CN113462653B - Monoclonal antibody of anti-pig Gasderm protein D, hybridoma cell strain secreting monoclonal antibody and application of monoclonal antibody - Google Patents

Abstract

The invention discloses a monoclonal antibody against porcine Gasderm D protein, a hybridoma cell strain secreting the monoclonal antibody and application of the monoclonal antibody. The monoclonal antibody for resisting the porcine Gasderm D protein is secreted and generated by a hybridoma cell strain with the preservation number of CGMCC No.22321. Experiments prove that the monoclonal antibody has reaction specificity on the porcine Gasdermin D protein, and the monoclonal antibody can detect not only the porcine GSDMD holoprotein but also the carboxyl-terminal segment of the activated and cut porcine GSDMD protein. The invention has important significance for the research of the biological function of the pig GSDMD.

Description

Monoclonal antibody of anti-pig Gasderm protein D, hybridoma cell strain secreting monoclonal antibody and application of monoclonal antibody

Technical Field

The invention relates to a monoclonal antibody, a hybridoma cell strain secreting the monoclonal antibody and application thereof, in particular to a monoclonal antibody for resisting a pig Gasderm min D protein and a hybridoma cell strain generating the monoclonal antibody, and further relates to a method for detecting the expression and cutting condition of the pig Gasderm min D protein in a tissue and cell culture of a pig. The invention belongs to the field of biotechnology.

Background

Cell apoptosis (apoptosis) is an important form of Programmed Cell Death (PCD). Unlike apoptosis, cellular apoptosis is a form of PCD with pro-inflammatory properties. Early studies found that caspase-1 (caspase-1), human caspase-4/5 and mouse caspase-11 are activated in cells during apoptosis. Meanwhile, the release of main proinflammatory cytokines such as IL-1 beta and IL-18 and inflammatory mediators such as high mobility group protein B1 (HMGB 1) can be increased in the process of cell apoptosis. Gasdermin D (GSDMD) is one of the effector molecules that mediate apoptosis of cells. The full-length GSDMD protein has two structural domains, namely an amino acid structural domain (GSDMD-N) and a carboxyl terminal structural domain (GSDMD-C), the two structural domains are connected through a flexible structure, and the flexible structure has a highly conserved enzyme cutting site of caspases. The complete GSDMD molecule, GSDMD-N and GSDMD-C, combine with each other to present self-inhibition state. GSDMDM can be cleaved between GSDMDM-N and GSDMDM-C when caspase-1, caspase-4/5, and caspase-11 are activated in the cells. Produces GSDMD-N with molecular weight of about 30kDa and GSDMD-C with molecular weight of about 20 kDa. GSDMDM-N can form transmembrane channels on cell membranes and mitochondrial membranes. The membrane-penetrating pore canal formed by the GSDMDM-N on the cell membrane is the main path for releasing cytokines such as IL-1 beta, IL-18, IL-33 and the like to the outside of the cell. When the cell membrane is seriously damaged by the membrane-penetrating pore canal formed by the GSDMD-N, the cell is subjected to scorching and apoptotic lysis, a large amount of contents are released, and the inflammatory reaction is aggravated. GSDMD can be cut into GSDMD-N and GSDMD-C sections by caspase-1, caspase-4/5 and caspase-11, and can also be cut by caspase-3 to obtain a peptide section with about 10kDa and a peptide section with about 43 kDa. The two polypeptide fragments generated by the GSDMD cut by caspase-3 do not show biological functions. Cleavage of GSDMD by caspase-3 is thought to be one of the important mechanisms for cells to regulate the way apoptosis occurs in itself. Activation of GSDMD has been shown to be closely linked to the development of a variety of diseases (e.g., tumors, autoimmune and infectious diseases, etc.) and is considered as a new therapeutic target for disease.

Currently, commercially available antibodies to GSDMD are both antibodies specific for human and mouse GSDMD, and the lack of antibodies specific for GSDMD from other organisms is disadvantageous for the work of investigating the function of GSDMD in other organisms. The invention aims to provide a mouse anti-porcine GSDMD protein monoclonal antibody and a method for carrying out immunoblot detection on GSDMD expression and cutting conditions in porcine cells and porcine tissues by using the monoclonal antibody.

Disclosure of Invention

One of the purposes of the invention is to provide a hybridoma cell strain secreting monoclonal antibody against porcine GSDMD protein;

the other purpose of the invention is to provide the monoclonal antibody of the anti-porcine GSDMD protein secreted and produced by the hybridoma cell strain;

it is a further object of the present invention to provide the use of said monoclonal antibodies.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

the hybridoma cell strain secreting the anti-pig Gasderm min D protein monoclonal antibody is named as 4G7-D7, is classified and named as the anti-pig Gasderm min D monoclonal antibody hybridoma cell strain, is preserved in the China center for type culture Collection, and is addressed to the microbial research institute of China academy of sciences No. 3 of the West Lu 1 Host North Chen of the sunward area in Beijing, and the strain preservation numbers are as follows: CGMCC No.22321, and preservation time of 2021, 05 months and 10 days.

The hybridoma cell strain is prepared by the following steps:

transforming the recombinant plasmid pET-30a (+) -pGSDMD into escherichia coli, obtaining recombinant porcine GSDMD protein (pGSDMD) expressed in an inclusion body form under the induction of IPTG, and purifying the protein; purified pGSDMD protein was used as immunogen to inoculate 5 week old BALB/c mice subcutaneously. The spleen cells of the immunized mice are fused with SP2/0 myeloma cells according to the conventional hybridoma cell fusion technology. And (3) establishing an indirect ELISA method by using the purified pGSDMD protein as a screening antigen, and finally screening 1 hybridoma cell strain 4G7-D7 which continuously secretes the anti-porcine GSDMD protein monoclonal antibody. The invention further obtains the monoclonal antibody of the anti-porcine GSDMD protein from the supernatant of the hybridoma cells or from the ascites of animals injected with the hybridoma cells.

Furthermore, the invention also provides a monoclonal antibody for resisting the porcine GSDMD protein, wherein the monoclonal antibody is secreted and generated by a hybridoma cell strain with the preservation number of CGMCC NO. 11188.

Furthermore, the invention also provides application of the monoclonal antibody for resisting the porcine GSDMD protein in preparing a reagent for detecting the porcine Gasderm min D protein.

Preferably, the reagent is used for immunoblotting detection. More preferably, the reagent is a reagent for ELISA detection.

Preferably, the reagent can be used for detecting GSDMD expression and cleavage condition.

Compared with the prior art, the invention has the beneficial effects that:

the invention provides a mouse anti-porcine GSDMD protein monoclonal antibody, and establishes a method for carrying out immunoblot detection on GSDMD expression and cutting conditions in porcine cells and porcine tissues by using the monoclonal antibody. Experiments prove that the monoclonal antibody has reaction specificity on the porcine Gasderm D protein, and the monoclonal antibody not only can detect the porcine GSDMD holoprotein, but also can detect the activated and cut carboxyl terminal segment of the porcine GSDMD protein. The invention has important significance for the research of the biological function of the pig GSDMD.

Drawings

FIG. 1 is a result diagram of the application of the monoclonal antibody against porcine GSDMD in immunoblotting method for detecting the expression and cleavage of GSDMD protein in porcine intestinal tissue;

FIG. 2 is a graph showing the results of the application of anti-porcine GSDMD monoclonal antibody in immunoblotting method for detecting the expression of GSDMD in porcine IPEC-J2 cells.

Detailed Description

The present invention is further illustrated by the following experiments in conjunction with examples, it being understood that these examples are for illustrative purposes only and in no way limit the scope of the present invention.

Example 1 preparation of monoclonal antibody against porcine Gasdermin D protein

1. Construction of recombinant plasmid pET-30a (+) -pgsdmd

Pig GSDMD (pig GSDMD, abbreviated as pgsdmd) whole gene synthesis was performed according to the published nucleotide Sequence of open reading frame of pig GSDMD protein-encoding gene (NCBI Reference Sequence: XM _ 021090504.1) and ligated into pUC57 plasmid. The gsdmd gene was amplified from pUC57-pgsdmd plasmid using the primers described in Table 1. The amplified product and pET-30a (+) vector plasmid are subjected to double enzyme digestion by Sal I and EcoR I respectively, and the enzyme digestion product is subjected to gel recovery. The recovered pig gsdmd gene was mixed with the pET-30a (+) fragment and ligated by ligase. Coli DH5 competent cells, and the transformation products were spread on LB agar plates containing kanamycin (30. Mu.g/mL). Picking single colony, amplifying and culturing, and extracting plasmid. The extracted plasmid is subjected to enzyme digestion, PCR and sequencing identification, and the plasmid which is successfully connected is identified and named as pET-30a (+) -pgsdmd, wherein the nucleotide sequence of the gsdmd gene is shown as SEQ ID NO. 1.

TABLE 1 primers for amplifying the pig gsdmd Gene

2. Preparation of recombinant porcine GSDMD protein (recombiant pig GSDMD, abbreviated rpGSDMD)

(1) Transformation of the recombinant plasmid pET-30a (+) -pgsdmd

1) 0.2dm of the recombinant plasmid was transformed into E.coli Rosetta (DE 3) TM competent cells and plated on LB agar plates containing kanamycin (in 30-state cells).

2) Randomly 1 transformant was selected and inoculated into LB medium (containing 50. Mu.g/mL kanamycin), followed by shaking culture at 37 ℃.

(2) Inducible expression of recombinant proteins

1) The positive bacteria were inoculated at 1% dose into 10mL LB medium (containing 50. Mu.g/mL kanamycin) and cultured at 37 ℃ at 220r/min for 2-3h.

2) Culturing until OD600nm reaches 0.4-0.6, and adding IPTG (final concentration of 1 mmol/L) to induce expression for 4-6 h.

3) Collecting thalli, carrying out ultrasonic disruption, centrifuging, taking supernatant and precipitate respectively, carrying out SDS-PAGE, and determining the expression condition of the target protein.

Finally, the rpGSDMD protein expressed in the form of inclusion bodies is obtained.

3. Purification of rpGSDMD

(1) Solution preparation:

1)LE Buffer

50mM NaH ₂ PO ₄ ·H ₂ o,300mM NaCl, 1L deionized water (pH adjusted to 8.0).

2) 10mM/mL imidazole

10mM imidazole, 1L LE Buffer (pH adjusted to 8.0).

3) 80mM/mL imidazole

80mM imidazole, 1L LE Buffer (adjusted pH to 8.0).

4)PBS

NaCl 8.0g，Na ₂ HPO ₄ ·12H ₂ O 2.9g，KCl 0.2g，KH ₂ PO ₄ 0.24g, deionized water 1L.

(2) Method of producing a composite material

The method comprises the following specific steps:

1) The positive bacteria were inoculated into 100mL of LB medium (containing 50. Mu.g/mL kanamycin) at a ratio of 1% according to the above-mentioned method, and scale-up induction expression of recombinant proteins was performed.

2) Purification of the recombinant protein was performed using Ni-NTA Purification System as described in the specification.

3) Collecting purified protein, dialyzing with 2L PBS glycerol, changing solution every 12 hr, and continuously dialyzing for 72 hr.

4) And (3) concentrating the dialyzed protein solution by using PEG6000, and determining the protein concentration by using a Biyunnan protein concentration determination kit.

5) The purified rpGSDMD protein with the concentration of 0.2-0.5mg/mL is obtained.

4. Determination of ELISA method

(1) Preparing a reagent:

1) Coating solution (carbonate buffer solution of pH9.6)

Anhydrous Na ₂ CO ₃ 0.1696g，NaHCO ₃ 0.2856g and 100mL of deionized water, and the mixture is completely dissolved to 4 ℃ and used within a week.

2) PBST formulations

NaCl 8.0g，Na ₂ HPO ₄ .·12H ₂ O 2.9g，KCl 0.2g，KH ₂ PO ₄ 0.24g, tween 20.5mL, deionized water 1L.

3) Stopping liquid

Concentrated sulfuric acid deionized water =1 (volume ratio), which was slowly added to sterile water with stirring to dissipate heat.

(2) Method of producing a composite material

The method comprises the following specific steps:

1) Coating quilt

ELISA plates were coated with 100. Mu.L of rpGSDMDM protein 100ng/mL per well using pH9.6 carbonate buffer as coating solution and overnight at 4 ℃.

2) Washing machine

And (3) discarding the coating solution after coating, and washing with PBST, wherein the method comprises the steps of filling each hole with PBST, standing for 5-10min, discarding the washing solution, filling again, repeatedly washing for 3-5 times, and finally patting dry the ELISA plate to wait for next step of sealing.

3) Sealing of

Taking the ELISA plate in the step 2) and PBST (containing 5% skim milk) as a blocking solution, wherein the volume of each hole is 300 mu L, and the temperature is 37 ℃ for 2h.

4) Washing machine

The ELISA plate after blocking is washed in the same manner as in step 2). Finally, patting dry to wait for adding primary antibody.

5) Adding a primary antibody

The primary antibody sample (hybridoma cell culture supernatant or mouse ascites purified monoclonal antibody) was diluted with PBST and added to the corresponding wells (100. Mu.L per well) and incubated at 37 ℃ for 1h.

6) Washing machine

The concrete steps are the same as the step 4).

7) Addition of HRP-labeled commercial Secondary antibody

Corresponding commercial HRP-labeled secondary antibodies were added and the secondary antibodies were diluted in PBST to appropriate fold with reference to secondary antibody instructions. The diluted secondary antibody was added to each well (100. Mu.L per well) and reacted at 37 ℃ for 1 hour.

8) Washing machine

The concrete steps are the same as the step 4).

9) Color development

After the step 8) is finished, 100 mu L of TMB substrate developing solution is added into each hole of the ELISA, and then the ELISA plate is placed at 37 ℃ for reaction for about 10min.

10 Terminate

The ELISA plate was removed, and a stop solution (1 mol/L sulfuric acid solution) was added to each well to stop the reaction, and immediately read on a microplate reader, and the detection wavelength was 450nm when TMB was used as a substrate.

5. Preparation of monoclonal antibodies

(1) Immunizing animals

The purified rpGSDMD protein was mixed with an equal volume of Freund's complete adjuvant, emulsified and injected subcutaneously into the back of the neck of mice (50. Mu.g/mouse). After two weeks, the purified rpGSDMD protein was mixed with an equal volume of incomplete freund's adjuvant and the mice were boosted after emulsification. Two more weeks apart, a third immunization was performed in the same manner as the second immunization. At 14d after the three-immunization, the mice were injected intraperitoneally with purified rpGSDMD protein (100. Mu.g/mouse), and after 3d, the mouse spleen cells were fused with SP2/0 myeloma cells.

(2) Establishment of positive hybridoma cell strain

Spleen of the above immunized mice was taken to prepare a cell suspension, and the cell suspension was fused with SP2/0 cells in logarithmic growth phase at a ratio of 8. When the fused cells grow to 1/3 of the bottom of the hole, positive hybridoma cell strains are screened out by the indirect ELISA, and then are continuously cloned for 2-3 times by using a limiting dilution method. And performing amplification culture until the positive rate is 100%, and freezing and storing in liquid nitrogen.

Finally obtaining 1 hybridoma cell strain, which is named as 4G7-D7 and is preserved in China center for type culture Collection with the preservation number as follows: CGMCC No.22321, and preservation date of 2021, 05 months and 10 days.

(3) Preparation of monoclonal antibodies

The hybridoma cells obtained by screening were expanded and cultured at 5X 10 ⁶ After injecting 1mL of Balb/c mice into the abdominal cavity of 0.5mL hybridoma cells and 1mL of 14d mice, collecting ascites by using an injector after the abdominal circumference of the mice is enlarged, centrifuging for 10min at 1000r/min, and filtering supernatant by using a 0.45 mu m filter to obtain the crude monoclonal antibody product. The crude single antibody was initially purified by ammonium sulfate precipitation followed by further purification of the single antibody by Protein A affinity chromatography.

Example 2 application of monoclonal antibody against porcine GSDMD protein in immunoblotting method for detecting GSDMD protein expression and cleavage condition in porcine tissue and porcine cell

1. Application of monoclonal antibody (prepared in example 1) resisting porcine GSDMD protein to detection of expression and cutting condition of GSDMD molecules in porcine tissues in immunoblotting experiment

1) Collecting kidney and intestine tissues of healthy and enteropathogenic escherichia coli inoculated piglets, homogenizing, performing protein electrophoresis, and transferring the protein onto an NC membrane.

2) NC membrane was blocked with 5% skim milk for 1h at room temperature with shaking. The membranes were washed 3 times 5 minutes each with TBST.

3) Anti-porcine GSDMD monoclonal antibodies were purified using TBST 1: at 2000 dilution, and incubated with NC membranes overnight at 4 ℃. The membranes were washed 3 times 5 minutes each with TBST.

4) HRP-labeled goat anti-rabbit IgG was purified using TBST 1: after 5000 dilution, the secondary antibody was incubated with NC membrane at room temperature for 1h. The membranes were washed 3 times for 5 minutes each with TBST.

5) And (3) dripping ECL luminous liquid on the NC membrane, detecting by using a chemiluminescence detection imager and taking a picture.

The results are shown in FIG. 1. The results show that the full-length GSDMD protein (with the molecular weight of about 53 kDa) can be detected in the tissue extract of a control pig by the above-mentioned immunoblotting method, and the carboxyl-terminal segment (with the molecular weight of about 20 kDa) of the GSDMD protein can be detected in the tissue extract of a pig inoculated with Escherichia coli, which indicates that the GSDMD is cleaved by the Escherichia coli.

2. Monoclonal antibody (prepared in example 1) resisting porcine GSDMD protein is used for detecting the expression condition of GSDMD molecules in porcine intestinal epithelial cells (IPEC-J2) and porcine kidney cells (PK-15) of a porcine pig by an immunoblotting experiment

1) Cultured IPEC-J2 and PK15 cells were cultured at 5X 10 ⁵ The density of each cell/well was seeded in 6-well cell culture plates and cultured at 37 ℃ until the cell confluence reached 90%. Meanwhile, mouse macrophage line (j774a.1 cells), bovine kidney cell line (MDBK cells), african green monkey kidney cell line (Vero cells), chicken macrophage line (HD 11 cells) and canine kidney cell line (MDCK cells) were cultured as controls.

2) Cells were lysed with RIPA lysate.

3) The cleavage products were subjected to protein electrophoresis, and the proteins were transferred onto NC membranes.

2) NC membrane was blocked with 5% skim milk for 1h at room temperature with shaking. The membranes were washed 3 times for 5 minutes each with TBST.

3) Monoclonal antibodies against porcine GSDMD protein were purified using TBST 1: diluted 2000 and incubated with NC membranes overnight at 4 ℃. The membranes were washed 3 times 5 minutes each with TBST.

4) HRP-labeled goat anti-mouse IgG was purified using TBST 1: after dilution with 5000, the cells were used as secondary antibodies and incubated with NC membranes for 1h at room temperature. The membranes were washed 3 times 5 minutes each with TBST.

5) And (3) dripping ECL luminous liquid on the NC film, detecting by using a chemiluminescence detection imager and taking a picture.

The results are shown in FIG. 2. The result shows that the full-length GSDMD protein (with the molecular weight of about 53 kDa) can be detected in the cells of pig origin relatively specifically by the immunoblotting method, and the corresponding protein cannot be detected in the cells of other animal origins, which indicates that the prepared monoclonal antibody can recognize the pig GSDMD relatively specifically.

The foregoing is merely a preferred embodiment of this invention, which is intended to be illustrative, not limiting; those skilled in the art will appreciate that many variations, modifications, and even equivalent variations are possible within the spirit and scope of the invention as defined in the appended claims.

Sequence listing

<110> northeast university of agriculture

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Claims (5)

1. A hybridoma cell strain secreting monoclonal antibody against porcine Gasderm D protein is characterized in that the hybridoma cell strain is named as 4G7-D7 and is preserved in China center for type culture collection, and the strain preservation number is as follows: CGMCC No.22321.

2. A monoclonal antibody against porcine Gasderm D protein, wherein the monoclonal antibody is secreted by the hybridoma cell strain of claim 1.

3. The use of the monoclonal antibody of claim 2 in the preparation of a reagent for detecting porcine Gasdermin D protein.

4. The use of claim 3, wherein the reagent is a reagent for immunoblotting detection.

5. The use according to any one of claims 3 to 4, wherein the agent is capable of being used for the detection of GSDMD expression and cleavage.

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