CN115806592A - Anti-snakehead pIgR antibody and preparation method and application thereof - Google Patents
Anti-snakehead pIgR antibody and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides an anti-snakehead pIgR antibody and a preparation method and application thereof, belonging to the technical field of fish molecular immunology. The dominant epitope polypeptide of the snakehead pIgR protein is obtained through in-depth analysis and research, and the dominant epitope polypeptide is used as an antigen to successfully prepare a snakehead pIgR polyclonal antibody, so that an important tool is provided for researching the structure, source and function of the snakehead pIgR protein and the application of the snakehead pIgR protein in relation with immunoglobulin, and an important technical means is provided for researching the effect of the snakehead pIgR in the mucosal immune defense process; in addition, the diagnosis of snakehead diseases and the evaluation of the using effect of the vaccine can be carried out by detecting the generation of the pIgR protein receptor in the snakehead mucus, so that the polyclonal antibody can be used for preparing a snakehead disease early diagnosis kit or constructing a vaccine using effect evaluation system and providing references for the prevention and treatment of other cultured fish diseases, and therefore, the polyclonal antibody has good practical application value.
Description
Technical Field
The invention belongs to the technical field of fish molecular immunology, and particularly relates to an anti-snakehead pIgR antibody, and a preparation method and application thereof.
Background
The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
In mammals, the polyimmunoglobulin receptor pIgR can form a compound with secretory polyimmunoglobulin (IgA or IgM), and the pIgR is a secretory component after being cleaved by protease, so that the secretory immunoglobulin is formed to prevent adhesion and invasion of viruses, bacteria, toxins and foreign bodies to mucosal tissues. Therefore, efficient secretion of pIgR is a prerequisite for IgA or IgM to exert mucosal defense functions.
Although fish is a lower vertebrate, it still has a more complete immune system similar to that of higher vertebrates. Research shows that the skin and intestinal tract of teleost fish and the intestinal tract of mammal adopt the same immunoglobulin transport system, i.e. tetrameric immunoglobulin can be transported into mucus by pIgR in epithelial cells to play the immune function. The polyclonal antibody of the anti-snakehead immunoglobulin receptor is prepared, and a powerful tool can be provided for clarifying the structure, the source and the function of the immunoglobulin receptor, the relation with immunoglobulin and the like; in addition, after fish is infected with a pathogen or inoculated with a vaccine, the fish body can produce pIgR and immunoglobulin compound, namely Secretory Immunoglobulin (SIGs). Therefore, by using the polyclonal antibody for resisting the snakehead poly-immunoglobulin receptor and detecting the generation of the poly-immunoglobulin receptor in the snakehead mucus, the early diagnosis of snakehead diseases and the evaluation of the using effect of the vaccine can be carried out, and the method has important theoretical and practical significance for the prevention and treatment of the snakehead diseases and has reference value for the prevention and treatment of other cultured fish diseases. However, the inventors found that there are currently few studies and reports on polyclonal antibodies against the snakehead immunoglobulin receptor.
Disclosure of Invention
Based on the defects of the prior art, the invention provides an anti-snakehead pIgR antibody and a preparation method and application thereof. The dominant epitope polypeptide of the snakehead pIgR protein is obtained through in-depth analysis and research, and the dominant epitope polypeptide is used as an antigen to successfully prepare a snakehead pIgR polyclonal antibody, so that an important tool is provided for researching the structure, source and function of the snakehead pIgR protein and the application of the snakehead pIgR protein in relation to immunoglobulin, and an important technical means is provided for researching the function of the snakehead pIgR in the mucosal immune defense process. The present invention has been completed based on the above results.
Specifically, the technical scheme of the invention is as follows:
in the first aspect of the invention, the specific amino acid sequence of the epitope polypeptide of the anti-snakehead pIgR antibody is SEEQQPETATDT (SEQ ID NO. 1).
The second aspect of the invention provides the application of the epitope polypeptide in preparing anti-snakehead pIgR antibody.
The anti-snakehead pIgR antibody can realize specific binding to snakehead pIgR protein, and further can be used for detecting the snakehead pIgR protein, wherein the detection comprises qualitative detection and quantitative detection.
In a third aspect of the invention, the invention provides an anti-snakehead pIgR antibody, which can specifically bind to snakehead pIgR protein and specifically recognize the epitope polypeptide.
In a fourth aspect of the present invention, a polynucleotide is provided, wherein the polynucleotide is a nucleic acid molecule capable of encoding the anti-snakehead pIgR antibody.
In a fifth aspect of the present invention, there is provided a method for producing the anti-pIgR antibody, wherein the method comprises immunizing a non-human animal with the epitope polypeptide.
In a sixth aspect of the invention, a product for detecting the snakehead pIgR protein is provided, and the product comprises the anti-snakehead pIgR antibody.
In a seventh aspect of the invention, a method for detecting the snakehead pIgR protein is provided, wherein the method comprises the following steps: and (3) detecting a sample to be detected by adopting the anti-snakehead pIgR antibody and/or the product.
In an eighth aspect of the invention, the epitope polypeptide, the anti-snakehead pIgR antibody, the product or the method are applied to any one or more of the following methods:
a) Basic research related to the snakehead pIgR protein;
b) Preparing a snakehead disease (early stage) diagnosis product;
c) And evaluating the using effect of the snakehead immune vaccine.
The beneficial technical effects of one or more technical schemes are as follows:
the technical scheme provides effective snakehead pIgR epitope polypeptide, and the effective snakehead pIgR epitope polypeptide is used as an antigen to successfully obtain an anti-snakehead pIgR polyclonal antibody, and the polyclonal antibody of the anti-snakehead pIgR protein can be specifically combined with the snakehead pIgR protein, and has high titer.
The invention shows that pIgR participates in the transportation of Ig to mucus in the teleost fish body through research, and is a necessary condition for the mucus to play the immune defense function. In addition to mediating and transporting immunoglobulins into the mucus, plgr also has a non-specific immune defense function. Particularly shows that pIgR can stimulate the synthesis of other immune factors; after pIgR is hydrolyzed by protease, free SC can prevent chemotaxis of neutrophil cells so as to reduce inflammatory reaction of an organism and protect epithelial cells; the free SC secretion fragment can bind to bacteria so as to effectively limit the infection of the bacteria to the body and the like. Therefore, pIgR plays a very key role in the immune defense function of organisms, particularly in the mucosal immune defense process. Therefore, the polyclonal antibody has good practical application value.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this application, are included to provide a further understanding of the application, and the description of the exemplary embodiments and illustrations of the application are intended to explain the application and are not intended to limit the application.
FIG. 1 is a diagram showing the hydropathy analysis of the snakehead pIgR protein in the example of the present invention;
FIG. 2 is an analysis diagram of transmembrane domain of snakehead pIgR protein in the embodiment of the invention;
FIG. 3 is a diagram of the predictive analysis of the signal peptide of the snakehead pIgR protein in the embodiment of the invention;
FIG. 4 is a diagram showing an antigenicity analysis of the snakehead pIgR protein in the example of the present invention;
FIG. 5 shows the HPLC analysis results of the polypeptides in the examples of the present invention;
FIG. 6 shows the results of MS analysis of polypeptides according to an embodiment of the present invention;
FIG. 7 is an SDS-PAGE analysis of purified antibodies in an example of the invention; wherein, M is the protein molecular mass standard; antibody purification assay results.
FIG. 8 shows the level of pIgR protein in the mucus and bile of snakehead detected by the antibody according to the present invention. Wherein A, B, C and D are ELISA results of snakehead skin, gill, intestinal mucus and bile pIgR respectively. The "+" marks represent the significant differences in protein expression levels (P < 0.05) at different times in each group after immunostimulation by inactivated aeromonas hydrophila.
FIG. 9 shows the immunoblotting results of antibody detection on snakehead serum, mucus and bile protein in the example of the present invention. Wherein, M: protein molecular mass standard; 1: skin mucus, 2: gill mucus, 3: intestinal mucus, 4: bile.
FIG. 10 shows the results of immunohistochemistry of intestine by antibody detection in the examples of the present invention. A and a are results of immunohistochemistry of snakehead intestine, rabbit anti-snakehead pIgR polyclonal antibody and non-immune rabbit serum respectively. Scale: 100 μm.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
As mentioned above, there are currently some studies and reports on polyclonal antibodies against the snakehead immunoglobulin receptor.
In view of the above, in an exemplary embodiment of the present invention, an epitope polypeptide of the anti-snakehead pIgR antibody is provided, and a specific amino acid sequence of the epitope polypeptide is seeqpetatdt (SEQ ID No. 1).
The invention discovers that an anti-pIgR polyclonal antibody prepared by using the epitope polypeptide as an antigen immune animal can perform specific reaction with pIgR, thereby realizing the analysis and detection of the pIgR and being an important tool for researching the structure and the source of a poly-immunoglobulin receptor and the function in mucosal immune response.
In another embodiment of the invention, the application of the epitope polypeptide in preparing anti-ophthalmia argus pIgR antibody is provided.
The antibody for resisting the snakehead pIgR can be a monoclonal antibody or a polyclonal antibody; in a specific embodiment of the invention, the anti-snakehead pIgR antibody is a polyclonal antibody.
The antibody for resisting the snakehead pIgR can realize specific binding to the snakehead pIgR protein, and further can be used for detecting the snakehead pIgR protein, wherein the detection comprises qualitative detection and quantitative detection.
In another embodiment of the invention, the anti-snakehead plgr antibody can specifically bind to snakehead plgr protein and specifically recognize the epitope polypeptide.
Specifically, the anti-snakehead pIgR antibody can be a monoclonal antibody or a polyclonal antibody; in a specific embodiment of the invention, the anti-snakehead pIgR antibody is a polyclonal antibody.
In another embodiment of the present invention, a polynucleotide is provided, which is specifically a nucleic acid molecule capable of encoding the anti-pIgR antibody of snakehead pIgR.
In another embodiment of the present invention, there is provided a method for preparing the anti-snakehead plgr antibody by immunizing a non-human animal with the epitope polypeptide.
The non-human animal includes a non-human mammal, and in one embodiment of the invention, the non-human mammal is a rabbit.
In another embodiment of the invention, a product for detecting the snakehead plgR protein is provided, and the product comprises the anti-snakehead plgR antibody.
The product may be a corresponding detection reagent, a detection kit, or a corresponding detection device, equipment, etc., and is not specifically limited herein, and the detection may be performed based on existing detection technologies such as ELISA, antigen-antibody reaction, a staining agent, a colloidal gold test strip, and a protein chip, and is not described herein again.
In another embodiment of the present invention, a method for detecting pIgR proteins of snakeheads is provided, which comprises: and (3) detecting a sample to be detected by adopting the anti-snakehead pIgR antibody and/or the product.
In another embodiment of the invention, the epitope polypeptide, the anti-snakehead pIgR antibody, the product or the method are applied to any one or more of the following methods:
a) Basic research related to the snakehead pIgR protein;
b) Preparing a snakehead disease (early stage) diagnosis product;
c) And evaluating the using effect of the snakehead immune vaccine.
Wherein, the basic research related to the snakehead pIgR protein includes but not limited to the research on the structure and the source of the pIgR receptor and the function of the pIgR receptor in mucosal immune response.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Examples
1. Sequence analysis of PIgR
The pIgR protein amino acid sequence is selected from GenBank KAF3696447.1 (Polymeric immunoglobulin receptor [ Channa argus ]), and the specific amino acid sequence is as follows: <xnotran> MKILHTLICCFFLSLQDGNFNFVNAQTFTRIKGTIIRVQCPFSSFGRRKYLCKEPCEQNILIETTNFNAQSGRYSIRYKKGSNLHVTITQLTGSDTGQYRCGLGNDNIPFGIIVVDALVDGNRGFSEDKRVQAKAGENLTVACSFTRTGTSKIICKDPCEKNLLVQTNGDTDRTGRHSILYLESSEAAFVHVTITQLRDSDSGLYYCGLGSSFHGFKIVITEAPSKATTPQTSTASFALPKFIDPSEEQQPETATDTEPSEEVYASIPESNQVYEEIGEKRQSRALPVEISAVYAHAKYAKPNEAEDKDANSFGSADCSQHKDQDEMNKLTYCEVNFFDRAAASSNGVLRGRDNEVVYSVLHVAVNSDDHGGEEPALCSSDP (SEQ ID NO. 2). </xnotran> Amino acid sequence: MW =42.1kd, pi =5.29. Analyzing the signal peptide structure, hydrophobicity analysis, transmembrane domain, antigenicity and the like of the polypeptide to finally obtain the epitope polypeptide sequence: SEEQQPETATDT-C (SEQ ID NO. 1), for further study.
2. Selecting peptide fragments according to the combined antigen epitope analysis result and the transmembrane analysis result, synthesizing the peptide fragments by adopting a chemical synthesis mode, and preparing the antibody after immunization.
Polypeptide sequence: SEEQQPETATDT-C (SEQ ID NO. 1) is the 246-257 amino acid sequence of the snakehead pIgR protein. The HPLC and MS spectra of the polypeptide are shown in FIG. 5 and FIG. 6.
3. Experimental protocol
Polypeptide synthesis, hapten coupling and quality control, animal immunization, titer detection, antiserum preparation, antibody purification and antibody verification
4. Polyclonal antiserum preparation
(1) Antigen preparation
Polypeptide synthesis and complete antigen preparation, wherein the polypeptide is synthesized and coupled with carrier protein, naked peptide provides MS and HPLC quality control reports, and the purity is more than 90%.
(2) Antigen quality control
And 6-7mg of complete antigen (-BSA) with the purity of more than 90 percent is detected by SDS-PAGE, and two rabbits are immunized.
(3) Animal immunization
After emulsification of the antigen, 2 rabbits were tested. The second immunization is carried out after 3 weeks of the first immunization, the third immunization is carried out after 2 weeks of the third immunization, the fourth immunization is carried out after 2 weeks of the third immunization, and the fifth immunization is carried out after 2 weeks of the fourth immunization.
(4) Potency detection
Blood is taken after the three-immunization to measure the titer, and serum WB is respectively taken during the immunization to detect positive tissue lysate.
(5) Antibody purification
The antibody was affinity purified using ProteinA/G.
(6) WB verification
4.1 animal immunization
| Number of immunizations | Immune cycle | Immunization dose | Immunologic adjuvant | Immune animal shape |
| |
1 day | 0.5mg | Complete Freund's adjuvant | Good effect |
| Second immunization | 14 days | 0.5mg | Incomplete fra | Is good |
| Third immunization | 28 days | 0.5mg | Incomplete fra | Good effect |
| Blood sampling without |
35 days | / | / | Normal blood sampling |
| The fourth immunization | 42 days | 0.5mg | Incomplete fujizuo | Is good |
| Blood sampling without taking blood | For 49 days | / | / | Normal blood sampling |
| Blood sampling for immunized animals | 56 days | / | / | Normal blood sampling |
4.2 antiserum ELISA detection
(1) Antigen coating: the antigen was diluted to 2ug/ml with the coating solution, added to a 96-well enzyme-labeled reaction plate at 100. Mu.L/well, and coated overnight at 4 ℃.
(2) Washing: the next day, the liquid in the wells was discarded and the wash solution was washed 3 times.
(3) And (3) sealing: add 200. Mu.L/well blocking solution and incubate at 37 ℃ for 2h.
(4) Washing: washed 3 times with the wash solution.
(5) Adding the sample to be tested (primary antibody): antisera were added (blood was taken at 4 ℃ for 10min by 4000rmp centrifugation and supernatant was taken) and the sera were diluted in duplicate (blank sera were used as negative controls) at 1.
(6) Washing: washed 3 times with the wash solution.
(7) Enzyme-labeled anti-antibody addition: HRP-labeled IgG secondary antibody was added to the cells at 100. Mu.l/well, and the cells were incubated at 37 ℃ for 40min. ( Goat anti-mouse-HRP 1:5000-1:10000, goat anti-rabbit-HRP 1:5000-1:10000 )
(8) Washing: washed 5 times with the washing solution and then patted dry.
(9) Color development: adding 100 mul/hole of freshly prepared substrate solution, and placing for 5-20 min at 37 ℃ in shade.
(10) Terminating reaction and carrying out color comparison: add 50. Mu.L/well stop solution. The color turns yellow; the absorbance of each well at 450nm was measured using a microplate reader.
4.3 antiserum titres
The titer is detected by ELISA, and the titer of the snakehead pIgR antibody is more than 870.4K.
TABLE 1 test results for purified antibodies
Initial dilution: 1 μ g/mL (i.e. 1
The titer, i.e. the highest dilution of the sample OD/blank OD being not less than 2.1
5. Antibody purification
5.1 antibody purification
(1) Pretreatment of a chromatographic column: the column is washed 3-5 times with 10 bed volumes of deionized water at a flow rate of 1ml/min. 0.02M PB +0.3M NaCl at 10 column volumes was washed 3-5 times with a flow rate of 1ml/min.
(2) Sample loading: 4-10ml of antiserum/ascites was diluted with 0.02M PB, filtered through a 0.22 μ M filter and applied to a column at a flow rate of 6 s/drop.
(3) Impurity washing: 0.02M PB was flushed until no protein was detected (G250 did not turn blue) and the flow rate was 2 s/drop.
(4) Antibody elution: 0.1m ph =3.0 glycine was eluted through the column at 3 s/drop, the eluate was collected and the eluted product was checked with G250 until no blue change occurred.
(5) Adjusting the pH value: saturated sodium carbonate adjusts the pH of the eluted product to neutrality.
(6) And (3) ultrafiltration concentration: and (4) performing ultrafiltration concentration to about 1-3ml by using a 10kDa ultrafiltration tube.
(7) And (3) dialysis: and (3) dialyzing the solution by using 5L,0.01M pH =7.4PBS overnight, changing the solution for 1 time on the next day, dialyzing for about 4-6h, running SDS gel, measuring the concentration of the antibody, filling the solution into a marked EP tube, and temporarily storing the solution at 4 ℃ for later use or directly storing the solution at-20 ℃.
(8) And (3) washing and storing the chromatographic column: washing the chromatographic column with deionized water, washing the chromatographic column with 5 times of 20% ethanol, and sealing at 4 deg.c.
5.2 antibody purification results
After purification, the antibody was subjected to SDS-PAGE and stained with Coomassie Brilliant blue. The purity of the purified antibody is more than 85%.
6. The invention relates to a polyclonal antibody ELISA method for detecting the change of the pIgR protein level in snakehead mucus and bile
1. Preparation of inactivated Aeromonas hydrophila
Aeromonas hydrophila strain (Aeromonas hydrophila) was cultured in LB medium at 28 ℃ with shaking for 48 hours. 100mL of a bacterial culture solution after being subjected to shaking culture at 28 ℃ for 48 hours is centrifuged at 11000r/min for 10min, the precipitate is washed three times with a sterilized phosphate buffer solution (PBS, pH7.2), resuspended with PBS containing 0.3% formalin, and placed at room temperature for 24 hours. The inactivated bacterial suspension is centrifuged again at 11000r/min for 10min, washed twice with sterile PBS to remove formalin, finally diluted to about 1.0X 108cells/mL with sterile PBS, and the prepared aeromonas hydrophila vaccine is placed in a refrigerator at 4 ℃ for later use.
2. Snakehead immunity and sample collection
Before experiment, the healthy snakeheads are randomly raised in a glass fiber reinforced plastic water tank with the volume of 2.0 multiplied by 0.5m, the water temperature is 20 +/-1 ℃, the pH value is 7.6-8.2, air is continuously filled during raising, the water is changed by 1/3-1/2 every day, the feed is fed for 2 times, and after the snakeheads are temporarily raised for one week, the snakeheads are divided into two groups for immunization. The first group is injected with 1 × 108CFU/mL concentration of aeromonas hydrophila inactivated vaccine diluted by PBS in the abdominal cavity, and 100 μ L of vaccine is injected in each tail; the second group is soaked in inactivated bacteria liquid diluted by cultivation and with the concentration of 1 × 108CFU/mL, and is continuously aerated and soaked for 30min.
3. Collection of mucus and bile of snakehead
Five snakehead snakeheads are randomly taken from each group of 1d, 3d, 5d, 7d, 10d, 14d, 21d, 28d, 35d and 49d before and after immunization respectively, and skin mucus, gill mucus, intestinal mucus and bile are collected respectively.
And (3) lightly scraping mucus on the skin surface of the snakeheads by using a clean and sterile glass slide, collecting the mucus, adding PBS with the same volume, and mixing the skin mucus of the five-tail fish.
Cutting gill of fish from blood-collected snakehead, washing with PBS for several times, placing into centrifuge tube containing PBS, standing at 4 deg.C for 2 hr, and shaking for several times.
Cutting fish intestines from the blood-collected snakeheads, carefully removing mesentery and blood vessels, washing with PBS for several times, cutting into small segments, longitudinally cutting, placing into a centrifuge tube filled with PBS, standing at 4 ℃ for 2h, and shaking for several times.
Taking out gallbladder from the bled snakeheads, carefully peeling tissues around the gallbladder, washing with PBS for several times, sucking surface water, puncturing the gallbladder to allow bile to flow out and collect, and mixing the bile of the five-tailed fish.
Centrifuging bile, skin, gill and intestinal mucus of the five-tailed fish at 4 deg.C for 15min, respectively, collecting supernatant, adjusting the concentration of each histone, and storing at-80 deg.C.
4. Detection of protein level change of pIgR in snakehead mucus and bile
In order to optimize the ELISA conditions, preliminary experiments were performed on the antigen coating concentration, the sample dilution factor and the antibody dilution factor before the actual experiments, and the optimal conditions were determined, which are given below.
(1) The collected skin mucus, gill mucus, intestinal mucus and bile concentrations were diluted to 0.1mg/mL in advance, added to a 96-well plate at 100. Mu.L/well, and placed in a refrigerator at 4 ℃ overnight.
(2) The next day, the membrane was washed three times with PBS (PBST) containing 0.05% Tween 20 for 5min, after which 200. Mu.L of PBS containing 5% BSA was added and blocked at 37 ℃ for 1h.
(3) The same method is washed three times, each hole is added with PBS diluted rabbit anti-snakehead pIgR multi-antibody 100u L (1 dilution 4000), 37 degrees C were incubated for 1h.
(4) After three PBST washes, 100 μ L of alkaline phosphatase-labeled goat anti-rabbit IgG antibody (1 diluted 5000) was added per well and incubated at 37 ℃ for 1h.
(5) PBST washing three times, after the last washing, adding pNPP buffer (35 mM NaHCO) containing 0.1% pNPP per well 3 ，15mM Na 2 CO 3 ，0.5mM MgCl 2 ·6H 2 O, pH 9.6), 100. Mu.L, development at room temperature for 30min, quenching of the color by NaOH, 2M, and determination of the average absorbance value (OD) at a wavelength of 405 nm. And (3) replacing rabbit anti-snakehead pIgR polyclonal antibody with non-immune rabbit serum as a negative control, calculating the OD value ratio P/N, and judging the positive condition when the P/N is more than or equal to 2.1.
5. As a result, the
5.1 changes in pIgR protein levels in Murass mucus and bile
After the aeromonas hydrophila is immunized in two different modes of intraperitoneal injection and soaking, the ELISA result shows that the pIgR protein level in external secretion such as skin mucus, gill mucus, intestinal mucus, bile and the like is firstly adjusted up within 28d and then is adjusted down to the level of a control group. After soaking immunization, the expression level of pIgR protein reaches a peak value 3d after immunization in skin mucus and gill mucus, the expression level of pIgR reaches a peak value 5d in intestinal mucus, and 7d is required to reach a peak value in bile; in the injection group, the protein level of pIgR in intestinal mucus and bile peaked 7d post-immunization, while in skin mucus and gill mucus, the protein level of pIgR peaked 5d post-immunization. The peak values of skin mucus, gill mucus and intestinal mucus were higher in the soaked group than in the injected group, while the peak value of bile was lower in the injected group.
7. The polyclonal antibody ELISA method of the invention detects pIgR in snakehead exosecretion and serum
1. Sample collection and pretreatment
Randomly taking a snakehead, collecting blood from the tail vein, standing at room temperature for 1h, standing overnight at 4 ℃, centrifuging at 12000g for 15min at 4 ℃ the next day, separating serum, and storing at-80 ℃ for later use.
And (3) performing ultrafiltration concentration on the taken serum and the snakehead skin mucus, gill mucus, intestinal mucus and bile collected in the experiment 3 through a 100kDa ultrafiltration tube, and finally removing part of small molecular weight protein and increasing the protein concentration.
2. Protein electrophoresis and western blot
(1) Preparation of SDS-PAGE gels
Note: the table is italicized for the 10% separation gel volume for this configuration.
Note: italics in the table are 5% concentrated gum volume for this configuration.
(2) Preparing the separating glue according to the formula, and adding the separating glue into the prepared rubber plate. Slowly add to the 2/3 position of the plate. Adding absolute ethyl alcohol to press glue. After the separation gel is solidified, adding the concentrated gel, and inserting a tooth comb.
(3) After the concentrated gel is completely solidified, 1 × electrophoretic solution is prepared.
(4) Putting the rack into the electrophoresis solution, pulling out the comb, and adding a certain volume of protein sample and Marker.
Protein loading amount:
(5) Preparing 1 × electrophoresis solution, immersing the gel plate with the 1 × electrophoresis solution, starting electrophoresis, compressing protein at 60V, and separating protein at 80V (120 minutes).
(6) When the strip runs to half of the rubber plate, 1 x of membrane transferring liquid is prepared and precooled.
(7) The PVDF membrane (about 1-2 cm) was cut according to the expected position of the strip and activated with methanol for 15s.
(8) And cutting the glue with proper size containing the internal reference or target strip.
(9) The "sandwich" (sponge-filter paper-gel-membrane-filter paper-sponge) was set in place.
(10) The "sandwich" was immersed in 1 × spin solution and spun at 300mA for 1h 20min.
Note: the table shows the electrophoresis and film transfer time for each index
(11) A3% skim milk block was prepared with 1 XTSST and blocked overnight.
(12) Rabbit anti-snakehead plgr dilutions (1.
(13) Washing the membrane, soaking in 1 × TBST for 10min, discarding 1 × TBST, and repeating for 3 times.
(14) 2 dilutions of anti-horseradish-enzyme-labeled goat anti-rabbit IgG (H + L) were prepared (1.
(15) Washing the membrane, soaking in 1 × TBST for 10min, discarding 1 × TBST, and repeating for 3 times.
(16) And preparing a luminescent solution, soaking the PVDF membrane by the luminescent solution, placing the PVDF membrane in a sample placing area of the ultra-high sensitivity chemiluminescence imaging system, and running a program to perform development imaging.
As a result: the polyclonal antibody of the polyclonal anti-rabbit anti-snakehead pIgR reacts with snakehead skin mucus, gill mucus, intestinal mucus and bile, and is all at the position with the relative molecular mass of about 47kD, but does not react with snakehead serum, which indicates that the pIgR exists in the skin mucus, gill mucus, intestinal mucus and bile, and the pIgR does not exist in the serum.
8. The invention relates to a polyclonal antibody immunohistochemical staining method for positioning pIgR in snakehead mucous membrane tissues
1. Preparation of snakehead tissue paraffin section
(1) Material taking: dissecting Channa argus with scalpel, scissors, gauze, and forceps, cutting skin and hindgut tissue into pieces of 0.5cm × 0.5cm × 0.5cm, washing with sterile PBS, and fixing with 4% paraformaldehyde for 24 hr.
(2) And (3) dehydrating and wax dipping: and (4) putting the dehydration box into a hanging basket, and dehydrating by sequentially gradient alcohol in the dehydration machine. The method comprises the following steps of 1h of 75% alcohol, 4h-85% alcohol, 2h-90% alcohol, 2h-95% alcohol, 1 h-absolute ethyl alcohol I, 30 min-absolute ethyl alcohol II, 30 min-alcohol benzene, 5-10 min-xylene I, 5-10 min-xylene II, 5-10min-65 DEG molten paraffin I, 1h-65 DEG molten paraffin II, 1h-65 DEG molten paraffin III.
(3) Embedding: embedding the tissues soaked with the wax in an embedding machine. Firstly, molten wax is put into an embedding frame, tissues are taken out from a dehydration box and put into the embedding frame according to the requirements of an embedding surface before the wax is solidified, and corresponding labels are attached. Cooling at-20 deg.C, solidifying wax, taking out the wax block from the embedding frame, and trimming the wax block.
(4) Slicing: the trimmed wax block was sliced in a paraffin slicer to a thickness of 4 μm. The slices float on a spreading machine at 40 ℃ warm water to flatten the tissues, a glass slide picks up the tissues, and the slices are baked in a 60 ℃ oven. Baking with water, drying with wax, baking, and storing at room temperature.
2. Immunohistochemical staining
(1) Antigen retrieval: placing the slices into a repairing box, adding antigen repairing solution (citric acid buffer solution), heating in a pressure cooker to automatically deflate, naturally cooling after leaving heat source after 2min, discarding the antigen repairing solution, and rinsing the slices with PBS. The formula of the antigen retrieval liquid comprises: weighing citric acidSodium Na 3 C 6 H 5 O 7 ·2H 2 O29.4 g, adding distilled water to 1L, weighing citric acid C 6 H 5 O 7 ·H 2 O21.0 g, adding distilled water to 1L; 16.2mL of sodium citrate solution and 3.8mL of citric acid solution are measured, distilled water is added to the solution to be 200mL, and the pH is adjusted to be 6.0.
(2) The sections were transferred to a wet box, freshly prepared 3% hydrogen peroxide was added to remove endogenous peroxidase blocking solution, incubated at room temperature for 10min, and rinsed thoroughly with pbs.
(3) And (3) sealing: washing the slide 3 times with PBS, 5min each time, blotting the PBS around the tissue with absorbent paper, adding 5% BSA dropwise to the slide, blocking at 37 deg.C for 30min.
(4) PBST was washed three times, and after spin-drying, rabbit anti-snakehead pIgR recombinant protein polyclonal antibody (1.
(5) PBST was washed three times, after which goat anti-rabbit IgG (H + L) secondary antibody (1.
(6) PBST is washed for three times, DAB is developed for 5-10min, the dyeing degree is controlled under a microscope, and tap water is used for washing for 1min.
(7) Counterstaining with hematoxylin for 3min, differentiating with hydrochloric acid and ethanol, and turning blue; washing with tap water for 1min, dehydrating, transparentizing, sealing, and examining under microscope.
(8) Non-immunized rabbit serum was used as a negative control instead of polyclonal antibody.
As a result: immunohistochemical staining is carried out on the hindgut of the snakehead mucosal immune tissue by using the anti-snakehead pIgR polyclonal antibody, and the result shows that in the hindgut of the snakehead, an obvious red positive signal appears in an experimental group, and no positive signal is observed in a control group.
Western blot results show that the polyclonal antibody can specifically react with pIgR, so that the polyclonal antibody can be used as an important tool for researching the structure and source of a polyimmunoglobulin receptor and the function of the polyimmunoglobulin receptor in mucosal immune response. In addition, the diagnosis of the snakehead disease and the evaluation of the using effect of the vaccine can be carried out by detecting the generation of the poly-immunoglobulin receptor in the mucus of the snakehead, so that the polyclonal antibody can be used for preparing an early diagnosis kit of the snakehead disease or constructing a system for evaluating the using effect of the vaccine, tools and technical means are provided for the prevention and treatment of the snakehead disease, and references are provided for the prevention and treatment of other diseases of cultured fishes.
It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and the present invention is not limited thereto, and although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications and equivalents can be made in the technical solutions described in the foregoing embodiments, or equivalents thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention. Although the present invention has been described with reference to the specific embodiments, it should be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.
Claims (10)
1. An epitope polypeptide of an anti-snakehead pIgR antibody is characterized in that the specific amino acid sequence of the epitope polypeptide is SEEQQPETATDT (SEQ ID NO. 1).
2. The application of the epitope polypeptide of claim 1 in preparing an anti-snakehead pIgR antibody; the anti-snakehead pIgR antibody is a monoclonal antibody or a polyclonal antibody.
3. An anti-snakehead pIgR antibody, which is characterized in that the anti-snakehead pIgR antibody specifically binds to snakehead pIgR protein and specifically recognizes the epitope polypeptide of claim 1;
the anti-snakehead pIgR antibody is a monoclonal antibody or a polyclonal antibody.
4. A polynucleotide, which is specifically a nucleic acid molecule encoding the anti-snakehead plgR antibody according to claim 3.
5. The method for producing an anti-snakehead plgr antibody according to claim 3, wherein the method is carried out by immunizing a non-human animal with the epitope polypeptide according to claim 1.
6. The method of claim 5, wherein the non-human animal comprises a non-human mammal, and further wherein the non-human mammal is a rabbit.
7. A product for detecting the snakehead plgr protein, which comprises the anti-snakehead plgr antibody of claim 3;
furthermore, the product is a corresponding detection reagent, a detection kit or a corresponding detection device and equipment; the detection is carried out based on ELISA, antigen-antibody reaction, a staining agent, a colloidal gold test strip and a protein chip.
8. A method for detecting the snakehead pIgR protein is characterized by comprising the following steps: and (3) detecting a sample to be detected by using the anti-snakehead PIgR antibody of claim 3 and/or the product of claim 7.
9. Use of the epitope polypeptide of claim 1, the anti-channa pIgR antibody of claim 3, the product of claim 7 or the method of claim 8 in any one or more of:
a) Basic research related to the snakehead pIgR protein;
b) Preparing a snakehead disease diagnosis product;
c) And evaluating the using effect of the snakehead immune vaccine.
10. The use of claim 9, wherein in a), basic studies on pIgR protein of snakeheads include studies on the structure, source and function of pIgR receptor in mucosal immune response.
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| CN116514945A (en) * | 2023-03-24 | 2023-08-01 | 山东省淡水渔业研究院（山东省淡水渔业监测中心） | Antigen epitope polypeptide screening of anti-micropterus salmoides pIgR antibody, polyclonal antibody preparation and application thereof |
| CN116514945B (en) * | 2023-03-24 | 2023-09-22 | 山东省淡水渔业研究院（山东省淡水渔业监测中心） | Antigen epitope polypeptide screening of anti-micropterus salmoides pIgR antibody, polyclonal antibody preparation and application thereof |
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