A method for preparing and applying a duck GRPR polypeptide and its polyclonal antibody

Abstract

This invention discloses a method for preparing a duck GRPR polypeptide and its polyclonal antibody, belonging to the fields of biochemistry and molecular immunology. This invention is the first to clone the duck GRPR gene and analyze its structure. Using the amino acid polypeptide at positions 350-368 of the carboxyl terminus of the duck GRPR protein as a hapten, the N-terminus of this polypeptide sequence was modified to obtain a duck GRPR-modified polypeptide. This modified polypeptide was then conjugated with the carrier protein keyhole hemocyanin to prepare a complete antigen. Animals were then immunized, and a polyclonal antibody against the duck GRPR polypeptide was isolated and purified from the antiserum. Experiments showed that this polyclonal antibody can specifically bind to the GRPR protein in duck tissues. This invention fills a gap in the research field of duck GRPR protein detection and lays the foundation for studying the function of GRPR in ducks.

Description

Technical Field

[0001] This invention relates to the fields of biochemistry and molecular immunology, and in particular to a duck GRPR polypeptide and its polyclonal antibody preparation method and application. Background Technology

[0002] The gastrin-releasing peptide receptor (GRPR) is a member of the bombesin (BN) receptor family and belongs to the G-protein-coupled receptor family. It has a typical seven-transmembrane domain and mainly exerts various biological functions by binding to its ligand, gastrin-releasing peptide (GRP), activating multiple intracellular signaling pathways mediated by G proteins. These functions include stimulating gastric acid secretion, regulating gastrointestinal motility, regulating the production of various hormones (gastrin, somatostatin, cholecystokinin, etc.), regulating immunity, stimulating lymphocyte proliferation, affecting lung development, regulating circadian rhythms, and mediating stress / anxiety / fear responses.

[0003] Since the discovery of GRPR, researchers both domestically and internationally have conducted extensive studies on the expression and distribution of GRPR in animals such as humans, mice, monkeys, pigs, and chickens, finding that GRPR is widely expressed in the central nervous system and multiple peripheral tissues and organs. However, research on duck GRPR genes is currently limited. Ducks are important waterfowl in my country, with delicious meat and eggs being important sources of animal protein, and their feathers also have significant economic value. Therefore, ensuring the health of ducks, improving their production performance, and preventing the occurrence and spread of duck diseases are important responsibilities of animal husbandry and veterinary work. Furthermore, research on ducks, as important waterfowl, provides a reference for research on other waterfowl. However, due to the lack of antibodies for detecting duck GRPR proteins on the market, coupled with the complex, costly, and time-consuming process of preparing duck GRPR monoclonal antibodies, research on GRPR in ducks has mainly focused on the gene level, severely restricting functional research on GRPR in ducks. Therefore, the preparation of antibodies for duck GRPR proteins is particularly important, as it can be used to study the physiological functions of GRPR in ducks and to meet the market demand for such antibodies. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing and applying a duck GRPR polypeptide and its polyclonal antibody, in order to solve the problems existing in the prior art. The polyclonal antibody has the characteristics of simple preparation method, low cost and high potency, and can specifically bind to GRPR protein in duck tissue.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] The present invention provides a duck GRPR polypeptide, the amino acid sequence of which is shown in SEQ ID NO.3.

[0007] The present invention also provides a duck GRPR modified polypeptide, the amino acid sequence of which is shown in SEQ ID NO.4; the sequence in SEQ ID NO.4 is formed by adding a cysteine ​​residue to the N-terminus of the sequence in SEQ ID NO.3.

[0008] The present invention also provides polyclonal antibodies obtained by immunizing animals with duck GRPR-modified peptides as antigens.

[0009] Preferably, the method for preparing the polyclonal antibody includes the following steps: conjugating the duck GRPR modified polypeptide with a carrier protein to obtain a complete antigen, immunizing animals, collecting blood from the immunized animals to prepare antiserum, and separating and purifying the polyclonal antibody from the antiserum.

[0010] Preferably, the carrier protein is keyhole hemocyanin.

[0011] Preferably, the duck GRPR-modified polypeptide is coupled to keyhole hemocyanin using the coupling agent Sulfo-SMCC to prepare the complete antigen.

[0012] The present invention also provides the application of the polyclonal antibody in the preparation of reagents for detecting GRPR protein levels in duck tissues.

[0013] Preferably, the method for detecting GRPR protein levels in duck tissue using the polyclonal antibody includes Western blotting and immunohistochemical methods.

[0014] The present invention also provides a reagent for detecting the level of GRPR protein in duck tissue, the reagent comprising the aforementioned polyclonal antibody.

[0015] The present invention also provides a method for detecting GRPR protein in duck tissue for non-diagnostic purposes, characterized in that the polyclonal antibody is used as a detection antibody to detect the expression level of GRPR protein in duck tissue.

[0016] The present invention discloses the following technical effects:

[0017] 1. Based on the laboratory-cloned duck GRPR gene sequence, this invention analyzes the transmembrane structure, hydrophilicity, surface accessibility, antigen index, and homology of the duck GRPR protein sequence, and screens out suitable peptide sequences as target sequences for artificial synthesis.

[0018] 2. The synthesized peptide sequence was N-terminally modified and coupled with the carrier protein keyhole hemocyanin to obtain duck GRPR-modified peptide-KLH-coupled protein.

[0019] 3. New Zealand white rabbits were immunized with the prepared duck GRPR-modified polypeptide-KLH coupled protein, and the titer of the antiserum was detected by indirect ELISA. The titer of the prepared antiserum was above 1:25600.

[0020] 4. The rabbit anti-duck GRPR polyclonal antibody prepared by this invention can specifically bind to GRPR protein in duck tissues, filling the gap in the field of duck GRPR protein detection research and laying the foundation for the study of GRPR function in ducks. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The results of the analysis of the transmembrane structure of the duck GRPR protein sequence using the online software TMHMM-2.0;

[0023] Figure 2 To detect the expression of the GRPR gene in multiple tissues and organs of ducks, including the pancreas, optic lobe, proventriculus, cerebellum, and duodenum, using quantitative real-time PCR;

[0024] Figure 3 The results of the analysis of hydrophilicity, antigenic index and surface accessibility of duck GRPR protein sequence using the bioinformatics software DNAstar (Protean) are shown.

[0025] Figure 4 The figure shows the HPLC purity detection results of the duck GRPR-modified peptide;

[0026] Figure 5 The mass spectrometry results of duck GRPR-modified peptides are shown in the image.

[0027] Figure 6 A graph showing the antiserum titer detected by the indirect ELISA method;

[0028] Figure 7 The image shows the Western Blot results for detecting the GRPR protein expression level in duck brain tissue using the rabbit anti-duck GRPR modified polypeptide polyclonal antibody of the present invention. M is the protein marker, and GRPR is the primary antibody used to treat brain tissue with rabbit anti-duck GRPR polyclonal antibody.

[0029] Figure 8The image shows the IHC detection results of the distribution and localization of GRPR protein in duck pancreatic tissue using the polyclonal antibody against the rabbit anti-duck GRPR modified peptide of this invention. Detailed Implementation

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0032] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0033] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0034] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0035] Example 1: Duck GRPR protein sequence analysis and design and synthesis of duck GRPR-modified peptides

[0036] Analysis of the duck GRPR gene sequence revealed that the open reading frame (ORF) of the duck GRPR gene is 1164 bp, encoding 387aa.

[0037] The nucleotide sequence of the open reading frame of the duck GRPR gene is shown in SEQ ID NO.1:

[0038]

[0039] The full-length amino acid sequence of the duck GRPR protein is shown in SEQ ID NO.2 below:

[0040] MASGECLLLDLETENFILYNISVNQSANLSILSDEWFYPAFLYAIPTIYGIIILIGLIGNITLIKIFCTVKSMRNVPNLFISSLALGDLLLLVTCVPVDASRYLADEWLFGRIGCKLIPFIQLTSVGVSVFTLTALSADRYKAIVRPMEIQASHALMKICVRAAIIWIVSMLLAIPEAAVFSDLHPFHDKGTNKT FISCAPYPHSDGLHPKIHSMASFLIFYIIPLSVISVYYYFIAKNLIRSAYNIPVEGNLHVRKQIESRKRLARTVLVFVCLFAFCWLPTHIIYLYRSYHYSEVDTSVLHFIASICARILAFTNSCVNPFALYLLSKSFRKQFNNQLFCCRARLLIRSQSMARSTTRMTSLKSTNHSLATFSLINGNHICHEGYV.

[0041] The transmembrane structure of the duck GRPR protein sequence was analyzed using the online software TMHMM-2.0, such as... Figure 1 As shown, the duck GRPR protein belongs to the G-protein-coupled receptor family and has seven transmembrane domains, including four extracellular domains (1-41, 99-117, 182-213, and 287-305), seven transmembrane domains (42-64, 76-98, 118-138, 159-181, 214-236, 264-286, and 306-328), and four intracellular domains (65-75, 139-158, 237-263, and 329-387). Furthermore, the duck GRPR gene and amino acid sequence share 94.07% and 98.45% homology with the chicken GRPR gene, respectively, and phylogenetic analysis shows that the duck GRPR gene is most closely related to the chicken GRPR gene. The expression of the GRPR gene in different tissues was detected by quantitative real-time PCR. The primer sequences required for quantitative real-time PCR detection were: upstream primer 5'-ACCTTCATCAGCTGTGCTCC-3', downstream primer 5'-TCCACGGGAATGTTGTAAGCA-3', amplified fragment of 167 bp, and annealed at 60℃. The reaction system and procedure followed the instructions for the FastReal PCR Premixed Reagent Kit (SYBR Green), purchased from Tiangen Biotech (Beijing) Co., Ltd. Results are as follows: Figure 2As shown, the GRPR gene was found to be expressed in multiple tissues and organs of ducks, including the pancreas, optic lobe, proventriculus, cerebellum, and duodenum, indicating that GRPR may also have important physiological functions in ducks.

[0042] The hydrophilicity, surface accessibility, and antigenic index of duck GRPR protein were analyzed using the Protean module in DNAstar software (see [link to DNAstar software]). Figure 3 By combining the transmembrane domain of the duck GRPR protein, a suitable sequence SQSMARSTTRMTSLKSTNH (SEQ ID NO.3) was selected as the fitted target sequence as antigen (350aa-368aa). This sequence is located in the intracellular region of the carboxyl terminus (C-terminus) of the duck GRPR protein and includes 19aa.

[0043] To enhance the immunogenicity of the peptide through coupling with the carrier protein, the selected peptide sequence was modified at the N-terminus by adding a cysteine ​​residue (Cys, C). Therefore, the final synthesized peptide sequence is CSQSMARSTTRMTSLKSTNH (SEQ ID NO. 4), i.e., the duck GRPR-modified peptide. The purity of the synthesized peptide was determined to be 95.46% by high-performance liquid chromatography (HPLC) (see...). Figure 4 The molecular weight of the synthesized peptide, as determined by mass spectrometry, was 2227.50 Da (see...). Figure 5 ).

[0044] Example 2: Coupling of duck GRPR-modified peptides with the carrier protein keyhole hemocyanin (KLH)

[0045] 5.0 mg of duck GRPR-modified peptide and 5.0 mg of carrier protein KLH were conjugated using the conjugating agent Sulfo-SMCC. The conjugation was performed by Jier Biochemical (Shanghai) Co., Ltd., to obtain duck GRPR-modified peptide-KLH conjugate protein, i.e., complete antigen.

[0046] Example 3: Immunization of experimental animals and preparation of antiserum

[0047] Male New Zealand white rabbits of appropriate age were selected as immunized animals. Blood was collected from the marginal ear vein of the rabbits before the first immunization (blood collection was carried out in the morning before feeding) to serve as control serum for subsequent ELISA testing.

[0048] For the initial immunization, 2 mg of complete antigen was dissolved in 2 mL of ultrapure water and emulsified thoroughly with an equal volume of Freund's complete adjuvant (Sigma-Aldrich, catalog number F5881) using a homogenization method. The emulsified antigen was injected subcutaneously at multiple sites (6-8 points) on the back of the rabbits at a dose of 0.4 mg / kg complete antigen. A second booster immunization was administered 14 days after the initial immunization. (For the booster immunization, 2 mg of complete antigen was dissolved in 2 mL of ultrapure water and emulsified thoroughly with an equal volume of Freund's incomplete adjuvant (Sigma-Aldrich, catalog number F5506). The emulsified antigen was then injected subcutaneously at multiple sites (6-8 points) on the back of the experimental animals at a dose of 0.4 mg / kg complete antigen.) Twelve days after the second booster immunization, blood was collected from the marginal ear vein of the rabbits, and the antibody titer was detected using an indirect ELISA method to determine whether further booster immunization was necessary, the required dose, and the number of booster immunizations. The invention involved two booster immunizations.

[0049] The rabbits were immunized for the second booster immunization at a dose of 0.36 mg / kg of complete antigen. Antiserum was collected 11 days after the third immunization. The rabbits were anesthetized, and a large amount of blood was collected by exsanguination of the abdominal aorta. The collected blood was tilted and allowed to stand at 37°C for about 1 hour, and then transferred to a 4°C refrigerator and allowed to stand for about 12 hours to allow for full separation of antiserum. The antiserum was separated by centrifugation at 2500 r / min for 20 minutes at 4°C, aliquoted and stored at -80°C for later use.

[0050] Example 4: Detection of antiserum titer using an indirect ELISA method

[0051] The duck GRPR-modified peptide was diluted to 10 μg / mL using coating buffer (pH 9.6, prepared by weighing 0.75 g sodium carbonate and 1.46 g sodium bicarbonate, dissolving in deionized water and bringing the volume to 500 mL). 100 μL of the diluted antigen solution was added to each well of the ELISA plate. The plate was then vacuum-sealed and incubated overnight at 4°C. Before use, it was incubated at 37°C for 30 min. The coating buffer was discarded, and the plate was washed with washing buffer (PBST, 0.05% Tween-20 PBS solution; PBS pH 9.6). 7.4 The 0.02 mol / L phosphate buffer is prepared as follows: Weigh 0.2 g potassium dihydrogen phosphate, 2.9 g disodium hydrogen phosphate, 8 g sodium chloride, and 0.2 g potassium chloride. Dissolve them in an appropriate amount of deionized water and bring the volume to 1000 mL. Add 200 μL to each well of the coated microplate (fill with filter paper). Wipe dry and wash 3 times, 3-5 min each time. Add 250 μL of blocking buffer (i.e., 1% BSA, diluted with 1 g of BSA to 100 mL) to each well of the microplate. PBST solution was used to incubate the ELISA plate in a humidified chamber at 37°C for 2 hours. The plate was washed as described above. For the blank control, antibody dilution buffer was added; for the negative control, unimmunized serum (1:100 dilution) was added; for the experimental groups, serially diluted 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:25600, 1:51200, 1:102400, 1:204800, 1:409600, and 1: Antiserum at dilutions of 819200 and 1:1638400 was added. The ELISA plate was then placed in a humidified chamber and incubated at 37°C for 1-2 hours. The plate was washed as above. 100 μL of horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (purchased from Wuhan Boster Biological Engineering Co., Ltd., catalog number BA1054) secondary antibody diluted 1:5000 was added to each well of the ELISA plate. The ELISA plate was placed in a humidified chamber and incubated at 37°C for 1-2 hours. The plate was washed as above. 100 μL of TMB chromogenic solution (purchased from Beyotime Biotechnology Co., Ltd., catalog number P0206) was added directly to each well of the ELISA plate and incubated at room temperature or 37°C for 5-30 minutes. Then, add 100 μL of 2M sulfuric acid stop solution (i.e., 2 mol / L sulfuric acid solution, prepared by adding 10.870 mL of 98% concentrated sulfuric acid to 60 mL of deionized water, bringing the volume to 100 mL, and storing at room temperature) to each well to terminate the reaction; place the ELISA plate in an ELISA reader and measure the absorbance at a wavelength of 450 nm. Calculate the antibody titer when the ratio to the negative control serum is greater than 2.1.

[0052] Test results as follows Figure 6 As shown: the antibody titer of the antiserum is 1:25600.

[0053] Example 5: Western blot detection of GRPR protein in duck tissues

[0054] Prepare a 10% SDS-PAGE gel according to standard methods. Add 40 μg of duck brain tissue lysis buffer to the sample well of a vertical electrophoresis tank. Run SDS-PAGE gel electrophoresis at 60V for 30 min, then adjust the voltage to 100V and stop electrophoresis when the bromophenol blue reaches approximately 1.5 cm from the bottom of the gel. After SDS-PAGE electrophoresis, transfer the proteins to a PVDF membrane using wet transfer. Block with 5% skim milk powder at room temperature for 2 h, then wash the membrane four times with TBST for 10 min each time. The membrane was incubated overnight at 4°C with primary antibody (rabbit anti-duck GRPR-modified polypeptide polyclonal antibody, diluted 1:2000) and washed 5 times with TBST. HRP-labeled goat anti-rabbit IgG was added as secondary antibody and incubated at room temperature for 2 hours, followed by washing 4 times with TBST. The PVDF membrane was incubated with a mixture of ECL chemiluminescence buffer (purchased from Xinsaimei Biotechnology Co., Ltd., catalog number P10300) A and B solutions, and reacted in the dark for 1 minute. Images were obtained by exposure using an imaging system.

[0055] The results are as follows Figure 7 As shown, the bands are clear, correctly positioned, and highly specific, indicating that GRPR protein is expressed at a high level in duck brain tissue.

[0056] Example 6: Immunohistochemical (IHC) detection of GRPR protein in duck tissues

[0057] Duck pancreatic tissue was fixed with 4% paraformaldehyde solution. Paraffin sections of the duck pancreatic tissue were prepared following the steps of tissue dehydration, clearing, paraffin infiltration, embedding, and sectioning, with a section thickness of 5 μm. Then, the paraffin sections of the duck pancreatic tissue were dewaxed by conventional xylene and graded alcohol infiltration. 0.1% Triton X-100 was dropped onto the tissue sections, permeabilized at 37°C for 10 min, and washed with distilled water for 10 min. Endogenous peroxidase was quenched by incubation in 3% H2O2 distilled water at room temperature for 8 min, followed by washing with PBS for 10 min. Heat antigen retrieval was performed using a microwave method with 0.01M citrate buffer, followed by cooling to room temperature. Blocking was performed by incubation with 5% BSA at 37°C for 30 min. The prepared rabbit anti-duck GRPR-modified polypeptide polyclonal antibody (1:100) was used as the primary antibody, incubated overnight at 4°C, and washed with PBS for 20 min. Biotin-labeled goat anti-rabbit IgG was used as the secondary antibody, incubated at 37°C for 1 h, and washed with PBS for 20 min. SABC was used as the secondary antibody. Incubate at 37℃ for 1 hour, wash with PBS for 20 minutes; perform color development with DAB, control the reaction time under a microscope, and stop the reaction with tap water; after staining the cell nuclei with hematoxylin, dehydrate, clear, and mount the slides as usual, and observe and photograph them under a microscope.

[0058] The results are as follows Figure 8 As shown, the detection results indicate the distribution of GRPR protein in pancreatic tissue cells.

[0059] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A duck GRPR polypeptide, characterized in that, The amino acid sequence of the duck GRPR polypeptide is shown in SEQ ID NO.

3.

2. A duck GRPR-modified polypeptide, characterized in that, The amino acid sequence of the duck GRPR-modified polypeptide is shown in SEQ ID NO.4.

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