Antigen epitope peptide of ca sr, vaccine, antibody and application thereof

Abstract

The application belongs to the technical field of bioactive peptides and antibodies, and discloses an antigen epitope peptide of CaSR, a vaccine, an antibody and application. The application aims to use polypeptide vaccine technology to construct yolk antibody targeting CaSR in the intestinal tract, improve calcium absorption efficiency of poultry, seek a scheme for efficient utilization of calcium elements of poultry, and realize calcium saving and emission reduction in production.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptides and antibodies, specifically to an antigenic epitope peptide of a calcium-sensitive receptor (CaSR), a vaccine, an antibody, and their application in improving calcium (Ca) absorption efficiency. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Calcium is the most abundant mineral in the body, with a wide range of biological functions, including bone mineralization, nerve conduction, and hormone secretion. Therefore, maintaining calcium ion metabolic balance is crucial. Age and physiological conditions in humans and animals affect calcium absorption and utilization. For example, taking broiler chickens as an example, with the development of modern breeding technology, the growth rate of broilers has continuously increased, and at 42 days of age, their weight can reach 2.80 kg. The rapid growth of broilers requires strong bone support, and the mortality rate of broilers due to abnormal bone mineralization is about 3%, accounting for 60% of all reported mortality rates in the broiler industry. Furthermore, in the later stages of layer hen farming, the egg breakage rate due to abnormal eggshell mineralization can reach 20%, causing huge economic losses to the industry. These problems are closely related to the low efficiency of feed calcium utilization in poultry. It has been reported that the apparent digestibility of dietary calcium in 7-week-old, 32-week-old, and 70-week-old layer hens is 38%, 49%, and 44%, respectively; while the apparent digestibility of dietary calcium in broilers is only about 53%. Therefore, improving the utilization efficiency of calcium in feed is of great importance.

[0004] In production, the calcium requirements of animals are often met by increasing the calcium supply level in the feed and adding vitamin D3. However, the addition of high levels of calcium and vitamin D3 in the feed does not fundamentally solve the problem of low calcium absorption and utilization efficiency. On the contrary, it can easily lead to constipation, bloating, and may even interfere with the homeostasis of calcium and phosphorus metabolism or the absorption and utilization of other mineral elements, which can have a negative impact on the health of poultry.

[0005] Calcium-sensitive receptors (CaSRs) are important calcium ion sensors that detect changes in extracellular calcium ion concentration and maintain calcium homeostasis by regulating calcium absorption and excretion. CaSRs exert their physiological functions by regulating parathyroid hormone (PTH) secretion, renal tubular calcium reabsorption, and insulin release. Studies have found that when the intraluminal calcium ion concentration increases, CaSRs distributed in the intestine can sense this change and, by downregulating the expression of calcium ion channels, feedback-inhibit intestinal calcium absorption. This may be an important reason for the low intestinal calcium utilization efficiency in humans and animals.

[0006] In existing technologies, some studies utilize calcimenors to mimic the action of calcium ions and enhance CaSR activity, which can be used to treat diseases such as hyperparathyroidism. Other studies use calcitonin to inhibit CaSR activity, reducing its sensitivity to calcium ions, and correcting abnormal blood calcium levels. However, these drugs are expensive, and large-scale use may cause a series of side effects such as increased liver burden and gastrointestinal discomfort. Due to the limitations of existing technologies, there is an urgent need to develop a CaSR modulator that targets intestinal CaSR to regulate intestinal calcium and phosphorus absorption efficiency, thereby improving the efficiency of intestinal calcium absorption and utilization. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide an antigenic epitope peptide, vaccine, antibody, and their applications related to CaSR. This invention aims to utilize peptide vaccine technology to construct egg yolk antibodies targeting intestinal CaSR, thereby improving calcium absorption efficiency in poultry, providing a solution for efficient calcium utilization in poultry, and achieving calcium conservation and emission reduction in production.

[0008] A first aspect of the present invention discloses an antigenic epitope peptide selected from one or more of the following polypeptide sequences, wherein the polypeptide sequences are SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5.

[0009] A second aspect of the present invention discloses a vaccine comprising the above-described antigenic epitope peptide.

[0010] A third aspect of the present invention discloses an antibody, which is an antibody produced in poultry after vaccination with the above-mentioned vaccine; preferably, the antibody is present in the yolk of the vaccinated poultry.

[0011] A fourth aspect of the present invention discloses a poultry feed comprising egg yolk containing the aforementioned antibodies.

[0012] In some embodiments, the poultry include, but are not limited to, chickens, turkeys, ducks, geese, quails, peacocks, emus, pheasants, and guinea fowl.

[0013] In some embodiments, the yolk is added at a mass fraction of 0.3-0.8%, preferably 0.5%.

[0014] The fifth aspect of the present invention discloses the use of the above-mentioned antigenic epitope peptides, vaccines, antibodies, or feeds in promoting calcium or phosphorus absorption in animals.

[0015] In some embodiments, the application is the use of the above-mentioned antibody in promoting calcium absorption in the intestinal tract of animals, preferably poultry.

[0016] In some embodiments, the application is applied during the egg-laying period, rapid growth and development period, and / or old age of poultry.

[0017] Beneficial effects

[0018] 1. Highly Efficient Antibody Preparation: This invention constructed five CaSR egg yolk antibodies using B-cell linear epitope prediction, achieving an antibody titer of up to 1:64000. Dietary supplementation with CaSR egg yolk antibodies promotes gene expression levels of key proteins involved in calcium absorption in the intestines and kidneys, improving intestinal calcium absorption and renal calcium reabsorption, thereby increasing calcium utilization efficiency and ultimately enhancing blood calcium levels and tibial mineralization.

[0019] 2. Innovative Targeting Mechanism: This invention is the first to propose a method for regulating intestinal calcium and phosphorus absorption efficiency by targeting the intestinal CaSR. By inhibiting the "calcium-sensing" function of the intestinal CaSR, the calcium absorption function of the poultry intestine is promoted, thereby improving the calcium absorption efficiency of poultry.

[0020] 3. Low-cost and high-efficiency production: This invention utilizes a simple, efficient, and extremely low-cost process for producing egg yolk antibodies from laying hens, making it suitable for large-scale production and application. Egg yolk antibodies offer advantages such as low cost, simple preparation, and high yield, and can precisely regulate target proteins in the intestine. Furthermore, egg yolk antibodies are not absorbed into the bloodstream, thus avoiding systemic burden on the body and exhibiting extremely high safety. In addition, since egg yolk antibodies primarily act on the intestine, they do not cause systemic side effects, making them suitable for long-term use.

[0021] 4. Wide Application Potential: This invention designs and prepares polypeptide yolk antibodies targeting CaSR to improve calcium and phosphorus absorption efficiency in poultry. This method not only has significant application potential in the field of poultry feed additives but may also provide new solutions for calcium and phosphorus absorption disorders in other animals and even humans. The core advantages of this technology lie in its innovative targeting mechanism, simple and low-cost production process, and high safety. Through the implementation of this patent, calcium utilization efficiency in poultry production can be significantly improved, calcium emissions into the environment can be reduced, and a win-win situation of economic and environmental benefits can be achieved. Attached Figure Description

[0022] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0023] Figure 1 This is a comparison diagram of the amino acid sequences of chicken and human CaSR in Example 1 of the present invention;

[0024] Figure 2 This is a three-dimensional crystal structure diagram of human CaSR from Embodiment 1 of the present invention;

[0025] Figure 3 This is a titer chart of CaSR egg yolk antibodies on day 35 after primary immunization in Example 1 of the present invention;

[0026] Figure 4 This is a graph showing the effect of immunization with CaSR polypeptide antigen on calcium and phosphorus levels in the blood of laying hens according to Example 1 of the present invention.

[0027] Figure 5 The figure shows the effect of the immunization with CaSR polypeptide antigen in Example 1 of the present invention on calcium and phosphorus excretion in laying hens; where (a) is the calcium excretion fraction of laying hens; (b) is the calcium excretion mass of laying hens; (c) is the phosphorus excretion fraction of laying hens; and (d) is the phosphorus excretion mass of laying hens.

[0028] Figure 6 The graph shows the effect of adding CaSR egg yolk antibody to the diet in Example 2 of this invention on blood calcium and phosphorus levels in broilers; where (a) represents the blood calcium level of broilers and (b) represents the blood phosphorus level of broilers.

[0029] Figure 7 This invention relates to the detection of antibody titers in mixed egg yolk powder and intestinal chyme IgY in Example 2 of this invention; wherein, (a) is the titer of different antibodies in the mixed egg yolk powder; (b) is the density of different antibodies detected in the duodenum; and (c) is the density of different antibodies detected in the ileum.

[0030] Figure 8 The effects of adding CaSR egg yolk antibody to the diet in Example 2 on blood hormones in broilers are shown in the following diagrams: (a) is a graph showing the relationship between the addition of egg yolk antibody and the 1,25(OH)2D3 in the blood of broilers; (b) is a graph showing the relationship between the addition of egg yolk antibody and the FGF-23 in the blood of broilers; and (c) is a graph showing the relationship between the addition of egg yolk antibody and the PTH in the blood of broilers.

[0031] Figure 9 The diagram shows the effect of dietary supplementation with CaSR egg yolk antibody on the expression of calcium transporter genes in the broiler intestine in Example 2 of this invention; wherein, (a) is a diagram showing the relationship between different antibodies and the expression of CALB1 and PMCA1b genes in the duodenum of broilers; (b) is a diagram showing the relationship between different antibodies and the expression of CALB1 genes in the jejunum of broilers; and (c) is a diagram showing the relationship between different antibodies and the expression of CALB1, PMCA1b, and NCX1 genes in the ileum. Detailed Implementation

[0032] To address the problem of low absorption and utilization rates of modified calcium in existing animal bodies, especially poultry, this invention proposes a CaSR egg yolk antibody that enhances intestinal calcium absorption, along with its preparation method and application.

[0033] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments. It should be noted that the materials, instruments and other items used in the present invention are all commercially available.

[0034] Example 1: Development and Application of CaSR Peptide Vaccine

[0035] In this embodiment, BepiPred 2.0 software was used to predict the antigenic epitopes of CaSR. Based on the three-dimensional structure of the CaSR protein, five epitope peptide sequences located on the protein surface were screened and conjugated to bovine gamma globulin (BgG) to create a peptide vaccine. This vaccine was then actively immunized in Hy-Line Brown laying hens during their peak immunization period to induce the production of antibodies against CaSR. The successful construction of the CaSR antibody was confirmed by detecting the CaSR antibody titer in egg yolks, the blood calcium level of the hens, and fecal calcium excretion.

[0036] 1. Peptide antigen design and vaccine preparation

[0037] 1.1 Selection of peptides

[0038] The amino acid sequences of chicken CaSR protein (NCBI sequence number: XP_416491.6) and human CaSR protein (code UniProtKB / Swiss-Prot:P41180.3) were obtained from the NCBI database (http: / / www.ncbi.nlm.nih.gov / ). See [link to NCBI database]. Figure 1 BLAST comparison of chicken and human CaSR sequences revealed high homology in their amino acid sequences. Therefore, the chicken CaSR amino acid sequence was imported into the BepiPred 2.0 online database for B-cell epitope prediction. Combined with the three-dimensional structure of the human CaSR protein, antigenic epitopes located on the surface of the chicken CaSR protein molecule were screened. Five antigenic epitope regions (8 amino acids in length) were initially screened and named YP1 (IAADDDYG), YP2 (SQYSDEEE), YP3 (ALKAGQIP), YP4 (TSVETPYM), and YP5 (GIIEGEPT). The peptide sequences were custom-synthesized by Nanjing Genscript Biotech Co., Ltd.

[0039] The amino acid sequences of the above polypeptides are denoted as follows:

[0040] The amino acid sequence of YP1 is SEQ ID NO.1, as follows: IAADDDYG;

[0041] The amino acid sequence of YP2 is SEQ ID NO.2, as follows: SQYSDEEE;

[0042] The amino acid sequence of YP3 is SEQ ID NO.3, as follows: ALKAGQIP;

[0043] The amino acid sequence of YP4 is SEQ ID NO.4, as follows: TSVETPYM;

[0044] The amino acid sequence of YP5 is SEQ ID NO.5, as follows: GIIEGEPT.

[0045] 1.2 Preparation of peptide vaccines

[0046] The above-mentioned peptide was conjugated with bovine gamma globulin (BgG) (peptide to carrier protein ratio of 1:1). The specific method is as follows: Weigh 4 mg of the synthesized CaSR peptide and dissolve it in 0.8 mL of ultrapure water; separately weigh 4 mg of BgG and dissolve it in 0.8 mL of 0.1 mol / L sodium acetate buffer. Mix the two solutions in a 10 mL centrifuge tube, slowly add 0.26 mL of 0.02 mol / L glutaraldehyde solution to the centrifuge tube, cap the tube, and place it on a shaker at room temperature for a slow reaction (60 rpm) for 3 hours; then add 10 mg of glycine and stop the reaction on the shaker for 1 hour. Next, transfer the mixture to a dialysis bag with a molecular weight of 6000–8000 for dialysis. One end of the dialysis bag was fixed with a dialysis clamp and placed in PBS (pH=7.6) buffer. Dialysis was performed at room temperature for 18 hours, with the PBS solution changed twice during the dialysis. The dialysate was collected the next morning, and the color, volume and clarity were recorded to obtain the peptide vaccine, which was stored at -20℃ for later use.

[0047] When immunizing laying hens, remove the prepared polypeptide vaccine from the refrigerator, add an appropriate amount of sterile physiological saline by volume, and mix with an equal volume of Freund's complete adjuvant (or Freund's incomplete adjuvant). Emulsify on ice using a tissue homogenizer for 8-10 minutes. After emulsification, transfer to a 5mL sterile syringe for later use. The standard for complete emulsification is that the vaccine does not disperse when placed in water.

[0048] 2 Immunity

[0049] 2.1 Immunized animals and diet composition

[0050] Thirty-six healthy Hy-Line Brown laying hens (30 weeks old) of similar weight were randomly divided into six groups of six. A 14-day pre-feeding period was implemented, during which the hens' condition, feed intake, and egg production were observed. After the pre-feeding period, the six groups were vaccinated with the control vaccine (CON) and CaSR polypeptide vaccines YP1, YP2, YP3, YP4, and YP5, respectively, and a 10-week rearing trial was conducted. The same diet was fed during both the pre-feeding and formal trial periods. The diet formulation was based on the 2004 Chinese Laying Hen Feeding Standard (NY / T33-2004), and the ingredient ratios and nutrient levels are shown in Table 1.

[0051] Table 1 Feed formulation and nutrient levels

[0052]

[0053] The premix provides the following per kilogram of feed: 8 mg copper (from copper sulfate), 60 mg iron (from ferrous sulfate), 80 mg zinc (from zinc sulfate), 60 mg manganese (from manganese sulfate), 0.3 mg selenium (from sodium selenite), 0.35 mg iodine (from calcium iodate), 8000 IU vitamin A, 1600 IU vitamin D3, 5 mg vitamin E, 0.5 mg vitamin K3, 0.8 mg thiamine, 2.5 mg riboflavin, 2.2 mg pantothenic acid, 20 mg nicotinamide, 3 mg pyridoxine, 0.1 mg biotin, and 0.25 mg folic acid.

[0054] 2.2 Feeding and Management

[0055] Individual cages (40cm long, 37cm deep, 33 / 38cm internal / external height) were used for rearing, with free access to drinking water and regular lighting. Lighting was provided from 5:30 AM to 9:30 PM daily, for 16 hours. Feed was provided twice daily, at 8:00 AM and 4:30 PM, approximately 70g each time (adjusted based on actual feed intake). Eggs were collected daily, weighed, and egg production recorded. Water lines were checked regularly, the coop was cleaned, and the coop was disinfected. The rearing trial lasted 10 weeks (32 to 42 weeks of age).

[0056] 2.3 Immunization Program

[0057] After the pre-feeding period, the initial immunization was administered, followed by booster immunizations on days 14 and 28. Immunization was carried out at 10:00 AM via intramuscular injection at four points on both legs and sides of the chest. The CON group received an adjuvant containing only saline solution, while the CaSR vaccine group received 600 μg of antigen per chicken. Freund's complete adjuvant was used for the initial immunization, while Freund's incomplete adjuvant was used for the booster immunization.

[0058] 3. Test Indicators and Measurement Methods

[0059] 3.1 Detection of CaSR egg yolk antibodies

[0060] Because antibody titers in the blood and egg yolks of laying hens peak one week after the second booster immunization and can remain high for more than three months, antibody titers in the egg yolks of each group of hens were tested on day 35 after the initial immunization.

[0061] (1) Pretreatment of egg yolk samples: On day 35 after the initial immunization, three eggs were collected from each group to detect the antibody titer in the egg yolk. The eggs were soaked in benzalkonium chloride solution for 1 minute, then wiped clean and air-dried. The eggs were carefully broken, the egg white was filtered out, and 2 mL of egg yolk liquid was drawn into a 10 mL centrifuge tube using a syringe without a needle. 2 mL of physiological saline was added, and the mixture was shaken well. Then, 4 mL of chloroform was added, and the mixture was shaken well again. The mixture was placed at room temperature for 2 hours, centrifuged at 3500 r / min for 20 minutes, and the supernatant was collected for enzyme-linked immunosorbent assay (ELISA) to detect the antibody titer.

[0062] (2) Preparation of coating antigen: The coating antigen was prepared by using ovalbumin (OVA) as the carrier protein to prepare a coating antigen with a concentration of 1 mg / mL and storing it at -80℃. The preparation method is based on existing technology.

[0063] (3) Egg yolk antibody ELISA titer detection, the specific operation steps are as follows:

[0064] 1) Coat a 96-well microplate with the coating antigen. Dilute the coating antigen to 10 μg / mL with coating buffer (pH=9.6), add 100 μL to each well, cap the plate, and incubate overnight at 4°C.

[0065] 2) Washing the plate. Shake off the coating solution from the wells of the microplate and gently pat it dry on a clean tissue. Then add 200 μL of 0.05% PBST washing buffer to each well. Let it stand for 4 minutes, pour out the washing buffer, and pat it dry on a tissue. Repeat 4 times.

[0066] 3) Blocking. Add 200 μL of 1% bovine serum albumin (BSA) blocking solution to each well and incubate at room temperature for 2 hours on a shaker at 80 rpm.

[0067] 4) Add samples. Dilute the yolk samples of the control group and the vaccine group with 1% BSA blocking solution at ratios of 1:500, 1000, 2000, 4000, 8000, 16000, 32000 and 64000. Add 50 μL of sample to each well, cover and incubate at room temperature on a shaker (80 r / min) for 1 hour. Use blocking solution as a blank control.

[0068] 5) Wash the plate. Repeat step 2, washing 4 times.

[0069] 6) Add enzyme-linked secondary antibody. Dilute the enzyme-labeled rabbit anti-chicken antibody 1:1000 with 1% BSA solution, add 50 μL to each well, cover the plate, gently tap the side of the plate to mix, and incubate at 37°C for 30 minutes.

[0070] 7) Wash the plate. Repeat step 2, washing 4 times, 4 minutes each time.

[0071] 8) Add substrate for colorimetric reaction. Add 125 μL of substrate solution to each well, cover and react in the dark for 3–5 minutes, then add 50 μL of 1.0 mol / L sulfuric acid to stop the reaction. Measure the OD value at 450 nm using a microplate reader. At the same dilution, a positive result is indicated when the OD value of the vaccine group egg yolk sample / the OD value of the control group egg yolk sample is ≥2.1, where the maximum dilution is the antibody titer. Conversely, a negative result is indicated when the OD value is lower.

[0072] 3.2 Blood calcium and phosphorus level measurement

[0073] Based on the calcium-phosphorus cycle rhythm observed in laying hens in our laboratory, blood samples were collected on the last day of the 10th week after initial immunization, immediately after the hens laid eggs. 5 mL of blood was collected from the wing vein using blood collection tubes containing an anticoagulant. The blood was allowed to stand for 1 hour until plasma separated. Then, the blood was centrifuged at 4°C and 3500 rpm for 15 minutes. The supernatant was collected, aliquoted into 1.5 mL centrifuge tubes, and stored at -80°C for later use. Calcium and phosphorus levels in the blood were measured using calcium and phosphorus assay kits, catalog numbers C004-2 and C006-3 (both purchased from Nanjing Jiancheng Bioengineering Institute).

[0074] 3.3 Determination of calcium and phosphorus levels in excrement

[0075] On the penultimate day after the experiment, excrement from each chicken was collected for 24 hours, from 8 PM to 8 PM the following day. The collected samples were immediately frozen at -20°C and then dried in a 65°C oven. After drying, the samples were cooled for 15–20 minutes and weighed. The samples were then pulverized and passed through a 40-mesh sieve, mixed thoroughly, and stored for later use. The methods for determining calcium and phosphorus content were based on "Feed Analysis and Feed Quality Testing Technology" (Zhang Liying, 3rd edition). The samples were ashed in a muffle furnace at 580°C for 5 hours, then removed and mixed with appropriate amounts of hydrochloric acid and nitric acid. The mixture was then digested and diluted to volume to prepare a decomposition solution for later use. Calcium content was determined using the EDTA rapid complexometric titration method, and phosphorus content in the excrement was determined using the molybdenum yellow colorimetric method.

[0076] 3.4 Data Statistics and Analysis

[0077] The experimental data were statistically analyzed using Office 2019. One-way ANOVA was performed using SPSS 22.0 (IBM, USA), and multiple comparisons between the CaSR vaccine group and the control group (CON) were conducted using the LSD method. A p-value < 0.05 was considered statistically significant and indicated by "#". The results are presented as mean ± standard error and plotted using GraphPad Prism 9 software.

[0078] 4. Experimental Results

[0079] 4.1 Prediction of CaSR epitope peptides

[0080] B-cell epitope analysis of the amino acid sequence of chicken CaSR was performed using BepiPred 2.0, combined with the protein structure and calcium-binding region of human CaSR (see...). Figure 2 Five polypeptide sequences located on the surface of CaSR molecules were screened out: YP1 (IAADDDYG), YP2 (SQYSDEEE), YP3 (ALKAGQIP), YP4 (TSVETPYM), and YP5 (GIIEGEPT).

[0081] 4.2 CaSR Egg Yolk Antibody Titer Detection

[0082] On day 35 post-primary immunization, antibody titers in egg yolks were measured using ELISA. Figure 3 The results showed that the yolk antibodies produced after immunizing laying hens with the five screened polypeptide antigens could specifically bind to the corresponding CaSR antigenic epitopes, and the antibody titers all reached 1:64000, indicating that the screened polypeptides possessed good immunogenicity. 4.3 Effects of immunization with CaSR polypeptide antigens on blood calcium and phosphorus levels in laying hens

[0083] Figure 4 This diagram illustrates the effect of immunization with the CaSR polypeptide antigen on blood calcium and phosphorus levels in laying hens in this embodiment. Figure 4 It was found that, compared with the CON group, immunization with CaSR polypeptide antigens YP1, YP2, and YP3 significantly increased the blood calcium level in laying hens (P<0.05). Simultaneously, immunization with CaSR polypeptide antigens YP1 and YP2 also significantly increased the blood phosphorus level in chickens (P<0.05). All vaccine groups showed statistically significant differences compared to the CON group (P<0.05), and the same applies below.

[0084] 4.4 Effects of immunization with CaSR polypeptide antigen on calcium and phosphorus excretion in laying hens

[0085] Figure 5 The graph shows the effect of immunization with CaSR polypeptide antigen on calcium and phosphorus excretion in laying hens. Figure 5 It can be seen that, compared with the CON group, immunization with the CaSR polypeptide antigen has a certain impact on the excretion of calcium and phosphorus in laying hens. From Figure 5 As shown in (a) and (b), immunization with YP5 significantly reduced the fractional calcium excretion in laying hens (P<0.05), and also reduced their total 24-hour calcium excretion (P<0.05). Immunization with YP2, YP3, and YP4 had no effect on the fractional calcium excretion, but significantly reduced the total 24-hour calcium excretion (P<0.05). Figure 5 (d) It can be seen that the immune YP2 significantly reduced the total excretion of fecal phosphorus in 24 hours (P<0.05).

[0086] 5 Discussions

[0087] The specificity of an antigen is not determined by the complete antigen molecule, but by specific sites on its surface, called antigenic determinants or epitopes. Antigens induce an immune response by binding to lymphocytes through epitopes, and also exert their immune effect by specifically binding to corresponding antibodies. Therefore, antigenic epitopes are widely used in antibody preparation, vaccine development, and drug development. With the development of bioinformatics and immunology, epitope prediction has become a research hotspot for peptide vaccines.

[0088] The five CaSR antigenic epitopes screened in this embodiment all exhibited good immunogenicity in their polypeptide sequences. After inducing CaSR antibody production in laying hens, they increased blood calcium levels and decreased fecal calcium excretion. These results indicate that CaSR antibodies have promising prospects for industrial applications.

[0089] Example 2: The regulatory effect of CaSR egg yolk antibody on calcium nutritional status in broilers

[0090] In this embodiment, the CaSR egg yolk antibody prepared in Example 1 was added to broiler feed. By observing indicators such as broiler production performance, tibia weight, blood calcium level, fecal calcium excretion, and intestinal calcium transport gene expression, its regulatory effect on broiler calcium nutritional status was explored.

[0091] 1. Materials and Methods

[0092] 1.1 Test Materials

[0093] The egg yolk powder containing CaSR antibody used in this experiment was prepared in Example 1. On day 35 after the initial immunization of laying hens, the antibody titer of CaSR in the egg yolk reached 1:64000. Therefore, eggs were collected from this point onwards, grouped together by category. After breaking the eggs and removing the egg white, the yolks were placed in a clean plastic dish and stirred evenly with disposable chopsticks. The mixture was then quick-frozen at -20°C, followed by freeze-drying in a vacuum freeze dryer to produce a mixed egg yolk antibody powder. This powder was then packaged in sealed bags, labeled, and stored at -20°C for later use.

[0094] 1.2 Grouping of experimental animals and composition of diet

[0095] Three hundred and sixty one-day-old Albert Irrigated (AA) broilers were randomly divided into six groups (six replicates per group, ten chickens per replicate). Each group was fed one of six egg yolk powders prepared in Example 1 (CON, YP1, YP2, YP3, YP4, and YP5), with a yolk powder addition of 0.5%. The feeding experiment lasted for 14 days. After the feeding experiment, three chickens were randomly selected from each replicate for a three-day metabolic test. Excrement was collected during this period for relevant indicator detection. The diet design for this experiment was based on the 2004 Chinese Broiler Feeding Standard (NY / T33-2004), and the diet composition and nutrient levels are shown in Table 2.

[0096] Table 2 Feed formulation and nutrient levels

[0097]

[0098] Among them, ① the premix provides the following per kilogram of complete feed: copper 8mg, iron 100mg, zinc 100mg, manganese 120mg, iodine 0.70mg, selenium 0.30mg, vitamin A 8000IU, vitamin D3 1000IU, vitamin K3 0.5mg, vitamin E 20mg, thiamine 2mg, riboflavin 8mg, pantothenic acid 10mg, niacin 35mg, pyridoxine 3.5mg, biotin 0.18mg, folic acid 0.55mg, and vitamin B12 0.01mg.

[0099] 1.3 Feeding and Management

[0100] This breeding experiment was conducted at the experimental teaching base of Northwest A&F University. One week before the experiment, the chicken coop was cleaned and thoroughly disinfected to ensure the temperature inside the coop was around 35℃, which was adjusted as needed based on the flock's condition. During the experiment, the chickens had free access to feed and water, and received 24 hours of light daily. The chicken coop was cleaned daily to ensure good ventilation. The flock's condition was monitored in real time, and the number and weight of dead chickens were recorded.

[0101] 1.4 Test Indicators and Detection Methods

[0102] At the end of the feeding trial, one broiler chicken was randomly selected from each replicate for slaughter and sampling. First, its live weight was recorded, and 4 mL of blood was collected from the wing vein using a disposable lancet. The chicken was then slaughtered, and the abdominal cavity was carefully opened. The intestines, kidneys, and left tibia were quickly removed. Each intestinal segment (duodenum, jejunum, and ileum) was divided into two sections. One section was used to collect chyme for detecting the distribution of CaSR yolk antibodies in the intestine. The other section was rinsed clean with sterile saline, longitudinally cut with surgical scissors, and the intestinal mucosa was gently scraped off using a clean glass slide and placed in cryovials. To prevent degradation of RNA and proteins, the kidneys, intestinal chyme, and mucosa were quickly transferred to liquid nitrogen for temporary storage. Upon returning to the laboratory, they were categorized according to labels and stored at -80°C. The attached material on the surface of the removed left tibia was removed, and then it was placed in a sealed plastic bag and stored at -20°C for later use.

[0103] 1.4.1 Growth performance

[0104] On day 14 of the feeding trial, the weight of the chickens and the weight of the remaining feed were recorded for each replicate. Feed intake, number of dead chickens and body weight during the trial period were recorded, and the average daily weight gain, average daily feed intake, feed conversion ratio and mortality rate during the trial period were calculated.

[0105] 1.4.2 Blood Biochemical Indicators

[0106] Blood samples were allowed to stand at room temperature for 1 hour. After plasma separation, the samples were centrifuged at 3500 rpm for 15 minutes at 4°C. The supernatant was collected, aliquoted into 1.5 mL centrifuge tubes, and stored at -80°C for later use. Blood calcium and phosphorus levels were measured using calcium and phosphorus assay kits (catalog numbers C004-2 and C006-3, respectively, purchased from Nanjing Jiancheng Biotechnology Institute), following the kit instructions. Blood hormones PTH, 1,25(OH)2D3, and FGF-23 were measured using ELISA kits (purchased from Shanghai Enzyme-Linked Biotechnology Co., Ltd.), following the instructions (catalog numbers ml00987411, ml00697414, and ml00321112, respectively).

[0107] 1.4.3 Determination of calcium and phosphorus content in feed, feces, and tibia

[0108] The feed sample or collected manure was dried in a 65℃ oven, pulverized through a 40-mesh sieve, mixed thoroughly, and packaged in a sealed plastic bag for later use. Referring to "Feed Analysis and Feed Quality Testing Technology" (Zhang Liying, 3rd Edition), 2g of sample was ashed at 580℃ for 5 hours, weighed, and then mixed with an appropriate amount of hydrochloric acid and a few drops of nitric acid. The mixture was then digested and diluted to volume to prepare the sample decomposition solution. The phosphorus content in the sample was determined using the molybdenum yellow colorimetric method, and the calcium content was determined using the EDTA rapid complexometric titration method.

[0109] For the determination of calcium and phosphorus in tibia, the sample needs to be dehydrated and defatted beforehand. The soft tissue on the surface of the tibia should be completely removed, and the sample should be dried in an oven at 105℃ for 12 hours, then dehydrated with anhydrous ethanol for 36 hours. After drying, it should be air-dried and defatted with anhydrous ether for another 36 hours. After air-drying in a fume hood, it should be dried in an oven at 105℃ for 4 hours, and the dry weight of the tibia should be recorded. The tibia should then be ashed in a muffle furnace (580℃) for 5 hours. Subsequent steps are the same as for the determination of calcium and phosphorus in feed and feces.

[0110] 1.4.4 Egg yolk powder IgY potency test

[0111] Take 2g of egg yolk powder into a 10mL centrifuge tube, add 4mL of physiological saline, shake vigorously to dissolve the egg yolk powder completely in the saline, then add 4mL of chloroform, mix thoroughly, and let stand at room temperature for 2 hours to allow the solution to separate into layers. Centrifuge at 3500r / min for 20 minutes, and collect the supernatant for ELISA antibody titer detection. The ELISA procedure is the same as in Example 1.

[0112] 1.4.5 Detection of IgY titer in chyme

[0113] Weigh 2g of the chyme, add 2mL of physiological saline, mix thoroughly, and incubate at 37℃ for 1 hour. After incubation, centrifuge at 8000r / min for 20 minutes, and collect the supernatant for ELISA detection. The ELISA procedure is the same as in Example 1.

[0114] 1.4.6 Relative expression levels of calcium and phosphorus transport-related proteins mRNA in the small intestine and kidney

[0115] (1) Total RNA extraction: Total RNA was extracted from tissues using TrizoL (purchased from Hunan Aikerui Biotechnology Co., Ltd.) to ensure no RNase contamination throughout the entire process. Specific steps are as follows:

[0116] 1) Remove the mucosal and kidney samples from different intestinal segments from the -80℃ freezer, and take 50-100 mg of the sample into a 1.5 mL RNase-free centrifuge tube. Add 1 mL of TRIzol, and homogenize the samples twice using a tissue homogenizer at 60 Hz for 10 s. After homogenization, let the samples stand for 5 min.

[0117] 2) Add 200 μL of chloroform to the above lysis buffer, shake vigorously, and let stand for 5 min to allow the solution to separate into layers. Centrifuge at 12000 r / min, 4℃ for 15 min.

[0118] 3) Take about 500 μL of the upper aqueous phase, add an equal volume of isopropanol, mix by inverting the container, and let stand for 10 min. Centrifuge at 12000 r / min, 4℃ for 10 min, and a precipitate will appear.

[0119] 4) Remove the supernatant, add 1 mL of 75% ethanol, and gently tap the precipitate. Centrifuge at 7500 rpm, 4°C for 5 minutes. Repeat twice.

[0120] 5) Remove the ethanol, invert the EP tube, let it air dry for about 20 minutes, and then add 80μL of DEPC water to dissolve it.

[0121] 6) Take 1 μL of RNA solution and quantify it using a Nano-Drop 2000 ultra-micro UV spectrophotometer. Determine the RNA quality based on OD 260 / OD280 and OD 260 / OD 230.

[0122] (2) RNA reverse transcription

[0123] The RNA concentration was diluted to 500 ng / μL, and reverse transcription was performed using EvoM-MLVRTPremix reagent (purchased from Hunan Aikerui Biotechnology Co., Ltd.) in a 10 μL reaction system.

[0124] (3) Real-time quantitative PCR

[0125] q-PCR was performed using SYBR Green reagent (purchased from Hunan Aikerui Biotechnology Co., Ltd.), with β-action as the internal reference gene, and 2... –△△Ct The relative expression levels of genes were calculated using a method. Primers were synthesized by Shanghai Sangon Biotech Co., Ltd., and the primer parameters are shown in Table 3.

[0126] Table 3. q-PCR Primer Information

[0127]

[0128] 1.4.7 Expression levels of calcium and phosphorus transport-related carrier proteins in the small intestine and kidney

[0129] The specific steps for tissue protein extraction and Western blotting are detailed in Appendix 2. The main procedures are: lysis with RIPA lysis buffer containing PMSF inhibitor for 30 minutes—protein concentration measurement—incubation at 100℃ for 10 minutes in a constant temperature metal bath—electrophoresis, membrane transfer—blocking—incubation with primary antibody—incubation with secondary antibody—development. The main reagents and consumables used include: BCA protein assay kit (Xi'an Hete Biotechnology Co., Ltd.), 30% AB acrylamide-bisacrylamide (Shanghai Sangon Biotech Co., Ltd.), 10% SDS (Shanghai Sangon Biotech Co., Ltd.), 1.5 mol / L TrisHCl (pH=8.8) separating gel (Shanghai Sangon Biotech Co., Ltd.), 1.0 mol / L TrisHCl (pH=6.8) stacking gel (Shanghai Sangon Biotech Co., Ltd.), Tween-20 (Shaanxi Xintai Biotechnology Development Co., Ltd.), BSA (Beijing Solarbio Technology Co., Ltd.), ECL luminescence solution (Bio-Rad, USA), and PVDF protein blot membrane (Roche). The antibodies used include: antibodies CALB1, TRPV5, NaPi-2a and NaPi-2b (purchased from Wuhan Ebotech Biotechnology Co., Ltd.), antibody β-actin (Beijing Kangwei Century Biotechnology Co., Ltd.), HRP-labeled goat anti-mouse (Beijing Biosen Biotechnology Co., Ltd.), and HRP-labeled goat anti-rabbit (Shanghai Diyi Biotechnology Co., Ltd.).

[0130] 2. Data Statistics and Analysis

[0131] The experimental data were statistically analyzed using Office 2019. One-way ANOVA was performed using SPSS 22.0, and multiple comparisons between CaSR and CON egg yolk powder were conducted using the LSD method. A p-value < 0.05 was considered statistically significant and indicated by "#". The experimental results are presented as mean ± standard error and plotted using GraphPad Prism 9 software.

[0132] 3. Experimental Results

[0133] 3.1 Effects of dietary supplementation with CaSR egg yolk antibody on broiler production performance

[0134] As shown in Table 4, compared with the CON group, the addition of five CaSR egg yolk antibodies (YP1, YP2, YP3, YP4 and YP5) to the diet had no significant effect on the body weight, daily feed intake, daily weight gain and feed conversion ratio of broilers aged 1-14 days (P>0.05).

[0135] Table 4. Effects of dietary supplementation with CaSR egg yolk antibody on broiler production performance.

[0136]

[0137] 3.2 Effects of dietary supplementation with CaSR egg yolk antibody on tibia quality in broilers

[0138] Table 5 shows that, compared with the CON group, dietary supplementation with CaSR egg yolk antibody YP2 significantly increased the ratio of tibia to body weight in broilers (P<0.05); supplementation with egg yolk antibodies YP3, YP4, and YP5 increased the ash calcium content of broiler tibia (P<0.05). Furthermore, supplementation with egg yolk antibodies YP3 and YP4 significantly increased the ash phosphorus content of broiler tibia (P<0.05), indicating that dietary supplementation with CaSR egg yolk antibodies can promote bone development and the deposition of calcium and phosphorus minerals.

[0139] Table 5. Effects of dietary supplementation with CaSR egg yolk antibody on tibia quality in broilers.

[0140]

[0141] Note: Compared with the CON group, the CaSR antibody group is always indicated by "#", which means there is a statistically significant difference (P<0.05). 3.3 Effect of dietary supplementation with CaSR egg yolk antibody on calcium and phosphorus excretion in broilers

[0142] Table 6 shows that, compared with the CON group, the addition of CaSR egg yolk antibodies YP2, YP3, and YP4 to the diet significantly reduced the fecal calcium excretion fraction of broilers (P<0.05), and the addition of egg yolk antibodies YP3 and YP4 increased the apparent digestibility of dietary calcium by 18.56% and 10.74%, respectively (P<0.05). Furthermore, the addition of CaSR egg yolk antibodies YP4 and YP5 reduced the fecal phosphorus excretion fraction of broilers (P<0.05), and the addition of antibodies YP3, YP4, and YP5 all significantly increased the apparent digestibility of dietary phosphorus (P<0.05).

[0143] Table 6. Effects of dietary supplementation with CaSR egg yolk antibody on calcium and phosphorus excretion in broilers.

[0144]

[0145] Note: Compared with the CON group, the CaSR antibody group is always compared with the CON group. "#" indicates a statistically significant difference, P<0.05. 3.4 Effect of dietary supplementation with CaSR egg yolk antibody on blood calcium and phosphorus levels in broilers

[0146] Depend on Figure 6 It was found that, compared with the CON group, dietary supplementation with CaSR egg yolk antibodies YP1, YP2, YP3, and YP4 significantly increased the blood calcium level of broilers (P<0.05). Figure 6 (a) ; The addition of egg yolk antibodies YP2, YP3 and YP4 significantly increased the blood phosphorus level in broilers (P<0.05). Figure 6 (b)). These results indicate that CaSR egg yolk antibodies can effectively increase blood calcium and phosphorus levels in broilers, thereby improving their calcium and phosphorus metabolism.

[0147] 3.5 Detection of antibody titer and intestinal digestive IgY in mixed egg yolk powder

[0148] Considering the combined effects of dietary supplementation with CaSR egg yolk antibodies on broiler tibia mass, excrement calcium content, and blood calcium and phosphorus levels, this study focused on egg yolk antibodies YP2, YP3, and YP4. Therefore, subsequent experimental indicators were measured around these three CaSR egg yolk antibodies.

[0149] First, the antibody titer of the mixed egg yolk powder was determined. Figure 7 (a) It can be seen that the mixed egg yolk powder containing CaSR egg yolk antibodies YP2, YP3, and YP4 still achieved an antibody titer of 1:32000. ELISA detection of antibodies against broiler small intestinal digesta... Figure 7 As shown in (b) and (c), the corresponding CaSR antibodies could be detected in the duodenum and jejunum, but not in the ileum (data not presented), with the OD value being higher in the duodenum.

[0150] 3.6 Effects of dietary supplementation with CaSR egg yolk antibody on broiler blood hormones

[0151] Figure 8 The effect of adding CaSR egg yolk antibody to the diet in this embodiment on blood hormones in broilers. Figure 8 (a) It can be seen that, compared with the CON group, dietary supplementation with CaSR egg yolk antibodies YP3 and YP4 significantly increased the 1,25(OH)2D3 level in broiler blood (P<0.05); Figure 8 (b) It can be seen that the addition of egg yolk antibody YP4 significantly increased the level of FGF-23 in the blood of broilers (P<0.05), while the addition of egg yolk antibodies YP2 and YP3 showed a trend of increasing blood FGF-23 (YP2, P=0.05; YP3, P=0.06); dietary addition of CaSR egg yolk antibody had no significant effect on blood PTH content (P>0.05). These results suggest that CaSR egg yolk antibody may affect calcium and phosphorus metabolism in broilers by regulating the levels of 1,25(OH)2D3 and FGF-23.

[0152] 3.7 Effects of dietary supplementation with CaSR egg yolk antibody on the expression of calcium-phosphorus transporter genes in the broiler intestine 3.7.1 Expression of intestinal calcium transporter genes

[0153] The gene levels of TRPV6, CALB1, PMCA1b, and NCX1, which are involved in the transcellular transport of calcium in the intestine, were measured. Transcriptional expression of TRPV6 was not detected in the duodenum, jejunum, or ileum in this study.

[0154] Figure 9The effect of dietary supplementation with CaSR egg yolk antibody on the expression of calcium transporter genes in the broiler intestine. Figure 9(a) shows that dietary supplementation with CaSR egg yolk antibody had no effect on the expression of CALB1 and PMCA1b genes in the duodenal region of broilers (P>0.05), but the supplementation with egg yolk antibodies YP2 and YP3 reduced the expression level of NCX1 gene (P<0.05). Figure 9 (b) It was found that dietary supplementation with egg yolk antibodies YP2 and YP3 both increased the gene expression of CALB1 in the jejunum (YP2, P < 0.05; YP3, P = 0.06), but had no significant effect on the gene expression of PMCA1b and NCX1 (P > 0.05). Figure 9 (c) It can be seen that the addition of CaSR egg yolk antibody to the diet had no significant effect on CALB1, PMCA1b and NCX1 in the ileum (P>0.05).

[0155] 3.7.2 Expression of intestinal phosphorus transporter genes

[0156] Intestinal phosphorus absorption is primarily mediated by sodium-phosphorus transporter type 2 (NaPi-2b), inorganic phosphorus transporter 1 (PiT-1), and 2 (PiT-2). Figures 3 to 5 As shown in (a), (b), and (c), compared with CON, dietary supplementation with CaSR egg yolk antibodies YP2 and YP3 significantly increased the gene expression levels of NaPi-2b in the duodenum, jejunum, and ileum (P<0.05), while supplementation with egg yolk antibody YP4 significantly decreased the gene expression level of NaPi-2b in the jejunum (P<0.05). Dietary supplementation with CaSR egg yolk antibodies had no effect on the PiT-1 gene expression in the duodenum, jejunum, and ileum (P>0.05); however, supplementation with egg yolk antibody YP3 significantly decreased the gene expression level of PiT-2 in the jejunum (P<0.05). These results indicate that CaSR egg yolk antibodies may affect the absorption and utilization of phosphorus in the broiler intestine by regulating the gene expression of NaPi-2b and PiT-2.

[0157] 4. Conclusion

[0158] This invention constructed five CaSR egg yolk antibodies using B-cell linear epitope prediction, with antibody titers reaching 1:64000. The recognized antigenic epitopes were: YP1, IAADDDYG; YP2, SQYSDEEE; YP3, ALKAGQIP; YP4, TSVETPYM; and YP5, GIIEGEPT. Dietary supplementation with CaSR egg yolk antibodies YP1, YP2, YP3, and YP4 significantly increased blood calcium levels in broilers (P<0.05) (Figure (a)); supplementation with egg yolk antibodies YP2, YP3, and YP4 significantly increased blood phosphorus levels in broilers (P<0.05) and improved calcium absorption efficiency and utilization in broilers.

[0159] Furthermore, the technical solutions and ideas of this invention are not limited to broiler chickens, but are also applicable to other breeds such as laying hens, and even to poultry such as turkeys, ducks, geese, quails, peacocks, emus, pheasants, and guinea fowl that have the same target protein in their intestines, and even to humans or animals.

[0160] 5. Scope of Patent Protection

[0161] The scope of protection of this invention is not limited to the above-described embodiments, but also includes any modifications, equivalent substitutions, or improvements made within the spirit and principles of this invention. All modifications, equivalent substitutions, or improvements made within the spirit and principles of this invention should be included within the scope of protection of this invention.

Claims

1. An antigenic epitope peptide, characterized in that, The polypeptide sequence of the antigenic epitope peptide is shown in SEQ ID NO.

3.

2. A vaccine, characterized in that, The vaccine includes the antigenic epitope peptide as described in claim 1.

3. An antibody, characterized in that, The antibody is an antibody produced in poultry after vaccination with the vaccine of claim 2, wherein the poultry is a laying hen, and the antibody is present in the yolk of the vaccinated poultry.

4. A poultry feed, characterized in that, The poultry feed includes egg yolk containing the antibody of claim 3, and the poultry is a chicken.

5. The poultry feed as described in claim 4, characterized in that, The added egg yolk has a mass fraction of 0.3%-0.8%.

6. The poultry feed as described in claim 5, characterized in that, The yolk added has a mass fraction of 0.5%.

7. The use of the antibody of claim 3 or the feed of claim 4 in promoting calcium or phosphorus absorption in an animal, wherein the animal is a chicken, and the use is for non-disease treatment purposes.

8. The application as described in claim 7, characterized in that, The application is the use of the antibody in promoting calcium absorption in the animal's intestines.

9. The application as described in claim 7, characterized in that, The application is for use during the egg-laying period, rapid growth and development period, and / or old age of chickens.

10. The application of the antigenic epitope peptide of claim 1 and the vaccine of claim 2 in promoting calcium absorption in animals, wherein the animal is a chicken and the application is for non-disease treatment purposes.

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