A method for preparing and applying a duck neurotransmitter B-modified polypeptide and its polyclonal antibody.

Abstract

This invention discloses a method for preparing and applying a duck interneuron B modified polypeptide and its polyclonal antibody, belonging to the fields of biochemistry and molecular immunology. The amino acid sequence of the duck interneuron B modified polypeptide is shown in SEQ ID NO.4. This invention screens a 19aa polypeptide sequence from the duck interneuron B protein sequence, adds a cysteine ​​residue (Cys, C) to its C-terminus, synthesizes it artificially in a solid phase, and uses it as an immunogenic antigen to immunize New Zealand white rabbits, obtaining a duck interneuron B polyclonal antibody with an antiserum titer of over 1:204800. The rabbit anti-duck interneuron B polyclonal antibody prepared by this invention can specifically bind to interneuron B protein in duck tissues, filling a gap in the research field of duck interneuron B protein detection and laying the foundation for the study of the function of interneuron B in ducks.

Description

Technical Field

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

[0002] Neuromedin B (NMB) is a member of the dermalin family. It exerts a variety of important physiological functions by binding to its receptors, such as regulating feeding, stimulating the contraction of smooth muscle in the gastrointestinal tract and urogenital tract, influencing behavioral responses such as anxiety / itching, regulating hormone synthesis / secretion, stimulating the growth of normal / tumor cells, mediating immune responses, and regulating reproduction.

[0003] Since the discovery of NMB, researchers both domestically and internationally have conducted extensive studies on the expression and distribution of NMB in animals such as humans, mice, pigs, sheep, rabbits, and chickens. These studies have revealed that NMB is widely expressed in the central nervous system and multiple peripheral tissues and organs. The homology of duck NMB gene and amino acid sequence with chicken NMB is 87.9% and 89.8%, respectively, and phylogenetic analysis also shows that duck NMB is most closely related to chicken NMB. The inventors previously used quantitative real-time PCR to find high expression levels of the NMB gene in multiple central nervous systems (cerebrum, cerebellum, optic lobe) and peripheral tissues and organs (trachea, lungs, pineal gland, thyroid gland, kidneys, and preen glands) in ducks, indicating that NMB may 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, as important waterfowl, provides a reference for research on other waterfowl. However, the lack of commercially available antibodies for detecting duck NMB protein, coupled with the complex, costly, and time-consuming process of preparing duck NMB monoclonal antibodies, has limited research on NMB in ducks, primarily focusing on the genetic level and severely restricting functional studies of NMB in ducks. Therefore, the preparation of antibodies targeting duck NMB protein is particularly important, as it can be used to study the physiological functions of NMB in ducks and meet 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 interneuron B modified polypeptides and their polyclonal antibodies, thereby addressing the problems existing in the prior art. The rabbit anti-duck interneuron B polyclonal antibody prepared by this invention can specifically bind to interneuron B protein in duck tissues, with an antiserum titer exceeding 1:204800. This fills a gap in the research field of duck interneuron B protein detection and lays the foundation for studying the function of interneuron B in ducks.

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

[0007] This invention provides a duck neurotransmitter B modified 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 neurotransmitter B modified polypeptide as an antigen in the preparation of duck neurotransmitter B polyclonal antibodies.

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

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

[0011] The duck neurotransmitter B-modified polypeptide was coupled with keyhole hemocyanin to obtain a complete antigen.

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

[0013] Optionally, the duck neurotransmitter B modified 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 duck neurotransmitter B polyclonal antibody obtained by the preparation method described above.

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

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

[0020] This invention screened a 19aa polypeptide sequence from the duck interneuron B protein sequence, added a cysteine ​​residue (Cys, C) to its C-terminus, synthesized it artificially in a solid phase, and used it as an immunogenic antigen to immunize New Zealand white rabbits, obtaining a duck interneuron B polyclonal antibody with an antiserum titer of over 1:204800. The rabbit anti-duck interneuron B polyclonal antibody prepared by this invention can specifically bind to interneuron B protein in duck tissues, filling a gap in the field of duck interneuron B protein detection research and laying the foundation for the study of the function of interneuron B 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 hydrophilicity, antigenic index and surface accessibility of duck neurotransmitter B precursor protein sequence;

[0023] Figure 2 The mass spectrometry results of duck neurotransmitter B-modified peptides are shown in the figure.

[0024] Figure 3 A graph showing the antiserum titer detected by the indirect ELISA method;

[0025] Figure 4 Figure 1 shows the Western blot results of the GST-duck NMB fusion protein.

[0026] Figure 5 The image shows the results of Western blotting analysis of the expression level of neurotransmitter B protein in duck kidney.

[0027] Figure 6 Figure 1 shows the IHC detection results for the distribution and localization of NMB protein in duck pituitary 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 NMB gene, which has an open reading frame (ORF) of 387 bp and encodes 128 amino acids. The ORF sequence of the duck NMB 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] ATGGCGGCGCTGCGCTGCCTCCTGCTGCTGCTGTGCGGAGCCGCGCTGGGGCCCGCCGTGCACCTCGACTTCGCCGAGCACCGCAGCCAGGCGGCCAAGATCAAGGTCAACCCCCGCGGAAACCTCTGGGCCACAGGACACTTCATGGGGAAGAAGAGCGTCACGGGCACCCCGCGCCTGGAGTCACCCGAAGA GCCCGCAGTGCCGATGGTTTTTGGTCCCTCGCTCAGAGCCTTGCTGGAGGACATGATGGAACTGCTCACCCGTGAGCTCCTGAAAATCCTCTTGCAAGAAAGACTTTTGGATGAGAACCAAGGAAAATATGACCTCGCTGATCAGGAGACTGGGCTTTTGACAAAGGTGCTGGAGAAGTATTTTTCAAACTGA.

[0036] SEQ ID NO.2:

[0037] MAALRCLLLLLCGAALGPAVHLDFAEHRSQAAKIKVNPRGNLWATGHFMGKKSVTGT PRLESPEEPAVPMVFGPSLRALLEDMMELLTRELLKILLQERLLDENQGKYDLADQETGLLT KVLEKYFSN.

[0038] In this embodiment of the invention, the BL21(DE3)-pET-N-GST-Precission-Duck NMB bacterium expressing the GST-duck NMB fusion protein (41.613 kDa) is a self-constructed engineered bacterium, and the construction method is as follows:

[0039] (1) Construction of pET-N-GST-Precission-duck NMB recombinant plasmid: EcoRI and XhoI were selected as the insertion restriction sites, and upstream primer 5'-CGGAATTCATGGCGGCGCTGCGCT-3' and downstream primer 5'-GCTCGAGGACTCCATCCTCCACTGCTCT-3' were designed and synthesized; the duck NMB target fragment with restriction sites was obtained by PCR amplification; the duck NMB target fragment and pET-N-GST-Precission plasmid (CS-01F97825) were double-digested with EcoRI and XhoI to obtain linearized fragments, and then ligated under the action of T4 DNA Ligase to construct pET-N-GST-Precission-duck NMB recombinant plasmid.

[0040] (2) Obtaining GST-Duck NMB fusion protein: The pET-N-GST-Precission-Duck NMB recombinant plasmid was transformed into BL21(DE3) competent cells to obtain the BL21(DE3)-pET-N-GST-Precission-Duck NMB expression strain; the GST-Duck NMB fusion protein could be obtained by inducing expression with 0.5mM IPTG.

[0041] Example 1: Sequence analysis of duck neurotransmitter B precursor protein and design and synthesis of duck neurotransmitter B modified peptides

[0042] The hydrophilicity, surface accessibility, and antigenic index of duck neurotransmitter B precursor protein (SEQ ID NO.2) were analyzed using the Protean module in DNAstar software. Figure 1 The sequences were analyzed for homology using NCBI BLAST's BLASTN tool, and a suitable sequence was selected as the target sequence for antigen fitting. This sequence includes 19 amino acids, located at positions 24-42 amino acids of the sequence shown in SEQ ID NO. 2. The specific sequence is as follows:

[0043] FAEHRSQAAKIKVNPRGNL (SEQ ID NO. 3).

[0044] To enhance the immunogenicity of the peptide by coupling with the carrier protein, the selected peptide sequence was modified at the C-terminus by adding a cysteine ​​residue (Cys, C). Therefore, the final synthesized peptide sequence is FAEHRSQAAKIKVNPRGNLC (SEQ ID NO. 4), which is a duck interleukin B-modified peptide. The purity of the synthesized peptide was determined to be 95.4% by high-performance liquid chromatography (HPLC), and the molecular weight of the synthesized peptide was determined to be 2239.55 Da by mass spectrometry. Figure 2 ).

[0045] Example 2: Coupling of duck neurotransmitter B modified peptide with carrier protein keyhole hemocyanin (KLH)

[0046] 5.0 mg of duck interleukin B modified peptide and 5.0 mg of carrier protein KLH were coupled using the coupling agent Sulfo-SMCC. The coupling was performed by Jier Biochemical (Shanghai) Co., Ltd., to obtain duck interleukin B modified peptide-KLH coupled protein, i.e., complete antigen.

[0047] Example 3: Immunization of experimental animals and preparation of antiserum

[0048] 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.

[0049] 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 the 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.

[0050] To boost immunization, 2 mg of complete antigen was dissolved in 2 mL of PBS and thoroughly emulsified with an equal volume of Freund's incomplete adjuvant (purchased from Sigma-Aldrich, catalog number F5506). After emulsification, the experimental animals were immunized subcutaneously at multiple points (6-8 points) on the back at a dose of 0.4 mg / kg of complete antigen.

[0051] Twelve days after the second booster immunization, blood was collected from the marginal ear vein of rabbits. Antibody titers were detected using indirect ELISA to determine whether further booster immunization was necessary, the required dose, and the number of booster immunizations. A total of two booster immunizations were administered. The dose for the second booster immunization (third immunization) was 0.36 mg / kg.

[0052] Antiserum was collected 11 days after the second booster immunization (third immunization). Rabbits were anesthetized, and a large amount of blood was collected by pleophoresis of the abdominal aorta. The collected blood was tilted and left to stand at 37°C for about 1 hour, and then transferred to a 4°C refrigerator and left 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, and then aliquoted and stored at -80°C for later use.

[0053] Example 4: Detection of antiserum titer using an indirect ELISA method

[0054] The duck interleukin B modified peptide was diluted to 10 μg / mL using coating buffer (i.e., 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 (i.e., PBST, a 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 in an appropriate amount of deionized water and bring the volume to 1000 mL. Add 200 μL to each well of the coated ELISA plate (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 BSA in 100 mL PBST solution) to each well of the ELISA plate. Place the ELISA plate in a humidified chamber and incubate at 37°C for 2 h. Wash the plate as above. Add antibody dilution to the blank control, add unimmunized serum (1:100 dilution) to the negative control, and add antiserum diluted at different ratios (1:200, 1:400, 1:800, 1:1600, 1:400, 1:800, 1:1600, 1:1 ... 3200, 1:6400, 1:12800, 1:25600, 1:51200, 1:102400, 1:204800, 1:409600, 1:819200 and 1:1638400) were used. Then, the microplate was 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 microplate. The microplate 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 microplate 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.

[0055] Test results as follows Figure 3 As shown: the antibody titer of the antiserum is 1:204800.

[0056] Example 5: Western Blot Detection of GST-Duck NMB Fusion Protein

[0057] Prepare a 10% SDS-PAGE gel according to standard methods. Add the BL21(DE3)-pET-N-GST-Precission-Duck NMB cell lysis buffer (laboratory stock) successfully expressing the GST-duck NMB fusion protein (41.613 kDa) to the sample wells 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, transfer the protein 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 neurotransmitter B modified polypeptide polyclonal antibody, diluted 1:5000) 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 for 1 minute in the dark, and the image was obtained by exposure using an imaging system.

[0058] The results are as follows Figure 4 As shown, the bands are clear and correctly positioned, indicating that the rabbit anti-duck interneuron B polyclonal antibody has good reactivity and can be used to detect duck interneuron B protein.

[0059] Example 6: Immunoblot detection of neurotransmitter B protein in duck tissues

[0060] Prepare a 12.5% ​​SDS-PAGE gel according to standard methods. Add 40 μg of duck kidney tissue lysate to the sample well of a vertical electrophoresis tank and run SDS-PAGE gel electrophoresis at 60V for 30 min. Then adjust the voltage to 100V and stop electrophoresis when the bromophenol blue reaches about 1.5 cm from the bottom of the gel. After SDS-PAGE electrophoresis, transfer the protein to a PVDF membrane using wet transfer. Block with 5% skim milk powder at room temperature for 2 h, and wash the membrane 4 times with TBST for 10 min each time. Add primary antibody (prepared rabbit anti-duck neurotransmitter B modified polypeptide polyclonal antibody, diluted 1:500), incubate overnight at 4℃, and wash the membrane 5 times with TBST. Add HRP-labeled goat anti-rabbit IgG as secondary antibody and incubate at room temperature for 2 h, and wash the membrane 4 times with TBST. Mix ECL chemiluminescence solutions A and B and incubate the PVDF membrane in the dark for 1 min, then expose and acquire images.

[0061] The results are as follows Figure 5As shown, the bands are clear, correctly positioned, and highly specific, indicating that the expression level of neurotransmitter B protein is high in duck kidneys.

[0062] Example 7 Immunohistochemical (IHC) Detection of Neurotransmitter B Protein in Duck Tissue

[0063] Duck pituitary tissue was fixed with 4% paraformaldehyde solution. Paraffin sections of the duck tissue were prepared following the steps of tissue dehydration, clearing, paraffin embedding, embedding, and sectioning, with a section thickness of 5 μm. Then, the duck pituitary tissue paraffin sections were dewaxed by conventional xylene and graded alcohol immersion. 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 neurotransmitter B 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.

[0064] The results are as follows Figure 6 As shown, the detection results indicate that neurotransmitter B protein is distributed in pituitary tissue cells.

[0065] 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 neurokinn B modified polypeptide, characterized in that, The amino acid sequence of the duck neurotransmitter B modified polypeptide is shown in SEQ ID NO.

4.

2. The use of the duck neurotransmitter B modified polypeptide as described in claim 1 as an antigen in the preparation of duck neurotransmitter B polyclonal antibodies.

3. A method for preparing a duck neurotransmitter B polyclonal antibody, characterized in that, The step includes immunizing animals with the duck neurotransmitter B modified polypeptide as described in claim 1 as an antigen.

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

5. The preparation method according to claim 4, characterized in that, The duck neurotransmitter B modified 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 duck neurotransmitter B polyclonal antibody obtained by the preparation method according to any one of claims 3-8.

10. The use of the duck neurotransmitter B polyclonal antibody according to claim 9 in detecting the expression level of neurotransmitter B protein in duck tissue.

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