CN114671943B - Preparation and application of fish ingestion regulating protein - Google Patents
Preparation and application of fish ingestion regulating protein Download PDFInfo
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Abstract
The invention discloses a preparation method of fish ingestion regulating protein, which relates to the technical field of genetic engineering and comprises the following steps: s1, extracting total RNA of the liver of the tilapia; s2, cloning the complete sequence of the tilapia angptl8 gene cDNA; s3, tissue expression distribution of tilapia angptl8; s4, the expression change of tilapia angptl8 in the feeding process; s5, preparing an angptl8 recombinant protein; s6, researching the influence of the angptl8 recombinant protein on tilapia ingestion by intraperitoneal injection. The fish angptl8 gene is expressed in a large amount in fish livers, and the mature protein generated by the gene has the function of regulating fish ingestion, can be developed and utilized by a biosynthesis method, is convenient to use, has obvious effect and has wide application value. In the scheme, the recombinant protein is prepared by prokaryotic expression, and the method 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 fish ingestion regulating protein.
Background
The existing phagostimulant has low activity, large dosage, high production cost and certain toxic and side effects. The invention aims at providing an angiopoietin-like 8 gene encoding a mature protein. The invention also aims to provide application of the fish angptl8 neuroprotein gene.
Disclosure of Invention
The invention aims at solving the existing problems and provides preparation and application of fish ingestion regulating protein.
The invention is realized by the following technical scheme:
a preparation method of fish ingestion regulating protein, comprising the following steps:
s1, extracting total RNA of tilapia liver:
taking a piece of healthy tilapia, dissecting and separating liver tissues, and obtaining total RNA of the liver of the tilapia by adopting a Trizol reagent method;
s2, cloning the complete sequence of the tilapia angptl8 gene cDNA:
s201, designing specific primers AN-F1 and AN-R1 by taking the synthesized first strand cDNA as a template, and performing PCR amplification;
s202, loading the obtained 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
Transforming DH5 alpha competent cells by Easy vector, and selecting positive clone for sequencing;
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 using a cDNA end rapid amplification technology;
s205, geneRacer according to RACE kit TM The Kit instruction book is used for dephosphorylating the total RNA of the liver of the tilapia mossambica, removing the 5' cap structure of the mRNA, connecting with the RNAoligo, and finally synthesizing a cDNA first chain through reverse transcription;
s206, 3'-RACE, 5' -RACE specific primers AN-F2, AN-F3 and AN-R2, AN-R3, and GeneRacer with Tilapia angptl8cDNA TM Amplifying a5 'end fragment and a 3' end fragment of the tilapia angptl8cDNA by using a universal primer of Kit;
s207, after recovery and purification of the obtained PCR product, connecting a T vector, transforming DH5 alpha competence, screening positive colonies by utilizing blue white spots, and performing DNA sequencing after colony PCR verification;
s3, tissue expression distribution of tilapia angptl 8:
detecting the expression of tilapia angptl8 in various tissues by using a real-time fluorescence quantitative PCR technology;
s301, extracting 12 tissue total RNAs of hypothalamus, end brain, optic cap, cerebellum, olfactory bulb, medulla oblongata, spinal cord, pituitary, liver, fat, intestine, muscle and the like of tilapia;
s302, after DNaseI treatment, 1 mug of RNA is taken for reverse transcription according to the iScript specification;
s303, diluting the reverse transcription product 10 times as cDNA template, referring to iQ TM 
The GreenSupermix instruction prepares a qPCR reaction system;
s304, determining the specificity of the amplified product by analyzing a qPCR dissolution curve;
s4, expression change of tilapia angptl8 in feeding process:
s401, randomly distributing tilapia with the weight of 2+/-0.5 g into four glass cylinders, wherein the four glass cylinders are respectively a control group and 3 feeding groups, and 6 tilapia are averagely arranged in each cylinder;
s402, feeding 1 time every day at 4 pm, wherein the feeding amount of food is 3% of the weight, and after two weeks of domestication, the experiment is formally started;
s403, sampling hypothalamus of normal feeding tilapia according to the following time points: 1h before ingestion, during ingestion (0 hr), 1hr after ingestion, and 3hr after ingestion;
s404, after the tilapia is anesthetized by 0.05% MS-222, the hypothalamus sample is killed and collected, and the tilapia is quickly frozen by liquid nitrogen and stored in an ultralow temperature refrigerator at the temperature of minus 80 ℃ for standby;
s405, detecting the change of the angptl8 gene expression level in the hypothalamus of the tilapia by a qPCR technology;
s5, preparation of an angptl8 recombinant protein:
s501, using the liver tissue cDNA of tilapia as a template, and amplifying the full length of the angptl8cDNA sequence by using a Taq enzyme kit;
s502, electrophoresis is carried out on the amplified product by using 1.5% agarose gel, and target fragment recovery is carried out by using a gel recovery kit;
s503, connecting the purified fragment with a pET30a (+) expression vector after double enzyme digestion, and converting BL21 (DE 3) competent cells;
s504, screening positive expression colonies by using an LB culture medium (Kana antibiotics: 50 mg/ml), and preserving seeds after bacterial liquid PCR verification;
s505, adding an angptl8-pET30a (+) -BL21 (DE 3) bacterial liquid according to the volume of 1% LB culture medium (Kana antibiotics: 50 mg/ml), shaking the mixture at 37 ℃ and 200rpm overnight, pouring the bacterial liquid into 100 times of the volume of LB culture medium (Kana antibiotics: 50 mg/ml), culturing at 37 ℃ and 200rpm until the bacterial liquid concentration OD600 apprxeq 0.5 (about 3 hr), adding IPTG to a final concentration of 0.1mM, and performing induction expression at 200rpm for 6hr;
s506, centrifuging the bacterial liquid with 5000 Xg for 2min, discarding supernatant, and rinsing with lysis solution for 2 times;
s507, adding a lysate according to 10% of the bacterial liquid volume, and carrying out ice bath ultrasonic crushing;
s508, centrifuging the crushed solution for 30min at 10,000Xg and 4 ℃ and filtering the supernatant by using a 0.45 mu m filter;
s509, purifying protein by using a His tag protein purification kit (Biyun Tian biotechnology company), wherein the elution conditions are as follows: washing the hybrid protein with 50mM imidazole and 100mM imidazole lysate, eluting the target protein with 500mM imidazole lysate;
s5011, concentrating the eluent by using a 3K ultrafiltration tube (Milopore), and measuring the final concentration of the target protein by using a BCA method;
s6, researching the influence of the angptl8 recombinant protein on tilapia ingestion by intraperitoneal injection:
s601, randomly distributing tilapia with a weight of 2+/-0.5 g into 2 glass cylinders, wherein the average weight of the tilapia is 6 per cylinder;
s602, feeding tilapia once every day at 4 pm, wherein the feeding amount of food is 3% of the weight, and after two weeks of domestication, starting a formal experiment;
s603, after the tilapia is anesthetized by MS-222, weighing the Body Weight (BW) of each group, and respectively injecting 0.7% of fish physiological saline (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 after 10 minutes of injection, and enabling each group of tilapia to eat freely for 2 hours;
and S605, respectively collecting and drying the rest food in each group, and calculating the ingestion amount of tilapia per unit weight in each group according to the weight of the rest food.
Further, the specific reaction procedure involved in qPCR described in step S303 is as follows: the pre-denaturation at 95℃for 2min, then denaturation at 95℃for 30s, annealing at 60℃for 30s and extension at 72℃for 30s were performed for 40 cycles.
Further, three parallel were set for each sample in the qPCR reaction in step S303.
Further, the ultrasound conditions described in step S507: 6mm probe, 40% power, 4s working, 9s rest, 10min working time.
Further, the specific primers AN-F1, AN-F2, AN-F3, AN-R1, AN-R2 and AN-R3 are respectively named as follows: SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 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, and the mature protein generated by the gene has the function of regulating fish ingestion, can be developed and utilized by a biosynthesis method, is convenient to use, has obvious effect and has wide application value. In the scheme, the recombinant protein is prepared by prokaryotic expression, and the method 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 the mature protein angptl8 coded by the fish angiopoietin-like 8 gene has the function of regulating fish ingestion. The feed additive has wide application value for developing a high-activity, nontoxic and low-cost phagostimulant which is used as a feed additive and promoting the development of farmed fish and the like.
Drawings
FIG. 1 shows the expression analysis of the fish angptl8 gene of the present invention in various tissues;
FIG. 2 shows the expression change of the fish angptl8 gene during feeding;
FIG. 3 is a diagram showing the purification of the recombinant protein of fish angptl8 of the present invention;
FIG. 4 shows the effect of the fish angptl8 intraperitoneal injection on tilapia ingestion according to the present invention;
Detailed Description
For a further explanation of the invention, reference is made to the accompanying drawings and specific embodiments.
A preparation method of fish ingestion regulating protein, comprising the following steps:
s1, extracting total RNA of tilapia liver:
taking a piece of healthy tilapia, dissecting and separating liver tissues, and obtaining total RNA of the tilapia liver by adopting a Trizol reagent method, wherein OD (OD) of the total RNA 260/280 ＝1.95，OD 260/230 =2.0. The electrophoresis result shows that the 28S rRNA,18S rRNA band is clear, the 28S band has the brightness of twice of 18S, and the obtained total RNA is not polluted by protein, phenol and genome DNA, so that the purity is high;
s2, cloning the complete sequence of the tilapia angptl8 gene cDNA:
s201, designing specific primers AN-F1 (5'atgatctggagcctgtgcttgc 3 ') and AN-R1 (ttacatatttccatgtcttctta ') by taking the synthesized first strand cDNA as a template, and performing PCR amplification;
s202, loading the obtained 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
Transforming DH5 alpha competent cells by using a vector, and selecting positive clones for sequencing;
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 using a cDNA end rapid amplification technology;
s205, geneRacer according to RACE kit TM Kit specification for dephosphorylating liver total RNA of tilapiaRemoving the mRNA5' cap structure, connecting with RNA Oligo, and finally synthesizing cDNA first strand by reverse transcription;
s206, 3'-RACE, 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 GeneRacer using tilapia angptl8cDNA TM Amplifying a5 'end fragment and a 3' end fragment of the tilapia angptl8cDNA by using a universal primer of Kit;
s207, after recovering and purifying the obtained PCR product, connecting a T vector, transforming DH5 alpha competence, screening positive colonies by utilizing blue white spots, performing DNA sequencing after colony PCR verification, and cloning in tilapia mossambica to obtain an angptl8cDNA sequence 1059bp, wherein an Open Reading Frame (ORF) sequence 321bp, the encoded angptl8 protein is 106 amino acids, the isoelectric point of a protein precursor is 9.37, the molecular weight is 12.04 kilodaltons, and N-terminal signal protein consists of 22 amino acids (a nucleic acid sequence table);
s3, tissue expression distribution of tilapia angptl 8:
detecting the expression of tilapia angptl8 in various tissues by using a real-time fluorescence quantitative PCR technology;
s301, extracting 12 tissue total RNAs of hypothalamus, end brain, optic cap, cerebellum, olfactory bulb, medulla oblongata, spinal cord, pituitary, liver, fat, intestine, muscle and the like of tilapia;
s302, after DNaseI treatment, 1 mug of RNA is taken for reverse transcription according to the iScript specification;
s303, diluting the reverse transcription product 10 times as cDNA template, referring to iQ TM 
The GreenSupermix specification prepares a qPCR reaction system, and the specific reaction procedure involved in qPCR is as follows: pre-denaturing at 95 ℃ for 2min, then denaturing at 95 ℃ for 30s, annealing at 60 ℃ for 30s, and extending at 72 ℃ for 30s, and performing 40 cycles in total;
s304, determining the specificity of the amplified product by analyzing a qPCR dissolution curve;
in order to ensure the credibility of the result, three parallel samples are arranged in each sample when qPCR reaction is carried out;
detecting the expression of the tilapia angptl8 gene in various tissues by adopting real-time fluorescence quantitative PCR, and displaying that the expression abundance is highest in livers and muscles; the expression level is lower in other tissues. Angptl8 was shown to have significant tissue expression specificity (fig. 1);
s4, expression change of tilapia angptl8 in feeding process:
s401, randomly distributing tilapia with the weight of 2+/-0.5 g into four glass cylinders, wherein the four glass cylinders are respectively a control group and 3 feeding groups, and 6 tilapia are averagely arranged in each cylinder;
s402, feeding 1 time every day at 4 pm, wherein the feeding amount of food is 3% of the weight, and after two weeks of domestication, the experiment is formally started;
s403, sampling hypothalamus of normal feeding tilapia according to the following time points: 1h before ingestion, during ingestion (0 hr), 1hr after ingestion, and 3hr after ingestion;
s404, after the tilapia is anesthetized by 0.05% MS-222, the hypothalamus sample is killed and collected, and the tilapia is quickly frozen by liquid nitrogen and stored in an ultralow temperature refrigerator at the temperature of minus 80 ℃ for standby;
s405, detecting the change of the angptl8 gene expression level in the hypothalamus of the tilapia by a qPCR technology;
the real-time fluorescence quantitative PCR is used for detecting the change of the tilapia gene in a fed state, and the result shows that: hypothalamic angptl8mRNA expression levels were significantly inhibited after 1 hour of feeding, indicating that angptl8 is associated with feeding regulation (fig. 2);
s5, preparation of an angptl8 recombinant protein:
s501, using the liver tissue cDNA of tilapia as a template, and amplifying the full length of the angptl8cDNA sequence by using a Taq enzyme kit;
s502, electrophoresis is carried out on the amplified product by using 1.5% agarose gel, and target fragment recovery is carried out by using a gel recovery kit;
s503, connecting the purified fragment with a pET30a (+) expression vector after double enzyme digestion, and converting BL21 (DE 3) competent cells;
s504, screening positive expression colonies by using an LB culture medium (Kana antibiotics: 50 mg/ml), and preserving seeds after bacterial liquid PCR verification;
s505, adding an angptl8-pET30a (+) -BL21 (DE 3) bacterial liquid according to the volume of 1% LB culture medium (Kana antibiotics: 50 mg/ml), shaking the mixture at 37 ℃ and 200rpm overnight, pouring the bacterial liquid into 100 times of the volume of LB culture medium (Kana antibiotics: 50 mg/ml), culturing at 37 ℃ and 200rpm until the bacterial liquid concentration OD600 apprxeq 0.5 (about 3 hr), adding IPTG to a final concentration of 0.1mM, and performing induction expression at 200rpm for 6hr;
s506, centrifuging the bacterial liquid with 5000 Xg for 2min, discarding supernatant, and rinsing with lysis solution for 2 times;
s507, adding a lysate according to the volume of 10% of the bacterial liquid, carrying out ice bath ultrasonic crushing, and carrying out ultrasonic conditions: a 6mm probe, 40% power, 4s working, 9s rest and 10min working time;
s508, centrifuging the crushed solution for 30min at 10,000Xg and 4 ℃ and filtering the supernatant by using a 0.45 mu m filter;
s509, purifying protein by using a His tag protein purification kit (Biyun Tian biotechnology company), wherein the elution conditions are as follows: washing the hybrid protein with 50mM imidazole and 100mM imidazole lysate, eluting the target protein with 500mM imidazole lysate;
s5011, concentrating the eluent by using a 3K ultrafiltration tube (Milpore), wherein the final concentration of the target protein is 1.2g/L (figure 3) by a BCA method;
the active protein is obtained by large-scale purification after prokaryotic expression of the recombinant protein angptl8 in vitro (figure 3);
s6, researching the influence of the angptl8 recombinant protein on tilapia ingestion by intraperitoneal injection:
s601, randomly distributing tilapia with a weight of 2+/-0.5 g into 2 glass cylinders, wherein the average weight of the tilapia is 6 per cylinder;
s602, feeding tilapia once every day at 4 pm, wherein the feeding amount of food is 3% of the weight, and after two weeks of domestication, starting a formal experiment;
s603, after the tilapia is anesthetized by MS-222, weighing the Body Weight (BW) of each group, and respectively injecting 0.7% of fish physiological saline (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 after 10 minutes of injection, and enabling each group of tilapia to eat freely for 2 hours;
s605, respectively collecting and drying the rest food in each group, and calculating the ingestion amount of tilapia in each group according to the weight of the rest food;
the actual application of the angptl8 mature protein expressed by the chemoprokaryote by intraperitoneal injection shows that the angptl8 can remarkably increase the feeding capacity of tilapia (figure 4).
Therefore, the mature protein angptl8 coded by the fish angptl8 gene can be used for inducing fish to ingest.
Sequence listing
<110> university of Sichuan
<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
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<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 (2)
1. The preparation method of the fish ingestion regulating protein is characterized by comprising the following steps:
(1) Using tilapia liver tissue cDNA as a template, and amplifying the full length of an angptl8cDNA sequence by using a Taq enzyme kit, wherein the angptl8cDNA sequence is shown as a sequence 7;
(2) Electrophoresis is carried out on the amplified product by using 1.5% agarose gel, and target fragment recovery is carried out by using a gel recovery kit;
(3) Double-enzyme cutting the purified fragment, connecting with a pET30a (+) expression vector, and converting BL21 (DE 3) competent cells;
(4) Screening positive expression colonies by utilizing an LB culture medium, and preserving seeds after bacterial liquid PCR verification;
(5) Adding angptl8-pET30a (+) -BL21 (DE 3) bacterial liquid according to the volume of 1% LB culture medium, pouring the bacterial liquid into 100 times of LB culture medium after shaking at 37 ℃ and 200rpm for culturing at 37 ℃ until the bacterial liquid concentration OD600 is approximately equal to 0.5, adding IPTG to the final concentration of 0.1mM, and inducing and expressing at 200rpm for 6hr at 37 ℃;
(6) Centrifuging the bacterial liquid with the concentration of 5000 Xg for 2min, discarding the supernatant, and rinsing with the lysate for 2 times;
(7) Adding a lysate according to 10% of the volume of the bacterial liquid, and carrying out ice bath ultrasonic crushing;
(8) The crushed solution was centrifuged at 10,000Xg for 30min at 4℃and the supernatant was filtered using a 0.45 μm filter;
(9) Purifying protein by using His tag protein purification kit, and eluting under the following conditions: washing the hybrid protein with 50mM imidazole and 100mM imidazole lysate, eluting the target protein with 500mM imidazole lysate;
(10) The eluate was concentrated using a 3K ultrafiltration tube and the final concentration of the target protein was measured by BCA method.
2. The method for preparing fish feeding control protein according to claim 1, wherein the ultrasonic conditions in step (7) are as follows: 6mm probe, 40% power, 4s working, 9s rest, 10min working time.
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| JP2002281942A (en) * | 2001-01-16 | 2002-10-02 | Kibun Food Chemifa Co Ltd | Texture-improving agent both for processed meat article and processed fish article |
| CN101948838A (en) * | 2010-08-24 | 2011-01-19 | 中山大学 | Fish RF neuropeptide gene, coded protein and application thereof |
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| FR2927335B1 (en) * | 2008-02-12 | 2012-04-20 | Cie Des Peches Saint Malo Sante | FISH PROTEIN HYDROLYSAT HAVING SATIETOGENIC ACTIVITY, NUTRACEUTICAL AND PHARMACOLOGICAL COMPOSITIONS COMPRISING SUCH HYDROLYSAT AND PROCESS FOR OBTAINING SAME |
| JP4593639B2 (en) * | 2008-03-04 | 2010-12-08 | 株式会社マルハニチロ食品 | Peptide-containing feeding regulator |
| US20110239318A1 (en) * | 2008-11-18 | 2011-09-29 | LiveFuels, Inc. | Methods for producing fish with high lipid content |
| CN103275204B (en) * | 2013-05-17 | 2015-04-22 | 中山大学 | Nile tilapia NPB29 (neuropeptide B29) peptide as well as encoding nucleic acid and application thereof |
| CN103319587A (en) * | 2013-06-24 | 2013-09-25 | 广东医学院附属医院 | Fish food intake promotion mature peptide and application thereof |
| KR102487839B1 (en) * | 2016-02-04 | 2023-01-12 | 리제너론 파마슈티칼스 인코포레이티드 | Non-human animals with an engineered ANGPTL8 gene |
| CA3020175A1 (en) * | 2016-04-08 | 2017-10-12 | Regeneron Pharmaceuticals, Inc. | Methods for treating hyperlipidemia with an angptl8 inhibitor and an angptl3 inhibitor |
| CN116425852A (en) * | 2018-04-27 | 2023-07-14 | 四川大学 | Mature peptide adropin for promoting fish ingestion, application thereof and fish phagostimulant |
| WO2019210268A2 (en) * | 2018-04-27 | 2019-10-31 | The Broad Institute, Inc. | Sequencing-based proteomics |
| CN108715851B (en) * | 2018-04-28 | 2023-05-16 | 中山大学 | Epinephelus coioides ingestion regulation related gene AgRP1 and application thereof |
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| JP2002281942A (en) * | 2001-01-16 | 2002-10-02 | Kibun Food Chemifa Co Ltd | Texture-improving agent both for processed meat article and processed fish article |
| CN101948838A (en) * | 2010-08-24 | 2011-01-19 | 中山大学 | Fish RF neuropeptide gene, coded protein and application thereof |
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