Duck NMBR polypeptide, polyclonal antibody and application

Abstract

The invention discloses a duck NMBR polypeptide, a polyclonal antibody and application, and belongs to the field of biochemistry and molecular immunology. The duck NMBR gene is cloned for the first time, and the structure of the duck NMBR gene is analyzed; amino acids at 341-359 sites at the carboxyl terminal of duck NMBR protein are used as haptens, the haptens are coupled with keyhole limpet hemocyanin to prepare complete antigens, then animals are immunized, and the polyclonal antibody of the duck NMBR polypeptide is separated and purified from antiserum. Experiments show that the polyclonal antibody can specifically bind to NMBR protein in duck tissue. According to the invention, the blank in the field of duck NMBR protein detection and research is filled, and a foundation is laid for research of the function of the NMBR in the duck body.

Description

TECHNICAL FIELD

[0001] The present application relates to the fields of biochemistry and molecular immunology, and particularly relates to a duck NMBR polypeptide, a polyclonal antibody and application. BACKGROUND

[0002] Neuromedin B receptor (NMBR) is a member of the bombesin receptor family, which belongs to G-protein coupled receptors, has a typical 7-transmembrane domain, and mainly plays a variety of biological functions by binding with its ligand neuromedin B (NMB) to activate G-protein-mediated intracellular signaling pathways, such as inhibiting feeding, stimulating smooth muscle contraction, reducing the impact of stress on the body, reducing the startle caused by fear and fear, regulating energy metabolism and hormone production, promoting cell growth, regulating immune response, etc.

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

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

[0005] To achieve the above purpose, the present application provides the following scheme:

[0006] The present application provides a duck NMBR polypeptide, and the amino acid sequence of the duck NMBR polypeptide is shown in SEQ ID NO. 3.

[0007] The application also provides a polyclonal antibody obtained by immunizing an animal with the duck NMBR polypeptide as an antigen.

[0008] Preferably, the method comprises the following steps: coupling the duck NMBR polypeptide with a carrier protein to prepare a complete antigen, immunizing an animal with the complete antigen, collecting blood of the immunized animal to prepare an antiserum, and separating and purifying the polyclonal antibody from the antiserum.

[0009] Preferably, the carrier protein is keyhole limpet hemocyanin.

[0010] Preferably, the N-terminus of the NMBR polypeptide is coupled with the keyhole limpet hemocyanin by a coupling agent Sulfo-SMCC to prepare the complete antigen.

[0011] The application also provides an application of the polyclonal antibody in preparing a reagent for detecting a duck tissue NMBR protein level.

[0012] Preferably, the method for detecting a duck tissue NMBR protein level by using the polyclonal antibody comprises an immunoblotting detection method and an immunohistochemical detection method.

[0013] The application also provides a reagent for detecting a duck tissue NMBR protein level, which comprises the polyclonal antibody.

[0014] The application also provides a method for detecting a duck tissue NMBR protein for non-diagnostic purposes, which uses the polyclonal antibody as a detection antibody to detect an expression level of a duck tissue NMBR protein.

[0015] The application discloses the following technical effects:

[0016] (1) The duck NMBR gene sequence is cloned for the first time, and the transmembrane structure, hydrophilicity, surface accessibility, antigen index and homology of the duck NMBR protein sequence are analyzed, a suitable peptide segment sequence is screened as a target sequence for artificial synthesis, and the duck NMBR polyclonal antibody is prepared.

[0017] (2) The N-terminus of the synthesized peptide segment sequence is coupled with keyhole limpet hemocyanin to obtain duck NMBR polypeptide-KLH coupling protein, that is, a complete antigen; and a New Zealand white rabbit is immunized with the complete antigen, the titer of the antiserum is detected by an indirect ELISA method, and the titer of the prepared antiserum is above 1:25600.

[0018] (3) The rabbit anti-duck NMBR polyclonal antibody prepared in the application can specifically bind to the NMBR protein in duck tissues, fills the blank in the field of duck NMBR protein detection research, and lays a foundation for the research on the function of NMBR in a duck. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the accompanying drawings needed in the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description only show some embodiments of the present application, and for those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0020] Figure 1 Analysis results of transmembrane structure of duck NMBR protein sequence by using online software TMHMM-2.0;

[0021] Figure 2 The expression of NMBR gene in multiple tissues and organs of duck such as cerebellum, brain, pancreas, pineal gland, pituitary, testis, heart, Hassal's gland and ileum was detected by fluorescence quantitative PCR;

[0022] Figure 3 Analysis results of hydrophilicity, antigen index and surface accessibility of duck NMBR protein sequence by using bioinformatics software DNAstar (Protean);

[0023] Figure 4 HPLC purity detection results chart of duck NMBR polypeptide;

[0024] Figure 5 Mass spectrum detection results chart of duck NMBR polypeptide;

[0025] Figure 6 Antiserum titer chart detected by indirect ELISA method;

[0026] Figure 7 Western Blot detection results chart of NMBR protein expression level in duck brain tissue by using rabbit anti-duck NMBR polypeptide polyclonal antibody, wherein M is a protein marker, and the duck brain tissue is treated by rabbit anti-duck NMBR polyclonal antibody as the primary antibody;

[0027] Figure 8 IHC detection results chart of NMBR protein distribution and positioning in duck testis tissue by using rabbit anti-duck NMBR polypeptide polyclonal antibody; NMBR is treated by rabbit anti-NMBR polyclonal antibody as the primary antibody, and the control is treated by PBS as the primary antibody. DETAILED DESCRIPTION

[0028] The various exemplary embodiments of the present application will now be described in detail below, which should not be considered as a limitation to the present application, but should be understood as a more detailed description of some aspects, characteristics and embodiments of the present application.

[0029] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, for a range of values of, for example, a parameter, an intermediate value of the parameter is encompassed if the intermediate value is within the range of values. Also, it is to be understood that any numerical range recited is intended to include all sub-ranges of the same numbers. These ranges are only intended to serve as a shorthand method of referring individually to each separate value and range falling within the overall range. Any individual value or sub-range not specifically recited can be included in the present application.

[0030] 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 application belongs. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe in detail the methods and / or materials which are related to the present application. In case of conflict, the content of the present specification will control.

[0031] Many modifications and variations of this application can be made in the light of the above teachings without departing from the spirit and scope thereof. Additional implementations 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 examples and embodiments described herein are exemplary only and are not intended to be limiting.

[0032] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean including, but not limited to.

[0033] Example 1 Duck NMBR protein sequence analysis and design and synthesis of duck NMBR polypeptide

[0034] The duck NMBR gene was cloned for the first time in the laboratory, and the duck NMBR gene sequence was analyzed, and it was found that the open reading frame (ORF) of the duck NMBR gene was 1173 bp, encoding 390 aa.

[0035] The nucleotide sequence of the open reading frame of the duck NMBR gene is shown in SEQ ID NO. 1:

[0036]

[0037] The full-length amino acid sequence of duck NMBR protein is shown in SEQ ID NO. 2 as follows:

[0038] MVLEAELLSELTAMFLEGNGSAQLVPDAVLLPPGQGTAMFTVRCVIPSLYLLIITVGLLGNVTLMKIFISNSAMRSVPNIFISSLAAGDLLLLVTCVPVDASRYFSEEWLFGEVGCKIIPAIQLTSVGVSVFTLTALSADRYKAIVNPMDIQTSSAVLWTCVKAITIWIISMLLAVPEAVFSELAHINNTDNATFKACIPYPMKDDMHPKIHSVLIFLVYFLIPLTIISIYYCHIAKSLIKSAHNIPGEYSEHSKRQMETRKRLAKIVLVFVGFFAICWFPNHVLYMYRSFNYNEIDSSIGHMVVTLVARVLSFCNSCVNPFALYFLSESFRRHFNNQLRCRKKSQQERSASYSHNSSAVQMTSLKSNTRNTVTSVTQLNGHNLKQEMSL.

[0039] The transmembrane structure of duck NMBR protein sequence was analyzed by online software TMHMM-2.0, as shown in Figure 1 The duck NMBR protein belongs to G-protein coupled receptor with seven transmembrane domains, including four extracellular regions (1-45, 100-117, 177-210 and 287-304), seven transmembrane regions (46-64, 77-99, 118-136, 154-176, 211-233, 264-286 and 305-327) and four intracellular regions (65-76, 137-153, 234-263 and 328-390). In addition, the homology of duck and chicken NMBR gene and amino acid sequence is 92.88% and 91.73%, respectively, and the phylogenetic tree analysis is also closest to chicken. The expression of NMBR gene in different tissues was detected by fluorescence quantitative PCR. The primer sequences required for fluorescence quantitative PCR are as follows: upper primer 5-TGCTGGGAAATGTCACCCTC-3, lower primer 5-GATGATCTTGCAGCCCACCT-3, the amplification fragment is 185 bp, and the annealing temperature is 60°C. The detection reaction system and reaction procedure are operated according to the operation manual of FastReal rapid fluorescence quantitative PCR premix reagent (SYBR Green) kit, which is purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. The results are shown in Figure 2As shown in the figure, the NMBR gene was found to be expressed in multiple tissues and organs of the duck, such as the cerebellum, brain, pancreas, pineal gland, pituitary, testis, heart, Hatshorn's gland and ileum, indicating that NMBR may also have important physiological functions in the duck.

[0040] The Protean module in the DNAstar software was used to analyze the hydrophilicity, surface accessibility, antigen index and other indicators of the duck NMBR protein (such as Figure 3 As shown in the figure, combined with the transmembrane structure analysis of the duck NMBR protein, a suitable sequence CRKKSQQERSASYSHNSSA (SEQ ID NO. 3) was screened out as the target sequence for synthesizing the antigen (341aa-359aa), which is located in the intracellular region of the carboxyl terminal (C-terminal) of the duck NMBR protein and includes 19 amino acids.

[0041] The duck NMBR polypeptide was artificially synthesized, and the purity of the synthesized polypeptide was 95.41% (see Figure 4 ) detected by high performance liquid chromatograph (HPLC), and the molecular weight of the synthesized polypeptide was 2154.28 Da (see Figure 5 ) detected by mass spectrometry.

[0042] Example 2: Coupling of duck NMBR polypeptide and carrier protein keyhole limpet hemocyanin (KLH)

[0043] The duck NMBR polypeptide sequence designed and synthesized in the present application is located in the carboxyl terminal (C-terminal) of the duck NMBR protein, and it is necessary to couple the carrier protein to the amino acid (N-terminal) of the synthesized duck NMBR polypeptide to increase the immunogenicity of the polypeptide. That is, 5.0 mg of duck NMBR polypeptide is coupled with 5.0 mg of carrier protein KLH by using the coupling agent Sulfo-SMCC, and the coupling is completed by Gilson Biochemical (Shanghai) Co., Ltd. to obtain duck NMBR polypeptide-KLH coupling protein, i.e. complete antigen.

[0044] Example 3: Immunization of experimental animals and preparation of antisera

[0045] A suitable age of male New Zealand white rabbits was selected as the immunized animals, and blood was collected from the marginal ear vein of the rabbits before the first immunization (blood collection was performed before feeding in the morning), which was used as the control serum for subsequent ELISA detection.

[0046] 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) by homogenization. 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 marginal ear 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, as well as the number of booster immunizations. A total of 2 booster immunizations were performed before and after the present application.

[0047] The second booster immunization was performed on the New Zealand white rabbits 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.

[0048] Example 4: Indirect ELISA method for detecting the titer of the antiserum

[0049] Dilute duck NMBR polypeptide to 10 μg / mL with coating solution (i.e. 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 making up to 500 mL); add 100 μL of the diluted antigen solution to each well of the enzyme-labeled plate, and seal the plate by vacuumizing with an enzyme-labeled plate sealer, then coat overnight at 4°C, and incubate in a 37°C incubator for 30 min; discard the coating solution, add 200 μL of washing solution (i.e. 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 making up to 1000 mL) to each well of the coated enzyme-labeled plate, and wash 3 times with filter paper, each for 3-5 min; add 250 μL of blocking solution (i.e. 1% BSA in PBST solution, 1 g of BSA in 100 mL of PBST solution) to each well of the enzyme-labeled plate, and incubate in a 37°C incubator for 2 h, then wash the plate as above; add the antibody diluent to the blank control, add the negative control with non-immune serum (diluted 1:100), and add the experimental group with antisera 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, then incubate the enzyme-labeled plate in a wet box at 37°C for 1-2 h, and wash the plate as above; add 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 to each well of the enzyme-labeled plate, and incubate the enzyme-labeled plate in a wet box at 37°C for 1-2 h, and wash the plate as above; add 100 μL of TMB color developing solution (purchased from Biyun Tian Biological Technology Co., Ltd., item number P0206) directly to each well of the enzyme-labeled plate, and incubate at room temperature or at 37°C for 5-30 min. Then, add 100 μL of 2 M 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 making up to 100 mL, and stored at room temperature) to each well to stop the reaction; place the enzyme-labeled plate in an enzyme-labeled plate reader, and measure the absorbance at 450 nm. When the ratio to the negative control serum is greater than 2.1, calculate the antibody titer.

[0050] The detection results are shown in Table 1. Figure 6 The antibody titer of the antisera is 1:25600.

[0051] Example 5: Immunoblotting detection of NMBR protein in duck tissues

[0052] Prepare a 10% SDS-PAGE gel according to the standard method, take 40 μg of duck brain tissue lysate and add it to the loading well of the vertical electrophoresis tank, run the SDS-PAGE gel electrophoresis, 60 V for 30 min, then adjust the voltage to 100 V, and terminate the electrophoresis when the bromophenol blue runs to about 1.5 cm from the bottom of the gel. After the SDS-PAGE electrophoresis is completed, transfer the protein to the PVDF membrane by wet transfer. Block with 5% skim milk, room temperature for 2 h, wash the membrane with TBST 4 times, 10 min each time. Add the primary antibody (rabbit anti-duck NMBR polyclonal antibody prepared in the application, 1:2000 dilution), incubate at 4°C overnight, wash the membrane with TBST 5 times; add HRP-labeled goat anti-rabbit IgG as the secondary antibody for incubation, incubate at room temperature for 2 h, wash the membrane with TBST 4 times; mix ECL luminescent solution (purchased from Sinobiological Technology Co., Ltd., Catalog No. P10300) A and B, incubate the PVDF membrane, avoid light for 1 min, and obtain the image by imaging system exposure.

[0053] The results are shown in Figure 7 The bands are clear, the positions are correct, the specificity is good, and the expression level of NMBR protein in duck brain tissue is high.

[0054] Example 6 Immunohistochemical (IHC) detection of NMBR protein in duck tissue

[0055] Take the duck testis tissue, fix it with 4% paraformaldehyde solution, and prepare the duck testis tissue paraffin section according to the steps of tissue dehydration, transparency, wax immersion, embedding, sectioning, etc. The section thickness is 5 μm. Then, the duck testis tissue paraffin section is deparaffinized by conventional dimethylbenzene and gradient alcohol; 0.1% Triton X-100 is dropped on the tissue section, 37°C for 10 min, and distilled water is washed for 10 min; 3% H2O2 is distilled water, incubated at room temperature for 8 min to inactivate endogenous peroxidase, and PBS is washed for 10 min; 0.01M citrate buffer is used for heat antigen repair by microwave method, and cooled to room temperature; 5% BSA is incubated at 37°C for 30 min for blocking; the rabbit anti-duck NMBR polyclonal antibody prepared in the application (1:50) is used as the primary antibody, incubated at 4°C overnight, and PBS is washed for 20 min; biotin-labeled goat anti-rabbit IgG is used as the secondary antibody, incubated at 37°C for 1 h, and PBS is washed for 20 min; SABC is incubated at 37°C for 1 h, and PBS is washed for 20 min; DAB is used for color development, and the reaction time is controlled under a microscope, and the reaction is terminated with tap water; after the cell nucleus is stained with hematoxylin, the conventional dehydration, transparency, and mounting are performed, and the observation and photography are performed by microscope.

[0056] The results are shown in Figure 8 The detection results show the distribution of NMBR protein in testicular cells.

[0057] The above-described embodiments are merely intended to describe the preferred modes of the present application, and are not intended to limit the scope of the present application. Various modifications and improvements of the present application made by those skilled in the art based on the above-described embodiments should fall within the scope of the present application defined by the claims.

Claims

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

3.

2. A polyclonal antibody obtained by immunizing an animal with the duck NMBR polypeptide of claim 1 as an antigen.

3. The method of claim 2, wherein the polyclonal antibody is prepared by immunizing a mammal with a mixture of antigens. The method comprises the following steps: After the duck NMBR polypeptide is coupled with a carrier protein to prepare a complete antigen, an animal is immunized, and the blood of the immunized animal is collected to prepare an antiserum, and a polyclonal antibody is separated and purified from the antiserum.

4. The production method according to claim 3, wherein The carrier protein is keyhole limpet hemocyanin.

5. The production method according to claim 4, wherein The N-terminal of the NMBR polypeptide is coupled with keyhole limpet hemocyanin through a coupling agent Sulfo-SMCC to prepare the complete antigen.

6. The polyclonal antibody of claim 2 is used for preparing a reagent for detecting the NMBR protein level in duck tissues.

7. Use according to claim 6, wherein The method for detecting the NMBR protein level in duck tissues by using the polyclonal antibody comprises an immunoblotting detection method and an immunohistochemical detection method.

8. A reagent for detecting the level of NMBR protein in duck tissues, characterized in that, The reagent comprises the polyclonal antibody of claim 2.

9. A method for detecting NMBR protein in duck tissues for non-diagnostic purposes, characterized by, The polyclonal antibody of claim 2 is used as a detection antibody to detect the expression level of the NMBR protein in duck tissues.

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