Preparation method and application of duck BRS3 polypeptide and polyclonal antibody thereof

Abstract

The application discloses a duck BRS3 polypeptide and a preparation method and application of a polyclonal antibody thereof, and belongs to the field of biochemistry and molecular immunology. The amino acid sequence of the duck BRS3 polypeptide is shown as SEQ ID NO. 3. A polypeptide sequence with a length of 20 aa is screened from an intracellular region of a duck BRS3 protein sequence, and after being artificially synthesized by a solid phase, the polypeptide sequence is used as an immunizing antigen to immunize New Zealand white rabbits, and a duck BRS3 protein polyclonal antibody with an antiserum titer of 1:25600 or above is obtained. The rabbit anti-duck BRS3 polyclonal antibody prepared in the application can specifically combine with the BRS3 protein in duck tissues, fills a blank in the field of duck BRS3 protein detection research, and lays a foundation for the research on the function of BRS3 in a duck body.

Description

Technical Field

[0001] This invention relates to the fields of biochemistry and molecular immunology, and in particular to a method for preparing and applying a duck BRS3 polypeptide and its polyclonal antibody. Background Technology

[0002] The bombesin receptor subtype 3 (BRS3) is a member of the bombesin (BN) receptor family, belonging to the G-protein-coupled receptor family. It possesses a typical seven-transmembrane domain and is also known as an orphan receptor because no endogenous ligand has been found for it. Studies have shown that BRS3 has multiple biological functions, including regulating energy balance and insulin secretion, influencing tumor growth and differentiation, regulating satiety through feeding, regulating body weight, body temperature, blood pressure, and heart rate, and regulating hormone secretion.

[0003] Since the discovery of BRS3, researchers both domestically and internationally have conducted extensive studies on its expression and distribution in animals such as humans, mice, pigs, monkeys, and chickens. These studies have revealed that BRS3 is widely expressed in the central nervous system and multiple peripheral tissues and organs. Duck BRS3 protein is similar to BRS3 proteins in other animals, belonging to the G-protein-coupled receptor family and possessing seven transmembrane domains. The transmembrane regions of BRS3 proteins from different species show high homology. Furthermore, the homology of the duck BRS3 gene and amino acid sequence with chickens is 94.21% and 96.21%, respectively, and phylogenetic analysis also shows that ducks are most closely related to chickens. The inventors previously discovered through quantitative real-time PCR that the BRS3 gene is highly expressed in multiple tissues and organs of ducks, including the brain, cerebellum, pancreas, trachea, and pituitary gland, suggesting that BRS3 may also play an important physiological role in ducks.

[0004] As an important waterfowl, ducks are prized for their delicious meat, and duck meat and eggs are important sources of animal protein. Their feathers also have significant economic value. Therefore, ensuring duck health, improving duck production performance, and preventing the occurrence and spread of duck diseases are crucial responsibilities of animal husbandry and veterinary medicine. Furthermore, research on ducks provides valuable insights for research on other waterfowl. However, the lack of commercially available antibodies to detect duck BRS3 protein, coupled with the complex, costly, and time-consuming process of preparing duck BRS3 monoclonal antibodies, has limited research on BRS3 in ducks, primarily focusing on the genetic level and severely restricting functional studies of BRS3 in ducks. Therefore, the development of antibodies targeting duck BRS3 protein is particularly important, both for studying the physiological functions of BRS3 in ducks and for meeting the market demand for such antibodies. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing and applying duck BRS3 polypeptide and its polyclonal antibody, thereby addressing the problems existing in the prior art. The duck BRS3 polyclonal antibody prepared by this invention can specifically bind to BRS3 protein in duck tissues, with an antiserum titer exceeding 1:25600, filling a gap in the research field of duck BRS3 protein detection and laying the foundation for the study of BRS3 function in ducks.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a duck BRS3 polypeptide, the amino acid sequence of which is shown in SEQ ID NO.3.

[0008] The present invention also provides the application of the duck BRS3 polypeptide as an antigen in the preparation of duck BRS3 protein polyclonal antibodies.

[0009] The present invention also provides a method for preparing a polyclonal antibody against duck BRS3 protein, comprising the step of immunizing animals with the duck BRS3 polypeptide as an antigen.

[0010] Optional, the specific steps are as follows:

[0011] The duck BRS3 polypeptide was coupled with keyhole hemocyanin to obtain a complete antigen.

[0012] Animals were immunized with the complete antigen, and serum containing anti-duck BRS3 polypeptide antibodies was collected to obtain the duck BRS3 protein polyclonal antibody.

[0013] Optionally, the duck BRS3 polypeptide is coupled to the keyhole hemocyanin at a mass ratio of 1:1.

[0014] Optionally, the immunization is performed by subcutaneous injection at multiple points on the back, followed by a second immunization 14 days after the first immunization, and a third immunization 12 days after the second immunization.

[0015] Optionally, the first vaccination uses Freund's complete adjuvant emulsification; the second and third vaccinations both use Freund's incomplete adjuvant emulsification.

[0016] Optionally, the immunization dose for the first and second immunizations is 0.4 mg / kg; and the immunization dose for the third immunization is 0.36 mg / kg.

[0017] The present invention also provides a polyclonal antibody against duck BRS3 protein obtained by the preparation method described above.

[0018] The present invention also provides the application of the aforementioned duck BRS3 protein polyclonal antibody in detecting the expression level of duck BRS3 protein in duck tissues.

[0019] The present invention discloses the following technical effects:

[0020] This invention screened a 20aa polypeptide sequence from the intracellular region of the duck BRS3 protein sequence, synthesized it artificially in a solid phase, and used it as an immunogenic antigen to immunize New Zealand white rabbits, obtaining a duck BRS3 protein polyclonal antibody with an antiserum titer of over 1:25600. The rabbit anti-duck BRS3 polyclonal antibody prepared by this invention can specifically bind to the BRS3 protein in duck tissues, filling a gap in the field of duck BRS3 protein detection research and laying the foundation for the study of BRS3 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 transmembrane structure analysis of the duck BRS3 protein sequence;

[0023] Figure 2 Results of hydrophilicity, antigenic index and surface accessibility analysis of duck BRS3 protein sequence;

[0024] Figure 3 The image shows the mass spectrometry results of duck BRS3 peptide detection.

[0025] Figure 4 A graph showing the antiserum titer detected by the indirect ELISA method;

[0026] Figure 5 The image shows the results of Western blotting analysis of BRS3 protein expression levels in duck brain tissue.

[0027] Figure 6 The image shows the IHC detection results for the distribution and localization of BRS3 protein in duck pancreatic tissue. Detailed Implementation

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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 obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0032] 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.

[0033] The inventors previously successfully cloned the duck BRS3 gene, which has an open reading frame (ORF) of 1191 bp, encodes 396 amino acids, and the predicted molecular weight of the duck BRS3 protein is 44.22 kDa. The ORF sequence of the duck BRS3 gene is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2.

[0034] SEQ ID NO.1:

[0035]

[0036] SEQ ID NO.2:

[0037] MSQVYLHSSNQTLCASTNGTELKSIIDNETTNEKWTEDSFPGLEILCTIYVTYAVIISVGLLGNAILIKVFFKIKSMQTVPNIFITSLAFGDLLLLLTCVPVDATRYIVDTWLFGRIGCKLLSFIQLTSVGVSVFTLTVLSADRYRAIVKPLELQTSDALLKTCCKAGSVWIVSMILAIPEAVFSDLYSFSNPEKNVT FEACAPYPVSEKILQEAHSLVCFLVFYIVPLAVISVYYFLIARTLYKSTFNMPAEEHGHARKQIESRKRVAKTVLVLVALFAFCWLPNHILYLYRSFTYHSSVDASTFHLIATIFSRALAFSNSCVNPFALYWLSKSFRQHFKKQVSCCKAKLRTKPPSATQSNSPTRALSITGSTHGSEISVTLLTDYSITKEEESV.

[0038] Example 1: Duck BRS3 protein sequence analysis and design and synthesis of duck BRS3 peptide

[0039] The transmembrane structure of duck BRS3 protein (SEQ ID NO.2) was analyzed using the online software TMHMM-2.0. The results are as follows: Figure 1 As shown, the duck BRS3 protein belongs to the G-protein coupled receptor family and has seven transmembrane domains, including four extracellular domains (1-44, 103-121, 185-217 and 292-310), seven transmembrane domains (45-67, 80-102, 122-141, 167-184, 218-240, 272-291 and 311-333), and four intracellular domains (68-79, 142-166, 241-271 and 334-396).

[0040] The hydrophilicity, surface accessibility, and antigenic index of duck BRS3 protein were analyzed using the Protean module in DNAstar software. Figure 2 Based on the transmembrane structure analysis of the duck BRS3 protein, a suitable sequence was screened from the intracellular region located at the C-terminus of the duck BRS3 protein to serve as the fitted target sequence for antigen. This sequence includes 20 amino acids, located at 347 amino acids to 366 amino acids of the sequence shown in SEQ ID NO.2. The specific sequence is as follows:

[0041] CKAKLRTKPPSATQSNSPTR (SEQ ID NO. 3).

[0042] A duck BRS3 peptide was synthesized artificially. The purity of the synthesized peptide was determined to be 95.42% by high-performance liquid chromatography (HPLC), and the molecular weight of the synthesized peptide was determined to be 2171.48 Da by mass spectrometry. Figure 3 ).

[0043] Example 2: Coupling of duck BRS3 polypeptide with carrier protein keyhole hemocyanin (KLH)

[0044] In Example 1, the synthesized duck BRS3 polypeptide sequence is located at the C-terminus of the duck BRS3 protein. A carrier protein needs to be coupled to the N-terminus of the synthesized duck BRS3 polypeptide to increase its immunogenicity. Specifically, 5.0 mg of duck BRS3 polypeptide was coupled to 5.0 mg of carrier protein KLH using the coupling agent Sulfo-SMCC. This coupling was performed by Jier Biochemical (Shanghai) Co., Ltd., yielding the duck BRS3 polypeptide-KLH coupled protein, i.e., the complete antigen.

[0045] Example 3: Immunization of experimental animals and preparation of antiserum

[0046] Five-month-old male New Zealand white rabbits 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.

[0047] 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 (purchased from 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 New Zealand white rabbits at a dose of 0.4 mg / kg complete antigen for the initial immunization. A second booster immunization (second vaccination) was administered 14 days after the initial immunization.

[0048] To boost immunization, 2 mg of complete antigen was dissolved in 2 mL of ultrapure water and thoroughly emulsified with an equal volume of Freund's incomplete adjuvant (purchased from Sigma-Aldrich, catalog number F5506). After emulsification, experimental animals were immunized by subcutaneous injection at multiple points (6-8 points) on the back at a dose of 0.4 mg / kg of complete antigen.

[0049] Twelve days after the second booster immunization, blood was collected from the marginal ear vein of rabbits. 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. A total of two booster immunizations were performed in this invention.

[0050] 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.

[0051] Example 4: Detection of antiserum titer using an indirect ELISA method

[0052] The duck BRS3 peptide was diluted to 10 μg / mL using coating buffer (0.05 mol / L phosphate buffer at 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 using an ELISA plate sealing machine and incubated overnight at 4°C. Before use, the plate 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. Antibody dilution was added to the blank control, and unimmunized serum (1:100 dilution) was added to the negative control. Different serial dilutions of antiserum (1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:25600, 1:51200, 1:102400, 1:204800, 1:409600, 1:819200, and 1:1638400) were added to the experimental groups. Then, place the ELISA plate in a humidified chamber and incubate at 37°C for 1-2 hours, washing the plate as above. Add 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 to each well of the ELISA plate, place the ELISA plate in a humidified chamber, and incubate at 37°C for 1-2 hours, washing the plate as above. Add 100 μL of TMB chromogenic solution (purchased from Beyotime Biotechnology Co., Ltd., catalog number P0206) directly to each well of the ELISA plate and incubate 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, and bringing the volume to 100 mL, 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. Antibody titer is calculated when the ratio of antibody titer to negative control serum is greater than 2.1.

[0053] Test results as follows Figure 4 As shown, the antibody titer of the antiserum is 1:25600.

[0054] Example 5: Western blot detection of BRS3 protein in duck tissues

[0055] 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 BRS3 polypeptide polyclonal antibody, diluted 1:500) 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.

[0056] The results are as follows Figure 5 As shown, the bands are clear, correctly positioned, and highly specific, indicating that BRS3 protein is expressed at a high level in duck brain tissue.

[0057] Example 6: Immunohistochemical (IHC) detection of BRS3 protein in duck tissues

[0058] 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 inactivated 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 BRS3 polypeptide polyclonal antibody (1:50) 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.

[0059] The results are as follows Figure 6 As shown, the detection results indicate that BRS3 protein is distributed in pancreatic islet cells.

[0060] 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 BRS3 polypeptide, characterized in that, The amino acid sequence of the duck BRS3 polypeptide is shown in SEQ ID NO.

3.

2. The application of the duck BRS3 polypeptide as described in claim 1 as an antigen in the preparation of duck BRS3 protein polyclonal antibodies.

3. A method for preparing a polyclonal antibody against duck BRS3 protein, characterized in that, The step includes immunizing animals with the duck BRS3 polypeptide of claim 1 as an antigen.

4. The preparation method according to claim 3, characterized in that, The specific steps are as follows: The duck BRS3 polypeptide was coupled with keyhole hemocyanin to obtain a complete antigen. Animals were immunized with the complete antigen, and serum containing anti-duck BRS3 polypeptide antibodies was collected to obtain the duck BRS3 protein polyclonal antibody.

5. The preparation method according to claim 4, characterized in that, The duck BRS3 polypeptide is coupled to the keyhole hemocyanin at a mass ratio of 1:

1.

6. The preparation method according to claim 4, characterized in that, The immunization was administered via subcutaneous injection at multiple points on the back, with a second immunization 14 days after the first, and a third immunization 12 days after the second.

7. The preparation method according to claim 6, characterized in that, The first vaccination uses Freund's complete adjuvant emulsification; the second and third vaccinations both use Freund's incomplete adjuvant emulsification.

8. The preparation method according to claim 6, characterized in that, The immunization dose for the first and second immunizations is 0.4 mg / kg; the immunization dose for the third immunization is 0.36 mg / kg.

9. A polyclonal antibody against duck BRS3 protein obtained by the preparation method according to any one of claims 3-8.

10. The use of the duck BRS3 protein polyclonal antibody of claim 9 in detecting the expression level of duck BRS3 protein in duck tissues.

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