CN107937408B

CN107937408B - Epinephelus coioidesinsulinGene, encoded protein and application thereof

Info

Publication number: CN107937408B
Application number: CN201711001284.6A
Authority: CN
Inventors: 李文笙; 杨国坤
Original assignee: Sun Yat Sen University
Current assignee: Sun Yat Sen University
Priority date: 2017-10-24
Filing date: 2017-10-24
Publication date: 2021-07-27
Anticipated expiration: 2037-10-24
Also published as: CN107937408A

Abstract

The invention belongs to the technical field of genetic engineering, and particularly discloses epinephelus coioidesinsulinGenes, encoded proteins and uses thereof. The above-mentionedinsulinThe nucleotide sequence of the gene is shown as SEQ ID NO. 1, and the amino acid sequence of the encoded insulin protein is shown as SEQ ID NO. 2. The invention clones to obtain the Epinephelus coioides for the first timeinsulinThe gene is used for constructing an expression vector and a recombinant escherichia coli strain, and a large amount of expressed recombinant epinephelus coioides indulin protein is obtained in vitro by using the recombinant escherichia coli strain, can promote glycolipid utilization and growth of marine fishes such as epinephelus coioides, can be used for preparing growth promoters or bait additives suitable for the marine fishes, and has a wide application prospect.

Description

Epinephelus coioidesinsulinGene, encoded protein and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to epinephelus coioidesinsulinGenes, encoded proteins and uses thereof.

Background

In the metabolic regulation of the body, insulin (insulin) plays an important regulatory role. The pancreas has been considered as an important organ for producing insulin, but recent studies have shown that organs other than the pancreas can also secrete insulin, which serves as an important hormone and plays an important role in the growth, cell differentiation and metabolism of the body. Fish insulin, one of the first insulin discovered, plays an important role in various aspects such as feeding, growth, development, and regulation of glycolipid metabolism in fish. After being combined with a receptor, insulin can trigger various downstream biological functions through PI3K/Akt, ERK and STAT/Jak-STAT signal pathways.

Bass family of genus Ammonidae (Serranidae) Genus Epinephelus (A)Epinephelus) Is warm water large and medium sized marine fishes. The grouper is rich in nutrition, tender and white in meat quality, similar to chicken, and called sea chicken as vegetarian. The grouper is a high-quality edible fish with low fat and high protein, is one of four major fishes in China in Hongkong and Australia regions, and is a necessary delicacy for high-grade banquet. At present, animal and plant proteins are used as main additives of the feed for the garrupa, however, the animal and plant proteins have higher cost, and if the utilization of glycolipids by the garrupa can be improved, the production cost can be greatly reduced. However, at present, no report that insulin protein is used as a bait additive or growth promoter for effectively promoting the utilization of glycolipids by Epinephelus coioides is found in artificial culture of Epinephelus coioides.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention provides the epinephelus coioidesinsulinA gene.

Another object of the present invention is to provide an epinephelus coioides insulin protein.

Another object of the present invention is to provide a method for producing a fish feed containing Epinephelus coioidesinsulinExpression vector of gene.

Another object of the present invention is to provide a recombinant strain of Escherichia coli transformed with the above expression vector.

The invention also aims to provide a method for producing recombinant epinephelus coioides indulin protein by using the escherichia coli recombinant strain.

The invention also aims to provide the recombinant epinephelus coioides indulin protein prepared by the method and application thereof in preparing growth promoters or bait additives for marine fishes.

In order to achieve the purpose, the invention is realized by the following scheme:

epinephelus coioidesinsulinThe nucleotide sequence of the gene is shown as SEQ ID NO. 1.

An epinephelus coioides insulin protein, the amino acid sequence of which is shown in SEQ ID NO 2.

The invention clones the Epinephelus coioides for the first timeinsulinGenes, and 3 'UTR and partial 5' UTR are obtained. The cloning method is a conventional gene cloning method, degenerate primers are designed by utilizing gene comparison of homologous species, an intermediate fragment is obtained after PCR, two downstream primers of 5'RACE and two upstream primers of 3' RACE are respectively designed according to the sequence of the intermediate fragment, and 5 'and 3' nested PCR is carried out by utilizing general primers AAP and AUAP to obtain the target productinsulinThe ORF, 5 'UTR and 3' UTR of (1). Finally, designing a primer for cloning ORF, connecting the obtained target gene to a PCR2.1 vector after PCR to obtain PCR2.1-insulin(ii) a Obtaining the Epinephelus coioides after gene sequencinginsulinThe nucleotide sequence of the gene is shown as SEQ ID NO. 1.

A health food containing the above Epinephelus coioidesinsulinThe expression vector of the gene can be any one of the Epinephelus coioides capable of efficiently expressing the Epinephelus coioidesinsulinA vector for the gene.

Preferably, the expression vector is an escherichia coli expression vector.

More preferably, the expression vector is an Escherichia coli expression vector pET22b-insulin。

The epinephelus coioides-containing fish constructed by the inventioninsulinEscherichia coli expression vector pET22b-insulinThe above-mentioned vector PCR2.1-insulinUsing the PCR method to synthesize the epinephelus coioides with the enzyme digestion joint as a templateinsulinThe gene is cut, separated and purified, and then is inserted between corresponding enzyme cutting sites (namely EcoRI and NotI) of the existing vector pET22b, thus constructing the needed epinephelus coioides containing geneinsulinExpression vector pET22b for geneinsulin。

A recombinant strain comprising the above expression vector.

Preferably, the strain is the recombinant Escherichia coli pET22b-insulin-BL 21; the strain can efficiently express recombinant epinephelus coioides indulin protein; the strain is prepared from the above Epinephelus coioidesinsulinExpression vector pET22b for geneinsulinThe strain obtained by transforming Escherichia coli BL21 was designated as pET22b-insulin-BL21。

The Epinephelus coioides has great economic value, and the Epinephelus coioides containing the Epinephelus coioides can be utilized by adopting the conventional genetic engineering technologyinsulinGenetic Escherichia coli recombinant strain pET22b-insulinBL21, producing recombinant epinephelus coioides insulin protein in large quantities.

In addition, the invention also provides a method for producing recombinant epinephelus coioides indulin protein by using the escherichia coli recombinant strain, which comprises the following steps: inoculating the recombinant Escherichia coli strain in LB liquid culture medium, and culturing at 37 deg.C and 200 rpm to OD₆₀₀And (3) when the value reaches 0.5-0.8, adding IPTG (isopropyl-beta-D-thiogalactoside) until the final concentration is 1mM, culturing at 37 ℃ and the rotation speed of 200 rpm for 4h, centrifuging at 5000 Xg for 10min to collect thalli, carrying out ultrasonication, centrifuging at 12000 Xg for 10min to collect supernatant, and separating and purifying to obtain the recombinant epinephelus coioides indulin protein.

The invention also requests to protect the recombinant epinephelus coioides indulin protein prepared by the method; the protein can improve the utilization of glycolipid by seawater fish and promote the growth of seawater fish.

The invention also provides application of the insulin protein or the recombinant epinephelus coioides insulin protein in preparation of growth promoters and/or bait additives for marine fishes.

A medicament and/or feed for promoting growth of marine fishes comprises the epinephelus coioides insulin protein or recombinant epinephelus coioides insulin protein.

Compared with the prior art, the invention has the following beneficial effects:

the invention clones to obtain the Epinephelus coioides for the first timeinsulinGene, and construction of expression vector and recombinant large intestine rod by using geneThe recombinant epinephelus coioides strain is utilized to obtain a large amount of expressed recombinant epinephelus coioides insulin protein in vitro, and the protein can remarkably inhibit the expression of gluconeogenesis related genes G6P and PEPCK, remarkably promote the gene expression of fat synthesis related genes ACC1 and FAS, and remarkably promote the gene expression of growth related genes IGF I and IGF II through research, namely the protein can promote the glycolipid utilization and growth of epinephelus coioides and other seawater fishes, can be used for preparing growth promoters or bait additives suitable for seawater fishes, reduces the use of animal and plant proteins, and reduces the production cost.

Drawings

FIG. 1 shows the PCR amplification of Brucella of Epinephelus coioides in example 1 using the reverse transcribed cDNA as templateinsulinGel electrophoresis analysis of the intermediate fragment product; wherein lane M is Marker, lane 1 is PCR amplification product, and lane NC is negative control.

FIG. 2 is a PCR electrophoretogram of 5'RACE and 3' RACE in example 1; a is the gel electrophoresis of the first product of 5' RACE, wherein lane M is Marker,

lanes

1, 2, 3 are PCR amplification products, and lane NC is negative control; b is the gel electrophoresis of the second round of 5' RACE products, wherein lane M is Marker,

lanes

1, 2, 3 are PCR amplification products, and lane NC is negative control; c is the gel electrophoresis of the first round of 3' RACE products, wherein lane M is Marker,

lanes

1, 2, 3 are PCR amplification products, and lane NC is negative control; d is the gel electrophoresis of the second round of 3' RACE products, wherein lane M is Marker,

lanes

1, 2, 3 are PCR amplification products, and lane NC is negative control; e is cloneinsulinGel electrophoresis of the product of ORF, wherein lane M is Marker,

lanes

1, 2, 3 are PCR amplification products, and lane NC is negative control.

FIG. 3: a is the site with enzyme in example 2insulinWherein Lane M is Marker, Lane 1 is PCR amplification product, and Lane NC is negative control; b is the expression vector pET22B-insulinThe restriction enzyme identification gel electrophoresis analysis map of (1), wherein lane M is Marker, lane 1 is pET22b-insulinEcoRI and NotI, lane 2 pET22b-insulinNo digestion was performed.

FIG. 4: a is the recombinant strain pET22b-insulinSDS-PAGE analysis of the insulin protein expressed in BL21, wherein lane M is protein Marker, lane NC is negative control, lane 1 is recombinant strain pET22b-insulin-BL21 expression product; b is the recombinant strain pET22B-insulinSDS-PAGE analysis of purified product of insulin protein expressed in BL21, wherein lane M is protein Marker, and

lanes

1, 2, 3 and 4 are purified product of insulin protein; c is the recombinant strain pET22b-insulinWestern blot identification of purified product of the insulin protein expressed in BL21, wherein Lane M is protein Marker, and

Lanes

1, 2, 3 and 4 are purified product of the insulin protein.

FIG. 5 shows the effect of recombinant epinephelus coioides insulin protein in application example 1 on glycolipid metabolism and growth related genes of primary hepatocytes of epinephelus coioides; a is the expression level of gluconeogenesis associated gene G6P; b is the expression level of gluconeogenesis related gene PEPCK; c is the expression level of the fat synthesis related gene ACC 1; d is the expression level of fat synthesis related gene FAS; e is the expression level of growth related gene IGF I; f is the expression level of growth related gene IGF II; wherein "-" represents P < 0.05; "x" indicates P < 0.01; ". indicates that P < 0.001.

Detailed Description

The present invention will be described in further detail with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

Example 1

Epinephelus coioidesinsulinCloning of a gene comprising the steps of:

1、insulincloning of intermediate fragments of genes

For reported nile tilapia (Oreochromis niloticus) Carp (C)Cyprinus carpio) Takifugu rubripes (takifugu rubripes) Of cyanKillifi (oryzias latipes) Obtained ininsulinComparing the sequences, finding out a relatively conserved region, and designing and cloning the Epinephelus coioidesinsulinA pair of degenerate primers of the middle segment of the sequence, wherein the upstream primer (SEQ ID NO: 3) is 5 '-GGHTTCTTCTAYAVCCCCARGAG-3', and the downstream primer (SEQ ID NO: 4) is 5 '-GTTRCAGTAGTTCTSSAGSTCRA-3'; and carrying out PCR by taking cDNA (complementary deoxyribonucleic acid) of the epinephelus coioides Brinell RNA after reverse transcription as a template. The PCR conditions were: 3min at 94 ℃; 30s at 94 ℃, 30s at 56 ℃, 30s at 72 ℃ and 35 cycles; 10min at 72 ℃; infinity at 4 ℃. The gel electrophoresis pattern after the PCR reaction is shown in FIG. 1.

2、insulinCloning of genes 5'RACE and 3' RACE

Two primers, 5'RACE and 3' RACE, are designed according to the obtained intermediate fragment sequence and are cloned by using common primers AUAP and AAP.

The 5' RACE upstream primer is:

AAP（SEQ ID NO:5）：5’-GGCCACGCGTCGACTAGTACGGGGGGGGGG-3’；

the downstream primers are respectively:

R1（SEQ ID NO:6）：5’-CGATGCCTCGCTTCACCA-3’；

R2（SEQ ID NO:7）：5’-CTCCGCCACCTCGTTCT-3’；

the 3' RACE upstream primers are respectively as follows:

F1（SEQ ID NO:8）：5’-CCCTCTGATGGGTTTCCTC-3’；

F2（SEQ ID NO:9）：5’-TTCGCCTACAAGGACCAGA-3’；

the downstream primer is AUAP (SEQ ID NO: 10): 5'-GGCCACGCGTCGACTAGTAC-3' are provided.

And carrying out PCR by using cDNA (complementary deoxyribonucleic acid) of the epinephelus coioides brucellosis corpuscle after reverse transcription as a template. The PCR conditions were: 3min at 94 ℃; 30s at 94 ℃, 30s at 56 ℃, 60s at 72 ℃ and 35 cycles; 10min at 72 ℃; infinity at 4 ℃. The gel electrophoresis pattern after the PCR reaction is shown in FIG. 2 as A, B, C, D.

3、insulinCloning of the ORF

Based on the results of

steps

1 and 2, a pair of clones was designedinsulinPrimer for ORF, upstream primer F-ORF (SEQ ID NO: 11) nucleusThe nucleotide sequence is 5'-ATGGCGGCTCTGTGGCTCCAGT-3', the nucleotide sequence of the downstream primer R-ORF (SEQ ID NO: 12) is 5'-TCAGTTGCAGTAGTTCTGCAGG-3', and the cDNA after the reverse transcription of the epinephelus coioides Brinell RNA is used as a template for PCR. The PCR conditions were: 3min at 94 ℃; 30s at 94 ℃, 30s at 56 ℃, 30s at 72 ℃ and 35 cycles; 10min at 72 ℃; infinity at 4 ℃. After PCR, the obtained product is connected with a PCR2.1 vector, namely PCR2.1-insulin. The gel electrophoresis pattern after the PCR reaction is shown in E in FIG. 2. Obtaining the Epinephelus coioides after gene sequencinginsulinThe nucleotide sequence of the gene is shown as SEQ ID NO. 1.

Example 2

Epinephelus coioidesinsulinEscherichia coli expression vector pET22b of gene-insulinThe method comprises the following steps:

1. epinephelus coioides with enzyme digestion siteinsulinSynthesis of genes

Epinephelus coioides cloned according to the aboveinsulinThe sequence of the gene, the recognition sequences of restriction endonucleases EcoRI and NotI and the protection bases, a pair of specific primers were designed and synthesized, the upstream primer F (SEQ ID NO: 13) was 5'-CGGAATTCACATCATCATCATCATCATGCCACGGCGCCGCAGC-3'insulinEcoRI enzyme digestion recognition sites and 6 XHIS labels are added in front of mature peptide sequences of the genes; the downstream primer R (SEQ ID NO: 14) is 5'-ATAGTTTAGCGGCCGCTCAGTTGCAGTAGTTCTG-3'insulinA termination codon and a NotI restriction enzyme cutting recognition site are added after the gene mature peptide sequence. PCR2.1-insulinCarrying out PCR reaction by using the template, wherein the PCR reaction conditions are as follows: 3min at 94 ℃; 30s at 94 ℃, 30s at 56 ℃, 40s at 72 ℃ and 35 cycles; 10min at 72 ℃; infinity at 4 ℃. The electrophoretic identification of the PCR amplification product is shown in A in FIG. 3.

2. And (3) carrying out double digestion on the PCR product in the step 1 by using restriction enzymes EcoRI and NotI, and carrying out E.Z.N.A on the digestion product.^®Recovering Gel Extraction Kit, performing agarose Gel electrophoresis, and separating and purifying Epinephelus coioidesinsulinA gene fragment; the vector plasmid pET22b is separated and purified after double digestion by restriction enzymes EcoRI and NotI, and the epinephelus coioides is separated and purifiedinsulinMixing gene fragments, connecting overnight at 16 ℃ by TOYOBO Ligtion High ligase, transferring into Escherichia coli DH5a by standard calcium chloride transformation method, screening transformants with Ampicillin resistance by LB plate, extracting plasmid by standard method, double-digesting recombinant plasmid by restriction enzymes EcoRI and NotI to obtain large and small fragments, the size of the two fragments is respectively equal to that of Epinephelus coioidesinsulinThe gene and the expression vector pET22b are the same in size, and prove that the epinephelus coioidesinsulinThe gene has been cloned into an Escherichia coli expression vector pET22b, and the recombinant plasmid (i.e., the expression vector) is named pET22b-insulin. The cleavage analysis patterns are shown in FIG. 3B, respectively.

Example 3

Escherichia coli recombinant strain pET22b capable of efficiently expressing recombinant epinephelus coioides indulin proteininsulinConstruction of BL21, steps as follows:

the recombinant plasmid pET22b prepared in example 2insulinColi BL21 competent cells were transformed by standard calcium chloride transformation and cultured on LB plates containing Ampicillin at 37 ℃. After overnight, a single clone grows out, and the single clone is selected to screen out an escherichia coli recombinant strain pET22b capable of efficiently expressing recombinant epinephelus coioides indulin protein through induction culture and identificationinsulin-BL21。

Example 4

A method for producing recombinant epinephelus coioides indulin protein by using the Escherichia coli recombinant strain obtained in example 3 comprises the following steps:

selecting an engineering recombinant strain pET22b of Escherichia coliinsulinThe single colony of-BL 21 was inoculated into 10mL LB liquid medium, cultured overnight at 37 ℃ at 200 rpm, then inoculated into 1L LB liquid medium at a ratio of 1:100, and cultured to OD at 37 ℃ at 200 rpm₆₀₀And (3) when the value reaches 0.5-0.8, adding IPTG (isopropyl-beta-D-thiogalactoside) to enable the final concentration to be 1mM, culturing for 4 hours at 37 ℃ and at the rotating speed of 200 rpm, collecting thalli through 5000 Xg centrifugation for 10min, collecting supernatant through 12000 Xg centrifugation for 10min after ultrasonication, and separating and purifying to obtain the recombinant epinephelus coioides indulin protein. The expression level of the insulin protein obtained by the method is 25 mg/L.

3 μ L of the suspension was added to 2 Xelectrophoresis loading buffer, boiled for 10 minutes and subjected to SDS-PAGE by standard methods, while negative control (suspension of a single clone grown after BL21 was transformed with unloaded plasmid pET22 b) was treated by the same method and subjected to SDS-PAGE, the result of which is shown as A in FIG. 4. pET22b-insulinThe supernatant of the expression of-BL 21 was purified by His-bind Resin from Novagen to obtain purified insulin protein, which was subjected to SDS-PAGE gel electrophoresis, and the results are shown in B in FIG. 4.

The recombinant epinephelus coioides indulin protein is identified by an immunoblotting (Western blot) method. The primary antibody was a murine anti-HIS monoclonal antibody (available from Novagen) and the secondary antibody was goat anti-murine IgG-HRP (available from Dr. Dermatology, Inc.). The result of Western blot identification is shown in FIG. 4C.

Application example 1

Example 4 functional study of the recombinant epinephelus coioides insulin protein: influence on glycolipid metabolism and growth related genes of primary hepatocytes of epinephelus coioides.

1. After isolation of primary hepatocytes from Epinephelus coioides, the cell concentration was adjusted to 5X 10 by adding 10% FBS in L15 medium containing phenol red⁵cells/mL, then adding 1mL per well in a 24-well plate, placing in a biochemical incubator at 25 ℃, after overnight culture, starving for 1h with 900 μ L of FBS-free L15 medium, then adding 100 μ L of the isolated and purified recombinant Epinephelus coioides indulin protein with concentration of 100, 1000, 10000ng/mL to each well of the treated group, adding an equal volume of serum-free medium to the control group, culturing for 12h in the biochemical incubator at 25 ℃, collecting culture supernatant, lysing the cells with Trizol (purchased from ThermoFisher Scientific Co.), and completely collecting the lysate after lysing the cells. Total RNA from the samples was extracted and reverse transcribed using M-MLV (purchased from ThermoFisher Scientific). The change in glycolipid metabolism and growth related genes between the control group and the treated group was calculated by performing fluorescent quantitative PCR reaction on LightCycler480II (purchased from Roche) using a fluorescent quantitative PCR kit (purchased from TOYOBO Co.), using 18S rRNA as an internal control.

2. The results show that the expression of gluconeogenesis related genes G6P and PEPCK can be remarkably inhibited after primary hepatocytes are treated by the insulin protein; significantly promoting the gene expression of the fat synthesis related genes ACC1 and FAS; and significantly promote the gene expression of growth-related genes IGF I and IGF II. The expression level of each gene is shown in FIG. 5. The insulin protein has obvious promotion effect on the average weight gain rate, survival rate, body length and the like of the epinephelus coioides.

The results show that the insulin protein can promote utilization and growth of saccharides and fat of the epinephelus coioides, and can be used for preparing growth promoters or bait additives suitable for seawater fishes.

Sequence listing

<110> Zhongshan university

<120> epinephelus coioides insulin gene, encoding protein and application thereof

<160> 14

<170> SIPOSequenceListing 1.0

<210> 1

<211> 351

<212> DNA

<213> Epinephelus coioides (Epinepheluscoides)

<400> 1

atggcggctc tgtggctcca gtctgtctct ctgctggtct tactggttgt gtcgtggccg 60

agctctcagg ccgccacggc gccgcagcac ctgtgtggct ctcacctggt ggacgcgctc 120

tacctggtct gtggggacag aggcttcttc tacaacccca agagagacgt ggaccctctg 180

atgggtttcc tccctccaaa ggtgggcgga gcagcagcag caggcggcga gaacgaggtg 240

gcggagttcg cctacaagga ccagatggag atgatggtga agcgaggcat cgtggagcag 300

tgctgccacc gaccctgcaa catcttcgac ctgcagaact actgcaactg a 351

<210> 2

<211> 116

<212> PRT

<213> Epinephelus coioides (Epinepheluscoides)

<400> 2

Met Ala Ala Leu Trp Leu Gln Ser Val Ser Leu Leu Val Leu Leu Val

1 5 10 15

Val Ser Trp Pro Ser Ser Gln Ala Ala Thr Ala Pro Gln His Leu Cys

20 25 30

Gly Ser His Leu Val Asp Ala Leu Tyr Leu Val Cys Gly Asp Arg Gly

35 40 45

Phe Phe Tyr Asn Pro Lys Arg Asp Val Asp Pro Leu Met Gly Phe Leu

50 55 60

Pro Pro Lys Val Gly Gly Ala Ala Ala Ala Gly Gly Glu Asn Glu Val

65 70 75 80

Ala Glu Phe Ala Tyr Lys Asp Gln Met Glu Met Met Val Lys Arg Gly

85 90 95

Ile Val Glu Gln Cys Cys His Arg Pro Cys Asn Ile Phe Asp Leu Gln

100 105 110

Asn Tyr Cys Asn

115

<210> 3

<211> 23

<212> DNA

<213> Epinephelus coioides (Epinepheluscoides)

<400> 3

gghttcttct ayavccccar gag 23

<210> 4

<211> 23

<212> DNA

<213> Epinephelus coioides (Epinepheluscoides)

<400> 4

gttrcagtag ttctssagst cra 23

<210> 5

<211> 30

<212> DNA

<213> Epinephelus coioides (Epinepheluscoides)

<400> 5

ggccacgcgt cgactagtac gggggggggg 30

<210> 6

<211> 18

<212> DNA

<213> Epinephelus coioides (Epinepheluscoides)

<400> 6

cgatgcctcg cttcacca 18

<210> 7

<211> 17

<212> DNA

<213> Epinephelus coioides (Epinepheluscoides)

<400> 7

ctccgccacc tcgttct 17

<210> 8

<211> 19

<212> DNA

<213> Epinephelus coioides (Epinepheluscoides)

<400> 8

ccctctgatg ggtttcctc 19

<210> 9

<211> 19

<212> DNA

<213> Epinephelus coioides (Epinepheluscoides)

<400> 9

ttcgcctaca aggaccaga 19

<210> 10

<211> 20

<212> DNA

<213> Epinephelus coioides (Epinepheluscoides)

<400> 10

ggccacgcgt cgactagtac 20

<210> 11

<211> 22

<212> DNA

<213> Epinephelus coioides (Epinepheluscoides)

<400> 11

atggcggctc tgtggctcca gt 22

<210> 12

<211> 22

<212> DNA

<213> Epinephelus coioides (Epinepheluscoides)

<400> 12

tcagttgcag tagttctgca gg 22

<210> 13

<211> 43

<212> DNA

<213> Epinephelus coioides (Epinepheluscoides)

<400> 13

cggaattcac atcatcatca tcatcatgcc acggcgccgc agc 43

<210> 14

<211> 34

<212> DNA

<213> Epinephelus coioides (Epinepheluscoides)

<400> 14

atagtttagc ggccgctcag ttgcagtagt tctg 34

Claims

1. The application of epinephelus coioides indulin protein or recombinant epinephelus coioides indulin protein in preparing growth promoters or bait additives for marine fishes;

the amino acid sequence of the epinephelus coioides indulin protein is shown in SEQ ID NO. 2;

the production method of the recombinant epinephelus coioides insulin protein comprises the following steps: inoculating the recombinant Escherichia coli strain in LB liquid culture medium, and culturing at 37 deg.C and 200 rpm to OD₆₀₀The value reaches 0.5-0.8, IPTG is added to the final concentration of 1mM, the mixture is cultured for 4 hours at 37 ℃ and the rotating speed of 200 revolutions per minute, thalli are collected by centrifugation at 5000 Xg for 10 minutes, supernatant is collected by centrifugation at 12000 Xg for 10 minutes after ultrasonic crushing, and recombinant epinephelus coioides indulin protein is obtained by separation and purification, wherein the escherichia coli recombinant strain is an expression strainThe Escherichia coli recombinant strain pET22b-insulin-BL21 formed by transferring the vector pET22b-insulin into Escherichia coli BL21, wherein the expression vector pET22b-insulin contains an epinephelus coioides insulin gene, and the nucleotide sequence of the epinephelus coioides insulin gene is shown as SEQ ID NO: 1.

2. A pharmaceutical agent and/or feed for promoting growth of marine fishes, which comprises the epinephelus coioides insulin protein of claim 1 or the recombinant epinephelus coioides insulin protein of claim 1.