CN114671943A - Preparation and application of fish ingestion regulation peptide - Google Patents
Preparation and application of fish ingestion regulation peptide Download PDFInfo
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- CN114671943A CN114671943A CN202210474927.3A CN202210474927A CN114671943A CN 114671943 A CN114671943 A CN 114671943A CN 202210474927 A CN202210474927 A CN 202210474927A CN 114671943 A CN114671943 A CN 114671943A
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/515—Angiogenesic factors; Angiogenin
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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Abstract
The invention discloses a preparation method of fish ingestion regulatory peptide, relating to the technical field of genetic engineering and comprising the following steps: s1, extracting total RNA of tilapia liver; cloning complete sequences of cDNA of S2 and tilapia angptl8 genes; (S3) tissue expression distribution of tilapia angptl 8; s4, the expression of tilapia angptl8 changes in the feeding process; preparing S5 and ANGPTL8 recombinant protein; s6, researching the influence of the ANGPTL8 recombinant protein on the tilapia feed by intraperitoneal injection. The fish angptl8 gene is expressed in a large amount in fish livers, mature peptides generated by the gene have the function of regulating fish ingestion, can be developed and utilized by a biosynthesis method, and has the advantages of convenience in use, remarkable effect and wide application value. The scheme adopts prokaryotic expression to prepare the recombinant protein, and has the advantages of high yield, low cost, convenient operation, easy repetition and the like.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to preparation and application of a fish ingestion regulation peptide.
Background
The existing phagostimulant has low activity, large dosage, higher production cost and certain toxic and side effects. The invention aims to provide an angiopoietin-like 8 gene coding mature peptide. The invention also aims to provide application of the fish ANGPTL8 neuropeptide gene.
Disclosure of Invention
The invention aims to provide preparation and application of a fish food intake control peptide aiming at the existing problems.
The invention is realized by the following technical scheme:
a preparation method of fish food intake control peptide comprises the following steps:
s1, extracting total tilapia liver RNA:
taking a healthy tilapia, dissecting and separating liver tissues, and obtaining total tilapia liver RNA by adopting a Trizol reagent method;
s2, cloning complete sequence of tilapia angptl8 gene cDNA:
s201, designing specific primers AN-F1 and AN-R1 by taking the synthesized first strand cDNA as a template, and carrying out PCR amplification;
s202, loading the PCR product to 1.5% agarose gel, separating DNA fragments by low-voltage electrophoresis, and purifying and recovering a target product from the gel;
s203, connecting the purified target product to
Vector, transforming DH5 alpha competent cell, selecting positive clone to sequence;
s204, designing a specific primer according to the obtained cDNA fragment sequence, and carrying out PCR amplification on the 3 'end and the 5' end of the target gene by utilizing a cDNA end rapid amplification technology;
s205, according to RACE kit GeneRacerTMThe Kit specification dephosphorizes total RNA of liver of tilapia and removes mRNA 5'A cap structure, which is connected with the RNA Oligo and finally synthesized into a first cDNA chain through reverse transcription;
s206, 3'-RACE using tilapia angptl8cDNA, 5' -RACE specific primers AN-F2, AN-F3 and AN-R2, AN-R3, and GeneRacerTMAmplifying a 5 'end fragment and a 3' end fragment of tilapia angptl8cDNA by using a universal primer of Kit;
s207, after the obtained PCR product is recovered and purified, connecting a T vector, transforming DH5 alpha competence, screening positive colonies by using a blue-white spot, and performing DNA sequencing after colony PCR verification;
s3 tissue expression profile of tilapia angptl 8:
detecting the expression of tilapia angptl8 in various tissues by using a real-time fluorescent quantitative PCR (polymerase chain reaction) technology;
s301, extracting total RNA of 12 tissues such as hypothalamus, telencephalon, optic cap, cerebellum, olfactory bulb, medulla oblongata, spinal cord, pituitary, liver, fat, intestine, muscle and the like of tilapia;
s302, after DNaseI treatment, taking 1 mu g of RNA for reverse transcription according to the specification of iScriptTM;
s303, diluting the reverse transcription product by 10 times to be used as a cDNA template, referring to iQTM 
GreenSupermix instructions prepare qPCR reaction systems;
s304, determining the specificity of the amplification product by analyzing a qPCR dissolution curve;
s4, the expression of tilapia angptl8 changes in the feeding process:
s401, randomly distributing tilapia with the weight of 2 +/-0.5 g into four glass jars, wherein the four glass jars are respectively a control group and 3 feeding groups, and 6 tilapia are averagely distributed in each jar;
s402, feeding for 1 time at 4 pm every day, wherein the food feeding amount is 3% of the body weight, and formally starting an experiment after two weeks of domestication;
s403, hypothalamus of normally fed tilapia was sampled at the following time points: 1h before ingestion, 0hr during ingestion, 1hr after ingestion, and 3hr after ingestion;
s404, after the tilapia is anesthetized by 0.05% MS-222, the tilapia is killed and collected with hypothalamic samples, and then the samples are subjected to quick freezing treatment by liquid nitrogen and stored in an ultra-low temperature refrigerator at minus 80 ℃ for later use;
s405, detecting the change of the angptl8 gene expression amount in the hypothalamus of tilapia by a qPCR (quantitative polymerase chain reaction) technology;
s5 and ANGPTL8 recombinant protein preparation:
s501, taking tilapia liver tissue cDNA as a template, and amplifying the full length of angptl8cDNA sequence by using a Taq enzyme kit;
s502, carrying out electrophoresis on the amplified product by using 1.5% agarose gel, and recovering the target fragment by using a gel recovery kit;
s503, carrying out double enzyme digestion on the purified fragment, connecting the purified fragment with a pET30a (+) expression vector, and converting BL21(DE3) competent cells;
s504, screening positive expression colonies by using an LB culture medium (Kana antibiotic: 50mg/ml), and preserving the strains after PCR verification of bacterial liquid;
s505, adding bacterial liquid of angptl8-pET30a (+) -BL21(DE3) according to the volume of 1% LB culture medium (Kana antibiotic: 50mg/ml), shaking overnight at 37 ℃ and 200rpm, pouring the bacterial liquid into 100 times of LB culture medium (Kana antibiotic: 50mg/ml), culturing at 37 ℃ and 200rpm until the bacterial liquid concentration OD600 is approximately equal to 0.5 (about 3hr), adding IPTG until the final concentration is 0.1mM, and inducing expression at 37 ℃ and 200rpm for 6 hr;
s506, centrifuging the bacterial liquid at 5000 Xg for 2min, then discarding the supernatant, and rinsing the supernatant for 2 times by using a lysis solution;
s507, adding lysis solution according to the volume of 10% of the bacterial solution, and carrying out ultrasonic crushing in ice bath;
s508, centrifuging the broken solution at 10,000 Xg 4 ℃ for 30min, and filtering the supernatant by using a 0.45-micron filter;
s509, purifying the protein by using a His tag protein purification kit (Biyuntian biotechnology company), wherein the elution conditions are as follows: washing the hybrid protein by 50mM imidazole and 100mM imidazole lysate, and eluting the target protein by 500mM imidazole lysate;
s5011, concentrating, eluting and removing a solution by using a 3K ultrafiltration tube (Milipore), and measuring the final concentration of the target protein by using a BCA method;
s6, researching the influence of the ANGPTL8 recombinant protein on the tilapia feed by intraperitoneal injection:
s601, randomly distributing tilapia with the weight of 2 +/-0.5 g into 2 glass jars, and averaging 6 tilapia in each jar;
s602, feeding tilapia once every 4 pm, wherein the feeding amount of food is 3% of the body weight, and starting formal experiments after two weeks of domestication;
s603, after tilapia is narcotized by MS-222, weighing individual Body Weight (BW) in each group, and injecting 0.7% fish normal saline (a control group) and 1 mug/g BW ANGPTL8 into the abdominal cavity according to the body weight;
s604, feeding food with fixed weight to each group of tilapia respectively after 10 minutes of injection, and allowing each group of tilapia to freely eat for 2 hours;
and S605, respectively collecting and drying the rest food of each group, and calculating the food intake of the single heavy tilapia in each group according to the weight of the rest food.
Further, the specific reaction procedures involved in qPCR described in step S303 are as follows: pre-denaturation at 95 ℃ for 2min, followed by denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s for a total of 40 cycles.
Further, three replicates of each sample were set up for the qPCR reaction in step S303.
Further, the ultrasonic conditions described in step S507 are: 6mm probe, 40% power, work for 4s, rest for 9s, and work time for 10 min.
Furthermore, the specific primers AN-F1, AN-F2, AN-F3, AN-R1, AN-R2 and AN-R3 are respectively named as: 1, 2, 3, 4, 5 and 6, the sequences of which are respectively:
SEQ ID NO:1 5’to3’atgatctggagcctgtgcttgc；
SEQ ID NO:2 5’to3’gcaagcacaggctccagatcat；
SEQ ID NO:3 5’to3’tgccaccttgtattccgtcttgctgg；
SEQ ID NO:4 5’to3’ttacatatttccatgtcttctta；
SEQ ID NO:5 5’to3’caggtaagaagacatggaaata；
SEQ ID NO:6 5’to3’cactgagagaactctccaaaagctga。
compared with the prior art, the invention has the following advantages:
1. the fish angptl8 gene is expressed in a large amount in fish livers, mature peptides generated by the gene have the function of regulating fish ingestion, can be developed and utilized by a biosynthesis method, and has the advantages of convenience in use, remarkable effect and wide application value. The scheme adopts prokaryotic expression to prepare the recombinant protein, and has the advantages of high yield, low cost, convenient operation, easy repetition and the like.
2. Based on theoretical research of tilapia, the invention identifies that mature peptide ANGPTL8 coded by the fish angiopoietin-like 8 gene has the function of regulating fish food intake. The attractant with high activity, no toxicity and low cost is developed and has wide application value as a feed additive for promoting the development of cultured fishes and the like.
Drawings
FIG. 1 shows the expression analysis of the angptl8 gene of fish of the invention in various tissues;
FIG. 2 shows the expression change of the angptl8 gene of fish of the invention during feeding;
FIG. 3 is a diagram showing the purification of recombinant fish ANGPTL8 protein according to the present invention;
FIG. 4 is a graph showing the effect of intraperitoneal injection of fish ANGPTL8 on tilapia feed;
Detailed Description
For further explanation of the invention, reference should be made to the following description taken in conjunction with the accompanying drawings.
A preparation method of fish food intake regulating peptide comprises the following steps:
s1, extracting total tilapia liver RNA:
taking a healthy tilapia, dissecting, separating liver tissue, and obtaining total tilapia liver RNA (OD) by using Trizol reagent method260/280＝1.95，OD260/2302.0. Electrophoresis results show that the 28S rRNA and 18S rRNA bands are clear, and the brightness of the 28S band is twice of that of 18S, which shows that the obtained total RNA is not polluted by protein, phenol and genome DNA and has high purity;
s2, cloning complete sequence of tilapia angptl8 gene cDNA:
s201, designing specific primers AN-F1(5' atgatctggagcctgtgcttgc 3') and AN-R1(ttacatatttccatgtcttctta 3') by using the synthesized first strand cDNA as a template, and carrying out PCR amplification;
s202, loading the PCR product to 1.5% agarose gel, separating DNA fragments by low-voltage electrophoresis, and purifying and recovering a target product from the gel;
s203, connecting the purified target product to
Vector, transforming DH5 alpha competent cell, selecting positive clone to sequence;
s204, designing a specific primer according to the obtained cDNA fragment sequence, and carrying out PCR amplification on the 3 'end and the 5' end of the target gene by utilizing a cDNA end rapid amplification technology;
s205, according to RACE kit GeneRacerTMThe Kit specification performs dephosphorylation on total RNA of the liver of the tilapia, removes a mRNA 5' cap structure, is connected with the RNA Oligo, and finally synthesizes a cDNA first chain through reverse transcription;
s206, 3'-RACE with tilapia angptl8cDNA, 5' -RACE specific primers AN-F2(5'gcaagcacaggctccagatcat 3'), AN-F3(5'tgccaccttgtattccgtcttgctgg 3') and AN-R2(5'caggtaagaagacatggaaata 3'), AN-R3(5'cactgagagaactctccaaaagctga 3'), and GeneRacerTMAmplifying a 5 'end fragment and a 3' end fragment of tilapia angptl8cDNA by using a universal primer of Kit;
s207, after the obtained PCR product is recovered and purified, connecting a T vector, converting DH5 alpha competence, screening positive colonies by using a blue white spot, performing DNA sequencing after colony PCR verification, and cloning in tilapia by a molecular cloning technology to obtain an ANGPTL8cDNA sequence 1059bp, wherein an Open Reading Frame (ORF) sequence 321bp, an encoded ANGPTL8 protein is 106 amino acids, a protein precursor isoelectric point is 9.37, the molecular weight is 12.04 kilodaltons, and an N-terminal signal peptide consists of 22 amino acids (a nucleic acid sequence table);
s3, tissue expression profile of tilapia angptl 8:
detecting the expression of tilapia angptl8 in various tissues by using a real-time fluorescent quantitative PCR (polymerase chain reaction) technology;
s301, extracting total RNA of 12 tissues such as hypothalamus, telencephalon, optic cap, cerebellum, olfactory bulb, medulla oblongata, spinal cord, pituitary, liver, fat, intestine, muscle and the like of tilapia;
s302, after DNaseI treatment, taking 1 mu g of RNA for reverse transcription according to the specification of iScriptTM;
s303, diluting the reverse transcription product by 10 times to be used as a cDNA template, referring to iQTM 
The GreenSupermix instruction prepares a qPCR reaction system, and the specific reaction procedures involved in qPCR are as follows: pre-denaturation at 95 ℃ for 2min, then denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s, and performing 40 cycles;
s304, determining the specificity of the amplification product by analyzing a qPCR dissolution curve;
in order to ensure the credibility of the result, three parallels are arranged for each sample when the qPCR reaction is carried out;
the real-time fluorescent quantitative PCR is adopted to detect the expression of tilapia angptl8 gene in various tissues, and the result shows that the expression abundance is highest in liver and muscle; the expression level is lower in other tissues. It was shown that angptl8 has significant tissue expression specificity (fig. 1);
s4, the expression of tilapia angptl8 changes in the feeding process:
s401, randomly distributing tilapia with the weight of 2 +/-0.5 g into four glass jars, wherein the four glass jars are respectively a control group and 3 feeding groups, and 6 tilapia are averagely distributed in each jar;
s402, feeding for 1 time at 4 pm every day, wherein the food feeding amount is 3% of the body weight, and formally starting an experiment after two weeks of domestication;
s403, hypothalamus of normally fed tilapia was sampled at the following time points: 1h before ingestion, 0hr during ingestion, 1hr after ingestion, and 3hr after ingestion;
s404, killing and collecting hypothalamus samples after the tilapia is anesthetized by 0.05% MS-222, carrying out liquid nitrogen quick freezing treatment, and storing in an ultra-low temperature refrigerator at minus 80 ℃ for later use;
s405, detecting the change of the angptl8 gene expression amount in the hypothalamus of tilapia by a qPCR (quantitative polymerase chain reaction) technology;
the real-time fluorescent quantitative PCR is used for detecting the change of tilapia gene in the eating state, and the result shows that: hypothalamic angptl8 mRNA expression levels were significantly inhibited after 1 hour of feeding, indicating that angptl8 is associated with feeding regulation (fig. 2);
s5 and ANGPTL8 recombinant protein preparation:
s501, taking tilapia liver tissue cDNA as a template, and amplifying the full length of angptl8cDNA sequence by using a Taq enzyme kit;
s502, performing electrophoresis on the amplified product by using 1.5% agarose gel, and recovering the target fragment by using a gel recovery kit;
s503, carrying out double enzyme digestion on the purified fragment, connecting the purified fragment with a pET30a (+) expression vector, and converting BL21(DE3) competent cells;
s504, screening positive expression colonies by using an LB (LB) culture medium (Kana antibiotic: 50mg/ml), and preserving the strains after PCR verification of a bacterial liquid;
s505, adding bacterial liquid of angptl8-pET30a (+) -BL21(DE3) according to the volume of 1% LB culture medium (Kana antibiotic: 50mg/ml), shaking overnight at 37 ℃ and 200rpm, pouring the bacterial liquid into 100 times of LB culture medium (Kana antibiotic: 50mg/ml), culturing at 37 ℃ and 200rpm until the bacterial liquid concentration OD600 is approximately equal to 0.5 (about 3hr), adding IPTG until the final concentration is 0.1mM, and inducing expression at 37 ℃ and 200rpm for 6 hr;
s506, centrifuging the bacterial liquid at 5000 Xg for 2min, then discarding the supernatant, and rinsing with a lysate for 2 times;
s507, adding lysis solution according to 10% of the volume of the bacterial solution, and carrying out ultrasonic crushing in ice bath under the ultrasonic condition: 6mm probe, 40% power, working for 4s, resting for 9s, and working time for 10 min;
s508, centrifuging the broken solution at 10,000 Xg 4 ℃ for 30min, and filtering the supernatant by using a 0.45-micron filter;
s509, purifying the protein by using a His tag protein purification kit (Biyuntian biotechnology company), wherein the elution conditions are as follows: washing the hybrid protein by 50mM imidazole and 100mM imidazole lysate, and eluting the target protein by 500mM imidazole lysate;
s5011, concentrating the elution and elution solution by using a 3K ultrafiltration tube (Milipore), and measuring the final concentration of the target protein to be 1.2g/L by using a BCA method (figure 3);
the active protein is obtained by purifying a large amount of in vitro recombinant protein ANGPTL8 prokaryotic expression (figure 3);
s6, researching the influence of the ANGPTL8 recombinant protein on the tilapia feed by intraperitoneal injection:
s601, randomly distributing tilapia with the weight of 2 +/-0.5 g into 2 glass jars, and averaging 6 tilapia in each jar;
s602, feeding tilapia once every 4 pm, wherein the feeding amount of food is 3% of the body weight, and starting formal experiments after two weeks of domestication;
s603, after tilapia is narcotized by MS-222, weighing individual Body Weight (BW) in each group, and injecting 0.7% fish normal saline (a control group) and 1 mug/g BW ANGPTL8 into the abdominal cavity according to the body weight;
s604, feeding food with fixed weight to each group of tilapia respectively after 10 minutes of injection, and allowing each group of tilapia to freely eat for 2 hours;
s605, respectively collecting and drying the rest foods of each group, and calculating the food intake of the unit heavy tilapia in each group according to the weight of the rest foods;
the application of intraperitoneal injection of the ANGPTL8 mature peptide expressed by chemical pronucleus shows that the ANGPTL8 can remarkably increase the food intake of tilapia (figure 4).
Therefore, the mature peptide angptl8 encoded by the fish angptl8 gene of the invention can be used to induce fish feeding.
Sequence listing
<110> Sichuan university
<120> preparation and application of fish ingestion regulating peptide
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
atgatctgga gcctgtgctt gc 22
<210> 2
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gcaagcacag gctccagatc at 22
<210> 3
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
tgccaccttg tattccgtct tgctgg 26
<210> 4
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ttacatattt ccatgtcttc tta 23
<210> 5
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
caggtaagaa gacatggaaa ta 22
<210> 6
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
cactgagaga actctccaaa agctga 26
<210> 7
<211> 1059
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
gctgaggggc tgatgacact gctatgacag acaagtgcct gtttatggct ttcatttttg 60
tctcatacat aaaaaaaaag ctttattttg cacaggcgct attgctgaaa gataaatcag 120
aaatatcttt tgaacatttg aaagaactga ggaaacaaca tttattatcg caatgatctg 180
gagcctgtgc ttgctttttg tggctggagc agtgcacgca agcccggtca ggaagaccag 240
caagacggaa tacaaggtgg caccgcaaga agaagtcaat gtgctcatgt ttggtgtctt 300
acagtttggc gaatccctaa actatgctta tgaaaccact gaggcaaaga tagcgagaat 360
cagccggtct ttgaagaaca ctgagagaac tctccaaaag ctggggaaac agactgagca 420
ggctgcagag gtggagaaac agatcaaaga agtgatacag ctgctacagg taagaagaca 480
tggaaatatg taaagaccac aaagactaaa gaatggctga ccagcctgga gaaggaagag 540
caggagctga ggacaaaagt gaaccggttg gagatgcacc tcagcaactc tgtacctcca 600
agcatcaagg agctgcagga gagagcagag gagcacgaca aagtcttgcg aggcttacag 660
cttttgactc agttccaaaa agaggatatt gagagtcaga atgagcagct tgccaaactg 720
cagaagatga gtgaagtgct gacatgatcc agatctcaac tccataaact cactgcactc 780
ccttccaacc ttgctatgga tgctatagat caactgtact ggcagaaaat atatattttg 840
ggtccttcat aattacatcc ttgttattgt gcctgtaatt tatattttgt gcattgtttt 900
tgtgtcttta catcagtttg ctttgtaaat ttgcttttta aaataagctg ttaaattatt 960
aaagattgtg tttggtgaac tagtattata tgtacacgct gtacatagaa taaaaacaat 1020
ttgtttgttt tttgtaaaaa aaaaaaaaaa aaaaaaaaa 1059
Claims (5)
1. A preparation method of fish feeding control peptide is characterized by comprising the following steps:
s1, extracting total tilapia liver RNA:
taking a healthy tilapia, dissecting and separating liver tissues, and obtaining total tilapia liver RNA by adopting a Trizol reagent method;
s2, cloning complete sequence of tilapia angptl8 gene cDNA:
s201, designing specific primers AN-F1 and AN-R1 by taking the synthesized first strand cDNA as a template, and carrying out PCR amplification;
s202, loading the PCR product to 1.5% agarose gel, separating DNA fragments by low-voltage electrophoresis, and purifying and recovering a target product from the gel;
s203, connecting the purified target product to
Easy vector, transforming DH5 alpha competent cell, selecting positive clone to sequence;
s204, designing a specific primer according to the obtained cDNA fragment sequence, and carrying out PCR amplification on the 3 'end and the 5' end of the target gene by utilizing a cDNA end rapid amplification technology;
s205, according to RACE kit GeneRacerTMThe Kit specification performs dephosphorylation on total RNA of the liver of the tilapia, removes a mRNA 5' cap structure, is connected with the RNA Oligo, and finally synthesizes a cDNA first chain through reverse transcription;
s206, 3'-RACE and 5' -RACE specific primers AN-F2, AN-F3, AN-R2, AN-R3 and GeneRacer of tilapia angptl8cDNATMAmplifying a 5 'end fragment and a 3' end fragment of tilapia angptl8cDNA by using universal primers of Kit;
s207, after the obtained PCR product is recovered and purified, connecting a T vector, transforming DH5 alpha competence, screening positive colonies by using blue white spots, and performing DNA sequencing after colony PCR verification;
s3 tissue expression profile of tilapia angptl 8:
detecting the expression of tilapia angptl8 in various tissues by using a real-time fluorescent quantitative PCR (polymerase chain reaction) technology;
s301, extracting total RNA of 12 tissues such as hypothalamus, telencephalon, optic cap, cerebellum, olfactory bulb, medulla oblongata, spinal cord, pituitary, liver, fat, intestine, muscle and the like of tilapia;
s302, after DNaseI treatment, taking 1 mu g of RNA for reverse transcription according to the specification of iScriptTM;
s303, diluting the reverse transcription product by 10 times to be used as a cDNA template, referring to iQTM 
GreenSupermix instructions preparation of qPCR reaction system;
s304, determining the specificity of the amplification product by analyzing a qPCR dissolution curve;
s4, the expression of tilapia angptl8 changes in the feeding process:
s401, randomly distributing tilapia with the weight of 2 +/-0.5 g into four glass jars, wherein the four glass jars are respectively a control group and 3 feeding groups, and 6 tilapia are averagely distributed in each jar;
s402, feeding for 1 time at 4 pm every day, wherein the food feeding amount is 3% of the body weight, and formally starting an experiment after two weeks of domestication;
s403, hypothalamus of normally fed tilapia was sampled at the following time points: 1h before ingestion, 0hr during ingestion, 1hr after ingestion, and 3hr after ingestion;
s404, after the tilapia is anesthetized by 0.05% MS-222, the tilapia is killed and collected with hypothalamic samples, and then the samples are subjected to quick freezing treatment by liquid nitrogen and stored in an ultra-low temperature refrigerator at minus 80 ℃ for later use;
s405, detecting the change of the angptl8 gene expression amount in the hypothalamus of tilapia by a qPCR (quantitative polymerase chain reaction) technology;
s5 and ANGPTL8 recombinant protein preparation:
s501, taking tilapia liver tissue cDNA as a template, and amplifying the full length of angptl8cDNA sequence by using a Taq enzyme kit;
s502, carrying out electrophoresis on the amplified product by using 1.5% agarose gel, and recovering the target fragment by using a gel recovery kit;
s503, carrying out double enzyme digestion on the purified fragment, connecting the purified fragment with a pET30a (+) expression vector, and converting BL21(DE3) competent cells;
s504, screening positive expression colonies by using an LB culture medium, and preserving the strains after PCR verification of a bacterial liquid;
s505, adding angptl8-pET30a (+) -BL21(DE3) bacterial liquid according to the volume of 1% LB culture medium, shaking overnight at 37 ℃ and 200rpm, pouring the bacterial liquid into 100-fold volume of LB culture medium, culturing at 37 ℃ and 200rpm until the bacterial liquid concentration OD600 is approximately equal to 0.5, adding IPTG until the final concentration is 0.1mM, and performing induction expression at 37 ℃ and 200rpm for 6 hours;
s506, centrifuging the bacterial liquid at 5000 Xg for 2min, then discarding the supernatant, and rinsing the supernatant for 2 times by using a lysis solution;
s507, adding lysis solution according to the volume of 10% of the bacterial solution, and carrying out ultrasonic crushing in ice bath;
s508, centrifuging the broken solution at 10,000 Xg 4 ℃ for 30min, and filtering the supernatant by using a 0.45-micron filter;
s509, purifying the protein by using a His tag protein purification kit, wherein the elution conditions are as follows: washing the hybrid protein by 50mM imidazole and 100mM imidazole lysate, and eluting the target protein by 500mM imidazole lysate;
s5011, concentrating, eluting and removing a solution by using a 3K ultrafiltration tube, and measuring the final concentration of the target protein by using a BCA method;
s6, researching the influence of the ANGPTL8 recombinant protein on the tilapia feed by intraperitoneal injection:
s601, randomly distributing tilapia with the weight of 2 +/-0.5 g into 2 glass jars, and averaging 6 tilapia in each jar;
s602, feeding tilapia once every 4 pm, wherein the feeding amount of food is 3% of the body weight, and starting formal experiments after two weeks of domestication;
s603, after tilapia is narcotized by MS-222, weighing individual weight of each group, and injecting 0.7% fish physiological saline and 1 microgram/g BW ANGPTL8 into the abdominal cavity according to the weight;
s604, feeding food with fixed weight to each group of tilapia respectively after 10 minutes of injection, and allowing each group of tilapia to freely eat for 2 hours;
and S605, respectively collecting and drying the rest food of each group, and calculating the food intake of the single heavy tilapia in each group according to the weight of the rest food.
2. The preparation of the fish feeding control peptide of claim 1, wherein the specific reaction sequence involved in qPCR in step S303 is as follows: pre-denaturation at 95 ℃ for 2min, followed by denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s for a total of 40 cycles.
3. The production of the fish feeding control peptide of claim 1, wherein three replicates of each sample are used in the qPCR reaction of step S303.
4. The method for preparing a fish feeding control peptide according to claim 1, wherein the ultrasonic conditions in step S507 are as follows: 6mm probe, 40% power, work for 4s, rest for 9s, and work time for 10 min.
5. The method for preparing fish feeding control peptide according to claim 1, wherein the specific primers AN-F1, AN-F2, AN-F3, AN-R1, AN-R2 and AN-R3 are respectively named as: 1, 2, 3, 4, 5 and 6, the sequences of which are respectively:
SEQ ID NO:1 5’to3’atgatctggagcctgtgcttgc；
SEQ ID NO:2 5’to3’gcaagcacaggctccagatcat；
SEQ ID NO:3 5’to3’tgccaccttgtattccgtcttgctgg；
SEQ ID NO:4 5’to3’ttacatatttccatgtcttctta；
SEQ ID NO:5 5’to3’caggtaagaagacatggaaata；
SEQ ID NO:6 5’to3’cactgagagaactctccaaaagctga。
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