CN111172116B - Pig-derived OPN monoclonal antibody, hybridoma cell strain and application thereof - Google Patents

Abstract

The invention discloses a swine OPN monoclonal antibody, a hybridoma cell strain and application thereof. The hybridoma cell strain secreting the swine OPN monoclonal antibody is classified and named as a hybridoma cell strain Cy2, is preserved in China center for type culture Collection (CCTCC for short) in 2019, 12 months and 19 days, and is preserved at the preservation address of Wuhan university, Wuhan, China with the preservation number of CCTCC NO: C2019293. the pig-derived OPN monoclonal antibody is secreted and generated by the hybridoma cell strain or a passage cell strain thereof. The monoclonal antibody is suitable for rapidly and accurately detecting the content of OPN protein in pig tissues, semen or porcine oocytes, so that the monoclonal antibody can be used for preparing related detection kits or colloidal gold test paper and the like, and a firm basis is laid for the in-depth research of porcine OPN.

Description

Pig-derived OPN monoclonal antibody, hybridoma cell strain and application thereof

Technical Field

The invention relates to the technical field of bioengineering, and particularly relates to a swine OPN monoclonal antibody, a hybridoma cell strain and application thereof.

Background

With the invasion of African swine fever, the swine industry has received a serious impact. Although the situation is controlled, the market for raising pigs is not optimistic, how to recover the production performance as quickly as possible, and how to improve the reproductive performance of boars? In the farm, boars with high fecundity are screened out by technical means for semen collection and hybridization, but the collection of data related to the fecundity is time-consuming and labor-consuming (Leno-Colorado et al, 2017). The best approach is to use new techniques to assist in propagation. Porcine seminal plasma and sperm membrane proteins are involved in the maturation and protection mechanisms of sperm (Dietrich et al, 2019; draart et al, 2019). These proteins can improve sperm quality and can be screened to identify other proteins that significantly improve sperm performance, and on the one hand, such proteins can be added directly to semen to increase the fertility of the entire population. On the other hand, the monoclonal antibody of the protein can be made into an ELISA kit, the content of the target protein in the semen of the boar can be directly detected, the boar with high reproductive capacity can be screened out, and the semen can be inseminated by using the high-reproductive semen, so that the reproductive capacity of the whole population can be greatly increased.

OPN is known as osteopontin, and is composed mainly of tripeptide sequences of arginine, glycine and aspartic acid. Originally isolated in bone matrix and teeth, it was primarily involved in the regulation of cell survival by acting through binding to receptors, directly or indirectly activating intracellular signaling pathways, mediating cell-to-cell, cell-to-extracellular matrix interactions. Through microsatellite positioning and PCR technology, OPN is found to be related to the breeding traits of pigs, particularly to the litter size of the Erhualian and Suzhong pigs, can be used as a genetic marker for increasing the litter size of the pigs (King et al, 2003; south wood et al, 1998; Short et al,1997 b; Liaw et al, 1999; Mengqing et al,2005), and the expression of the protein is found in the oviduct, uterus and placenta of the sows through protein immunoblotting; meanwhile, it was found that OPN protein was expressed in testis and spermatocytes of boars and also in small amounts in the supporting cells by Western blotting and immunofluorescence detection (Garlow et al, 2002; Hernandez et al, 2013). When the relationship between OPN and boar sperms is researched, the OPN is found to be significantly and positively correlated with the boar sperm motility, and the OPN protein can promote the in vitro fertilization porcine embryo division rate and reduce the polyspermia egg rate (Lin et al, 2006; Hao et al, 2008). The research shows that the OPN plays an indispensable role in improving the reproductive capacity of boars, and the research on pig-derived OPN needs to be carried out more closely.

At present, no report of a swine OPN protein monoclonal antibody expressed by eukaryotic cells exists at home and abroad, and compared with a prokaryotic system, the protein expressed by the eukaryotic system has higher controllability, a structure closer to natural protein and higher biological activity for the expression of recombinant protein. For antibodies, monoclonal antibodies are more specific and the results are more consistent than polyclonal antibodies. The porcine OPN protein is sheared by MMP-2, and two protein bands appear: 33.7kDa and 64.7 kDa. At present, a plurality of antigenic determinants can be identified by a plurality of porcine OPN polyclonal antibodies, and a plurality of nonspecific strips are generated when immunoblotting is carried out; when immunohistochemistry is carried out, the background is deeper, and the probability of false positive is higher; when immunofluorescence is carried out, the recognized parts are not single. Therefore, it is necessary to prepare monoclonal antibodies with good reactivity, and further to realize more intensive research on the function of the pig-derived OPN protein.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a hybridoma cell strain secreting the pig-derived OPN monoclonal antibody, which can be used as a constant regeneration source of the monoclonal antibody.

The invention also aims to provide a swine OPN monoclonal antibody which can be used for rapidly and accurately detecting the content of OPN protein in porcine tissues and semen.

The invention further aims to provide application of the porcine OPN monoclonal antibody.

The purpose of the invention is realized by the following technical scheme: a hybridoma cell strain secreting pig-derived OPN monoclonal antibodies is classified and named as a hybridoma cell strain Cy2, and is preserved in China center for type culture Collection (CCTCC for short) in 2019, 12 months and 19 days, wherein the preservation address is China, Wuhan university, and the preservation number is CCTCC NO: C2019293.

the swine OPN monoclonal antibody is secreted and produced by the hybridoma cell strain or a subculture cell strain thereof.

A detection kit, which contains the hybridoma cell strain and/or the swine OPN monoclonal antibody.

The kit comprises an enzyme linked immunosorbent assay kit, a fluorescence immunoassay kit and the like.

A colloidal gold test paper, which contains the hybridoma cell strain and/or the pig-derived OPN monoclonal antibody.

The hybridoma cell strain, the swine OPN monoclonal antibody, the detection kit or the colloidal gold test paper are applied to detection of OPN protein for non-diagnosis purposes.

The non-diagnosis purpose comprises the detection of the content of the recombinant OPN protein, the detection of the content of the OPN protein in isolated pig tissues, porcine oocytes and/or porcine semen and the like.

The hybridoma cell strain and/or the swine OPN monoclonal antibody are applied to preparation of a detection kit or test paper for detecting the content of OPN protein.

The OPN protein is derived from pig tissues, porcine oocytes and/or porcine semen.

The test paper comprises colloidal gold test paper and the like.

Compared with the prior art, the invention has the following advantages and effects:

the monoclonal hybridoma cell capable of secreting the specific antibody is obtained, can be used as a constant regeneration source of the monoclonal antibody, and meanwhile, the monoclonal antibody capable of specifically reacting with the porcine OPN protein is also obtained, so that the monoclonal antibody is suitable for quickly and accurately detecting the OPN protein in porcine tissues and seminal fluid, an antibody is provided for an ELISA box and a colloidal gold test paper for detecting the porcine OPN protein in the next step, a firm basis is laid for the deep research of the porcine OPN, an important technical support is provided for screening high-speed boars and improving the reproductive performance of a population, and the monoclonal antibody has high application value.

Drawings

FIG. 1 is a drawing showing the Western blot identification of the OPN monoclonal cells of example 1 (in the drawing, clone cells are used as a transfected group, and control is used as an untransfected group).

FIG. 2 is an SDS-PAGE identification of porcine OPN protein in the supernatant of example 1.

FIG. 3 is a diagram showing the state of culture of the positive hybridoma cell line Cy2 in example 2.

FIG. 4 is a graph showing the subtype identification of the monoclonal antibody in example 3 (in the graph, Cy2 is a monoclonal antibody group, and control is a blank control group).

FIG. 5 is a graph of the monoclonal antibody titer ELISA identification in example 3.

FIG. 6 is a Western immunoblot of the detection of monoclonal antibody properties in example 3.

FIG. 7 is an immunohistochemical chart of detection of monoclonal antibody properties in example 3; wherein, A is testis negative control; b is the result of testis immunohistochemistry; c is an ovary negative control; d is ovarian immunohistochemistry result.

FIG. 8 is an immunofluorescence plot of porcine mature oocytes assayed for monoclonal antibody properties in example 3; wherein A and D are OPN protein staining patterns, B and E are nuclear staining patterns, and C and F are OPN protein staining patterns and nuclear staining Merge patterns.

FIG. 9 is a pig sperm cell immunofluorescence plot showing detection of monoclonal antibody properties in example 3, wherein A and B are Merge plots of OPN protein staining, nuclear staining and sperm acrosome staining (A is a control group, B is an experimental group).

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, reagents and starting materials for use in the present invention are commercially available.

In the following examples, a rabbit anti-human OPN antibody (ab8448) was derived from abcam; the mouse monoclonal antibody subtype identification kit is from proteintech company; puromycin (Puro), Freund's complete adjuvant, Freund's incomplete adjuvant, polyethylene glycol (PEG4000), pristane, FITC-PSA and the drugs used in the pig in vitro fertilization culture system were purchased from SIGMA company; fetal Bovine Serum (FBS), HAT Media Supplement (50X), HT Media Supplement (50X), PBS buffer, DPBS powder, Hochest33342 (nuclear staining solution), Alexa Fluor goat anti-mouse fluorescent secondary antibody, and RPMI-1640 medium were purchased from Thermo Fisher Scientific Co; plasmids pMD2.G, psPAX2, pIRES2-EGFP and pCDH-CMV-MCS-EF1-GFP + Puro were purchased from Youbao organisms; trypan blue, formalin, paraformaldehyde, hematoxylin, an anti-quencher and reagents (such as ELISA antibody diluent) used for ELISA detection are all purchased from Beijing Rayleigh biotechnologies Inc.; lysis buffer and ECL (extracellular matrix) hypersensitive luminescence kit are purchased from Biyun sky; nitrocellulose membranes were purchased from Millipore; the 180kDa protein Marker was purchased from SMOBIO; rabbit serum was purchased from bosch de organisms; bovine Serum Albumin (BSA) was purchased from scout subfamily science and technology ltd; HRP-labeled goat anti-mouse IgG antibodies were purchased from EarthOx, peroxidase-labeled streptavidin-biotin and diaminobenzidine were purchased from beijing baiolabeckojiu, ltd, absolute ethanol and xylene were purchased from tianjin mao chemical agents.

In the following examples, HAT selection medium: RPMI-1640 medium containing 20% (v/v) fetal bovine serum and HAT Media Supplement (1 ×); HT selection culture solution: RPMI-1640 medium containing 20% (v/v) fetal bovine serum and HT Media Supplement (1X).

In the following examples, Sp2/0 cells, 293FT cells and CHO-K1 cell line were from ATCC company; BABL/c female mice (6-8 weeks, 18g-22g in body weight) were purchased from the center of medical laboratory animals in Guangdong province [ license number: SCXK (Yue) 2013-.

Example 1: in vitro acquisition of porcine-derived OPN protein

Referring to examples 1 and 2 in Chinese patent application 201811476996.8 "a recombinant lentivirus vector, recombinant lentivirus and its application", a recombinant protein of pig source OPN was obtained. The method specifically comprises the following steps: and predicting the signal peptide of the OPN protein by using signal peptide prediction software SignalP 4.1Server, removing the signal peptide of the OPN protein, and adding a 6 'His sequence in front of the gene sequence, namely the final sequence is a 6' His-OPN sequence. And connecting the target sequence to a lentivirus modified vector pCDH-CMV-HSA-MCS-6His-EF1-GFP + Puro to obtain a recombinant lentivirus vector pCDH-CMV-HSA-6' His-OPN-6His-EF1-GFP + Puro. The packaging plasmids (pMD2.G and psPAX2) and the lentivirus recombinant vector are co-transfected into 293FT cells through a lentivirus packaging system to obtain the virus containing the target gene. Infecting the virus into a CHO-K1 cell line, screening monoclonal cells by puromycin (Puro) drugs, identifying the monoclonal (figure 1), expanding the population for reserving the seeds, collecting the supernatant, carrying out SDS-PAGE (figure 2) identification and purification to obtain the porcine OPN recombinant protein, and carrying out mass spectrum identification and Western blot identification on the porcine OPN recombinant protein.

Example 2: preparation of porcine OPN murine monoclonal antibody

Mice were immunized with the purified recombinant porcine OPN protein expressed from eukaryotic system as immunogen in example 1. 6-8 weeks SPF grade pure breed BABL/c female mice are selected to immunize pig OPN recombinant protein. The first immunization is performed by subcutaneous multi-point injection of 100 mu g of pig OPN recombinant protein and Freund's complete adjuvant (equal volume mixing); three weeks apart, the second immunization of 100. mu.g of the porcine OPN recombinant protein was injected subcutaneously in multiple spots with Freund's incomplete adjuvant, and the total volume was 200. mu.L; three weeks apart, the third immunization dose is halved, namely 50 mug protein is not added with adjuvant, the intraperitoneal injection is carried out, blood is collected in orbit after 7 days, the titer is measured by an ELISA method, and the boosting immunization is carried out after the titer reaches a certain level; three weeks apart, the fourth immunization was a booster, i.e., 200 μ g of protein without adjuvant, i.e., intraperitoneal injection. After 3 days, spleen cells are taken and fused with a mouse myeloma cell line Sp2/0 according to the ratio of the number of the cells to 1:5 to prepare hybridoma cells, positive hybridoma cells are obtained by ELISA detection after screening, and the hybridoma cells are injected into the abdominal cavity of a mouse to obtain the monoclonal antibody. The method comprises the following specific steps:

1. preparation of feeder cells

Mouse abdominal cavity macrophage is taken and cultured to about 2x10 by 96 pore plates⁴～10⁵Cells/well as feeder cells.

The preparation process comprises the following steps:

taking a mouse of the same strain as the immunized mouse, carrying out neck dislocation to kill the mouse, soaking the mouse in 75% alcohol, and disinfecting for 5 min; in a clean bench, the skin was cut with sterile scissors, the peritoneum was exposed, 6mL of pre-cooled RPMI-1640 medium (without puncturing the bowel) was injected with a sterile syringe, and the wash repeated. Aspirating the washing solution, centrifuging at 1200rpm for 6min, resuspending in RPMI-1640 medium containing 20 (v/v)% fetal calf serum, and adjusting the cell count to 1X10⁵cells/mL, added to 96-well plates, 100. mu.L/well, placed in CO at 37 ℃₂Culturing in an incubator.

2. Preparation of immune spleen cells

Killing BABL/c female mice after four immunizations by dislocation of neck, soaking in 75% alcohol for disinfection, and taking spleen from an ultra-clean bench; the spleen was placed in a petri dish containing 4mL of RPMI-1640 medium, ground on a sterile mesh screen using a latex head of a sterile syringe, and placed under the mesh in a petri dish containing 4mL of RPMI-1640 medium. Washing the screen with culture solution to clarify, collecting the liquid, centrifuging at 1200rpm/min for 6min twice, resuspending the cell precipitate with culture solution, counting, and collecting 10⁸Spleen lymphocyte suspension is ready for use.

Preparation of Sp2/0 cells

Centrifuging logarithmic growth Sp2/0 cells at 1200rpm/min for 6min, washing with RPMI-1640 medium for 2 times, counting to obtain 1X10⁷The cells are ready for use.

4. Cell fusion

Sp2/0 cells were mixed with splenocytes at a cell number ratio of 1:5, washing the mixture for 1 time by using an RPMI-1640 culture medium in a 50mL centrifuge tube, centrifuging the mixture at 1200rpm/min, and centrifuging the mixture for 8 min; discard the supernatant and use pipette to clean the residual liquid to avoid affecting the concentration of polyethylene glycol (PEG). Lightly flick the bottom of the tube to loosen the cell pellet. Adding 1mL 45% (v/v) PEG (molecular weight 4000) solution pre-warmed at 37 ℃ within 1.5min while slightly shaking, and performing water bath at 37 ℃ for 1.5 min; the PEG action was stopped by adding RPMI-1640 medium pre-warmed at 37 ℃ and added at intervals of 2min to the medium at 1mL, 2mL, 3mL, 4mL, 5mL and 6mL, respectively. Centrifuging at 800rpm for 6 min; discarding the supernatant, and resuspending with HAT selective culture medium; adding the cells into a 96-well plate with a feeder cell layer, and adding 100 mu L of the cells into each well; the plates were incubated at 37 ℃ with 5% CO₂Culturing in an incubator.

5. Selection of hybridoma cells and antibody detection

(1) HAT selection of hybridoma cells

Culturing for 1-2 days with HAT selective culture medium, killing a large amount of tumor cells, eliminating the tumor cells after 3-4 days, forming small colonies by hybrid cells, maintaining the HAT selective culture medium for 7-10 days, then replacing with HT culture medium, maintaining for 2 weeks, and replacing with RPMI-1640 culture medium of 20% (v/v) fetal calf serum. During the selective culture, when the hybridoma cells spread over the area of the bottom 1/10 of the well, the detection of specific antibodies can be started, and positive hybridoma cell lines can be screened. Half of the culture solution is changed every 2-3 days during the selective culture period. Detecting cell culture supernatant by enzyme-linked immunosorbent assay (ELISA), and selecting positive hybridoma cell strain according to ELISA result (hybridoma cell strain OD450 is more than or equal to 0.5, negative control OD450 is less than 0.2, and positive control OD450 is more than 1.0). The method comprises the following specific steps:

1) coating: the purified porcine OPN recombinant protein (prepared in example 1) and a lentivirus recombinant expression vector His tag protein (the His tag protein is a lentivirus recombinant expression empty vector pCDH-CMV-HSA-MCS-6His-EF1-GFP + Puro packaged lentivirus, CHO-K1 cells are infected to obtain a monoclonal, the supernatant obtained after the monoclonal amplification and p-culture is purified to obtain the tag protein; the specific preparation method refers to example 1) are respectively coated with ELISA enzyme label plates, the coating amount of each hole is 1 mug/100 mug, and the ELISA enzyme label plates are coated overnight at 4 ℃.

2) Washing: the plate was washed 4 times 200. mu.L/well with PBST containing 0.5% o (v/v) Tween-20.

3) And (3) sealing: blocking with 1% (w/v) gelatin-containing blocking solution, and incubating at 37 ℃ for 2h at 120. mu.L/well.

4) Washing: the plate was washed 4 times with 200. mu.L/well of PBST containing 0.5% Tween-20.

5) Adding cell culture supernatant: adding cell culture supernatant (HAT selection culture medium, HT culture solution and RPMI-1640 culture medium cultured "cell culture supernatant") into enzyme labeling plate coated with pig OPN recombinant protein and His tag protein, respectively, adding blank control, and acting at 37 deg.C for 1 hr, wherein each well has a volume of 50 μ L.

6) Washing: the plate was washed 4 times 200. mu.L/well with PBST containing 0.5% o (v/v) Tween-20.

7) Adding a secondary antibody: enzyme-labeled Alexa Fluor goat anti mouse fluorescent secondary antibody was diluted with ELISA antibody as 1: 10000 dilution, 50 microliter per well adding enzyme label plate, 37 degrees C incubation for 40 min.

8) Washing: the plate was washed 4 times 200. mu.L/well with PBST containing 0.5% o (v/v) Tween-20.

9) Color development: add 50. mu.L of TMB to each well for color development and incubate for 15min at room temperature in the dark.

10) And (4) terminating: add 50. mu.L of 2M H per well₂SO₄And (3) solution.

11) And (3) detecting an OD value: OD450 values were measured by a microplate reader.

After double ELISA screening of the porcine OPN recombinant protein and the lentivirus recombinant expression vector His tag protein, selecting clones with negative reaction of the vector tag protein and positive reaction of the recombinant OPN protein, carrying out subcloning for 4 times after amplification culture, and carrying out double ELISA screening and identifying positive clones after each subcloning. 4 hybridoma cell strains Cy1, Cy2, Cy3 and Cy4 which stably secrete monoclonal antibodies are obtained after 4 times of subcloning, and the strains are expanded and cultured. The most preferred hybridoma cell Cy2 is preserved in the China center for type culture Collection (CCTCC for short, address: Wuhan, Wuhan university Collection) in 2019, 12 months and 19 days, with the preservation number of CCTCC NO: c2019293, its classification name is hybridoma cell strain Cy 2. The hybridoma cell strain Cy2 is used for preparing monoclonal antibody ascites after cryopreservation, resuscitation, passage and expanded culture.

(2) Amplification culture of hybridoma cell line Cy2

When the positive clones in the 96-well plate grow to the bottom of the well, gently blowing and beating the uniformly mixed cells by using a pipette, transferring the cells into a 15mL centrifuge tube, and centrifuging at 1200rpm/min for 3 min; discarding supernatant, adding RPMI-1640 culture medium, washing again, removing residual liquid with pipette gun, adding prepared frozen stock solution (RPMI-1640 culture medium: FBS: DMSO (dimethyl sulfoxide): 4:5:1), and adjusting density of positive hybridoma cell strain Cy2 to 1x10⁶And (3) sucking 1mL of cell sap, adding the cell sap into a 1.8mL cryopreservation tube, marking the cryopreservation tube, performing temperature gradient cryopreservation, standing at 4 ℃ for 10min, standing at-20 ℃ for 20min, standing at-80 ℃ in a refrigerator overnight, and then transferring into liquid nitrogen.

Freezing once per passage, after 1 week of 10 th freezing, recovering hybridoma cells, taking out a freezing tube from a liquid nitrogen tank, thawing the cells in water bath at 37 ℃, transferring the freezing solution containing the cells into a centrifuge tube containing 3mL of RPMI-1640 culture medium, centrifuging at 1200rpm/min for 3 min; the supernatant was discarded, and RPMI-1640 medium was added thereto and washed once more, and the residual liquid was aspirated, and 2mL of 20% (v/v) FBS-containing RPMI-1640 medium was added thereto, and cultured in a 96-well plate at 100. mu.L/well. Expanding culture at a ratio of 1:3 (from 1 hole to 3 holes) every other day, and preparing monoclonal antibody ascites.

(3) Preparation of monoclonal antibody ascites

A8-week-old female BABL/c mouse is selected, and 400 mu L of pristane is intraperitoneally injected. One week after the immunization with pristane, positive hybridoma cells in logarithmic growth phase (FIG. 3) were harvested at 1200rpm and centrifuged for 8 min. Sampling, staining with trypan blue, counting viable cells, resuspending in RPMI-1640 basal medium, inoculating the abdominal cavity of mice, 1X10⁷Cell/cell. Observing the change of the abdomen of the mouse after one week, collecting ascites if the abdomen is swollen, and collecting ascites at 4000rpmCentrifuging for 10min, and collecting supernatant for purification.

EXAMPLE 3 monoclonal antibody purification

1. Coarse extraction by caprylic acid-ammonium sulfate precipitation

Adding 2 parts of 0.06mol/L acetic acid buffer solution with pH of 5.0 into 1 part of ascites after centrifugal pretreatment, and adjusting the pH to 4.8 by using 1mol/L HCl solution; adding 11 mu L of octanoic acid into diluted ascites per ml, dropwise adding octanoic acid under stirring at room temperature within 30min, standing at 4 deg.C for 2h, centrifuging at 2000rpm for 30min, and removing precipitate; filtering the supernatant with a nylon sieve (125 μm), adding 1/10 volumes of 0.01mol/L PBS buffer solution, and adjusting the pH to 7.2 with 1mol/L NaOH; adding saturated ammonium sulfate at 4 deg.C to 45% saturation, acting for 30min, and standing for 1 hr; centrifuging at 12000rpm for 30min, and discarding the supernatant; dissolving the precipitate in a proper amount of PBS (containing 137mmol/L NaCl, 2.6mol/L KCl and 0.2mmol/L EDTA (ethylene diamine tetraacetic acid)), dialyzing in 50-100 times volume of PBS, and changing water for more than 3 times at 4 ℃ overnight; centrifuging at 12000rpm for 30min, removing insoluble precipitate, determining protein content by BCA method, packaging, and freezing at-80 deg.C.

HiTrap rProtein AFF Columns purification

1) Desalting: redissolving the crude monoclonal antibody, desalting by using a GE HiTrap desalting column at the flow rate of 5ml/min, and collecting a sample for later use;

2) GE 5mL HiTrap rProtein AFF Columns purification

Add 200. mu.L of 1M Tris-HCl, pH9.0 (1 mL sample per tube) to the collection tube in advance; removing ethanol from the column using 5 column volumes of distilled water; 5 column volumes of binding buffer (20mM sodium phosphate, pH7.0) were used, flow rate 5 mL/min; sampling, 0.5 mL/min; washing the column by using a large amount of binding buffer solution, wherein the volume of the column is 5-10 times that of the column until no substance is eluted, and the flow rate is 5 mL/min; performing linear elution with elution buffer (0.1M glycine-hydrochloric acid buffer, pH2.7) at flow rate of 5 mL/min; collecting an elution sample and carrying out SDS-PAGE identification according to the ultraviolet detection value; regenerating the column, determining the protein content (2mg/mL) by using a BCA method, subpackaging the pig OPN monoclonal antibody (monoclonal antibody for short) and freezing and storing at-80 ℃ for later use.

Example 4 identification of monoclonal antibody types and subclasses

And (3) identifying the subtype of the antibody by using a mouse monoclonal antibody subtype identification kit (marked by HRP) and referring to an instruction book, wherein the subtype corresponding to the deepest hole in color is the corresponding subtype. The results are shown in FIG. 4, in which the monoclonal antibody Cy2 is IgG1 and the light chain types are all Kappa.

Example 5 ELISA Titer identification of monoclonal antibodies to porcine OPN protein

The monoclonal antibody prepared in example 3 was used as a primary antibody, and the ELISA titer of the monoclonal antibody was detected by an indirect ELISA method, which was identical to the ELISA method for detecting the supernatant, and the primary antibody was a monoclonal antibody diluted 1:1000 times in PBS buffer, and a blank control was set.

As shown in FIG. 5, the maximum OD of the hybridoma cells prepared in example 3 obtained after immunization of the mouse was 1.75 at 450 nm. In FIG. 5, MAb is a porcine OPN monoclonal antibody; the positive serum is BABL/c mouse serum after 4 weeks of immunization of the purified pig OPN recombinant protein; the negative serum is the serum of an unimmunized BABL/c mouse; the His tag protein is obtained after the induction and purification of the lentivirus recombinant expression vector.

Example 6 specific reaction of porcine OPN monoclonal antibody

The monoclonal antibody prepared in example 3 (concentration 2mg/mL) was used as a primary antibody to examine the specific reactivity of the pig OPN monoclonal antibody by WB, IHC and IF. The specific operation is as follows:

1. western Blotting (WB)

Cutting 0.1-0.2 mg of pig testis/ovary by operation, cutting to pieces as much as possible, adding 0.5mL lysine buffer, homogenizing by a homogenizer, carrying out centrifugation for 10min at 4 ℃, 12000rpm, taking supernatant, transferring to a new tube, and detecting and collecting protein concentration by using a BCA method. 5 Xloading buffer is mixed according to the volume ratio of 4:1, boiled for 10min and stored at-20 ℃ for later use.

20 mu g of equivalent total protein is loaded for SDS-PAGE electrophoresis, and after the electrophoresis is finished, the protein is transferred to a nitrocellulose membrane (NC membrane) by a Berlole semi-dry membrane transfer instrument at a voltage of 90V for 1 h. After the transfer printing is finished, carrying out subsequent experiments, and specifically operating as follows:

1) and (3) sealing: the NC membrane was blocked with 5% (w/v) skim milk in TBST for 1 hour at room temperature.

2) Washing: the NC membranes were washed 2 times with TBST containing 0.5% o (v/v) Tween-20, 1 time with TBS, 10 min/time.

3) Adding a primary antibody: the monoclonal antibody (2mg/mL) prepared in example 3 was diluted at a volume ratio of 1:500 into TBST buffer solution containing 5% (w/v) skim milk and incubated overnight at 4 ℃ with a rabbit anti-human OPN antibody (ab8448, 82mg/mL) as a positive control at a dilution ratio of 1:1000 (volume ratio).

4) Washing: the NC membranes were washed 2 times with TBST containing 0.5% o (v/v) Tween-20, 1 time with TBS, 10 min/time.

5) Adding a secondary antibody: goat anti-mouse IgG antibodies were labeled with HRP, and the resulting mixture was diluted with TBST buffer solution containing 5% (w/v) skim milk at a volume ratio of 1: diluting 3000 times, and acting at room temperature for 1 h.

6) Washing: the NC membranes were washed 2 times with TBST containing 0.5% o (v/v) Tween-20, 1 time with TBS, 10 min/time.

7) And (3) developing: and carrying out exposure development by adopting an ECL (Ecl hypersensitive luminescence) kit.

As shown in FIG. 6, the monoclonal antibody prepared in example 3 was more specific than the commercial OPN polyclonal antibody. In FIG. 6, the first two protein bands are testis tissue and the last two protein bands are ovary tissue.

2. Immunohistochemical detection (IHC)

1) And (3) slicing preparation: fresh testis of adult boar and fresh ovary of sow are obtained, immediately fixed in formalin, dehydrated in gradient (xylene I5 min, xylene II 5min, xylene III 5min (3 times elution with "xylene"), 100% alcohol 5min, 85% alcohol 5min, 75% alcohol 5min), embedded in paraffin, sliced to a thickness of 4 μm.

2) Antigen retrieval: antigen retrieval was performed by microwave pretreatment for 10min, blocked with 3% (v/v) rabbit serum (TBS solution dilution), and incubated at room temperature for 30 min.

3) Adding a primary antibody: the sections were placed in TBST buffer containing 5% (w/v) skim milk containing the mAb prepared in example 3 (1:500 dilution) overnight at 4 ℃ while setting a negative control (secondary antibody alone).

4) Adding a secondary antibody: sections were washed 3 times 5 min/time in PBS. Spin-drying, adding a secondary goat anti-mouse antibody (HRP-labeled goat anti-mouse IgG antibody), and incubating at 37 ℃ for 1 h.

5) Color development: sections were washed 3 times 5 min/time in PBS buffer. Spin-drying, staining with peroxidase-labeled streptavidin-biotin and Diaminobenzidine (DAB), observing under microscope, and washing the slices with tap water to stop color development.

6) Counterstaining cell nuclei: the sections were counterstained with hematoxylin for 3min, washed with tap water, differentiated with hematoxylin differentiation solution for 5s, washed with tap water, rewetted with hematoxylin, and washed with running water.

7) Dewatering and sealing: dewatering the slices in 75% alcohol (5min), 85% alcohol (5min), 100% alcohol (5min), and xylene (5min) to obtain transparent slices, air drying, and sealing with neutral gum.

8) And (3) photographing: the slide was examined by olympus BX41 fluorescence microscopy for image acquisition analysis.

The results are shown in fig. 7, and the porcine OPN mab prepared in example 3 has good specificity and is clearly expressed in the spermatocyte of testis and the granulosa cell (cytoplasm) of ovary. In fig. 7, fig. 7A and 7C are negative controls of testis and ovary, respectively, and fig. 7B and 7D are immunohistochemical results of testis and ovary, respectively.

3. Immunofluorescence (IF)

(1) Immunofluorescence of oocytes

1) 50 oocytes after in vitro maturation are arranged, and each group comprises 25 oocytes, and a control group and an experimental group are arranged; digesting and removing granular cells by 0.1% (w/v) hyaluronidase, and washing for 3 times and 15 min/time by using a DPBS-PVA solution at 37 ℃; then removing the zona pellucida with a pH of 2.5, and washing with DPBS-PVA in the same manner (3 times for washing, 15 min/time); wherein

The preparation method of the DPBS-PVA solution is as follows: heating and dissolving 1g of PVA (polyvinyl alcohol with the molecular weight of 30000-70000) in 100mL of Milli-Q water, cooling, adding 9.55g of DPBS powder (Du's phosphate buffer), adding Milli-Q water, and keeping the volume to 1L;

2) fixing with 4% (v/v) paraformaldehyde for 30 min; washing with DPBS-PVA solution in the same way;

3) 10% (v/v) rabbit serum blocking for 1h (note: sealing without washing);

4) OPN monoclonal antibody (1:200) prepared in example 3 was added diluted with 1% (v/v) BSA while setting negative control (same volume of PBS buffer) at 4 ℃ overnight; washing with DPBS-PVA solution in the same way;

5) adding 1% (v/v) of Alexa Fluor goat anti-mouse fluorescent secondary antibody (1:1000) diluted by BSA, and reacting for 2h at 37 ℃ in a dark place; washing with DPBS-PVA solution in the same way;

6) dyeing 10 mu g/mL Hochest33342 at normal temperature in the dark for 5min, washing with a DPBS-PVA solution, and performing the same method;

7) anti-quenching mounting, laser scanning and taking pictures by a fluorescence microscope.

(2) Sperm immunofluorescence

1) Taking 2mL of fresh pig semen, centrifuging at 12000rpm for 10min, removing the semen, washing with PBS buffer solution for 3 times and 5 min/time;

2) sperm count, cell density 5X10⁷Setting a control group and an experimental group per mL; taking 100 mu L of sperm, placing on a glass slide, and drying on ice for 40 min;

3) fixing with 4% (v/v) paraformaldehyde for 20 min; washing with PBS buffer solution in the same manner as above (3 times for washing, 5 min/time);

4) 10% (v/v) rabbit serum block for 45min (note: sealing without washing);

5) OPN monoclonal antibody (1:200) prepared in example 3 was added diluted with 1% (v/v) BSA while setting negative control (same volume of PBS buffer) at 4 ℃ overnight; washing with PBS buffer solution in the same manner as above;

6) adding 1% (v/v) of Alexa Fluor goat anti-mouse fluorescent secondary antibody (1:1000) diluted by BSA, and reacting for 1h at 37 ℃ in a dark place; washing with PBS buffer solution in the same manner as above;

7) culturing 10 μ g/mL FITC-PSA at 37 deg.C in the dark for 30min, washing with PBS buffer solution, and performing the same method;

8) staining 10 μ g/mL Hochest33342 at normal temperature in the dark for 5min, washing with PBS buffer solution, and performing the same method;

9) anti-quenching mounting, laser scanning and taking pictures by a fluorescence microscope.

As shown in FIGS. 8 and 9, the porcine OPN monoclonal antibody prepared in example 3 was highly specific, expressed in oocytes, and specifically expressed at the equator of sperm. In FIG. 8, FIGS. 8A and 8D are OPN protein staining patterns, FIGS. 8B and 8E are nuclear staining patterns, and FIGS. 8C and 8F are Merge patterns of OPN protein staining patterns and nuclear staining patterns. In fig. 9, fig. 9A is a control group, fig. 9B is an experimental group, fig. 9A and 9B are large images of OPN protein staining, nuclear staining and sperm acrosome staining, red represents OPN protein staining, blue represents nuclear staining, and green represents sperm acrosome staining.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims (9)

1. A hybridoma cell strain secreting pig-derived OPN monoclonal antibodies is characterized in that: the hybridoma cell strain is classified and named as a hybridoma cell strain Cy2, is preserved in the China center for type culture Collection in 2019, 12 months and 19 days, and has the preservation address of China, Wuhan university and the preservation number of CCTCC NO: C2019293.

2. a swine OPN monoclonal antibody, which is characterized in that: the porcine OPN monoclonal antibody is secreted and produced by the hybridoma cell strain or the passage cell strain thereof of claim 1.

3. A detection kit, characterized in that: which comprises the hybridoma cell strain of claim 1 and/or the pig OPN monoclonal antibody of claim 2.

4. The detection kit according to claim 3, characterized in that:

the kit is an enzyme linked immunosorbent assay kit or a fluorescence immunoassay kit.

5. A colloidal gold test paper is characterized in that: which comprises the hybridoma cell strain of claim 1 and/or the porcine OPN monoclonal antibody of claim 2.

6. The use of the hybridoma cell line of claim 1, the porcine OPN monoclonal antibody of claim 2, the detection kit of any one of claims 3 to 4, or the colloidal gold strip of claim 5 for the detection of OPN protein for non-diagnostic purposes.

7. Use according to claim 6, characterized in that:

the non-diagnosis purpose comprises the detection of the content of the recombinant OPN protein, and the detection of the content of the OPN protein in-vitro pig tissues, porcine oocytes and/or porcine semen.

8. The hybridoma cell strain of claim 1 and/or the pig-derived OPN monoclonal antibody of claim 2, and application thereof in preparing a detection kit or test paper for detecting the content of OPN protein.

9. Use according to claim 8, characterized in that:

the OPN protein is derived from pig tissues, porcine oocytes and/or porcine semen.

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