Duck GRPR polypeptide as well as preparation method and application of polyclonal antibody of duck GRPR polypeptide

Abstract

The invention discloses a duck GRPR polypeptide and a preparation method and application of a polyclonal antibody of the duck GRPR polypeptide, and belongs to the field of biochemistry and molecular immunology. The duck GRPR gene is cloned for the first time, and the structure of the duck GRPR gene is analyzed; the preparation method comprises the following steps: taking amino acid polypeptide at 350-368 sites of a carboxyl terminal of duck GRPR protein as a hapten, and carrying out N-terminal modification on a polypeptide sequence to obtain duck GRPR modified polypeptide; and coupling the modified polypeptide with a carrier protein keyhole limpet hemocyanin to prepare a complete antigen, immunizing an animal, and separating and purifying from antiserum to obtain the polyclonal antibody of the duck GRPR polypeptide. Experiments show that the polyclonal antibody can be specifically combined with GRPR protein in duck tissue. According to the invention, the blank in the field of duck GRPR protein detection and research is filled, and a foundation is laid for research of the functions of the GRPR in the duck body.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biochemistry and molecular immunology, in particular to a duck GRPR polypeptide and a preparation method and application of a polyclonal antibody thereof. BACKGROUND

[0002] Gastrin-releasing peptide receptor (GRPR) is a member of the bombesin receptor family, which belongs to G-protein coupled receptors, has a typical 7-transmembrane domain, and mainly activates G-protein-mediated intracellular signaling pathways by binding with its ligand gastrin-releasing peptide (GRP) to exert various biological functions, such as stimulating gastric acid secretion, regulating gastrointestinal peristalsis, regulating the production of various hormones (gastrin, somatostatin, cholecystokinin, etc.), regulating immunity, stimulating lymphocyte proliferation, affecting lung development, regulating circadian rhythm, and mediating stress / anxiety / fear response.

[0003] Since the discovery of GRPR, researchers at home and abroad have conducted a large number of studies on the expression and distribution of GRPR in animals such as humans, mice, monkeys, pigs, and chickens in vivo, and found that GRPR is widely expressed in the central nervous system and multiple peripheral tissues and organs. However, there is currently little research on duck GRPR genes. Duck is an important waterfowl in China, with delicious meat, and duck meat and eggs are important animal proteins. Its feathers also have important economic value. Therefore, it is an important responsibility for animal husbandry and veterinary workers to ensure the health of ducks, improve their production performance, and prevent the occurrence and spread of diseases. At the same time, as an important waterfowl, research on ducks can also provide a reference for the study of other waterfowl. However, due to the lack of antibodies for detecting duck GRPR protein in the market, combined with the complexity, high cost, and long time of preparing duck GRPR monoclonal antibodies, the research on GRPR in ducks is mainly at the gene level, which seriously restricts the functional research of GRPR in ducks. Therefore, it is particularly important to prepare antibodies for duck GRPR protein, which can be used to study the physiological functions of GRPR in ducks and meet the market demand for such antibodies. SUMMARY

[0004] The purpose of the present application is to provide a duck GRPR polypeptide and a preparation method and application of a polyclonal antibody thereof, which has the characteristics of simple preparation method, low cost, and high titer, and can specifically bind to GRPR protein in duck tissues, to solve the problems existing in the prior art.

[0005] To achieve the above-mentioned purpose, the present application provides the following solutions:

[0006] The present invention provides a duck GRPR polypeptide, the amino acid sequence of the duck GRPR polypeptide is shown as 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 SEQ ID NO.4 sequence is obtained by adding a cysteine ​​to the N-terminus of the SEQ ID NO.3 sequence.

[0008] The present invention also provides a polyclonal antibody obtained by immunizing an animal with the duck GRPR modified polypeptide as an antigen.

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

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

[0011] Preferably, the duck GRPR modified polypeptide is coupled to keyhole limpet hemocyanin via a coupling agent Sulfo-SMCC to obtain the complete antigen.

[0012] The present invention also provides application of the polyclonal antibody in preparing a reagent for detecting the GRPR protein level in duck tissue.

[0013] Preferably, the method for detecting the GRPR protein level in duck tissues using the polyclonal antibody includes immunoblotting detection and immunohistochemistry detection.

[0014] The present invention also provides a reagent for detecting the GRPR protein level in duck tissues, wherein the reagent comprises the polyclonal antibody.

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

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

[0017] 1. Based on the laboratory cloning of duck GRPR gene sequence, the present invention analyzes the transmembrane structure, hydrophilicity, surface accessibility, antigenic 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 limpet hemocyanin to obtain duck GRPR modified polypeptide-KLH coupled protein.

[0019] 3. The prepared duck GRPR modified polypeptide-KLH conjugate protein was used to immunize New Zealand white rabbits, and the titer of the antisera was detected by indirect ELISA method, and the titer of the prepared antisera was above 1:25600.

[0020] 4. The prepared rabbit anti-duck GRPR polyclonal antibody can specifically bind to the GRPR protein in duck tissues, fills the blank in the field of duck GRPR protein detection research, and lays a foundation for the research on the function of GRPR in the duck body. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0022] Figure 1 It is the analysis result of the transmembrane structure of the duck GRPR protein sequence by using online software TMHMM-2.0;

[0023] Figure 2 It is the expression of the GRPR gene in the pancreas, optic lobe, glandular stomach, cerebellum and duodenum of the duck, and other multiple tissue organs detected by fluorescence quantitative PCR;

[0024] Figure 3 It is the analysis result of the hydrophilicity, antigen index and surface accessibility of the duck GRPR protein sequence by using bioinformatics software DNAstar (Protean);

[0025] Figure 4 It is the HPLC purity detection result graph of the duck GRPR modified polypeptide;

[0026] Figure 5 It is the mass spectrum detection result graph of the duck GRPR modified polypeptide;

[0027] Figure 6 It is the titer graph of the antisera detected by indirect ELISA method;

[0028] Figure 7 It is the Western Blot detection result graph of the application of the rabbit anti-duck GRPR modified polypeptide polyclonal antibody to detect the expression level of the GRPR protein in the duck brain tissue, wherein M is a protein marker, and GRPR is the primary antibody treated with the rabbit anti-duck GRPR polyclonal antibody;

[0029] Figure 8The IHC detection result map of the application of the rabbit anti-duck GRPR modified polypeptide polyclonal antibody to detect the distribution and positioning of GRPR protein in duck pancreas tissue. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present application will now be described in detail, which should be considered to be illustrative of the application and not restrictive of the application. It will be appreciated that the detailed description is not limited to the specific embodiments described herein but encompasses all alternatives falling within the scope of the application.

[0031] It should be understood that the terms used in the present application merely describe particular embodiments and are not intended to limit the present application. In addition, for numerical ranges in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between any stated value or stated range, and any other stated value or intermediate value in the stated range, is also encompassed within the present application. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0032] Unless otherwise defined, 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 application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, preferred methods and materials are described. All publications mentioned in this specification are herein incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference in this specification is not intended as an admission that the reference is prior art, but rather that the reference is part of the technical literature that is relevant to A person of ordinary skill in the art.

[0033] Many modifications and variations of this application specification of the present application can be made in light of the above teachings. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only. It is intended that the scope of the application be limited only by the appended claims.

[0034] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" and the like are open-ended terms that are intended to mean "including but not limited to".

[0035] Example 1: Analysis of duck GRPR protein sequence and design and synthesis of duck GRPR modified polypeptide

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

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

[0038]

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

[0040] MASGECLLLDLETENFILYNISVNQSANLSILSDEWFYPAFLYAIPTIYGIIILIGLIGNITLIKIFCTVKSMRNVPNLFISSLALGDLLLLVTCVPVDASRYLADEWLFGRIGCKLIPFIQLTSVGVSVFTLTALSADRYKAIVRPMEIQASHALMKICVRAAIIWIVSMLLAIPEAVFSDLHPFHDKGTNKTFISCAPYPHSDGLHPKIHSMASFLIFYIIPLSVISVYYYFIAKNLIRSAYNIPVEGNLHVRKQIESRKRLARTVLVFVCLFAFCWLPTHIIYLYRSYHYSEVDTSVLHFIASICARILAFTNSCVNPFALYLLSKSFRKQFNNQLFCCRARLLIRSQSMARSTTRMTSLKSTNHSLATFSLINGNHICHEGYV.

[0041] The transmembrane structure of duck GRPR protein sequence was analyzed by online software TMHMM-2.0, as shown in Figure 1 The duck GRPR protein belongs to G-protein coupled receptor with seven transmembrane domains, including four extracellular regions (1-41, 99-117, 182-213 and 287-305), seven transmembrane regions (42-64, 76-98, 118-138, 159-181, 214-236, 264-286 and 306-328) and four intracellular regions (65-75, 139-158, 237-263 and 329-387). In addition, the homology of duck and chicken GRPR gene and amino acid sequence is 94.07% and 98.45%, respectively, and the phylogenetic tree analysis is also closest to chicken. The expression of GRPR gene in different tissues was detected by fluorescence quantitative PCR. The primer sequences required for fluorescence quantitative PCR detection are as follows: upstream primer 5'-ACCTTCATCAGCTGTGCTCC-3', downstream primer 5'-TCCACGGGAATGTTGTAAGCA-3', the amplification fragment is 167 bp, and the annealing temperature is 60℃. The detection reaction system and reaction procedure were operated according to the instructions of FastReal rapid fluorescence quantitative PCR premix reagent (SYBR Green) kit, which was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. The results are shown in Figure 2As shown, the GRPR gene was found to be expressed in multiple tissues and organs of the duck, such as the pancreas, optic lobe, glandular stomach, cerebellum and duodenum, indicating that GRPR may also have important physiological functions in the duck.

[0042] The Protean module in the DNAstar software was used to analyze the hydrophilicity, surface accessibility, antigen index and other indicators of the duck GRPR protein (see Figure 3 ), and a suitable sequence SQSMARSTTRMTSLKSTNH (SEQ ID NO. 3) was screened out as the target sequence for synthesizing the antigen (350aa-368aa), which is located in the intracellular region of the carboxyl terminal (C-terminal) of the duck GRPR protein and includes 19 aa.

[0043] In order to be coupled with the carrier protein to increase the immunogenicity of the polypeptide, the selected polypeptide sequence was modified by adding cysteine (Cys, C) at the N-terminal, so the final synthesized polypeptide sequence was CSQSMARSTTRMTSLKSTNH (SEQ ID NO. 4), i.e., the duck GRPR modified polypeptide. The purity of the synthesized polypeptide was 95.46% detected by high performance liquid chromatograph (HPLC) (see Figure 4 ), and the molecular weight of the synthesized polypeptide was 2227.50 Da detected by mass spectrometry (see Figure 5 ).

[0044] Example 2 Coupling of duck GRPR modified polypeptide with carrier protein keyhole limpet hemocyanin (KLH)

[0045] 5.0 mg of duck GRPR modified polypeptide was coupled with 5.0 mg of carrier protein KLH by coupling agent Sulfo-SMCC, and the coupling was completed by Gilson Biochemical (Shanghai) Co., Ltd. to obtain duck GRPR modified polypeptide-KLH coupled protein, i.e., complete antigen.

[0046] Example 3 Immunization of experimental animals and preparation of antisera

[0047] A suitable age male New Zealand white rabbit was selected as the immunized animal, and blood was collected from the rabbit ear marginal vein before the first immunization (blood collection was performed before feeding in the morning), which was used as the control serum for subsequent ELISA detection.

[0048] The first immunization, 2 mg of the complete antigen was dissolved in 2 mL of ultrapure water, and was emulsified with an equal volume of Freund's complete adjuvant (purchased from Sigma-Aldrich, item number F5881) by homogenization. The emulsified antigen was injected into the New Zealand white rabbits by subcutaneous injection of multiple points (6-8 points) on the back at a dose of 0.4 mg / kg of the complete antigen. The first booster immunization (second immunization) was performed 14 days after the first immunization. (The booster immunization, 2 mg of the complete antigen was dissolved in 2 mL of ultrapure water, and was emulsified with an equal volume of Freund's incomplete adjuvant (purchased from Sigma-Aldrich, item number F5506), and the emulsified antigen was injected into the experimental animals by subcutaneous injection of multiple points (6-8 points) on the back at a dose of 0.4 mg / kg of the complete antigen.) The rabbit ear marginal vein was bled 12 days after the booster immunization (second immunization), and the antibody titer was detected by indirect ELISA to determine whether to continue the booster immunization and the required dose of immunization, and the number of booster immunizations. A total of 2 booster immunizations were performed before and after the present application.

[0049] The second booster immunization was performed at a dose of 0.36 mg / kg of the complete antigen, and the antiserum was collected 11 days after the third immunization. The rabbit was anesthetized, and a large amount of blood was collected by abdominal aortic blood collection. The blood was collected at 37°C and inclined for about 1 h, and then was transferred to a 4°C refrigerator and inclined for about 12 h to allow the antiserum to be analyzed. The antiserum was separated by centrifugation at 2500 r / min for 20 min at 4°C, and was stored at -80°C for use.

[0050] Example 4: Indirect ELISA method for detecting the titer of the antiserum

[0051] Duck GRPR modified polypeptide was diluted to 10 μg / mL with coating solution (0.05 mol / L phosphate buffer solution at pH 9.6, prepared by weighing 0.75 g of sodium carbonate and 1.46 g of sodium bicarbonate, dissolving in deionized water and diluting to 500 mL); 100 μL of the diluted antigen solution was added to each well of the enzyme-labeled plate, which was vacuum-sealed with an enzyme-labeled plate sealing machine and coated at 4°C overnight, and in the meantime, incubated in a 37°C incubator for 30 min; the coating solution was discarded, and the coated enzyme-labeled plate was washed with washing solution (0.05% Tween-20 in PBS solution; PBS is 0.02 mol / L phosphate buffer solution at pH 7.4, prepared by weighing 0.2 g of potassium dihydrogen phosphate, 2.9 g of sodium phosphate dibasic, 8 g of sodium chloride, and 0.2 g of potassium chloride, dissolving in deionized water, and diluting to 1000 mL) by adding 200 μL to the full amount of the coated enzyme-labeled plate, blotting with filter paper, and washing for 3 times, each for 3-5 min; 250 μL of blocking solution (1% BSA in 100 mL of PBST solution) was added to each well of the enzyme-labeled plate, which was placed in a wet box and incubated at 37°C for 2 h, and the plate was washed as above; the blank control was added with the antibody diluent, the negative control was added with non-immune serum (diluted 1:100), and the experimental group was added with antiserum at dilutions of 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, 1:1638400, and then, the enzyme-labeled plate was placed in a wet box and incubated at 37°C for 1-2 h, and the plate was washed as above; 100 μL of horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (purchased from Wuhan Dr. Deki Biological Engineering Co., Ltd., item number BA1054) secondary antibody at a dilution of 1:5000 was added to each well of the enzyme-labeled plate, which was placed in a wet box and incubated at 37°C for 1-2 h, and the plate was washed as above; 100 μL of TMB color developing solution (purchased from Biyun Tian Biological Technology Co., Ltd., item number P0206) was directly added to each well of the enzyme-labeled plate, which was incubated at room temperature or at 37°C for 5-30 min. Then, 100 μL of 2 M sulfuric acid stop solution (2 mol / L sulfuric acid solution, prepared by adding 10.870 mL of 98% concentrated sulfuric acid to 60 mL of deionized water and diluting to 100 mL, and stored at room temperature) was added to each well to stop the reaction; the enzyme-labeled plate was placed in an enzyme-labeled plate reader, and the absorbance at 450 nm was measured. When the ratio to the negative control serum was greater than 2.1, the antibody titer was calculated.

[0052] The detection results are shown in Table 1. Figure 6 The antibody titer of the antiserum was 1:25600.

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

[0054] A 10% SDS-PAGE gel was prepared according to standard methods, 40 μg of duck brain tissue lysate was added to the loading well of a vertical electrophoresis tank, and an SDS-PAGE gel electrophoresis was performed, 60 V for 30 min, then the voltage was adjusted to 100 V, and the electrophoresis was terminated when the bromophenol blue ran to about 1.5 cm from the bottom of the gel. After the SDS-PAGE electrophoresis was completed, wet transfer was used for membrane transfer, and the protein was transferred to a PVDF membrane. Blocking was performed with 5% skim milk, and the membrane was washed with TBST 4 times for 10 min each time. The primary antibody (prepared rabbit anti-duck GRPR modified polypeptide polyclonal antibody, diluted 1:2000) was added, and incubated at 4°C overnight, and the membrane was washed with TBST 5 times. HRP-labeled goat anti-rabbit IgG was added as a secondary antibody for incubation, and the membrane was washed with TBST 4 times for 10 min each time. ECL luminescent solution (purchased from Xin Saimei Biotechnology Co., Ltd., product number P10300) A and B were mixed to incubate the PVDF membrane, and the reaction was carried out in the dark for 1 min. An image was obtained by imaging system exposure.

[0055] The results are shown in Figure 7 The bands were clear, the positions were correct, the specificity was good, and the expression level of GRPR protein in duck brain tissue was high.

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

[0057] Duck pancreas tissue was fixed with 4% paraformaldehyde solution, and paraffin sections of duck pancreas tissue were prepared according to the steps of tissue dehydration, transparency, wax immersion, embedding, sectioning, etc. The section thickness was 5 μm. Then, the paraffin sections of duck pancreas tissue were deparaffinized by conventional dimethylbenzene and gradient alcohol; 0.1% Triton X-100 was dropped on the tissue sections, and the membrane was permeated at 37°C for 10 min, and then washed with distilled water for 10 min; 3% H2O2 was distilled water was used to incubate at room temperature for 8 min to inactivate endogenous peroxidase, and PBS was washed for 10 min; 0.01M citrate buffer was used for heat antigen repair by microwave method, and cooled to room temperature; 5% BSA was used for incubation at 37°C for 30 min for blocking; the prepared rabbit anti-duck GRPR modified polypeptide polyclonal antibody (1:100) was used as the primary antibody, and incubated at 4°C overnight, and PBS was washed for 20 min; biotin-labeled goat anti-rabbit IgG was used as the secondary antibody, and incubated at 37°C for 1 h, and PBS was washed for 20 min; SABC was used for incubation at 37°C for 1 h, and PBS was washed for 20 min; DAB was used for color development, and the reaction time was controlled under a microscope, and tap water was used to terminate the reaction; after the cell nucleus was stained with hematoxylin, conventional dehydration, transparency, and mounting were performed, and observation and photography were performed by microscope.

[0058] The results are shown inFigure 8 As shown, the detection results show the distribution of GRPR protein in pancreatic tissue cells.

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

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.

3. A polyclonal antibody obtained by immunizing an animal with the duck GRPR modified polypeptide according to claim 2 as an antigen.

4. The method for preparing a polyclonal antibody according to claim 3, wherein: The preparation method comprises the following steps: coupling the duck GRPR modified polypeptide with a carrier protein to obtain a complete antigen, then immunizing an animal, collecting blood from the immunized animal to prepare antiserum, and separating and purifying the antiserum to obtain polyclonal antibodies.

5. The preparation method according to claim 4, wherein The carrier protein is keyhole limpet hemocyanin.

6. The preparation method according to claim 5, wherein The GRPR modified polypeptide is coupled to keyhole limpet hemocyanin via a coupling agent Sulfo-SMCC to prepare the complete antigen.

7. Use of the polyclonal antibody according to claim 3 in the preparation of a reagent for detecting GRPR protein levels in duck tissues.

8. The use according to claim 7, characterized in that The method for detecting the GRPR protein level in duck tissues by utilizing the polyclonal antibody comprises an immunoblotting detection method and an immunohistochemical detection method.

9. A reagent for detecting the level of GRPR protein in duck tissue, characterized in that: The reagent comprises the polyclonal antibody according to claim 3.

10. A method for detecting GRPR protein in duck tissue for non-diagnostic purposes, characterized in that: The polyclonal antibody according to claim 3 is used as a detection antibody to detect the expression level of GRPR protein in duck tissue.

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