titer of the monoclonal antibody secreted by the hybridoma cells.

[0077] The type of antibody secreted by the hybridoma cell line was detected using a mouse monoclonal antibody Ig class / subclass / identification ELISA kit. Negative and positive controls were also set up. After color development, D 450nm When the sample D 450nm When the value is <0.15, it is judged as negative; when the sample D 450nm >Negative Control D 450nm Then, ascites was prepared by in vivo ascites induction method, antibodies were purified by ammonium octanoate sulfate method, and the titer of monoclonal antibodies in ascites was determined by ELISA.

[0078] The results showed that the fusion rate between the spleen of immunized mice and SP2 / 0 myeloma cells was 33%, and the antibody positive rate was 99%. After 3 to 5 subcloning, a total of 9 hybridoma cell lines (1E8, 1F4, 2D3, 2C10, 2F3, 2D10, 3F4, 3D4 and 3D5) that stably secreted porcine IL-15-specific monoclonal antibodies were obtained. Identified by the mouse monoclonal antibody Ig class / subclass / identification ELISA kit, the 9 monoclonal antibodies all reacted with anti-IgG2b antibodies and anti-κ light chain antibodies, belonging to the IgG2b class κ chain, namely IgG2bκ. The antibody titers in the culture supernatants of the 9 hybridoma cells were between 1:3200 and 1:12800 ( Figure 3 A). Hybridoma cell lines 2C10, 2D10, 2F3, and 3D5 were selected to prepare ascites, and the titer of ascites antibody was between 400,000 and 3,200,000 ( Figure 3 B).

[0079] 1.3 Identification of monoclonal antibodies

[0080] After SDS-PAGE electrophoresis of the recombinant porcine IL-15 protein, the monoclonal antibody was identified by Western blot, and the product induced by transforming E. coli with empty plasmid pET28α(+) was used as a negative serum control. During Western blot identification, the monoclonal antibody to be tested was diluted 1:500, and the goat anti-mouse IgG-HRP was diluted 1:5000.

[0081] The eukaryotic expression plasmid pCDNA3.1-porcine IL-15 was transfected into PK-15 cells by liposome transfection. 48 hours after transfection, the monoclonal antibody was identified by IFA. During the identification, the dilution multiple of the monoclonal antibody to be tested was 1:200, and the dilution multiple of FITC-labeled goat anti-mouse IgG was 1:300. After the reaction was completed, the results were observed under a fluorescence microscope.

[0082] 1.4 Specificity detection of monoclonal antibodies

[0083] Nine different proteins, IL-1, IL-2, IL-4, IL-6, IL-10, IL-12, Gzms-B, IFN-γ, and TNF-α, were coated on ELISA plates, and the cross-reactivity of the prepared porcine IL-15-specific monoclonal antibody with the above protein antigens was detected by indirect ELISA to verify the specificity of the monoclonal antibody.

[0084] Western blot analysis showed that when monoclonal antibodies 1E8, 1F4, 2D3, 2C10, 2F3, 2D10, 3F4, 3D4 and 3D5 reacted with porcine IL-15 protein, a clear specific reaction band appeared in the corresponding lanes, but no reaction band appeared in the corresponding lanes after reacting with the induced product of empty plasmid transformed bacteria ( Figure 4 ). Further IFA results showed that after the PK-15 cells transfected with the pcDNA3.1-porcine IL-15 recombinant plasmid reacted with various monoclonal antibodies, obvious green fluorescence appeared in the cells ( Figure 5 ).

[0085] ELISA results showed that the monoclonal antibody obtained only specifically bound to porcine IL-15 protein and had no cross-reaction with protein antigens ( Figure 6 ), indicating that the monoclonal antibody obtained by the present invention has good specificity.

[0086] Example 2 Establishment and evaluation of the porcine IL-15 double antibody sandwich ELISA detection method

[0087] 2.1 Affinity determination of monoclonal antibodies

[0088] The affinity of monoclonal antibodies was identified by indirect ELISA method. The specific method was as follows: 0.5 and 0.25 μg / mL of porcine IL-15 protein were used to coat the ELISA plate, and the selected monoclonal antibodies 2C10, 2D10, 2F3, and 3D5 were diluted to 10, 5, 2.5, 1.25, 0.625, 0.313, 0.156, and 0.078 μg / mL, respectively. After color development, D 450nm The antibody-antigen binding reaction curve was drawn, and the maximum D value of each antigen concentration (Ag) was obtained by plotting. 450nm Half of the value (i.e. 50% D 450nm ) and calculate the affinity constant (Kaff).

[0089] Kaff=(n-1) / 2(n Ab′-Ab)

[0090] Wherein: Ab and Ab′ represent the antibody (Ab) concentration (mol / L) of the half-absorbance value produced when the antigen (Ag) concentration is 0.5 μg / mL and 0.25 μg / mL, respectively, n = Ag / Ag′;

[0091] The average value of Kaff values ​​is calculated as the affinity constant Kaff of the monoclonal antibody. The dissociation constant Kd of the monoclonal antibody is the reciprocal of the affinity constant Kaff.

[0092] D after the reaction of various monoclonal antibodies with porcine IL-15 protein at different dilutions determined by ELISA450nm The dissociation constants of monoclonal antibodies 2C10, 2D10, 2F3, and 3D5 were 8.32, 12.4, 8.33, and 8.32 nmol / L, respectively, indicating that the four monoclonal antibodies have a high affinity for porcine IL-15 ( Figure 7 ).

[0093] 2.2 Monoclonal Antibody Pairing Test

[0094] Refer to LinKine TM Instructions for the horseradish peroxidase coupling kit, label 4 purified monoclonal antibodies (2C10, 2D10, 2F3, 3D5). Then use sandwich ELISA to pair the 4 antibodies in pairs. The specific method is: use unlabeled 2C10, 2D10, 2F3 or 3D5 as the capture antibody to coat the ELISA plate (0.5μg / well), incubate the ELISA plate at 37℃ for 1h, and transfer to 4℃ overnight; wash the plate 3 times, 5min / time; add blocking solution, block at 37℃ for 1h; add 1μg / mL recombinant porcine IL-15 protein, react at 37℃ for 1h, and set up other proteins as negative controls; wash the plate, add different HRP-labeled antibodies respectively, incubate at 37℃ for 1h (combination pairing); add TMB substrate colorimetric solution to color at room temperature in the dark for 15min, add 2mol / L concentrated sulfuric acid to terminate the reaction, and measure D 450 nm The test results are shown in Table 1.

[0095] Table 1 Combination pairing and detection results of monoclonal antibodies

[0096]

[0097] As shown in Table 1, after 2D10 and HRP-2C10 were combined, the positive and negative controls D 450 nm The ratio of the values ​​(P / N value) was the highest (12.61), so 2D10 was selected as the capture antibody and HRP-2C10 as the enzyme-labeled antibody.

[0098] 2.3 Determination of antibody coating concentration

[0099] The ELISA plate was coated with monoclonal antibody 2D10 at 0.1, 0.5, and 1 μg / mL, respectively, and 1 μg / mL recombinant porcine IL-15 protein was used as the positive sample. After reacting with HRP-2C10 (1:4000 dilution), TMB was added to develop color at room temperature in the dark for 15 minutes, and D 450 nm value, calculate the P / N value.

[0100] The results showed that the P / N value was the largest when 2D10 was coated at 5 μg / mL ( Figure 8 A), so 5 μg / mL was used as the antibody coating concentration.

[0101] 2.4 Determination of blocking solution

[0102] To determine the optimal antibody coating concentration, the cells were blocked with PBST (PBS containing 0.05% Tween-20, 0.01 mol / L, pH 7.4) containing 5% skimmed milk powder, 5% BSA, 2% BSA, 5% calf serum and 1% gelatin, and then ELISA was performed and the P / N value was calculated.

[0103] The results showed that the P / N ratio was the highest when the blocking solution was 2% BSA-PBST, so 2% BSA-PBST was selected as the blocking solution ( Figure 8 B).

[0104] 2.5 Determination of the dilution multiple of enzyme-labeled monoclonal antibodies

[0105] The enzyme-labeled antibody was diluted 1:1000, 1:2000, 1:4000, and 1:8000, and sandwich ELISA was performed to calculate the P / N value.

[0106] The results showed that when HRP-2C10 was diluted 1:2000, the P / N value was higher than that of other dilutions ( Figure 8 C), so the enzyme-labeled antibody was diluted 1:2000.

[0107] 2.6 Establishment of standard curve and sensitivity analysis of sandwich ELISA

[0108] Absolute quantitative method was used to detect different concentrations (1000, 500, 250, 125, 62.5, 31.2, 15.6, 0 ng / mL) of porcine IL-15 standard by sandwich ELISA. The concentration of the standard was used as the horizontal axis, and D 450 nm The standard curve was drawn with the value as the ordinate, and the regression equation was obtained and the correlation coefficient (R 2 ) to determine the linear range of the standard curve.

[0109] The standard curve drawn according to the results showed that when the porcine IL-15 protein concentration was between 15 and 1000 ng / mL, D 450 nm The value showed a good linear relationship with the sample concentration, and R 2 is 0.99( Fig. 9 A).

[0110] Porcine IL-15 (2 μg / mL) was diluted 2-fold (8 dilutions in total) for sandwich ELISA, and other proteins were set up as negative controls. The maximum dilution when the P / N value was >2.1 was used as the detection line of the established double antibody sandwich ELISA method, and the minimum detection concentration of porcine IL-15 was calculated based on the standard curve.

[0111] The results showed that when the concentration of porcine IL-15 was 31.25 ng / mL, the P / N value was 6.05>2.1 ( Fig. 9 B), indicating that the sensitivity of the established sandwich ELISA was 31.25 ng / mL.

[0112] 2.7 Specificity test

[0113] According to the established ELISA procedure, the established double antibody sandwich ELISA method was used to simultaneously detect porcine IL-15 and other cytokines or proteins (IL-1, IL-2, IL-4, IL-6, IL-10, IL-12, Gzms-B, IFN-γ, TNF-α).

[0114] The results showed that except for the porcine IL-15 sample, the other protein test results were negative ( Fig.10 ), indicating that this method does not cross-react with other cytokines or proteins.

[0115] 2.8 Repeatability test

[0116] ELISA was performed using 2D10 capture antibodies from the same batch (intra-group repetitions) and three batches (inter-group repetitions) to detect six lymph node tissue fluids and two negative protein samples. The coefficient of variation (CV) of the intra-group and inter-group test results was calculated to evaluate the repeatability of the method. The results are shown in Tables 2 and 3.

[0117] Table 2 Intra-batch repeatability experiment

[0118]

[0119]

[0120] Table 3 Inter-batch repeatability experiment

[0121]

[0122] The average CV of the intra-batch repeatability test was 2.51%, and the average CV of the inter-batch repeatability test was 3.15%, indicating that the sandwich ELISA established by the present invention has good repeatability.

[0123] The present invention amplified the IL-15 gene from the pig lymph node, and the coding region thereof was 498 bp in total, and the consistency with the reference sequence (GenBank ID: EU234503) was 96%, indicating that the present invention successfully obtained the IL-15 gene.

[0124] The fusion rate of mouse spleen cells and SP / 20 cells is directly related to the screening of hybridoma cells. The cell fusion rate of the hybridoma cell of the present invention is 33%, and the antibody positive rate is 99%, which are higher than the fusion rate (24.91%) of the mouse anti-human IL-15 specific monoclonal antibody in the prior art, and the antibody positive rate is 1.0%.

[0125] Such a high antibody positive rate may affect the specificity of the test results. For this reason, the present invention uses the induced product of Escherichia coli transformed with an empty plasmid as a coating antigen to rescreen the positive hybridoma cells screened by the IL-15 protein antigen. After 3 to 5 rounds of cloning screening, 9 hybridoma cell lines were obtained, and the monoclonal antibody type secreted by them was IgG2bκ. This antibody type is different from the subclass (IgM) of the mouse anti-human IL-15 specific monoclonal antibody in the prior art, and the subclass (IgG1 and IgG2a) of the mouse anti-human 4 IL-15 specific monoclonal antibody.

[0126] In order to identify and prove the specificity of the monoclonal antibody obtained in the present invention binding to the IL-15 protein, the present invention simultaneously constructed an IL-15 eukaryotic expression plasmid, and further identified the obtained monoclonal antibody using PK-15 cells transfected with the IL-15 expression plasmid. Further, the cross-reaction with other cytokines or proteins was used to prove the specific binding of the obtained monoclonal antibody to IL-15.

[0127] ELISA is one of the most commonly used methods for antigen and antibody detection. In order to provide a commercial detection tool for porcine IL-15, the present invention has conducted a mutual pairing test between monoclonal antibodies, and finally selected 2D10 as the capture antibody and 2C10 as the enzyme-labeled antibody to establish a porcine IL-15 protein quantitative sandwich ELISA method. The results of the standard curve drawing and its linear range related to the quantitative detection show that the standard curve established by the recombinant porcine IL-15 protein standard prepared by the present invention has a good linear relationship with the OD value of ELISA (R 2 =0.99), which is similar to the linear relationship of the standard curves of commercial cytokine kits such as IL-4, IL-10, IFN-γ, and TNF-α.

[0128] In summary, the IL-15 sandwich ELISA established by the present invention can detect a minimum of 31.25 ng / mL of porcine IL-1, has good specificity and sensitivity, and can be applied to the quantitative detection of porcine IL-15 in biological samples. The preparation of porcine IL-15-specific monoclonal antibodies and the establishment and evaluation of the sandwich ELISA method of the present invention can provide important material support for the study of infection immunity related to porcine IL-15.

[0129] Example 3 Application of the pig IL-15 double antibody sandwich ELISA detection method

[0130] The inguinal lymph nodes of 16 piglets were collected, cut into pieces, and 500 g / mL of type IV collagenase and 200 μg / mL of DNAse I were added for digestion for 15 minutes; the cell suspension after digestion was filtered through a 70 nm filter and centrifuged at 1000 r / min for 10 minutes; the cell precipitate was collected, resuspended with 10% FBS1×RPMI-1640 nutrient solution, and dendritic cells (DC) were separated by the wall adhesion method. Then, DC cells were stimulated with 10 μg / mL of LPS for 18 hours, and the cell supernatant and cells were collected respectively. The IL-15 concentration in the collected supernatant was detected by the pig IL-15 double antibody sandwich ELISA detection method established by the present invention, and the IL-15 content in DC was detected by Western blot method.

[0131] The main steps of the Western blot experiment were as follows: inguinal lymph node tissue was taken for SDS-PAGE, and the protein was transferred to a PVDF membrane; the membrane was blocked overnight at 4°C; after washing the membrane, 2D10 monoclonal antibody was added at a dilution of 1:50,000, and incubated at room temperature for 2 hours; the membrane was washed, goat anti-mouse IgG-HRP was added at a dilution of 1:5000, and incubated at room temperature for 1.5 hours; the membrane was washed, CEL substrate colorimetric solution was added, and the reaction between the antibody and the expressed protein was observed. The results are shown in Tables 4 and Fig.11 shown.

[0132] Table 4 IL-15 content detection results

[0133]

[0134]

[0135] From Table 4 and Fig.11 It can be seen that the IL-15 content expressed by porcine DC detected by the porcine IL-15 double antibody sandwich ELISA detection method established by the present invention is between 1.4 and 56 ng / mL, which is consistent with the Western blot detection result. This result shows that the porcine IL-15 double antibody sandwich ELISA detection method can be used for the quantitative detection of IL-15.

[0136] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A porcine IL-15 monoclonal antibody, characterized in that: The hybridoma cell line with the deposit number of CCTCC NO: C2024127 was used for preparation.

2. A method for preparing a porcine IL-15 monoclonal antibody as claimed in claim 1, characterized in that: The following steps are involved: S21, extract nucleic acid from porcine inguinal lymph node tissue, reverse transcribe cDNA and perform PCR amplification. The porcine IL-15 primers are: ePig IL-15U: 5′-CTAGAATTCGCCACCATGAGAATTTTAAAAC-3′ ePorcine IL-15L: 5′-GACCTCGAGTCAAGAAGGGTTGATG-3′; The PCR product, pET28α(+) and pcDNA3.1(+) were digested with restriction endonucleases, the digested PCR product was connected with the plasmid to construct the recombinant plasmids pET-28α-porcine IL-15 and pcDNA3.1-porcine IL-15, the pET-28α-porcine IL-15 recombinant plasmid was transformed into Escherichia coli Rosetta (DE3) competent cells, and the pcDNA3.1-porcine IL-15 recombinant plasmid was transfected into PK-15 cells; S22, recombinant porcine IL-15 protein, use purified porcine IL-15 protein to immunize mice and then boost immunization, prepare spleen cell suspension and fuse spleen cells with SP2 / 0 myeloma cells, screen hybridoma cell lines that stably secrete porcine IL-15-specific monoclonal antibodies, prepare ascites, purify antibodies using the ammonium octanoate sulfate method, and complete the preparation of porcine IL-15 monoclonal antibodies.

3. The preparation method according to claim 2, characterized in that: The expression of porcine IL-15 protein in Escherichia coli was demonstrated by SDS-PAGE and Western Blot analysis; the expression of porcine IL-15 was verified by Western Blot and IFA experiments.

4. The preparation method according to claim 2, characterized in that: After the hybridoma cell line that stably secretes porcine IL-15-specific monoclonal antibodies is screened, at least one of subcloning and antibody titer determination, monoclonal antibody class / subclass identification, monoclonal antibody identification, and specificity detection is performed.

5. The preparation method according to claim 2, characterized in that: Western Blot was used to confirm the expression of recombinant porcine IL-15 protein and the specificity of the monoclonal antibody. Fluorescence microscopy was used to observe the intracellular fluorescence signal to verify the expression of porcine IL-15 in PK-15 cells and the binding ability of the monoclonal antibody. Cross-reactivity detection was used to ensure the specificity of the monoclonal antibody to porcine IL-15.

6. A double antibody sandwich ELISA detection method for quantitatively detecting the content of porcine IL-15 in biological samples, characterized in that: The method is carried out using the porcine IL-15 monoclonal antibody according to any one of claims 1 to 5.

7. A method for establishing and evaluating the detection method as claimed in claim 5, characterized in that: The following steps are involved: S11, affinity determination of monoclonal antibodies; S12, performing a monoclonal antibody pairing test; S13, determination of antibody coating concentration; S14. Determination of blocking solution; S15. Determination of dilution multiple of enzyme-labeled monoclonal antibody; S16, determining the minimum detectable concentration of porcine IL-15; S17, evaluate the specificity of the double antibody sandwich ELISA method; S18. Evaluate the repeatability of the double antibody sandwich ELISA method.

8. The method according to claim 7, characterized in that The double antibody comprises a capture antibody which can specifically bind to porcine IL-15 and an enzyme-labeled antibody.

9. The method according to claim 7, characterized in that: The antibody coating concentration is the concentration at which the P / N value is the highest.

10. The method according to claim 7, characterized in that The method for determining the blocking solution is as follows: at a specific antibody coating concentration, after blocking treatment with different blocking solutions, ELISA is performed and the P / N value is calculated. The optimal blocking solution is the one with the maximum P / N value.

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