Anti-porcine cholecystokinin and somatostatin dual yolk antibody and preparation method thereof

Abstract

The present invention co-expresses CCK and SS for the first time, constructs pCold-pCCK-SS by selecting a cold shock vector pCold GST DNA, and prepares Transetta (DE3)-pCold-pCCK-SS recombinant bacteria, which can efficiently express pCCK-SS recombinant protein. The protein is used for laying hen immunization to obtain egg yolk antibodies with a maximum titer of more than 1:102400. The egg yolk antibodies are used in pig feed to effectively increase the feed intake and average daily weight gain of piglets, reduce the feed-to-meat ratio, and greatly improve the economic benefits of breeding.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to an anti-porcine cholecystokinin and somatostatin dual yolk antibody and a preparation method thereof. Background Art

[0002] The growth rate of animals and the quality of meat products have always been critical issues for animal husbandry and breeding, and have always been a hot topic for biologists. Generally speaking, each tissue and organ in an animal's body will produce inhibitory factors that can specifically inhibit the growth of the animal to prevent its own excessive growth. This process is generally achieved through the inhibitory substances produced by tissues and organs during their growth and development. The normal growth of tissues and organs also benefits from the accumulation of inhibitory substances to a certain extent.

[0003] Cholecystokinin (CCK) is a brain gut peptide that can be secreted by both central nervous cells and proximal I cells of the small intestine. It is widely present in the digestive system, nerve center and peripheral nervous system of organisms. It can promote the secretion of pancreatic enzymes and the contraction of the gallbladder. It can also stimulate pancreatic secretion to enhance gastrointestinal motility. In terms of biological function, CCK can cause a sense of fullness and has a strong appetite-inhibiting effect. The usual mechanism of action is to directly inhibit gastric acid secretion through the regulation of the gastric secretion system, slowing down the digestion and emptying of food in the stomach after eating. As an endogenous satiety signal, it plays a very critical role in appetite regulation. Studies have also shown that cholecystokinin also plays a certain role in the psychological emotions of organisms. As a major anxiety-causing mediator, it plays a regulatory role in the nervous anxiety of animals. At present, the research on cholecystokinin is mainly focused on animal production. By using the immune system of pigs, CCK with a certain content and concentration can significantly increase the daily weight gain and total feed intake of pigs. According to the immunoneutralization technology of CCK, the satiety effect of CCK can be significantly reduced. Since animals cannot know whether they are full or not, they can increase their food intake and promote their growth. Egg yolk containing CCK antibodies can effectively improve the growth performance of weaned piglets and reduce the CCK content in serum. At present, most researchers prefer CCK-8 as an antigen. Usually, this CCK-8 is obtained by chemical synthesis, including sulfated CCK (S CCK) and non-sulfated CCK (NS CCK). The disadvantage is that the above two synthetic CCK-8 are very expensive, and the affinity of sulfated CCK binding to CCK-AR is 500-1000 times higher than that of non-sulfated CCK, and the preparation is complicated, so S CCK is more expensive. From the perspective of the economic benefit ratio of the monomer prepared by CCK, the chemical synthesis method cannot be applied to large-scale production.

[0004] Somatostatin (SS) is a regulatory neuropeptide that can inhibit the secretion of growth hormone. SS combines the characteristics of neurohormones, neurotransmitters and hormones, making its biological effects more extensive than cholecystokinin. In terms of nervous system regulation, somatostatin has a significant inhibitory effect on hypothalamus, cerebral cortex and spinal cord neurons, causing changes in basic biological functions such as movement, sleep, memory, and energy consumption of animals; in terms of the endocrine system, it can inhibit the secretion of multiple hormones released by the pituitary gland, such as calcitonin, parathyroid hormone, and thyrotropin-releasing hormone; somatostatin also has an effect on neurotransmitters. According to studies, if administered to the somatostatin secretion center of animals, it will significantly affect neurotransmitters such as NE energy, NMDA energy, DA energy and Ach energy. As an inhibitory factor, SS regulates the digestive function of animals and also has a significant inhibitory effect on gastric motility and the secretory function of the pancreas and gastrointestinal tract. At present, some studies have used SS or synthetic SS analogs for active immunization, so that animals can produce anti-SS antibodies and neutralize SS in the body, thereby eliminating the inhibition of growth hormone and promoting animal growth. Since the SS peptide chain is very short and the immunogenicity is very weak, it needs to be connected with other carrier proteins to enhance immunogenicity. Huang et al. used xylanase and SS genes for fusion expression to prepare xylanase-SS fusion protein with good immunogenicity and antigenicity, which can be used as a bifunctional vaccine feeding enzyme protein; Han et al. mediated by attenuated Salmonella cholerae C500 to construct a DNA vaccine of SS gene, and the immunization results showed that the vaccine can effectively induce mice to produce SS-specific antibodies; Zhou Zichao's previous research showed that genetic immunization with somatostatin can induce animals to produce anti-somatostatin antibodies and neutralize endogenous somatostatin, block the interaction between somatostatin and its receptors, and promote the secretion and release of multiple hormones such as growth hormone and insulin-like factor-1, thereby promoting animal growth and improving animal production performance. At present, using genetic immunization technology to increase the growth rate of livestock is an ideal way. Using the somatostatin gene to construct a DNA vaccine can effectively promote animal growth. This effect has been reported in many studies. It induces the body to produce somatostatin antibodies to neutralize endogenous somatostatin and eliminate the growth inhibitory effect of somatostatin. However, this method is costly and difficult to preserve.

[0005] The preparation of CCK receptors is relatively expensive and not suitable for mass production. In addition, the SS peptide chain is short and has weak immunogenicity. It needs to be connected to other carrier proteins to enhance immunogenicity. Therefore, it is not very realistic to implement it widely in animal husbandry. Compared with other production methods, the preparation of antigen proteins through molecular cloning and prokaryotic expression technology has the following advantages: first, it is economical and has good development prospects. Second, antibody proteins are easily digested and decomposed by the animal intestine after entering the animal body, and there will be no problem of residues in the body. Third, hormones such as growth hormone have a short half-life and a short duration of use, but antibodies do not have this problem. Summary of the invention

[0006] In order to solve the problems of high cost of CCK and short half-life and weak immunogenicity of SS in the prior art, the present invention expresses CCK and SS in tandem to obtain recombinant porcine cholecystokinin and somatostatin fusion protein, which can be used for active or passive immunization of animals, specific egg yolk antibodies and provide a basic raw material for the development of porcine growth-promoting preparations.

[0007] The present invention adopts the following specific scheme:

[0008] A recombinant porcine cholecystokinin and somatostatin fusion protein, characterized in that the amino acid sequence of the recombinant porcine cholecystokinin and somatostatin fusion protein is shown in SEQ ID No: 2.

[0009] The method for preparing the recombinant porcine cholecystokinin and somatostatin fusion protein is characterized by comprising the following steps: (1) constructing a pCold-pCCK-SS recombinant vector; (2) transforming into competent Escherichia coli Transetta (DE3) to prepare a recombinant strain Transetta (DE3)-pCold-pCCK-SS; (3) inducing expression of the recombinant strain Transetta (DE3)-pCold-pCCK-SS to prepare the recombinant porcine cholecystokinin and somatostatin fusion protein.

[0010] Preferably, the pCold-pCCK-SS recombinant vector is constructed by ligating the nucleotide sequence shown in SEQ ID No: 2 after enzyme digestion with the pCold plasmid after enzyme digestion.

[0011] The specific method of inducing expression is to place the activated Transetta (DE3) -pCold-pCCK-SS bacterial solution in an ice-water mixture, let it stand for 30 minutes, cool the bacterial solution temperature to 15 ° C, add 300 μL IPTG so that the final concentration of IPTG is 1 mmol / L, 15 ° C, 220 r / min induction for 16 to 18 hours.

[0012] The present invention also claims protection for an anti-porcine cholecystokinin and somatostatin dual yolk antibody, characterized in that it is prepared by immunizing laying hens with the recombinant porcine cholecystokinin and somatostatin fusion protein prepared according to any one of claims 2-4 as an immunogen.

[0013] Preferably, the method for preparing the egg yolk antibody comprises the following steps:

[0014] (1) Vaccine preparation: The recombinant porcine cholecystokinin and somatostatin fusion protein prepared according to any one of claims 2 to 4 is used as the immunogen, and the vaccine adjuvant is MONTANIDE ISA 71 VG. The antigen is added to the adjuvant MONTANIDETMISA 71VG at a mass ratio of 30% and emulsified. The shear parameters are: 7000 r / min, below 15°C, and emulsification for 30 minutes.

[0015] (2) Animal immunization: Layers at the peak of egg production are injected intramuscularly on both sides of the chest at a dose of 1 mL / feather, 0.5 mL on each side. Each chicken is immunized three times, with the first and second immunizations separated by three weeks, and the second and third immunizations separated by one month.

[0016] (3) Extraction of yolk antibody (IgY).

[0017] The present invention also requests protection for an anti-porcine cholecystokinin and somatostatin dual yolk antibody preparation, characterized in that the yolk antibody preparation is prepared by selecting immune eggs with an antibody titer ≥1:12800, separating the egg white and the yolk, collecting the yolk for homogenization, adding an equal mass of maltodextrin, and then low-temperature drying at 45-50°C to a water content of ≤10%, and then crushing to obtain yolk powder.

[0018] The present invention also requests protection for the use of the anti-porcine cholecystokinin and somatostatin dual yolk antibody in the preparation of pig feed, characterized in that adding the anti-porcine cholecystokinin and somatostatin dual yolk antibody to the feed can effectively increase the pig's feed intake, weight gain, and reduce the feed-to-meat ratio.

[0019] Preferably, the pig is a piglet. Preferably, the amount of the anti-porcine cholecystokinin and somatostatin dual yolk antibody added to the feed is 1% (w / w).

[0020] Based on the above technical solution, the present invention has the following advantages and beneficial effects:

[0021] First, the present invention co-expresses CCK and SS for the first time, constructs recombinant expression vectors pET28a-pCCK-SS and pCold-pCCK-SS, and prepares corresponding recombinant strains. However, it is found that in the pET28a vector, the recombinant protein pCCK-SS is expressed in a small amount in the inclusion body, and is negative by Western blot detection, which may be due to the reduced protein expression efficiency in pET28a or inappropriate expression conditions. Based on the above research, the present invention uses the cold shock vector pCold GST DNA as a vector connected to pCCK-SS. The plasmid pCold GST is used to transform the competent cell Transetta (DE3) of Escherichia coli, screen the monoclonal strain, identify it as positive by enzyme digestion, purify the enzyme digestion product to obtain the linearized vector pCold, and the results show that the pCold purified product band is bright and has no impurities, indicating that the plasmid has a high copying ability and a good purification test effect, and successfully expresses the recombinant protein pCCK-SS in the supernatant and precipitate.

[0022] Secondly, the present invention prepared the recombinant protein pCCK-SS for the first time, and used it as an immunogen to immunize laying hens to obtain anti-porcine cholecystokinin and somatostatin dual yolk antibody. After testing, when the titer of CCK-SS antibody reached a peak, the highest titer of the yolk antibody Ig Y prepared by the present invention was 1:102400 or more. The present invention further prepared it into a preparation for the preparation of pig feed. The test showed that the feed intake of the test group fed with the feed containing anti-porcine cholecystokinin and somatostatin dual yolk antibody increased by 18.64% relative to the blank control group, and increased by 16.75% relative to the control group fed with ordinary yolk powder; and the average daily weight gain of piglets also reached the highest, which increased by 38.62% relative to the blank control group and increased by 25.46% compared to the control group. The feed-to-meat ratio was significantly reduced, which was reduced by 14.41% relative to the blank control group and reduced by 6.95% compared to the control group. Based on the above test results, it can be seen that adding the anti-porcine cholecystokinin and somatostatin dual yolk antibody of the present invention to the piglet diet can effectively increase the feed intake and average daily weight gain of the piglets, reduce the feed-to-meat ratio, and greatly improve the economic benefits of breeding.

[0023] In summary, the present invention co-expresses CCK and SS for the first time, constructs pCold-pCCK-SS by selecting the cold shock vector pCold GST DNA, and prepares the Transetta (DE3) -pCold-pCCK-SS recombinant bacteria, which can efficiently express the pCCK-SS recombinant protein, and the protein is used for laying hen immunization to obtain yolk antibodies with a maximum titer of 1:102400 or more. The yolk antibodies are used in pig feed, which can effectively increase the feed intake and average daily weight gain of piglets, reduce the feed-to-meat ratio, and greatly improve the economic benefits of breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 . Enzyme digestion identification of recombinant expression vectors, Figure 1 -A is the enzyme digestion identification of the recombinant expression vector pET28a- pCCK-SS, where M: DL2000 DNA Marker; 1~2: double enzyme digestion products of the recombinant vector pET28a- pCCK-SS. Figure 1 -B is the double restriction enzyme digestion identification of the recombinant plasmid pCold-pCCK-SS, where M: DL5000 Marker; 1 is the extracted plasmid; 2 is the product of the double restriction enzyme digestion of the plasmid by BamH I and Hind III.

[0025] Figure 2 . SDS-PAGE detection of recombinant porcine cholecystokinin and somatostatin fusion protein rpCCK-SS, where M: protein marker; 1~2: 1~2 are the supernatant and inclusion body of the expression product of the recombinant strain BL21-pET28a-pCCK-SS respectively;

[0026] 3: BL21(DE3) bacterial protein; 4: BL21(DE3) bacterial protein transformed with the empty vector BL21-pET28a.

[0027] Figure 3 . Western blot detection of recombinant porcine cholecystokinin and somatostatin fusion protein rpCCK-SS, M: protein marker; 1~2: 1~2 are the supernatant and inclusion body of the expression product of the recombinant strain BL21-pET28a- pCCK-SS respectively;

[0028] 3: BL21(DE3) bacterial protein; 4: BL21(DE3) bacterial protein transformed with the empty vector BL21-pET28a.

[0029] Figure 4.SDS-PAGE detection and Western blot identification of the medium-level expression of the recombinant protein rpCCK-SS, where M. protein marker; 1. ultrasonic supernatant of induced-expression Transetta(DE3)-pCold-pCCK-SS; 2. ultrasonic precipitation of induced-expression Transetta(DE3)-pCold-pCCK-SS; 3. ultrasonic supernatant of induced-expression Transetta(DE3)-pCold.

[0030] Figure 5 . SDS-PAGE test results of egg yolk antibody extracted from eggs 14 days after the second immunization, where M: protein marker; 1-8: ①-⑧ IgY stock solution.

[0031] Figure 6 . Effect of anti-porcine cholecystokinin and somatostatin dual yolk antibody on piglet feed intake, where group 1 was the experimental group and group 2 was the control group. DETAILED DESCRIPTION

[0032] Example 1. Prokaryotic expression and identification of porcine cholecystokinin and somatostatin

[0033] 1. Synthesis of pCCK-SS nucleotide sequence and construction of vector

[0034] According to the gene sequences of CCK and SS, their tandem gene sequences were designed, and BamHI and Hind III restriction sites were set at their 5' and 3' ends, respectively, that is, the sequence was: BamHI+pCCK+SS+TAA+Hind III. The specific nucleotide sequence is shown in SEQID No: 1, and the corresponding encoded protein sequence is shown in SEQID No: 2, with a total of 230 amino acids and a molecular weight of approximately 25.2 kDa.

[0035] The sequence was synthesized by Takara and cloned into pMD18-T vector to obtain pMD18T-pCCK-SS.

[0036] Construction of recombinant prokaryotic expression vector pET28a-CCK-SS

[0037] 2.1 Reagent digestion and recovery of pCCK-SS target gene

[0038] The recombinant vector pMD18T-pCCK-SS was double-digested with BamH I and Hind III restriction endonucleases in a 40 µL system, and the pCCK-SS gene was subsequently recovered and purified by agarose gel electrophoresis.

[0039] Ligation of gene fragments with pET28a and pCold vectors

[0040] The recovered target gene fragment pCCK-SS was connected to the pET28a vector fragment after enzyme digestion, and transformed into competent Escherichia coli BL21 (DE3) to prepare the recombinant strain BL21 (DE3)-pET28a-pCCK-SS. The recovered target gene fragment pCCK-SS was connected to the pCold vector fragment after enzyme digestion, and transformed into competent Escherichia coli Transetta (DE3) to prepare the recombinant strain Transetta (DE3)-pCold-pCCK-SS.

[0041] Identification

[0042] The cloned colonies on the agar were positively identified, and 5-6 positive clones were selected. The colonies were cultured overnight in a shaker and expanded. After the plasmid DNA was extracted by boiling, double restriction digestion was performed at 37°C for 1 h. The target gene pCCK-SS was connected to the expression vector pET28a by electrophoresis. The specific results are as follows: Figure 1 A and Figure 1 B. Based on Figure 1 A and Figure 1 The double enzyme digestion results of B showed that the present invention successfully constructed the recombinant strains BL21 (DE3) -pET28a-pCCK-SS and Transetta (DE3) -pCold-pCCK-SS.

[0043] Prokaryotic expression and identification of

[0044] 3.1 Inducible expression of BL21(DE3)-pET28a-pCCK-SS

[0045] (1) Bacterial activation: 10 µL of the bacterial solution of the recombinant porcine cholecystokinin and somatostatin fusion protein rpCCK-SS expression strain (BL21(DE3)-pET28a-pCCK-SS) and the empty vector solution to be transformed (BL21(DE3)-pET28a) were taken respectively, and then inoculated into 5 mL of LB medium supplemented with kanamycin (Kanr) at a final concentration of 200 ng·mL-1. The cells were cultured overnight at 37°C with shaking at 240 r·min-1 for 15-16 h.

[0046] (2) Reactivation: After shaking culture, the above 0.6 mL of bacterial solution was inoculated into 6 mL of LB medium containing Kanr (final concentration was 200 ng·mL-1) at an inoculation ratio of 1:10, and then the bacterial solution was shaken and cultured at 37°C and 240 r·min-1 for 2.5 h;

[0047] (3) IPTG induction: add 1 mmol·L-1 IPTG and induce at 40℃ and 240 r·min-1 for 4.5 h;

[0048] (4) Bacterial cell collection: Use a 1.5 mL EP tube to collect 1 mL of the induced bacterial solution, then centrifuge the EP tube at 1400 r·min-1 for 30 s, aspirate and discard the supernatant, add 1 mL of bacterial solution to the tube, and repeat the above experimental operation until the total volume of 5.5 mL of bacterial solution is concentrated to 1.5 mL;

[0049] (5) Extraction and collection: The bacterial cells were broken by ultrasonic disruption and the bacterial proteins were extracted. The samples were shaken for 30 min and centrifuged at 4000 r / min at 4°C. -1 The inclusion body protein and supernatant protein were separated by centrifugation. The supernatant was pipetted into a new 1.5 mL EP tube, labeled as "pET28a-pCCK-SS-U", and stored at -80°C for SDS-PAGE analysis. The precipitate at the bottom of the tube was the inclusion body protein. 100 µL of PBS was added to each EP tube to suspend the inclusion body precipitate protein, which was labeled as "pET28a-pCCK-SS-D" and stored at -80°C for SDS-PAGE analysis. The specific results are shown in the figure. Figure 2 shown.

[0050] Identification of induced products

[0051] First, the recombinant porcine cholecystokinin and somatostatin fusion protein rpCCK-SS was purified by electrophoresis, the voltage was adjusted to 10 V, and the transfer time was 25 min; then it was blocked with BSA. It was best to cover the entire NC membrane with the liquid during blocking. The blocking conditions were 37°C, 60 r•min-1, and the time was 2 h; the shaking speed was set to 60 r•min-1, the NC membrane was washed with PBST for 5 min, and then the blocking solution was recovered; Mouse Anti-His mAb primary antibody solution diluted 1:2000 with 2% skim milk powder-PBS was added. In order to better bind to the antigen protein, it is best to shake and oscillate for 30 min; the NC membrane was washed with 10 mL of PBST, 5 min / time, repeated 3 times, and shaken for 30 min, and the primary antibody was recovered; 2% skim milk powder-PBS, 1:2000 diluted secondary antibody solution HRP-SPA, also shaken and oscillated for 30 min, the secondary antibody was recovered, and the NC membrane was washed in the same way as the primary antibody, and the PBST solution was discarded; the color developing solution 2 was prepared mL, add the color developer, and shake continuously until the target band can be clearly seen. Then wash the membrane repeatedly with distilled water, and finally take a picture to record the color reaction. Figure 3 shown.

[0052] Combination Figure 2 and Figure 3 It can be seen that after induction of BL21(DE3)-pET28a-pCCK-SS, the recombinant porcine cholecystokinin and somatostatin fusion protein mainly exists in the inclusion body, and the target protein does not exist in the supernatant. The molecular weight of the recombinant porcine cholecystokinin and somatostatin fusion protein rpCCK-SS is about 25 kDa. The bacterial protein of BL21(DE3) with empty vector pET28a and the protein expressed by empty bacteria BL21(DE3) in the two control groups did not show a band of the same size near the target protein at the relevant position, which indicates that the pCCK-SS gene was successfully induced and expressed. However, in Western blot identification, according to the expected size of the target protein rpCCK-SS, the Western blot color development result should have a color development imprint near 25 kDa, but due to Figure 3 It can be seen that there is no color development at 25 kDa, so it is determined that the recombinant porcine cholecystokinin and somatostatin fusion protein rpCCK-SS cannot be effectively prepared by BL21(DE3)-pET28a-pCCK-SS.

[0053] Low temperature induced expression

[0054] 20 μL of the control strain Transetta(DE3)-pCold and the recombinant expression strain Transetta(DE3)-pCold-pCCK-SS were taken and inoculated into 4 mL of 100 μg mL -1 Amp in liquid LB medium, labeled, 37°C, 220 r·min -1 Culture for 16-18 h. The next day, inoculate all the above bacterial solution in 16 mL containing 100 µg mL -1 In liquid LB medium containing ampicillin, 37 °C, 220 r·min -1 , 4-6 h. Take the above bacterial solution and immediately cool it on ice for 30 min. Then add a final concentration of 1 mmol·L -1 IPTG, 15 °C, 220 r·min -1 Induce for 12-18 hours. Centrifuge to enrich the cells and label them. Suspend the precipitate fully with PBS and perform two sonications at 12000r·min-1 for 10 min. Aspirate all the supernatant and store it in EP tubes. Suspend the precipitate fully with inclusion body dissolving solution and store it at -20℃ for subsequent SDS-PAGE and Western blot detection.

[0055] Test results such as Figure 4As shown in the figure, the positively identified Transetta(DE3)-pCold-pCCK-SS strain was subjected to IPTG low-temperature induction for small-scale expression, the bacteria were collected by centrifugation, and the protein was extracted by adding protein extract for SDS-PAGE and WB identification. The results showed that the No. 1 and No. 4 recombinant strains Transetta(DE3)-pCold-pCCK-SS had bands near 50 kDa in the supernatant and precipitate, which was consistent with the expected molecular weight. Western blot identification showed that the band could specifically bind to the primary antibody. The uninduced bacterial protein of the empty vector strain Transetta(DE3)-pCold had basically no specific binding to the primary antibody. The protein induced by the empty vector strain Transetta(DE3)-pCold could partially bind to the primary antibody, but the bound protein band did not match the expected molecular weight. Therefore, it can be determined that the target protein has been expressed and is mainly expressed in the inclusion body.

[0056] Example 2. Optimization of fusion expression of porcine cholecystokinin and somatostatin

[0057] 1.1 First activation of the strain: Take 60 µL of Amp and add it to 60 mL / bottle of fresh LB medium (containing Amp at a final concentration of 100 µg / mL), then take 60 µL of recombinant bacteria Transetta(DE3)-pCold-pCCK-SS and add it to 60 mL / bottle of LB medium containing Amp (bacteria solution: LB=1:1000), and culture at 37°C, 220 r / min overnight (16-18 h).

[0058] 1.2 Secondary activation of bacteria: The next day, add 240 µL of Amp (final concentration 100 µg / mL) to 240 mL / bottle of fresh LB, then add 60 mL of the activated bacteria in step 2.1 to 240 mL / bottle of LB medium containing Amp at a ratio of 1:4, and incubate at 37 °C, 220 r / min, and shake for 6 h.

[0059] 1.3 Ice bath: Take out the secondary activated bacterial solution and immediately place it in an ice-water mixture for 30 min to cool the bacterial solution to 15 °C to ensure the accuracy of low-temperature induction.

[0060] 1.4 IPTG induction: Add 300 µL IPTG (final concentration 1 mmol / L) and induce at 15 °C, 220 r / min for 16-18 h.

[0061] 1.5 Collect and concentrate the cells: Collect the cells in each conical flask by centrifugation. Concentrate every 300 mL of bacterial solution in a 35 mL centrifuge tube. Centrifugation parameters: 4 °C, 12000 r / min for 5 min.

[0062] 1.6 Wash the cells: Add 30 mL of PBS to each tube, pipette repeatedly to mix well, vortex for 1 min, suspend the cells, centrifuge at 4°C, 12,000 r / min for 5 min, and discard the supernatant.

[0063] 1.7 Ultrasonic disruption once: add 30 mL of PBS to each centrifuge tube, pipette repeatedly to mix well, then vortex for 1 min, and perform ultrasonication on ice at 60% (300 W) power, ultrasonicate for 3 s each time, with an interval of 5 s, for a total of 1 h per tube.

[0064] 1.8 Centrifugation: 4 ℃, 12000 r / min for 15 min. Collect the supernatant and store at -20 ℃.

[0065] 1.9 Second ultrasonic disruption: Use 30 mL PBS to suspend the precipitate after the first ultrasonic treatment, blow it evenly with a pipette, vortex it for 1 min, and ultrasonicate it on ice for 3 s at a time with an interval of 5 s for a total of 1 h.

[0066] 1.10 Collect the purified inclusion body protein: centrifuge at 4°C, 12000 r / min for 15 min, collect the supernatant and store it at -20°C. The precipitate is the inclusion body protein to be collected.

[0067] 1.11 Dissolution of inclusion body protein: Use an appropriate amount of inclusion body dissolution solution (20-30 mL / 300 mL of induced bacteria) to dissolve the inclusion body protein until it is completely dissolved (using ultrasound: 3 s, 5 s interval, 20 min in total to accelerate dissolution) until the liquid is clear. Mark well and store at -80 ℃.

[0068] 1.12 Protein purity test: Take 20 μL of protein sample and mix with 20 μL of 6× Loading Buffer, boil in boiling water for 10 minutes, take 10 μL of sample for SDS-PAGE analysis, take pictures to record purity.

[0069] Example 3. Preparation of anti-porcine cholecystokinin and somatostatin dual yolk antibody

[0070] 1. Vaccine preparation and animal immunization

[0071] 1.1 Vaccine preparation: The vaccine adjuvant is MONTANIDE ISA 71VG. Emulsification method: follow the instructions, add 30% by mass of the antigen to the adjuvant MONTANIDE ISA 71VG for emulsification, shear parameters: 7000 r / min, below 15°C, emulsification for 30 min.

[0072] 1.2 Animal immunization: Laying hens at the peak of egg production should be immunized with 1 mL / bird by intramuscular injection on both sides of the chest, 0.5 mL on each side. Each chicken should be immunized three times, with the first and second immunizations three weeks apart, and the second and third immunizations one month apart.

[0073] Extraction of yolk antibody (IgY)

[0074] 2.1 Separate the yolk: wipe the egg shell with alcohol cotton to clean it; separate the yolk into a beaker and number the beaker; stir the yolk with a glass rod, measure 10 ml and put it into a labeled beaker and weigh it, and keep a record (the weight of the yolk = the total weight of the beaker and the yolk - the weight of the beaker);

[0075] 2.2 Dilution: Mix 10 ml egg yolk with sterile PBS buffer (pH 7.4) at a ratio of 1:2 (V / V), ensure thorough mixing, and transfer to a centrifuge tube;

[0076] 2.3 First precipitation: Add 3.5% (W / V) PEG-6000 (weighing error range 0.01), mix thoroughly, heat in a 37 ℃ water bath for 15-30 min, take out and let stand after fully dissolved, and let stand at room temperature for 1 h (the longer the better, it can be left overnight; during the standing period, the solution will be stratified);

[0077] 2.4 Centrifugation: 12000 r / min, 4 ℃, 30 min centrifugation, the stratification appears, from top to bottom are yellow fat layer, clear layer (IgY) and semi-solid layer;

[0078] 2.5 Filtration: Pour out the supernatant, filter with rapid filter paper, collect the filtrate and record its volume, and discard the precipitate;

[0079] 2.6 Second precipitation: Add 8.5% (W / V) PEG-6000 according to the volume of the filtrate, shake well, and place in a 37 ℃ water bath for 15-30 min to fully dissolve;

[0080] 2.7 Centrifugation: 12000 r / min, 4°C, 30 min;

[0081] 2.8 Dissolve: Discard the supernatant and add 10 mL of sterile PBS buffer (pH 7.4) to dissolve the precipitate, ensuring that the precipitate is completely suspended, dispersed and dissolved;

[0082] 2.9 Third precipitation: add 12% (W / V) (1.2 g) PEG-6000, shake well, and place in a 37 ℃ water bath for 15-30 min to fully dissolve;

[0083] 2.10 Centrifugation: 12000 r / min, 4°C, 30 min;

[0084] 2.11 Dissolution: Discard the supernatant and add 1 mL of sterile PBS buffer (pH 7.4) to dissolve the precipitate (if the liquid is not clear after dissolution, add 1 mL of sterile PBS buffer (pH 7.4) to dissolve it);

[0085] 2.12 Packaging: Dispense the above dissolved solution into suitable EP tubes.

[0086] Gel electrophoresis to detect the molecular integrity and purity of egg yolk antibodies

[0087] The sample was taken for SDS-PAGE detection. The test results showed that there were obvious heavy chain and light chain bands at 70 kDa and 25 kDa, and the purity was high. The specific results are as follows Figure 5 As shown, it indicates that the antibody molecules have good integrity, high content, and less impurity proteins.

[0088] Detection of yolk antibody IgY titer

[0089] (1) Electrophoresis and membrane transfer: The CCK-SS fusion protein was subjected to SDS-PAGE electrophoresis and transferred to a PVDF membrane.

[0090] (2) Blocking: Place the membrane with the front side facing up in a box filled with 2% skimmed milk powder-PBS (the amount is just enough to cover the membrane) and place it on a shaker at 37°C, 40 rpm, for 1 h.

[0091] (3) Washing: Pour away 2% skimmed milk powder PBS, add PBST, wash three times, shake at 40 r / min, 5 min / time, and discard the washing solution.

[0092] (4) Cutting the membrane: On a clean aluminum box, use a scalpel to cut the membrane into single strips according to the principle of one membrane per lane.

[0093] (5) Primary antibody incubation:

[0094] a. Antibody dilution: prepare 10 EP tubes, marked 1- / 12, add 4 mL PBS to tube 1, and add 2 mL 2% skim milk powder-PBS to the other tubes. Add 40 μL IgY to tube 1, turn it upside down and mix thoroughly, take 2 mL from tube 1 and add it to tube 2, turn it upside down and mix thoroughly, and perform 2× gradient dilution in the same way for the other tubes. The dilution multiples in tubes 1-12 are: 100, 200, 400, 800, 1600, 3200, 6400, 12800, 25600, 51200, 102400, 204800 times.

[0095] b. Primary antibody incubation: Add diluted antibody to each membrane, incubate at 37°C, 30 rpm, for 1 h;

[0096] (6) Washing: Discard the primary antibody incubation solution, wash three times with PBST, shake at 30 r / min, 5 min / time, and discard the washing solution.

[0097] (7) Secondary antibody incubation:

[0098] a. Dilution of secondary antibody: Take 1 μL of goat anti-chicken secondary antibody labeled with horseradish peroxidase and add it to 6 mL of 2% skim milk powder-PBS, mix well, and dilute it 6000 times.

[0099] b. Secondary antibody incubation: 37 ℃, 30 r / min, 1 h;

[0100] (8) Washing: Discard the secondary antibody incubation solution, wash three times with PBST, shake on a shaker at 30 r / min, 5 min / time, and discard the washing solution. (The washing solution does not need to be discarded for the third wash)

[0101] (9) Color development: Prepare 1 mL of color development solution according to the instructions. Color development: Pour out the PBST solution, use a gun to suck the color development solution and gently blow it onto the membrane. Repeat this operation 10 times until the target band is colored. Rinse with tap water to terminate the reaction.

[0102] (10) Record the results.

[0103] It has been determined that when the CCK antibody titer reaches a peak, the highest titer of the egg yolk antibody Ig Y prepared by the present invention is above 1:102400.

[0104] Example 4. Application of anti-porcine cholecystokinin and somatostatin dual yolk antibody

[0105] 1. Preparation of anti-porcine cholecystokinin and somatostatin dual yolk antibody preparation

[0106] Select immune eggs with an antibody titer of ≥1:12800, separate the egg white and the yolk, collect the yolk for homogenization, add an equal mass of maltodextrin, and then low-temperature dry at 45-50°C to a moisture content of ≤10%, and prepare the yolk powder by crushing.

[0107] 2. Effect of anti-cholecystokinin and somatostatin dual yolk antibody on piglet feed intake

[0108] Two healthy hybrid piglets were selected and randomly divided into two groups. One of them was the experimental group, and 1% of the anti-porcine cholecystokinin and somatostatin dual yolk antibody preparation was added to its basic diet; Group 2 was the control group, and 1% of the negative yolk powder (eggs produced by non-immunized laying hens, the negative yolk powder prepared by the method 1 above) was added to the basic diet. The same feeding method was used for feeding 5 times a day, and the feeding situation was checked every two hours. Feed was supplemented as needed, and the feed intake of each piglet was recorded. The observation lasted for 14 days. The specific results are as follows Figure 6 As shown, on the third day after adding anti-swine cholecystokinin and somatostatin dual yolk antibodies to the feed, the pigs' feed intake was greatly increased, and after the fifth day of feeding, the feed intake remained at a relatively stable level, and its feed intake was significantly increased compared with the control group.

[0109] Application of anti-porcine cholecystokinin and somatostatin dual yolk antibody in piglet feed

[0110] 3.1 Experimental animals and design

[0111] Fifteen crossbred piglets with similar birth weight were selected and randomly divided into three groups, each with 5 piglets, wherein Group 1 was a blank control group, which was fed with a basic diet; Group 2 was a control group, which was fed with 1% negative yolk powder (eggs produced by non-immunized laying hens, and negative yolk powder prepared by the method 1 above) on the basis of the basic diet; Group 3 was a test group, which was fed with 1% of the anti-porcine cholecystokinin and somatostatin dual yolk antibody preparation prepared by the present invention on the basis of the basic diet. The basic diet consisted of corn, soybean meal and premix.

[0112] 3.2 Effects of anti-cholecystokinin and somatostatin dual yolk antibodies on feed intake and growth performance of piglets

[0113] During the experiment, the breeding ground was kept clean, and the piglets were allowed to drink water and eat freely. They were fed 5 times a day, and their feeding situation was checked every two hours. Feed was supplemented as needed, and the feed intake of each piglet was recorded. The observation lasted for 20 days. The fasting weight of the piglets was recorded before and after the experiment, and the average daily weight gain, average daily feed intake and feed-to-meat ratio of the piglets were calculated. The specific test results are shown in Table 1:

[0114] Table 1 Effects of anti-cholecystokinin and somatostatin dual yolk antibody on feed intake and growth performance of piglets

[0115] Blank control group Control group Experimental Group Average daily feed intake of piglets (kg / head) 0.7098 0.7213 0.8421 Average daily weight gain (kg / head) 0.391 0.432 0.542 Feed to meat ratio (F / G) 1.815 1.670 1.554

[0116] Based on the above experiments, it can be known that the test group fed with a feed containing anti-porcine cholecystokinin and somatostatin dual yolk antibodies of the present invention has a feed intake that is 18.64% higher than that of the blank control group, and a feed intake that is 16.75% higher than that of the control group fed with ordinary yolk powder; and the average daily weight gain of the piglets also reached the highest, which was 38.62% higher than that of the blank control group and 25.46% higher than that of the control group. The feed-to-meat ratio was significantly reduced, which was 14.41% lower than that of the blank control group and 6.95% lower than that of the control group.

[0117] Based on the above test results, it can be seen that adding the anti-porcine cholecystokinin and somatostatin dual yolk antibody of the present invention to the piglet diet can effectively increase the feed intake and average daily weight gain of the piglets, reduce the feed-to-meat ratio, and greatly improve the economic benefits of breeding.

Claims

1. A method for preparing a recombinant porcine cholecystokinin and somatostatin fusion protein, characterized in that: The amino acid sequence of the recombinant porcine cholecystokinin and somatostatin fusion protein is shown in SEQ ID No:

2. The method comprises the following steps: (1) constructing a pCold-pCCK-SS recombinant vector, wherein the recombinant vector is obtained by ligating the nucleotide sequence shown in SEQ ID No: 1 after enzyme digestion with the pCold plasmid after enzyme digestion; (2) Transforming the pCold-pCCK-SS recombinant vector into competent Escherichia coli Transetta (DE3) to prepare the recombinant strain Transetta (DE3)-pCold-pCCK-SS; (3) inducing the expression of the recombinant strain Transetta (DE3)-pCold-pCCK-SS to prepare the recombinant porcine cholecystokinin and somatostatin fusion protein, The specific method of inducing expression is to place the activated Transetta (DE3) -pCold-pCCK-SS bacterial solution in an ice-water mixture, let it stand for 30 minutes to cool the bacterial solution to 15°C, add 300 µL IPTG so that the final concentration of IPTG is 1mmol / L, and induce at 15°C and 220 r / min for 16 to 18 hours; The recombinant porcine cholecystokinin and somatostatin fusion protein is used to prepare anti-porcine cholecystokinin and somatostatin dual yolk antibody, and the highest titer of the prepared yolk antibody Ig Y is 1:102400.

2. An anti-porcine cholecystokinin and somatostatin dual yolk antibody, characterized in that: The yolk antibody is prepared by immunizing laying hens with the recombinant porcine cholecystokinin and somatostatin fusion protein prepared by the method of claim 1 as an immunogen. The preparation method of the egg yolk antibody comprises the following steps: (1) Vaccine preparation: The recombinant porcine cholecystokinin and somatostatin fusion protein prepared in claim 1 is used as the immunogen, and the vaccine adjuvant is MONTANIDE ISA 71 VG. The antigen is added to the adjuvant MONTANIDE ISA 71 VG at a mass ratio of 30% and emulsified. The shear parameters are: 7000 r / min, below 15°C, and emulsification for 30 minutes. (2) Animal immunization: Layers at the peak of egg production are injected intramuscularly on both sides of the chest at a dose of 1 mL / feather, 0.5 mL on each side. Each chicken is immunized three times, with the first and second immunizations separated by three weeks, and the second and third immunizations separated by one month. (3) Extraction of egg yolk antibody IgY. After measurement, the highest titer of the prepared egg yolk antibody Ig Y was 1:102400.

3. An anti-porcine cholecystokinin and somatostatin dual yolk antibody preparation, characterized in that: According to the method of claim 2, immune eggs are prepared, and immune eggs with an antibody titer of ≥1:12800 are selected, the egg white and the yolk are separated, the yolk is collected and homogenized, an equal mass of maltodextrin is added, and then low-temperature drying is performed at 45-50°C to a moisture content of ≤10%, and the yolk powder is prepared by crushing.

4. The use of the anti-porcine cholecystokinin and somatostatin dual yolk antibody in the preparation of pig feed according to claim 2, characterized in that: Adding the anti-porcine cholecystokinin and somatostatin dual yolk antibody to feed can effectively increase pig feed intake, weight gain, and reduce feed-to-meat ratio. The addition amount of the anti-porcine cholecystokinin and somatostatin dual yolk antibody to feed is 1% w / w.

5. The use according to claim 4, characterized in that: The pigs are piglets.

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