CaSR antigen epitope peptide, vaccine, antibody and application thereof

Abstract

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

Description

Technical Field

[0001] The present invention relates to the technical field of bioactive peptides and antibodies, and in particular to an antigenic epitope peptide, a vaccine, an antibody of a calcium-sensitive receptor (CaSR) and applications thereof in improving calcium (Ca) absorption efficiency. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Calcium is the most abundant mineral in the body and has a wide range of biological functions, including bone mineralization, nerve conduction and hormone secretion. Therefore, it is crucial to maintain the balance of calcium ion metabolism in the body. The age and physiological conditions of humans or animals will affect the absorption and utilization of calcium. For example, taking broilers as an example, with the development of modern breeding technology, the growth rate of broilers has been continuously improved. At the age of 42 days, the weight of broilers can reach 2.80kg. The rapid growth of broilers requires strong bone support. The mortality rate of broilers caused by abnormal bone mineralization is about 3%, accounting for 60% of all mortality rates reported in the broiler industry. In addition, in the later stage of laying hen breeding, the egg breakage rate caused by abnormal eggshell mineralization can reach 20%, causing huge economic losses to the industry. The above problems are closely related to the low efficiency of poultry in utilizing calcium in feed. It is reported that the apparent digestibility of calcium in feed for laying hens at 7 weeks, 32 weeks and 70 weeks of age is 38%, 49% and 44% respectively; while the apparent digestibility of calcium in feed for broilers is only about 53%. Therefore, how to improve the utilization efficiency of calcium in feed plays an important role.

[0004] In production, the calcium needs 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. Instead, it can easily lead to constipation and bloating, and may even interfere with the homeostasis of calcium and phosphorus metabolism or the absorption and utilization of other mineral elements, which has a negative impact on poultry health.

[0005] The calcium-sensing receptor (CaSR) is an important calcium ion sensor that can sense changes in extracellular calcium ion concentration and maintain the body's calcium homeostasis by regulating calcium absorption and excretion. CaSR exerts its physiological function by regulating parathyroid hormone (PTH) secretion, renal tubular calcium reabsorption, and insulin release. Studies have found that when the calcium ion concentration in the intestinal cavity increases, the calcium-sensing receptor (CaSR) distributed in the intestine can sense its changes and inhibit the intestinal absorption of calcium by feedback by downregulating the expression of calcium ion channels. This may be an important reason for the low efficiency of intestinal calcium utilization in humans or animals.

[0006] In the prior art, some studies have used calcimimetic agents to simulate the effects of calcium ions and enhance CaSR activity for the treatment of diseases such as hyperparathyroidism. Some studies have used calcilytic agents to inhibit the activity of CaSR and reduce its sensitivity to calcium ions for the correction of abnormal blood calcium levels. However, the cost of such drugs is high, and their large-scale use may cause a series of side effects such as increased liver burden and gastrointestinal discomfort. Due to the shortcomings of the prior art, there is an urgent need to develop a CaSR regulator that regulates the intestinal calcium and phosphorus absorption efficiency by targeting the intestinal CaSR, thereby improving the intestinal absorption and utilization efficiency of calcium. Summary of the invention

[0007] In order to solve the deficiencies of the prior art, the purpose of the present invention is to provide a CaSR antigen epitope peptide, vaccine, antibody and application thereof. The present invention aims to use polypeptide vaccine technology to construct egg yolk antibodies targeting intestinal CaSR, thereby improving the calcium absorption efficiency of poultry, providing a solution for efficient utilization of calcium elements in poultry, and achieving calcium conservation and emission reduction in production.

[0008] The first aspect of the present invention discloses an antigenic epitope peptide, wherein the antigenic epitope peptide is 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] The second aspect of the present invention discloses a vaccine, which comprises the above-mentioned antigen epitope peptide.

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

[0011] The fourth aspect of the present invention discloses a poultry feed, which comprises egg yolk containing the above-mentioned antibody.

[0012] In some embodiments, the poultry includes but is not limited to chicken, turkey, duck, goose, quail, peacock, emu, pheasant, guinea fowl.

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

[0014] The fifth aspect of the present invention discloses the use of the above-mentioned antigen epitope peptide, vaccine, antibody or feed in promoting calcium absorption or phosphorus absorption in animals.

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

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

[0017] Beneficial Effects

[0018] 1. Preparation of efficient antibodies: The present invention constructs 5 kinds of CaSR yolk antibodies through B cell linear epitope prediction, and the antibody titer can reach 1:64000. Adding CaSR yolk antibodies to feed is beneficial to promote the gene expression level of key calcium absorption proteins in the intestine and kidney, which can improve intestinal calcium absorption and kidney calcium reabsorption function, improve calcium utilization efficiency, and thus improve the body's blood calcium level and tibia mineralization quality.

[0019] 2. Innovative targeting mechanism: This invention proposes for the first time a method to regulate the intestinal calcium and phosphorus absorption efficiency by targeting the intestinal CaSR. By inhibiting the "calcium sensing" function of the intestinal CaSR, the intestinal calcium absorption function of poultry is promoted, thereby improving the calcium absorption efficiency of poultry.

[0020] 3. Low-cost and efficient production: The process of producing yolk antibodies by laying hens is simple, efficient and extremely low-cost, and is suitable for large-scale production and application. Yolk antibodies have the advantages of low cost, simple preparation, and high yield, and can accurately regulate target proteins in the intestine. Yolk antibodies will not be absorbed into the blood, so they will not cause systemic burden on the body and have extremely high safety. In addition, since yolk antibodies mainly act on the intestine, they will not cause systemic side effects and are suitable for long-term use.

[0021] 4. Potential for wide application: The present invention improves the calcium and phosphorus absorption efficiency of poultry by designing and preparing polypeptide yolk antibodies targeting CaSR. 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, the calcium utilization efficiency in poultry production can be significantly improved, the discharge of calcium in the environment can be reduced, and a win-win situation of economic and environmental benefits can be achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

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

[0024] Figure 2 The three-dimensional crystal structure of human CaSR in Example 1 of the present invention;

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

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

[0027] Figure 5 The figure is a graph showing the effect of the immune CaSR polypeptide antigen of Example 1 of the present invention on calcium and phosphorus excretion of laying hens; wherein (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 This is a graph showing the effect of adding CaSR yolk antibodies to the diet of Example 2 of the present invention on the blood calcium and phosphorus levels of broilers; wherein (a) is the blood calcium level of broilers, and (b) is the blood phosphorus level of broilers;

[0029] Figure 7 The antibody titer of the mixed egg yolk powder and the intestinal chyme IgY detection in Example 2 of the present 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; (c) is the density of different antibodies detected in the ileum;

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

[0031] Fig. 9 This is a graph showing the effect of adding CaSR yolk antibodies to the feed of Example 2 of the present invention on the gene expression of calcium transporters in the intestine of broiler chickens; wherein, (a) is a graph showing the relationship between different antibodies and the gene expression of CALB1 and PMCA1b in the duodenum of broiler chickens; (b) is a graph showing the relationship between different antibodies and the gene expression of CALB1 in the jejunum of broiler chickens; (c) is a graph showing the relationship between different antibodies and the gene expression of CALB1, PMCA1b and NCX1 in the ileum. DETAILED DESCRIPTION

[0032] In view of the problem that the absorption and utilization rate of calcium in existing animals, especially poultry, is low, the present invention provides a CaSR yolk antibody for improving intestinal calcium absorption and a preparation method and application thereof.

[0033] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments. It should be noted that the materials, instruments, etc. used in the present invention are all commonly available on the market.

[0034] Example 1: Development and application of CaSR polypeptide vaccine

[0035] In this example, BepiPred2.0 software was used to predict the antigenic epitope of CaSR, and combined with the three-dimensional structure of CaSR protein, 5 epitope peptide sequences located on the surface of the protein were screened, and they were coupled with bovine gamma globulin (BgG) to make peptide vaccines, which were used to actively immunize peak-stage Hy-Line brown-shell laying hens to induce the production of antibodies against CaSR. The successful construction of CaSR antibodies was confirmed by detecting the titer of CaSR antibodies in egg yolk, blood calcium levels of chickens, 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 (number UniProtKB / Swiss-Prot: P41180.3) were obtained through the NCBI (http: / / www.ncbi.nlm.nih.gov / ) database. Figure 1 . BLAST comparison of chicken and human CaSR sequences revealed that the amino acid sequences of the two were highly homologous. Therefore, the chicken CaSR amino acid sequence was imported into the BepiPred2.0 online website for B cell epitope prediction, and the antigenic epitopes located on the surface of the chicken CaSR protein molecule were screened in combination with the three-dimensional structure of the human CaSR protein. Five antigenic epitope regions (8 amino acids in length) were preliminarily screened and named YP1 (IAADDDYG), YP2 (SQYSDEEE), YP3 (ALKAGQIP), YP4 (TSVETPYM) and YP5 (GIIEGEPT). The polypeptide sequences were custom-synthesized by Nanjing GenScript Biotechnology Co., Ltd.

[0039] The amino acid sequences of the above polypeptides are respectively recorded as:

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

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

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

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

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

[0045] 1.2 Preparation of peptide vaccines

[0046] The above-mentioned polypeptide was coupled with bovine immunoglobulin gamma (BgG) (the ratio of polypeptide to carrier protein was 1:1). The specific method is as follows: weigh 4 mg of the above-mentioned synthesized CaSR polypeptide and dissolve it in 0.8 mL of ultrapure water; weigh another 4 mg of BgG and dissolve it in 0.8 mL of 0.1 moL / L sodium acetate buffer. Mix the above two solutions in a 10 mL centrifuge tube, slowly add 0.26 mL of 0.02 moL / L glutaraldehyde solution to the centrifuge tube, cover the lid, place it on a shaker at room temperature, and slowly react (60 r / min) for 3 hours; then add 10 mg of glycine and stop the reaction on a shaker for 1 hour. Next, transfer the mixed solution 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. The PBS solution was replaced twice during the dialysis. The dialysate was collected the next morning, and the color, volume and clarity were recorded to obtain the polypeptide vaccine, which was stored at -20°C for future use.

[0047] When immunizing laying hens, take out the prepared polypeptide vaccine from the refrigerator, add an appropriate amount of sterile saline by volume, and mix with an equal volume of Freund's complete adjuvant (or Freund's incomplete adjuvant), and emulsify it on ice using a tissue disruptor for 8 to 10 minutes. After emulsification is completed, transfer it to a 5mL sterile syringe for use. The standard for 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 Hailan brown-shell laying hens (30 weeks old) with similar body weight were selected and randomly divided into 6 groups, with 6 chickens in each group. The chickens were pre-fed for 14 days, during which the chickens' condition, feed intake and egg production rate were observed. After the pre-feeding period, the 6 groups of chickens were vaccinated with control vaccine (CON) and CaSR peptide vaccines YP1, YP2, YP3, YP4 and YP5, respectively, and a 10-week breeding experiment was carried out. The same feed was fed during the pre-feeding period and the formal trial period. The feed formula was formulated with reference to the 2004 Chinese Laying Hen Feeding Standard (NY / T33-2004). The raw material proportions and nutritional levels are shown in Table 1.

[0051] Table 1 Feed formula and nutritional level

[0052]

[0053] Among them, ① the premix provides per kilogram of feed: copper (derived from copper sulfate) 8 mg, iron (derived from ferrous sulfate) 60 mg, zinc (derived from zinc sulfate) 80 mg, manganese (derived from manganese sulfate) 60 mg, selenium (derived from sodium selenite) 0.3 mg, iodine (derived from calcium iodate) 0.35 mg, vitamin A 8000 IU, vitamin D3 1600 IU, vitamin E 5 mg, vitamin K3 0.5 mg, thiamine 0.8 mg, riboflavin 2.5 mg, pantothenic acid 2.2 mg, niacinamide 20 mg, pyridoxine 3 mg, biotin 0.1 mg, and folic acid 0.25 mg.

[0054] 2.2 Feeding and management

[0055] The chickens were raised in single cages (40 cm long, 37 cm deep, 33 / 38 cm high inside and outside), with free drinking water and regular lighting. The lighting time was 5:30 am to 9:30 pm, with a lighting period of 16 hours. Feed was added once every morning at 8:00 am and 4:30 pm, with each feed adding about 70 g (depending on the actual feeding situation). Eggs were collected daily, weighed and egg production was recorded, and the water line was checked regularly, and the chicken coop was cleaned and disinfected. The breeding experiment lasted for 10 weeks (32 to 42 weeks of age).

[0056] 2.3 Immunization Procedure

[0057] After the pre-feeding period, the first immunization was carried out, and booster immunization was carried out on the 14th and 28th days after the first immunization. The immunization time was 10:00 in the morning, and 4 intramuscular injections were performed on both sides of the legs and chest of the chickens. The CON group was injected with an adjuvant containing only saline, and the immunization dose of the CaSR vaccine group was 600μg of antigen per chicken. Freund's complete adjuvant was used for the first immunization; Freund's incomplete adjuvant was used for the booster immunization.

[0058] 3 Test indicators and measurement methods

[0059] 3.1 Detection of CaSR yolk antibodies

[0060] Since the antibody titer in the blood and yolk of laying hens reaches a peak one week after the second booster immunization and can last for more than 3 months, the antibody titer in the yolk of each group of chickens was tested on the 35th day after the initial immunization.

[0061] (1) Pretreatment of egg yolk samples: On the 35th day 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 a chlorhexidine solution for 1 minute, and the surface was wiped clean after being taken out and 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 with a syringe without a needle. 2 mL of physiological saline was added, and after shaking evenly, 4 mL of chloroform was added, and the mixture was shaken again. The mixture was placed at room temperature for 2 hours, and centrifuged at 3500 r / min for 20 minutes. The supernatant was drawn for enzyme-linked immunosorbent assay (ELISA) to detect the antibody titer.

[0062] (2) Preparation of coating antigen: The coating antigen was prepared using chicken ovalbumin (OVA) as a carrier protein to prepare a coating antigen with a concentration of 1 mg / mL and stored at -80°C. The preparation method thereof refers to the prior art.

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

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

[0065] 2) Wash the plate. Shake out the coating solution in the wells of the ELISA plate, tap gently on clean toilet paper, and then add 200 μL of 0.05% PBST washing solution to each well. Let it stand for 4 minutes, pour out the washing solution and tap dry on toilet paper, 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 egg yolk samples of the control group and vaccine group in advance with 1% BSA blocking solution at 1:500, 1000, 2000, 4000, 8000, 16000, 32000 and 64000, add 50 μL of sample to each well, cover the lid, incubate on a shaker (80 r / min) at room temperature for 1 hour, and use the blocking solution as a blank control.

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

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

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

[0071] 8) Add substrate for color development reaction. Add 125 μL substrate solution to each well, cover and protect from light for 3 to 5 minutes, and add 50 μL 1.0 mol / L sulfuric acid stop solution. Detect its OD value at a wavelength of 450nm on an ELISA reader. At the same dilution multiple, when the OD value of the vaccine group egg yolk sample / the OD value of the control group egg yolk sample is ≥2.1, it indicates a positive result, where the maximum dilution multiple is the antibody titer. Otherwise, the result is negative.

[0072] 3.2 Blood calcium and phosphorus levels

[0073] According to the calcium-phosphorus cycle rhythm of laying hens discovered by our laboratory, blood samples were collected on the last day of the 10th week after primary immunization, when the chickens just laid eggs. 5 mL of blood was collected from the wing vein using a blood collection tube containing an anticoagulant, and allowed to stand for 1 hour. When the plasma was precipitated, it was centrifuged at 4°C and 3500r / min for 15 minutes, the supernatant was aspirated, and dispensed into 1.5 mL centrifuge tubes, and stored at -80°C for later use. The calcium and phosphorus content in the blood were determined using calcium-phosphorus determination kits, with the item numbers C004-2 and C006-3 (both purchased from Nanjing Jiancheng Bioengineering Research Institute).

[0074] 3.3 Determination of excreta calcium and phosphorus levels

[0075] On the penultimate day after the end of the experiment, the excrement of each chicken for 24 hours was collected from 8 pm to 8 pm the next day. The collected samples were immediately frozen at -20℃ and then dried in a 65℃ oven. After cooling for 15 to 20 minutes after drying, the air-dried weight of the sample was weighed. The sample was crushed by a grinder and passed through a 40-mesh sieve, mixed evenly and stored for later use. The determination method of calcium and phosphorus content refers to "Feed Analysis and Feed Quality Testing Technology Determination" (Zhang Liying, third edition). The sample was ashed at 580℃ in a muffle furnace for 5 hours, and an appropriate amount of hydrochloric acid and nitric acid were added after removal. After digestion and constant volume, the decomposition solution was prepared for use. The calcium content was determined by EDTA rapid complexometric titration, and the phosphorus content of the excrement was determined by molybdenum yellow colorimetry.

[0076] 3.4 Data Statistics and Analysis

[0077] Office 2019 was used to analyze the experimental data, SPSS 22.0 (IBM, USA) was used for one-way analysis of variance, and the LSD method was used for multiple comparisons between the CaSR vaccine group and the control group CON. P < 0.05 indicated a statistically significant difference, indicated by "#". The experimental results were presented as mean ± standard error, and GraphPad Prism 9 software was used for plotting.

[0078] 4 Test results

[0079] 4.1 Prediction of CaSR epitope peptide results

[0080] The amino acid sequence of chicken CaSR was analyzed by BepiPred2.0 for B cell epitope, combined with the protein structure and calcium ion binding region of human CaSR (see Figure 2 ) and screened out five polypeptide sequences located on the surface of the CaSR molecule, namely YP1 (IAADDDYG), YP2 (SQYSDEEE), YP3 (ALKAGQIP), YP4 (TSVETPYM) and YP5 (GIIEGEPT).

[0081] 4.2 CaSR yolk antibody titer detection

[0082] On the 35th day after the initial immunization, the antibody titer in the egg yolk was tested by ELISA. Figure 3 The results showed that the yolk antibodies produced by immunizing laying hens with the five selected polypeptide antigens could specifically bind to the corresponding CaSR antigen epitopes, and the antibody titer reached 1:64000, indicating that the selected polypeptides have good immunogenicity.

[0083] Figure 4 This is a graph showing the effect of CaSR polypeptide antigen immunization on blood calcium and phosphorus in laying hens in this example. Figure 4 It can be seen that compared with the CON group, the blood calcium level of laying hens was significantly increased after immunization with CaSR polypeptide antigens YP1, YP2 and YP3 (P<0.05). At the same time, the blood phosphorus level of chickens was also significantly increased after immunization with CaSR polypeptide antigens YP1 and YP2 (P<0.05). Among them, all vaccine groups were compared with CON, P<0.05, indicating significant differences, the same below.

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

[0085] Figure 5 The effect of immunization with CaSR polypeptide antigen on calcium and phosphorus excretion in laying hens is shown in Figure 2. Figure 5 It can be seen that compared with the CON group, immunization with CaSR polypeptide antigen had a certain effect on the excretion of calcium and phosphorus in laying hens. Figure 5 (a) and (b) show that immunization with YP5 significantly reduced the calcium excretion fraction of laying hens (P<0.05), and also reduced the total calcium excretion in 24 hours (P<0.05). Immunization with YP2, YP3 and YP4 had no effect on the calcium excretion fraction, but significantly reduced the total calcium excretion in 24 hours (P<0.05). Figure 5 (d) It can be seen that immunization with 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 some specific sites on the surface of the molecule. These specific sites are called antigenic determinants, also known as antigenic epitopes. Antigens induce immune responses by binding to lymphocytes through epitopes. In addition, they also exert immune effects by specifically binding to corresponding antibodies through epitopes. 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 polypeptide sequences of the five CaSR antigen epitopes screened in this example all have good immunogenicity. After inducing laying hens to produce CaSR antibodies, the blood calcium level of laying hens was increased, and the excretion of fecal calcium was reduced. These results show that CaSR antibodies have good prospects in industrial applications.

[0089] Example 2: Effect of CaSR yolk antibody on calcium nutritional status of broiler chickens

[0090] In this example, the CaSR yolk antibody prepared in Example 1 was added to the broiler diet, and its regulatory effect on the calcium nutritional status of broilers was explored by observing indicators such as broiler production performance, tibia quality, blood calcium level, fecal calcium excretion, and intestinal calcium transport gene expression.

[0091] 1 Materials and Methods

[0092] 1.1 Test materials

[0093] The egg yolk powder containing CaSR antibodies used in this experiment was prepared by Example 1. On the 35th day after the initial immunization of laying hens, the antibody titer of CaSR detected in the egg yolk could reach 1:64000. Therefore, eggs were collected from this time, and all eggs were collected together in groups. After breaking the eggs and removing the egg white, the egg yolk was placed in a clean plastic dish, stirred evenly with disposable chopsticks, placed in a -20°C refrigerator for quick freezing, and then transferred to a vacuum freeze dryer for freeze drying. The mixed egg yolk antibody powder was placed in a sealed bag, sealed and labeled, and stored at -20°C for use.

[0094] 1.2 Experimental animal groups and diet composition

[0095] The experimental animals were 360 ​​1-day-old AA broilers, which were randomly divided into 6 groups (6 replicates per group, 10 chickens per replicate), and fed with 6 kinds of egg yolk powder (i.e., CON, YP1, YP2, YP3, YP4, and YP5) prepared in Example 1, respectively, with the addition of 0.5% egg yolk powder, and the feeding experiment lasted for 14 days. After the feeding experiment, 3 chickens were randomly selected from each replicate for a 3-day metabolic experiment, and the excrement of the chickens during the collection period was used for relevant index detection. The feed formula design of this experiment refers to the 2004 Chinese broiler feeding standard (NY / T33-2004), and the feed composition and nutritional level are shown in Table 2.

[0096] Table 2 Feed formula and nutritional level

[0097]

[0098] Among them, ① the premix provides, 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, vitamin B12 0.01mg.

[0099] 1.3 Feeding and management

[0100] This breeding experiment was conducted at the experimental teaching base of Northwest Agriculture and Forestry University. One week before the experiment, the chicken house was cleaned and fully disinfected to ensure that the temperature of the chickens was around 35°C. The temperature was adjusted appropriately according to the status of the chickens in the later stage. During the experiment, the chickens were free to eat and drink, and the light was on for 24 hours a day. The chicken house was cleaned daily to ensure good ventilation. The status of the chickens was observed 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 experiment, one broiler 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 blood collection needle. Then the chicken was slaughtered, the broiler's abdominal cavity was carefully opened, and the intestinal segment, kidney and left tibia of the chicken were quickly removed. Each intestinal segment (duodenum, jejunum and ileum) was divided into two sections. One section collected chyme to detect the distribution of CaSR yolk antibodies in the intestinal segment, and the other section was rinsed with sterile saline, cut longitudinally along the intestinal segment with surgical scissors, and the intestinal mucosa was gently scraped with a clean slide and placed in a cryopreservation tube. To prevent RNA and protein degradation, the kidney, intestinal chyme and mucosa were quickly transferred to liquid nitrogen for temporary storage. After returning to the laboratory, they were classified according to the labels and stored in a -80℃ refrigerator. The surface attachments of the removed left tibia were shaved off, then put into a plastic bag and stored at -20℃ for later use.

[0103] 1.4.1 Growth performance

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

[0105] 1.4.2 Blood biochemical indicators

[0106] The collected blood was left to stand at room temperature for 1 hour. After the plasma was separated, it was centrifuged at 3500r / min for 15 minutes at 4°C. The supernatant was aspirated and dispensed into 1.5mL centrifuge tubes and stored at -80°C for later use. The blood calcium and phosphorus contents were determined using calcium and phosphorus determination kits, with the item numbers C004-2 and C006-3 (both purchased from Nanjing Jiancheng Bioengineering Institute), and the test was performed according to the instructions of the kit. The blood hormones PTH, 1,25(OH)2D3 and FGF-23 were determined using ELISA kits (purchased from Shanghai Enzyme-Linked Biotechnology Co., Ltd.). The specific operation steps refer to the instructions, and the item numbers are 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 feces is dried in an oven at 65°C, crushed by a grinder and passed through a 40-mesh sieve, mixed evenly, and packed into a plastic bag for later use. Referring to "Feed Analysis and Feed Quality Testing Technology Determination" (Zhang Liying, third edition), 2g of sample is ashed at 580°C for 5 hours, weighed, and an appropriate amount of hydrochloric acid and a few drops of nitric acid are added. After digestion and constant volume, the sample decomposition solution is prepared. The phosphorus content in the sample is determined by the molybdenum yellow colorimetric method, and the calcium content in the sample is determined by the EDTA rapid complexometric titration method.

[0109] For the determination of calcium and phosphorus in tibia, the sample must be dehydrated and defatted in advance. Remove the soft tissue on the surface of the tibia, dry it in an oven at 105°C for 12 hours, dehydrate it with anhydrous ethanol for 36 hours, take it out and dry it, continue to degrease it with anhydrous ether for 36 hours, take it out and dry it in a fume hood, and then put it in an oven at 105°C for 4 hours, and record the dry weight of the tibia after drying. Then put the tibia in a muffle furnace (580°C) for ash treatment for 5 hours, and the subsequent steps are the same as the determination of calcium and phosphorus in feed and feces.

[0110] 1.4.4 Egg yolk powder IgY titer detection

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

[0112] 1.4.5 IgY titer detection in chyme

[0113] Weigh 2 g of chyme, add 2 mL of physiological saline, mix the two thoroughly, incubate at 37°C for 1 hour, centrifuge at 8000 r / min for 20 minutes, and take out the supernatant for ELISA detection. The ELISA operation steps are the same as those in Example 1.

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

[0115] (1) Total RNA extraction: TrizoL (purchased from Hunan Aikerui Biotechnology Co., Ltd.) was used to extract total RNA from tissues to ensure that there was no RNase contamination during the entire operation. The specific steps are as follows:

[0116] 1) Take out the intestinal mucosa and kidney samples from different segments from the -80℃ freezer, take 50-100 mg of the sample and put it into a 1.5mL RNase-free centrifuge tube. Add 1mL of TRIzol and use a tissue homogenizer at 60 Hz for 10s, perform twice, and let it stand for 5 minutes after taking it out.

[0117] 2) Add 200 μL of chloroform to the above lysate, shake vigorously, let stand for 5 minutes to allow the solution to separate into layers, and centrifuge at 12000 r / min, 4°C for 15 minutes.

[0118] 3) Take about 500 μL of the upper aqueous phase, add an equal volume of isopropanol, mix by inversion, let stand for 10 minutes, and centrifuge at 12000 r / min, 4°C for 10 minutes until a precipitate appears.

[0119] 4) Remove the supernatant, add 1 mL of 75% ethanol, gently flick the precipitate, and centrifuge at 7500 r / min, 4°C for 5 minutes. Repeat twice.

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

[0121] 6) Take 1 μL of RNA solution and use Nano-Drop 2000 ultra-micro UV spectrophotometer for quantification, and judge the RNA quality according to OD 260 / OD 280 and OD 260 / OD 230.

[0122] (2) RNA reverse transcription

[0123] The RNA concentration was diluted to 500 ng / μL, and 10 μL was used as the reaction system. EvoM-MLVRTPremix reagent (purchased from Hunan Aikerui Bioengineering Co., Ltd.) was used for reverse transcription.

[0124] (3) Real-time fluorescence quantitative PCR

[0125] SYBRGreen reagent (purchased from Hunan Aikerui Biotechnology Co., Ltd.) was used for q-PCR, with β-action as the internal reference gene and 2 –△△Ct The relative expression of genes was calculated by the method. The primers were synthesized by Shanghai Shenggong Biotechnology Co., Ltd., and the primer parameters are shown in Table 3.

[0126] Table 3q-PCR primer information

[0127]

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

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

[0130] 2. Data Statistics and Analysis

[0131] Office 2019 was used to statistically analyze the experimental data, SPSS 22.0 one-way analysis of variance was used, and the LSD method was used for multiple comparisons between CaSR egg yolk powder and CON egg yolk powder. P < 0.05 indicated a statistically significant difference, indicated by "#". The experimental results were presented as mean ± standard error, and GraphPad Prism 9 software was used to draw the graph.

[0132] 3 Test results

[0133] 3.1 Effect of dietary CaSR yolk antibodies on broiler performance

[0134] As can be seen from Table 4, compared with the CON group, the addition of five CaSR 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-to-meat ratio of broilers at 1-14 days of age (P>0.05).

[0135] Table 4 Effects of dietary CaSR yolk antibodies on broiler production performance

[0136]

[0137] 3.2 Effect of dietary CaSR yolk antibody on tibia quality in broiler chickens

[0138] As shown in Table 5, compared with the CON group, the addition of CaSR yolk antibody YP2 to the diet significantly increased the ratio of tibia to body weight of broilers (P<0.05); the addition of yolk antibodies YP3, YP4 and YP5 increased the ash calcium content of tibia of broilers (P<0.05). In addition, the addition of yolk antibodies YP3 and YP4 significantly increased the ash phosphorus content of tibia of broilers (P<0.05), indicating that the addition of CaSR yolk antibodies to the diet can promote bone development and the deposition of mineral calcium and phosphorus.

[0139] Table 5 Effect of dietary CaSR yolk antibody on tibia quality of broiler chickens

[0140]

[0141] Note: The CaSR antibody groups were compared with the CON group. “#” indicates statistical difference compared with CON, P<0.05. 3.3 Effect of dietary CaSR yolk antibody on calcium and phosphorus excretion in broilers

[0142] As shown in Table 6, compared with the CON group, the addition of CaSR 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 yolk antibodies YP3 and YP4 increased the apparent digestibility of dietary calcium by 18.56% and 10.74%, respectively (P<0.05). In addition, the addition of CaSR 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 significantly increased the apparent digestibility of dietary phosphorus (P<0.05).

[0143] Table 6 Effects of dietary CaSR yolk antibodies on calcium and phosphorus excretion in broilers

[0144]

[0145] Note: The CaSR antibody group was compared with the CON group. “#” indicates statistical difference with CON, P<0.05. 3.4 Effect of dietary CaSR yolk antibody on blood calcium and phosphorus levels in broilers

[0146] Depend on Figure 6 It can be seen that compared with the CON group, the addition of CaSR yolk antibodies YP1, YP2, YP3 and YP4 to the diet 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 of broilers (P<0.05) ( Figure 6 (b)). These results indicate that CaSR yolk antibodies can effectively increase blood calcium and phosphorus levels in broiler chickens, thereby improving their calcium and phosphorus metabolism.

[0147] 3.5 Mixed egg yolk powder antibody titer and intestinal chyme IgY detection

[0148] Taking into account the effects of dietary CaSR yolk antibodies on tibia quality, excrement calcium content, and blood calcium and phosphorus content in broiler chickens, this study focused on yolk antibodies YP2, YP3, and YP4. Therefore, subsequent test indicators were measured around these three CaSR yolk antibodies.

[0149] First, the antibody titer of the mixed egg yolk powder was determined. Figure 7 (a) It can be seen that the antibody titer of the mixed egg yolk powder of CaSR yolk antibodies YP2, YP3 and YP4 still reached 1:32000. ELISA test of broiler small intestinal chyme antibodies Figure 7 (b) and (c) show that the corresponding CaSR antibodies can be detected in the duodenum and jejunum, but not in the ileum (data not shown), among which the OD value of the duodenum is higher.

[0150] 3.6 Effects of dietary CaSR yolk antibodies on blood hormones in broilers

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

[0152] 3.7 Effect of dietary CaSR yolk antibody on intestinal calcium-phosphorus transporter gene expression in broiler chickens 3.7.1 Intestinal calcium transporter gene expression

[0153] The gene levels of intestinal calcium transcellular transporters TRPV6, CALB1, PMCA1b and NCX1 were measured. In this study, no transcriptional expression of TRPV6 was detected in the duodenum, jejunum and ileum.

[0154] Fig. 9The figure shows the effect of adding CaSR yolk antibodies to the diet on the expression of calcium transporter genes in the intestine of broiler chickens. As shown in Figure 9(a), adding CaSR yolk antibodies to the diet had no effect on the gene expression of CALB1 and PMCA1b in the duodenum of broiler chickens (P>0.05), but adding yolk antibodies YP2 and YP3 reduced the gene expression of NCX1 (P<0.05). Fig. 9 (b) It can be seen that the addition of yolk antibodies YP2 and YP3 to the diet 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). Fig. 9 (c) It can be seen that the addition of CaSR yolk antibodies to the diet had no significant effect on ileal CALB1, PMCA1b and NCX1 (P>0.05).

[0155] 3.7.2 Intestinal phosphorus transporter gene expression

[0156] Intestinal absorption of phosphorus is mainly mediated by sodium phosphate transporter type 2 (NaPi-2b), inorganic phosphate transporter 1 (PiT-1) and 2 (PiT-2). Figures 3 to 5 (a), (b) and (c) show that compared with CON, the addition of CaSR yolk antibodies YP2 and YP3 to the diet significantly increased the gene expression of NaPi-2b in the duodenum, jejunum and ileum (P<0.05), while the addition of yolk antibody YP4 significantly reduced the gene expression of NaPi-2b in the jejunum (P<0.05); the addition of CaSR yolk antibodies to the diet had no effect on the gene expression of PiT-1 in the duodenum, jejunum and ileum (P>0.05); while the addition of yolk antibody YP3 significantly reduced the gene expression of PiT-2 in the jejunum (P<0.05). These results indicate that CaSR yolk antibodies may affect the absorption and utilization of phosphorus in the intestine of broilers by regulating the gene expression of NaPi-2b and PiT-2.

[0157] 4 Conclusion

[0158] The present invention constructs 5 kinds of CaSR egg yolk antibodies through B cell linear epitope prediction, and the antibody titer can reach 1:64000. The recognized antigen epitopes are: YP1, IAADDDYG; YP2, SQYSDEEE; YP3, ALKAGQIP; YP4, TSVETPYM; YP5, GIIEGEPT. The addition of CaSR egg yolk antibodies YP1, YP2, YP3 and YP4 to the diet significantly increases the blood calcium level of broilers (P<0.05) (Figure (a)); the addition of egg yolk antibodies YP2, YP3 and YP4 significantly increases the blood phosphorus level of broilers (P<0.05), and improves the absorption efficiency and utilization rate of calcium in broilers.

[0159] In addition, the technical solutions and ideas of the present invention are not limited to broilers, but are also applicable to other breeds such as laying hens, and even to poultry with the same target protein in the intestines such as turkeys, ducks, geese, quails, peacocks, emus, pheasants, guinea fowls, and even humans or animals.

[0160] 5 Scope of Patent Protection

[0161] The protection scope of the present invention is not limited to the above-mentioned implementation cases, but also includes any modification, equivalent substitution, improvement, etc. made within the spirit and principle of the present invention. Any modification, equivalent substitution, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An antigen epitope peptide, characterized in that: The antigen epitope peptide is selected from one or more of the following polypeptide sequences, and the polypeptide sequence is SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.

5.

2. A vaccine, characterized in that: The vaccine comprises the antigen epitope peptide according to claim 1.

3. An antibody, characterized in that The antibodies are antibodies produced after poultry is vaccinated with the vaccine according to claim 2; preferably, the antibodies are present in the yolk of the vaccinated poultry.

4. A poultry feed, characterized in that: The poultry feed comprises egg yolk containing the antibody according to claim 3.

5. The poultry feed according to claim 4, characterized in that The poultry include, but are not limited to, chickens, turkeys, ducks, geese, quails, peacocks, emus, pheasants, and guinea fowls.

6. The poultry feed according to claim 4, characterized in that: The added mass fraction of the egg yolk is 0.3-0.8%, preferably 0.5%.

7. Use of the antigen epitope peptide according to claim 1, the vaccine according to claim 2, the antibody according to claim 3 or the feed according to claim 4 in promoting calcium absorption or phosphorus absorption in animals.

8. The use according to claim 7, characterized in that The application is the application of the antibody in promoting intestinal calcium absorption in animals.

9. The use according to claim 7, characterized in that The animal body is poultry.

10. The use according to claim 7, characterized in that The application is application in the egg-laying period, rapid growth and development period and / or old age period of poultry.

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