CN115806592B - Anti-snakehead pIgR antibody and preparation method and application thereof - Google Patents
Anti-snakehead pIgR antibody and preparation method and application thereof Download PDFInfo
- Publication number
- CN115806592B CN115806592B CN202211378441.6A CN202211378441A CN115806592B CN 115806592 B CN115806592 B CN 115806592B CN 202211378441 A CN202211378441 A CN 202211378441A CN 115806592 B CN115806592 B CN 115806592B
- Authority
- CN
- China
- Prior art keywords
- snakehead
- pigr
- antibody
- protein
- mucus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention provides an anti-snakehead pIgR antibody, and a preparation method and application thereof, and belongs to the technical field of fish molecular immunology. The dominant epitope polypeptide of the snakehead pIgR protein is obtained through deep analysis and research, and is used as an antigen to successfully prepare the snakehead pIgR polyclonal antibody, so that an important tool is provided for researching the structure, the source and the 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 action of the snakehead pIgR in the process of mucosal immune defense; in addition, the diagnosis of the 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 the snakehead disease early diagnosis kit or constructing a vaccine using effect evaluation system, and provides references for the prevention and treatment of other cultured fish diseases, thereby having 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 disclosure of this background section is only intended to increase the 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 already known to those of ordinary skill in the art.
In mammals, the poly-immunoglobulin receptor pIgR is capable of forming a complex with a secretory poly-immunoglobulin (IgA or IgM), which is cleaved by protease to form a secretory component, thereby forming a secretory immunoglobulin that prevents adhesion and invasion of viruses, bacteria, toxins and foreign substances to mucosal tissues. Thus, efficient secretion of pIgR is a necessary condition for IgA or IgM to exert a mucosal defense function.
Although fish are lower vertebrates, it still has a more complete immune system similar to higher vertebrates. Studies have shown that the skin and intestinal tracts of teleost fish and the intestinal tracts of mammals employ the same immunoglobulin transport system, i.e. can exert their immune function by transporting tetrameric immunoglobulins into mucus via the pIgR in epithelial cells. The preparation of polyclonal antibodies against the snakehead poly-immunoglobulin receptor can provide powerful tools for elucidating the structure, source, function, relationship with immunoglobulin and the like of the poly-immunoglobulin receptor; in addition, fish can produce a pIgR and immunoglobulin complex, i.e., secreted immunoglobulins (SIgs), after infection with a pathogen or vaccination. Therefore, by utilizing polyclonal antibodies against the snakehead poly-immunoglobulin receptor, early diagnosis of snakehead diseases and evaluation of vaccine use effects can be performed by detecting the generation of the poly-immunoglobulin receptor in snakehead mucus, and the method has important theoretical and practical significance for preventing and treating the snakehead diseases and has reference value for preventing and treating other cultured fish diseases. However, the inventors found that there are currently few studies and reports on polyclonal antibodies against the snakehead poly-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 deep analysis and research, and is used as an antigen to successfully prepare the snakehead pIgR polyclonal antibody, so that an important tool is provided for researching the structure, the source and the 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 action of the snakehead pIgR in the mucosal immune defense process. Based on the above results, the present invention has been completed.
Specifically, the technical scheme of the invention is as follows:
in a first aspect of the invention, there is provided an epitope polypeptide of an anti-snakehead pIgR antibody, the specific amino acid sequence of the epitope polypeptide is SEEQQPETATDT (SEQ ID NO. 1).
In a second aspect, the invention provides application of the epitope polypeptide in preparation of an anti-snakehead pIgR antibody.
The anti-snakehead pIgR antibody can realize the specific binding of the snakehead pIgR protein, and is further used for detecting the snakehead pIgR protein, wherein the detection comprises qualitative and quantitative detection.
In a third aspect of the present invention, there is provided an anti-snakehead pIgR antibody capable of specifically binding to a snakehead pIgR protein and specifically recognizing the above epitope polypeptide.
In a fourth aspect of the invention, there is provided a polynucleotide, in particular a nucleic acid molecule capable of encoding an anti-snakehead pIgR antibody as described above.
In a fifth aspect of the invention, there is provided a method for preparing the above-described anti-snakehead pIgR antibody by immunizing a non-human animal with the above-described epitope polypeptide.
In a sixth aspect of the invention, there is provided a product for detecting the snakehead pIgR protein, the product comprising the above-described anti-snakehead pIgR antibody.
In a seventh aspect of the present invention, there is provided a method for detecting the pIgR protein of snakehead, the method comprising: and detecting the sample to be detected by adopting the anti-snakehead pIgR antibody and/or the product.
In an eighth aspect of the invention, there is provided the use of an epitope polypeptide, an anti-snakehead pIgR antibody, a product or a method as described above in any one or more of the following:
a) Basic research related to snakehead pIgR protein;
b) Preparing a snakehead disease (early) diagnosis product;
c) And (5) evaluating the using effect of the snakehead immune vaccine.
The beneficial technical effects of one or more of the technical schemes are as follows:
the technical scheme provides the effective snakehead pIgR epitope polypeptide, and the snakehead pIgR polyclonal antibody can be successfully obtained by taking the snakehead pIgR epitope polypeptide as an antigen, and the snakehead pIgR protein-resistant polyclonal antibody can be specifically combined with the snakehead pIgR protein and has high titer.
According to the invention, researches show that the pIgR participates in Ig transportation into mucus in teleost bodies, and is a necessary condition for mucus to exert immune defense function. In addition to mediating and transporting immunoglobulins into mucus, pIgR also has a non-specific immune defence function. In particular in that the pIgR is capable of stimulating the synthesis of other immune factors; after pIgR is hydrolyzed by protease, free SC can prevent chemotaxis of neutrophil, thereby reducing inflammatory reaction of organism and protecting epithelial cells; the free SC secretory fragments are capable of binding bacteria to effectively limit bacterial infection of the body, and the like. It can be seen that pIgR plays a very critical role in the immune defenses of the organism itself, in particular in the mucosal immune defenses. Therefore, the polyclonal antibody has good practical application value.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiments of the application and together with the description serve to explain the application and do not constitute an undue limitation to the application.
FIG. 1 is a graph showing the hydrophilic-hydrophobic analysis of the snakehead pIgR protein in the embodiment of the invention;
FIG. 2 is a graph showing the analysis of the transmembrane domain of the snakehead pIgR protein in the embodiment of the invention;
FIG. 3 is a graph showing the predictive analysis of the signal peptide of the snakehead pIgR protein in the embodiment of the invention;
FIG. 4 is an antigenicity analysis chart of the snakehead pIgR protein in the embodiment of the invention;
FIG. 5 shows the results of HPLC analysis of polypeptides in an embodiment of the present invention;
FIG. 6 shows the MS analysis results of the polypeptides according to the embodiment of the invention;
FIG. 7 shows SDS-PAGE analysis of purified antibodies in the examples of the present invention; wherein M is a protein molecular mass standard; 1, antibody purification analysis results.
FIG. 8 shows the detection of pIgR protein level changes in snakehead mucus and bile by the antibodies according to the examples of the present invention. Wherein A, B, C and D are ELISA results of snakehead skin, gill, intestinal mucus and bile pIgR respectively. The "+" numbers represent significant differences in protein expression levels (P < 0.05) for each group at different times following immune stimulation by inactivated aeromonas hydrophila.
FIG. 9 shows immunoblotting results of antibodies against snakehead serum, mucus and bile proteins in an 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 intestinal immunohistochemistry for antibody detection in the examples of the present invention. A, a are the results of immunohistochemical treatment of snakehead intestines and rabbit anti-snakehead pIgR polyclonal antibodies and non-immune rabbit serum respectively. Ruler: 100 μm.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention. 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 in accordance with the present application. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.
As described above, there are few studies and reports on polyclonal antibodies against the snakehead poly-immunoglobulin receptor.
Accordingly, in one exemplary embodiment of the present invention, an epitope polypeptide of an anti-snakehead pIgR antibody is provided, the specific amino acid sequence of which is SEEQQPETATDT (SEQ ID No. 1).
The antigen epitope polypeptide is 246-257 amino acid sequences of snakehead pIgR protein, has excellent immunogenicity and specificity, and is found through research, the antigen epitope polypeptide is used as an anti-pIgR polyclonal antibody prepared by immunizing animals with antigen, and can specifically react with pIgR, so that the analysis and detection of the pIgR are realized, and the structure, the source and the important tool of functions in mucosal immune response of a poly immunoglobulin receptor can be researched.
In still another embodiment of the present invention, there is provided an application of the above epitope polypeptide in preparing an anti-snakehead pIgR antibody.
Wherein, the anti-snakehead pIgR antibody can be a monoclonal antibody or a polyclonal antibody; in one embodiment of the invention, the anti-snakehead pIgR antibody is a polyclonal antibody.
The anti-snakehead pIgR antibody can realize the specific binding of the snakehead pIgR protein, and is further used for detecting the snakehead pIgR protein, wherein the detection comprises qualitative and quantitative detection.
In yet another embodiment of the present invention, there is provided an anti-snakehead pIgR antibody capable of specifically binding to a snakehead pIgR protein and specifically recognizing the above epitope polypeptide.
Specifically, the anti-snakehead pIgR antibody can be a monoclonal antibody or a polyclonal antibody; in one embodiment of the invention, the anti-snakehead pIgR antibody is a polyclonal antibody.
In yet another embodiment of the present invention, there is provided a polynucleotide, specifically a nucleic acid molecule capable of encoding the above-described anti-snakehead pIgR antibody.
In still another embodiment of the present invention, there is provided a method for producing the above-mentioned anti-snakehead pIgR antibody, wherein the method comprises immunizing a non-human animal with the above-mentioned 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 still another embodiment of the present invention, there is provided a product for detecting the snakehead pIgR protein, the product comprising the above-mentioned anti-snakehead pIgR antibody.
The product may be a corresponding detection reagent, a detection kit, or a corresponding detection device, apparatus, etc., which are not specifically limited herein, and the detection may be performed based on existing detection technologies such as ELISA, antigen-antibody reaction, stain, colloidal gold test strip, and protein chip, and will not be described herein.
In yet another embodiment of the present invention, there is provided a method for detecting the pIgR protein of snakehead, the method comprising: and detecting the sample to be detected by adopting the anti-snakehead pIgR antibody and/or the product.
In yet another embodiment of the present invention, there is provided the use of the above epitope polypeptide, anti-snakehead pIgR antibody, product or method in any one or more of the following:
a) Basic research related to snakehead pIgR protein;
b) Preparing a snakehead disease (early) diagnosis product;
c) And (5) evaluating the using effect of the snakehead immune vaccine.
Wherein the snakehead pIgR protein related basic study includes, but is not limited to, a study of the structure, source and 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. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention.
Examples
1. Sequence analysis of PIgR
The amino acid sequence of the pIgR protein is selected from GenBank KAF3696447.1 (Polymeric immunoglobulin receptor [ Channa argus ]), and the specific amino acid sequence is as follows: MKILHTLICCFFLSLQDGNFNFVNAQTFTRIKGTIIRVQCPFSSFGRRKYLCKEPCEQNILIETTNFNAQSGRYSIRYKKGSNLHVTITQLTGSDTGQYRCGLGNDNIPFGIIVVDALVDGNRGFSEDKRVQAKAGENLTVACSFTRTGTSKIICKDPCEKNLLVQTNGDTDRTGRHSILYLESSEAAFVHVTITQLRDSDSGLYYCGLGSSFHGFKIVITEAPSKATTPQTSTASFALPKFIDPSEEQQPETATDTEPSEEVYASIPESNQVYEEIGEKRQSRALPVEISAVYAHAKYAKPNEAEDKDANSFGSADCSQHKDQDEMNKLTYCEVNFFDRAAASSNGVLRGRDNEVVYSVLHVAVNSDDHGGEEPALCSSDP (SEQ ID NO. 2). Amino acid sequence: mw=42.1 kd, pi=5.29. Analyzing the signal peptide structure, hydrophobicity analysis, transmembrane domain, antigenicity and the like of the polypeptide to finally obtain an epitope polypeptide sequence: SEEQQPETATDT-C (SEQ ID NO. 1) for the next study.
2. Peptide fragments are selected according to the epitope analysis result and the transmembrane analysis result, the peptide fragments are synthesized in a chemical synthesis mode, and antibodies are prepared after immunization.
Polypeptide sequence: SEEQQPETATDT-C (SEQ ID NO. 1) is the 246 th-257 th amino acid sequence of the snakehead pIgR protein. HPLC and MS spectra of the polypeptides are shown in FIGS. 5 and 6.
3. Experimental protocol
Polypeptide synthesis, hapten coupling, quality control, animal immunity, titer detection, antiserum preparation, antibody purification and antibody verification
4. Polyclonal antiserum preparation
(1) Antigen preparation
Polypeptide synthesis and complete antigen preparation, polypeptide synthesis and coupling carrier protein, and naked peptide providing MS and HPLC quality control report with purity of more than 90%.
(2) Antigen quality control
SDS-PAGE detection, complete antigen (-BSA) 6-7mg, purity greater than 90%, two rabbits were immunized.
(3) Immunization of animals
After emulsification of the antigen 2 experimental rabbits were immunized. One was given a second, 2 weeks apart, then a third, 2 weeks apart, four, 2 weeks apart, and five days apart.
(4) Potency detection
Blood is taken for measuring titer after three-immunity, and serum WB is taken for detecting positive tissue lysate during the immunization period.
(5) Antibody purification
Antibodies were affinity purified using ProteinA/G.
(6) WB validation
4.1 immunization of animals
| Number of immunizations | Immune cycle | Immunization dose | Immunoadjuvant | Immunization of animals |
| First immunization | For 1 day | 0.5mg | Complete Freund's adjuvant | Good quality |
| Second immunization | 14 days | 0.5mg | Incomplete Freund's adjuvant | Good quality |
| Third immunization | For 28 days | 0.5mg | Incomplete Freund's adjuvant | Good quality |
| Blood taking with three-free function | For 35 days | / | / | Normal blood sampling |
| Fourth immunization | 42 days | 0.5mg | Incomplete Freund's adjuvant | Good quality |
| Four-way blood taking | 49 days | / | / | Normal blood sampling |
| Blood collection of immunized animals | For 56 days | / | / | Normal blood sampling |
4.2 antiserum ELISA detection
(1) Antigen coating: the antigen was diluted to 2ug/ml with coating solution, 100. Mu.L/well was added to a 96-well enzyme-labeled reaction plate and coated overnight at 4 ℃.
(2) Washing: the next day the liquid in the wells was discarded and the wash liquid was washed 3 times.
(3) Closing: 200. Mu.L/well of blocking solution was added and incubated at 37℃for 2h.
(4) Washing: washing with the washing liquid for 3 times.
(5) Adding a sample to be tested (primary antibody): antisera (blood was collected at 4 ℃ C. And 4000rmp was centrifuged for 10min to collect supernatant), and the serum was incubated with the diluent at 1:2000,1:4000,1:8000,1:16000,1:32000,1:64000,1:128000.
(6) Washing: washing with the washing liquid for 3 times.
(7) Adding enzyme-labeled anti-antibody: HRP-labeled IgG secondary antibody was added and incubated at 37℃for 40min at 100. Mu.l/well. ( Goat anti-mouse-HRP 1:5000-1:10000, goat anti-rabbit-HRP 1:5000-1:10000 )
(8) Washing: washing with washing liquid for 5 times, and drying.
(9) Color development: adding 100 mu L/hole of freshly prepared substrate solution, and shading and standing for 5-20 min at 37 ℃.
(10) Termination reaction, colorimetric: add 50. Mu.L/Kong Zhongzhi solution. The color turns yellow; the absorbance of each well at 450nm was measured with a microplate reader.
4.3 antiserum titers
The ELISA detection titer shows that the snakehead pIgR antibody titer is more than 870.4K.
TABLE 1 detection results of purified antibodies
Initial dilution: 1 μg/mL (i.e., 1:850 dilution initiation)
The titer, i.e. the highest dilution of the sample OD/blank OD is more than or equal to 2.1
5. Antibody purification
5.1 antibody purification
(1) Pretreatment of a chromatographic column: the deionized water with 10 times of bed volume is washed 3-5 times with the flow rate of 1ml/min. The column was flushed 3-5 times at a flow rate of 1ml/min at 0.02M PB+0.3M NaCl which was 10 column volumes.
(2) Sample loading: 4-10ml of antiserum/ascites, diluted with 0.02M PB, filtered through a 0.22 μm filter and applied to a column, the flow rate was adjusted to 6 s/drop.
(3) Washing: 0.02M PB wash until no protein was run off (G250 does not change blue), flow rate 2 s/drop.
(4) Antibody elution: 0.1M ph=3.0 glycine was eluted at 3 s/drop through the column, and the eluate was collected and the eluted product was detected with G250 until it did not turn blue.
(5) pH value adjustment: saturated sodium carbonate adjusts the pH of the eluted product to neutral.
(6) Ultrafiltration concentration: the 10kDa ultrafiltration tube is subjected to ultrafiltration concentration to about 1-3 ml.
(7) And (3) dialysis: 5L,0.01M PH=7.4 PBS dialyses overnight, after changing liquid 1 time the next day, dialyses about 4-6 hours, runs SDS gel, and packs into the marked EP tube after measuring the antibody concentration, can be temporarily stored at 4 ℃ for standby or directly stored at-20 ℃.
(8) Washing and storing the chromatographic column: washing the chromatographic column with deionized water, washing the chromatographic column with 20% ethanol with 5 times of the bed volume, and sealing at 4deg.C.
5.2 antibody purification results
After purification, the antibodies were subjected to SDS-PAGE and stained with Coomassie blue. The purity of the purified antibody is above 85%.
6. The polyclonal antibody ELISA method of the invention detects the pIgR protein level change in snakehead mucus and bile
1. Preparation of inactivated aeromonas hydrophila
Aeromonas hydrophila strain (Aeromonas hydrophila) was shake-cultured in LB medium at 28℃for 48 hours. 100mL of the bacterial culture solution after 48h of shaking culture at 28℃was centrifuged at 11000r/min for 10min, and the pellet was washed three times with sterilized phosphate buffer (PBS, pH 7.2), resuspended in PBS containing 0.3% formalin, and left at room temperature for 24h. The inactivated bacterial suspension is centrifuged again at 11000r/min for 10min, washed twice with sterilized PBS to remove formalin, and finally diluted to about 1.0X108 cells/mL with sterilized PBS, and the prepared aeromonas hydrophila vaccine is placed in a refrigerator at 4 ℃ for standby.
2. Snakehead immunity and sample collection
Healthy snakeheads are randomly fed into a glass fiber reinforced plastic water tank with the volume of 2.0X2.0X0.5 m before the experiment, the water temperature is 20+/-1 ℃, the pH is 7.6-8.2, the continuous aeration is carried out during the feeding period, the water is changed 1/3-1/2 every day, the feed is fed for 2 times, and after temporary culture for one week, the fishes are divided into two groups for immunization. The first group was intraperitoneally injected with inactivated vaccine of Aeromonas hydrophila diluted in PBS at a concentration of 1X 108CFU/mL, 100. Mu.L per tail; the second group is soaked in inactivated bacteria liquid with the concentration of 1X 108CFU/mL which is diluted by culture, and is continuously aerated and soaked for 30min.
3. Collection of snakehead mucus and bile
The five snakehead tails are randomly taken from each group of 1d, 3d, 5d, 7d, 10d, 14d, 21d, 28d, 35d and 49d before and after immunization, and skin mucus, gill mucus, intestinal mucus and bile are collected respectively.
The mucus on the skin surface of snakehead is gently scraped with a clean and sterile glass slide, collected, added with PBS with equal volume, and mixed with the skin mucus of five tail fish.
Shearing fish gills from the snakehead after blood collection, cutting into a plurality of sections, flushing the sections with PBS for a plurality of times, placing the sections into a centrifuge tube filled with PBS, and standing the sections for 2 hours at 4 ℃ and vibrating the sections for a plurality of times.
Cutting off fish intestines from blood-collected snakeheads, carefully removing mesenteries and blood vessels, flushing with PBS for several times, cutting into small sections, cutting longitudinally, placing into a centrifuge tube filled with PBS, standing at 4 ℃ for 2 hours, and vibrating for several times.
Taking out gallbladder from the blood-taken snakehead, carefully peeling the tissues around the gallbladder, flushing the snakehead with PBS for several times, sucking surface water, puncturing the gallbladder, allowing bile to flow out and collect, and mixing the bile of the five-tail fish.
Centrifuging the bile and skin, gill and intestinal mucus of the Penaeus vannamei Boone at each time point at 12000g for 15min at 4deg.C, collecting supernatant, adjusting the concentration of each group protein to be consistent, and storing at-80deg.C for use.
4. Detection of protein level change of pIgR in snakehead mucus and bile
In order to optimize the conditions of ELISA, pre-experiments were performed on the antigen coating concentration, the dilution of the sample and the dilution of the antibody before the official experiments, and the optimum 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 and added to 96-well plates with 100. Mu.L of each well, and placed in a refrigerator at 4℃overnight.
(2) The samples were washed three times with PBS (PBST) containing 0.05% Tween 20 for 5min, 200. Mu.L of PBS containing 5% BSA was added thereto, and the mixture was blocked at 37℃for 1 hour.
(3) The cells were washed three times in the same manner, 100. Mu.L of the rabbit anti-snakehead pIgR polyclonal antibody diluted in PBS was added to each well (dilution of 1:4000), and incubated at 37℃for 1 hour.
(4) After three washes of PBST, 100. Mu.L of alkaline phosphatase-labeled goat anti-rabbit IgG antibody (1:5000 dilution) was added to each well and incubated at 37℃for 1h.
(5) PBST is washed three times, and after the last washing is finished, each hole is added with the solution containing 0.1 percentpNPP buffer of pNPP (35 mM NaHCO) 3 ，15mM Na 2 CO 3 ，0.5mM MgCl 2 ·6H 2 O, pH 9.6) 100. Mu.L, developed at room temperature for 30min,2M NaOH stopped the development and the average absorbance (OD) was measured at 405 nm. The non-immunized rabbit serum replaces the rabbit anti-snakehead pIgR polyclonal antibody to be used as a negative control, the OD value proportion P/N is calculated, and positive is judged when the P/N is more than or equal to 2.1.
5. Results
5.1 changes in pIgR protein levels in snakehead mucus and bile
After the aeromonas hydrophila is immunized by intraperitoneal injection and soaking, ELISA results show that the pIgR protein level in skin mucus, gill mucus, intestinal mucus, bile and other exocrine fluids is firstly up-regulated within 28d and then down-regulated to the level of a control group. After immersion immunization, the expression level of the pIgR protein peaks at 3d after immunization in skin mucus and gill mucus, at 5d in intestinal mucus, and at 7d in bile; in the injected group, the protein level of pIgR in intestinal mucus and bile peaked 7d after immunization, whereas in skin mucus and gill mucus, the protein level of pIgR peaked 5d after immunization. The peak values of skin mucus, gill mucus and intestinal mucus in the soaked group were higher than those in the injected group, while the peak values of bile were lower than those in the injected group.
7. The polyclonal antibody ELISA method of the invention detects pIgR in the snakehead exocrine fluid and serum
1. Sample collection and pretreatment
Randomly taking snakehead, taking blood from tail vein, standing at room temperature for 1h, standing at 4deg.C overnight, centrifuging at 12000g for 15min at 4deg.C the next day, separating serum, and storing at-80deg.C for use.
And (3) carrying out ultrafiltration concentration on the serum and the snakehead skin mucus, gill mucus, intestine 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) Configuration of SDS-PAGE gels
Note that: the italics in the table indicate 10% of the separator volume for this configuration.
Note that: the table is italicized with 5% concentrate volume for this configuration.
(2) And (3) preparing the separating glue according to the formula, and adding the separating glue into the already-erected glue-making plate. Slowly add to the plate at the 2/3 position. Adding absolute ethyl alcohol to press the gel. After the separating glue is solidified, adding the concentrated glue, and inserting a tooth comb.
(3) After the gel concentrate was completely coagulated, a 1 Xelectrophoresis solution was prepared.
(4) And (3) placing the rack into electrophoresis liquid, removing the comb teeth, and adding a certain volume of protein sample and Marker.
Protein loading amount:
(5) 1 Xthe electrophoresis solution was prepared, the gel plate was immersed in the 1 Xthe electrophoresis solution, electrophoresis was started, 60V was performed to compress the protein, and 80V was performed to isolate the protein (120 minutes).
(6) When the strip runs to half of the rubber plate, 1X film transfer liquid is prepared and precooled.
(7) PVDF membrane (about 1-2 cm) was cut according to the position of the expected strip and methanol was activated for 15s.
(8) The glue is cut to size suitable for containing the internal reference or target strip.
(9) The "sandwich" (sponge-filter-glue-membrane-filter-sponge) was placed.
(10) The "sandwiches" were immersed in 1 Xtransfer solution and transferred to the membrane for 1 hour and 20min with 300mA constant flow.
Note that: the table shows the electrophoresis and transfer time of each index
(11) 3% skim milk blocking solution was prepared with 1 XTBST and blocked overnight.
(12) Rabbit anti-snakehead pIgR dilutions (1:3000) were prepared and PVDF membranes were incubated for 3 hours against primary antibodies.
(13) The membrane was washed, soaked with 1 XTBE for 10min, and then 1 XTST was discarded and repeated 3 times.
(14) A 2-anti-horseradish enzyme labeled goat anti-rabbit IgG (h+l) dilution (1:5000 dilution) was prepared and PVDF membranes were incubated for 2 hours.
(15) The membrane was washed, soaked with 1 XTBE for 10min, and then 1 XTST was discarded and repeated 3 times.
(16) Preparing a luminescent liquid, soaking a PVDF film by the luminescent liquid, and then placing the PVDF film in a sample placing area of an ultra-high sensitivity chemiluminescent imaging system for developing and imaging by a running program.
Results: the polyclonal antibody of the immune gill mucus, intestinal mucus and bile reaction, and are all at a position of about 47kD relative to the molecular mass, but did not react with snakehead serum, indicating that there was pIgR in skin mucus, gill mucus, intestinal mucus and bile, whereas there was no pIgR in serum.
8. The polyclonal antibody immunohistochemical staining method of the invention locates pIgR in snakehead mucous membrane tissue
1. Preparation of snakehead tissue paraffin section
(1) Drawing materials: dissecting the snakehead with a dissecting knife, scissors, gauze, forceps and other tools, cutting the skin and the hindgut tissue into small pieces with the size of 0.5cm multiplied by 0.5cm, washing with sterile PBS, and then placing into 4% paraformaldehyde for fixing for 24 hours.
(2) Dewatering and immersing wax: and placing the dehydration box into a basket, and sequentially carrying out gradient alcohol dehydration in a dehydrator. 75% alcohol 4h-85% alcohol 2h-90% alcohol 2h-95% alcohol 1 h-absolute alcohol I30 min-absolute alcohol II 30 min-alcohol benzene 5-10 min-xylene I5-10 min-xylene II 5-10 min-65% melted paraffin I1 h-65% melted paraffin II 1 h-65% melted paraffin III 1 h-65%.
(3) Embedding: embedding the wax-soaked tissue in an embedding machine. Firstly, putting melted wax into an embedding frame, taking out tissues from a dehydration box before the wax is solidified, putting the tissues into the embedding frame according to the requirement of an embedding surface, and attaching corresponding labels. Cooling at-20 deg.c, solidifying, taking out the wax block from embedding frame and trimming.
(4) Slicing: the trimmed wax block was sliced in a paraffin slicer to a thickness of 4 μm. The slices float on warm water at 40 ℃ of a slice spreading machine to flatten the tissues, the glass slide drags the tissues out, and the slices are baked in a baking oven at 60 ℃. And (5) baking the water, drying the wax, baking, taking out and preserving at normal temperature for standby.
2. Immunohistochemical staining
(1) Antigen retrieval: placing the slices into a repairing box, adding antigen repairing liquid (citric acid buffer solution), heating the slices in an autoclave to automatically deflate, naturally cooling the slices after 2 minutes, discarding the antigen repairing liquid, and leaching the slices with PBS. The antigen retrieval liquid comprises the following formula: weighing sodium citrate Na 3 C 6 H 5 O 7 ·2H 2 29.4g of O, adding distilled water to 1L, and weighing citric acid C 6 H 5 O 7 ·H 2 O21.0 g, distilled water was added to 1L; 16.2mL of sodium citrate solution, 3.8mL of citric acid solution, and distilled water to 200mL were measured and the pH was adjusted to 6.0.
(2) The sections were transferred into a wet box, fresh 3% hydrogen peroxide was added to remove endogenous peroxidase blocking solution, incubated for 10min at room temperature, and rinsed thoroughly with pbs.
(3) Closing: the slides were washed 3 times with PBS for 5min each, the PBS surrounding the tissue was blotted with absorbent paper, 5% BSA was added dropwise to the slides, and the slides were blocked at 37℃for 30min.
(4) PBST is washed three times, and after spin-drying, rabbit anti-snakehead pIgR recombinant protein polyclonal antibody (1:500) is respectively dripped, and incubated for 1h at 37 ℃.
(5) PBST was washed three times, and then goat anti-rabbit IgG (H+L) secondary antibody (1:100) working solution was added dropwise, followed by incubation at 37℃for 30min.
(6) PBST is washed three times, DAB is developed for 5-10min, the dyeing degree is mastered under a microscope, and tap water is used for washing for 1min.
(7) Hematoxylin counterstain for 3min, hydrochloric acid alcohol differentiation, and blue returning; washing with tap water for 1min, dehydrating, transparency, sealing, and microscopic examination.
(8) Non-immunized rabbit serum was used as a negative control instead of polyclonal antibody.
Results: immunohistochemical staining of the snakehead mucosa immune tissue hindgut with the anti-snakehead pIgR polyclonal antibody showed that in the snakehead hindgut, the experimental group showed a clear red positive signal, whereas no positive signal was observed in the control group.
Western blot results show that the polyclonal antibody can specifically react with pIgR, so that the polyclonal antibody can be proved to be an important tool for researching the structure, the source and the function of a multimeric immunoglobulin receptor in mucosal immune response. In addition, the diagnosis of the snakehead diseases 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 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, provides tools and technical means for the prevention and treatment of the snakehead diseases and provides references for the prevention and treatment of other cultured fish diseases.
Finally, it should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and the present invention is not limited to the above-mentioned embodiments, but may be modified or substituted for some of them by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention. While the foregoing describes the embodiments of the present invention, it should be understood that the present invention is not limited to the embodiments, and that various modifications and changes can be made by those skilled in the art without any inventive effort.
Claims (10)
1. An epitope polypeptide of an anti-snakehead pIgR antibody, which is characterized in that the specific amino acid sequence of the epitope polypeptide is SEEQQPETATDT (SEQ ID NO. 1).
2. The use of the epitope polypeptide of claim 1 for 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 specifically binds to a snakehead pIgR protein and specifically recognizes the epitope polypeptide of claim 1;
the anti-snakehead pIgR antibody is a polyclonal antibody.
4. A polynucleotide, wherein said polynucleotide is specifically a nucleic acid molecule encoding the anti-snakehead plgr antibody of claim 3.
5. A method of producing an anti-snakehead pIgR antibody according to claim 3, wherein the method is immunization of a non-human animal with the epitope polypeptide according to claim 1.
6. The method of claim 5, wherein the non-human animal is a non-human mammal.
7. The method of claim 6, wherein the non-human mammal is a rabbit.
8. A detection reagent or detection kit for detecting the snakehead pIgR protein, characterized in that the detection reagent or detection kit comprises the anti-snakehead pIgR antibody of claim 3.
9. The detection reagent or detection kit according to claim 8, wherein the detection is performed based on ELISA, colloidal gold test strips or protein chips.
10. Use of the epitope polypeptide of claim 1, the anti-snakehead pIgR antibody of claim 3, the detection reagent or the detection kit of any one of claims 8-9 in the preparation of a snakehead disease diagnosis product.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211378441.6A CN115806592B (en) | 2022-11-04 | 2022-11-04 | Anti-snakehead pIgR antibody and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211378441.6A CN115806592B (en) | 2022-11-04 | 2022-11-04 | Anti-snakehead pIgR antibody and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115806592A CN115806592A (en) | 2023-03-17 |
| CN115806592B true CN115806592B (en) | 2023-06-09 |
Family
ID=85483135
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211378441.6A Active CN115806592B (en) | 2022-11-04 | 2022-11-04 | Anti-snakehead pIgR antibody and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115806592B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2009137859A (en) * | 2007-12-05 | 2009-06-25 | Univ Nihon | Monoclonal antibody reactive with mouse polymeric immunoglobulin receptor |
| CN106565842A (en) * | 2016-11-10 | 2017-04-19 | 中山大学 | Universal polyclonal antibody of freshwater fish as well as preparation method and application of the universal polyclonal antibody |
| CN106478822B (en) * | 2016-12-14 | 2019-06-28 | 长江大学 | A kind of preparation method of swamp eel aldehyde ketone reductase polyclonal antibody |
-
2022
- 2022-11-04 CN CN202211378441.6A patent/CN115806592B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN115806592A (en) | 2023-03-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US7022328B1 (en) | Therapeutic and diagnostic compositions | |
| Proudman et al. | Correlation of antigen specific IgG and IgG (T) responses with Anoplocephala perfoliata infection intensity in the horse | |
| Dunne et al. | Human antibody responses to Schistosoma mansoni: the influence of epitopes shared between different life‐cycle stages on the response to the schistosomulum | |
| CN115806592B (en) | Anti-snakehead pIgR antibody and preparation method and application thereof | |
| Ellis et al. | Glycans as targets for monoclonal antibodies that protect rats against Trichinella spiralis | |
| Estévez et al. | Monoclonal antibodies to turbot (Scophthalmus maximus) immunoglobulins: characterization and applicability in immunoassays | |
| CN111925436B (en) | Monoclonal antibody of African swine fever virus P30 protein and application thereof | |
| Shaw et al. | Identification and characterisation of an aspartyl protease inhibitor homologue as a major allergen of Trichostrongylus colubriformis | |
| CN113265003A (en) | anti-TdT protein monoclonal antibody, cell strain thereof, preparation method and application | |
| Ogunremi et al. | Diagnosis of Taenia saginata cysticercosis by immunohistochemical test on formalin-fixed and paraffin-embedded bovine lesions | |
| CN106947744A (en) | The hybridoma cell strain of one plant of anti-flatfoot protein monoclonal antibody of secretion and its application | |
| CN116514945B (en) | Antigen epitope polypeptide screening of anti-micropterus salmoides pIgR antibody, polyclonal antibody preparation and application thereof | |
| Tomer et al. | Comparative analysis of the outer membrane protein profiles of isolates of the Pasteurella multocida (B: 2) associated with haemorrhagic septicaemia | |
| Tsutsumi et al. | Iocalized amyloidosis of the seminal vesicle: Identification of lactoferrin immunoreactivity in the amyloid material | |
| CN113845592B (en) | anti-CK 5/6 protein monoclonal antibody, cell strain thereof, preparation method and application | |
| CN112552405A (en) | Preparation method and application of anti-grass carp pIgR polyclonal antibody | |
| CN117143225A (en) | Anti-micropterus salmoides mucus IgM antibody and preparation method and application thereof | |
| EP0381427B1 (en) | Vaccine Composition | |
| US5650154A (en) | Protective antigens against disease pathogens | |
| Luciani et al. | Selection of a monoclonal antibody by ELISA, immunoblotting and Quartz Crystal Microbalance technology for immunohistochemical detection of Mycoplasma mycoides subsp. mycoides. | |
| EP2561362B1 (en) | Method for detecting a salmonella infection | |
| Tegtmeier et al. | Development of a peroxidase‐antiperoxidase (PAP) technique for the identification of Haemophilus somnus in pneumonic calf lungs in Denmark | |
| CN116769019B (en) | ASFVp30 protein monoclonal antibody and application thereof | |
| CN113999304B (en) | Anti-enterobacterin monoclonal antibody mAb4 and application thereof in enterobacterin detection | |
| Neelam et al. | Development of monoclonal antibodies to iron-regulated outer membrane proteins and lipopolysaccharide of Aeromonas salmonicida |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |