A protein for promoting sturgeon feeding, its encoding gene and use

Abstract

The present invention discloses a protein for promoting the feeding of sturgeons, a coding gene and a use thereof, and belongs to the technical field of molecular biology. The amino acid sequence of the protein is shown in SEQ ID NO.2, and the nucleotide sequence of the gene encoding the protein is shown in SEQ ID NO.1. The present invention obtains a protein for promoting the feeding of Siberian sturgeons, which can not only increase the feeding amount of Siberian sturgeons, but also activate the expression and release of multiple appetite-promoting factors in the hypothalamus, and is expected to be used as a potential attractant to enhance its feeding, thereby facilitating the rapid growth of young fish.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and in particular relates to a protein for promoting sturgeon feeding, a coding gene and uses thereof. Background Art

[0002] Feeding is an important physiological activity of animals, and the amount of food intake directly affects the growth performance, metabolic state and reproductive performance of animals. In the breeding process, due to frequent environmental changes and disease occurrence, animals are prone to appetite suppression and reduced food intake, which is not conducive to health and growth, and reduces economic benefits. Animal feeding is mainly regulated by hormones and neuropeptides secreted by the feeding center (brain) and peripheral tissues (such as liver, stomach, intestines, etc.). However, current research on the application of appetite-stimulating proteins in breeding production is relatively limited. Therefore, it is critical to explore potential genes and their functional proteins that improve feeding.

[0003] Acipenser siberianus Acipenser baerii Brandt is one of the important sturgeon aquaculture varieties in my country. Its meat is rich in polyunsaturated fatty acids such as DHA and EPA, and caviar is known as "black gold" and is deeply loved by consumers. However, problems such as low feeding rate, abnormal metabolism, and slow growth in the seedling stage limit the efficient development of sturgeon aquaculture. Therefore, it is of great significance to screen and develop functional genes and target proteins that can increase the feeding amount of sturgeons. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the present invention provides a protein for promoting sturgeon feeding, a coding gene and uses thereof, which can not only increase the food intake of sturgeons, but also strongly stimulate the expression and release of other central and peripheral appetite-stimulating factors, and is expected to serve as a functional peptide that can potentially induce sturgeon feeding, thereby facilitating the rapid growth of young fish.

[0005] To achieve the above purpose, the technical solution adopted by the present invention to solve the technical problem is: One object of the present invention is to provide a protein for promoting feeding of sturgeons, the amino acid sequence of the protein is shown as SEQ ID NO.2.

[0006] Another object of the present invention is to provide a gene encoding the above protein, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0007] Another object of the present invention is to provide an expression vector comprising the above gene.

[0008] Another object of the present invention is to provide the use of the above gene or expression vector in regulating fish feeding.

[0009] Another object of the present invention is to provide the use of the above protein in preparing a fish anorectic agent having the effect of promoting feeding.

[0010] Another object of the present invention is to provide the use of the above protein in preparing fish feed additives.

[0011] Another object of the present invention is to provide a method for promoting feeding of sturgeons, wherein the above protein is used as a feeding promoter to feed sturgeons, or the above protein is promoted to be overexpressed in sturgeons.

[0012] Furthermore, the sturgeon is Acipenser Siberianus.

[0013] Furthermore, the effective amount of protein is 100~200ng / g BW.

[0014] Further, the effective amount of the protein is 100, 120, 150, 180 or 200 ng / g BW.

[0015] Beneficial effects of the present invention: The present invention obtains a protein that can promote the feeding of Siberian sturgeon, which can not only increase the feeding amount of Siberian sturgeon, but also activate the expression and release of multiple appetite-stimulating factors in the hypothalamus, and is expected to serve as a potential attractant to enhance its feeding, thereby facilitating the rapid growth of young fish. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The electrophoresis pattern of the PCR product of the target gene in the coding functional region; Figure 2 This is the result of the solubility test of the recombinant functional protein; M: Marker; 1: mixed solution after the recombinant protein is broken; 2: precipitate after the recombinant protein is broken; 3: supernatant after the recombinant protein is broken; Figure 3 The results of the time point screening for induced protein expression; M: protein marker; 1: sample without induction of the recombinant vector; 2-8: samples after the recombinant vector-transformed bacteria were induced for 2, 4, 6, 8, 10, 12 and 24 hours respectively; Figure 4 The figure shows the results of IPTG concentration screening for inducing protein expression; M: protein marker; 1-8: IPTG concentrations of 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 and 1.4 mmol / L respectively induce recombinant bacterial expression; Figure 5 The results of Ni-NTA purification of His-tagged recombinant proteins; M: Marker; 1: stock solution before column; 2: column effluent; 3-9: 8MUrea eluate containing different concentrations of imidazole (10, 20, 50, 100, 150, 200 and 300mM) Figure 6 This is the SDS-PAGE image of protease cleavage purification; M: marker; 1: recombinant protease cleavage mixture; 2: column effluent; 3: 50mM imidazole elution; 4: column effluent after concentration; Figure 7 Western-blotting diagram of proteinase cleavage and purification; M: Marker; 1: Recombinant protein; 2: Enzyme cleavage mixture; 3: Purified protein after enzymatic cleavage; 4: 50mM imidazole eluent; 5: Target protein concentrate Figure 8 To investigate the effect of injection of target protein on the food intake of Siberian sturgeon; Fig. 9 Effects of injected protein on the expression of appetite genes in the hypothalamus and stomach of Siberian sturgeon. DETAILED DESCRIPTION

[0017] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0018] Example 1 1. Cloning of target gene Total RNA was extracted from the liver tissue of Siberian sturgeon using a total RNA extraction kit, and then the first-strand cDNA was synthesized by reverse transcription using a 047A reverse transcription kit (TaKaRa). Then, prokaryotic expression primers with restriction sites were designed: Upstream Primer-F: 5'-C GGATCC GACGACGACGACAAACGCAGAAGC-3'; Downstream Primer-R: 5'-CCCCCC AAGCTT TTAATGAAGAATAATTTGGA TT-3′; Add protective base (C), BamHI restriction site (GGATCC), and enterokinase cleavage site (GATGACGATGACAAA) to the 5' end of the upstream primer, and add protective base and HindⅢ restriction site (AAGCTT) to the 5' end of the downstream primer. PCR amplification conditions: 94℃, 5min denaturation, 35 cycles (94℃, 30s; 64℃, 30s; 72℃, 50s), 72℃ extension for 10min. The PCR amplification product was purified and recovered using a gel recovery kit, connected to the pMD19-T vector at 4℃ overnight, heat-shocked and transformed into DH5α competent cells, and the positive clones were selected and sent to Shanghai Biotech for sequencing verification. The gel electrophoresis results are as follows: Figure 1 shown.

[0019] The nucleotide sequence of the functional region of the target gene is as follows: CGCAGAAGCACCAATGGAACAGAAGCTGAATCTCAGAACGACTCGCAGGATGAAAATGAGAGCATCAGCCTTGCCAGTGTGGACATTGAAGAACCTCTAGTCTTCAGCTTGAACATCACTGACCTGGACAAAAAAGACCACATCCTGGAGCTGGTACCAGCGCTCACCGCACTGACTAACCACGTGCGATACTTGATTGATTTTGGAAACGAGGAT GGCTTCTTCAAAATCAACCAGAAAGAAGGAATAAGCTATCTTCACCTGACCAAGAAGAAACCCCATACCTGGAGCCTATTCGTTACAAATCAGCAGTGTCCCGCTGTATAAAAAGAAGGAACTTGACAATCTGGAGGATAAACATGACAAAGACTACCTCACTGGTGAGCTCGGTGACATCCTTAAAATGAAAATCCAAATTATTCTTCATTAA (SEQ ID NO.1).

[0020] The amino acid sequence of the target protein is as follows, and its molecular mass is 17kDa: RRSTNGTEAESQNDSQDENESISLASVDIEEPLVFSLNITDLDKKDHILELVPALTNHVRYLIDFGNEDGFFKINQKEGISYLHLTKKKPIPGAYSLQISSVPLYKKKELDNLEDKHDKDYLTGELGDILKMKIQIILH (SEQ ID NO. 2).

[0021] 2. Protein expression and purification (1) Construction of recombinant plasmid The correctly sequenced cloning plasmid and expression vector pET-32a were double-digested with BamHI and HindⅢ restriction endonucleases, the target fragment and the large vector fragment were recovered, and the recombinant expression plasmid was obtained by ligating with T4 enzyme at 4°C overnight. The recombinant expression plasmid was transformed into DH5α competent cells, and PCR detection and double-enzyme digestion verification were performed after bacterial culture, and the correct recombinant plasmid was sequenced.

[0022] (2) Expression of recombinant plasmid in expression bacteria Transform the correctly sequenced recombinant plasmid into the expression strain E. coli BL21 (DE3) was cultured in LB liquid medium (containing Amp) until OD 600 The value reached 0.4~0.6 (37℃, 200 rpm / min), and isopropyl-β-D-thiogalactoside (IPTG, 0.8 mmol / L) was added to induce protein expression. The estimated size of the recombinant expressed protein was about 37kD.

[0023] (3) Recombinant protein solubility test The bacterial culture was cultured to OD 600 When the value was 0.4-0.6, 1.0 mM IPTG was added to induce expression for 10 hours, and the precipitate was collected by centrifugation at 12000 rpm for 1 minute. The precipitate was resuspended and washed twice by adding 1 ml PBS, and lysozyme (0.1 mg / ml) was added to digest the reaction at room temperature for half an hour. The supernatant and precipitate were collected after repeated freeze-thaw for 4 times, and centrifuged (12000 rpm, 5 minutes). SDS-PAGE electrophoresis analysis was performed to analyze whether the recombinant protein in the expression product was mainly present in the supernatant or precipitate, and then it was judged whether the expressed protein was soluble protein or inclusion body protein. The results are as follows Figure 2 As shown, the recombinant protein was mainly present in the precipitate, indicating that its main expression form was inclusion body protein.

[0024] (4) Optimization of induction conditions The bacterial culture solution was cultured to OD 600 When the value was 0.4-0.6, IPTG (1.0 mmol / L) was added to stimulate protein expression, and the culture was shaken at 37°C for 0, 2, 4, 6, 8, 10, 12 and 14 h. The bacterial precipitate was collected and subjected to SDS-PAGE electrophoresis to explore the effect of induction time on protein expression. Figure 3 As shown, a large amount of recombinant protein can be obtained after induction for 10-24 hours.

[0025] In addition, the bacterial culture was cultured to OD 600When the value was 0.4-0.6, different volumes of IPTG stock solution were added to the LB medium with final concentrations of 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 and 1.4 mmol / L, and the medium was cultured on a shaker at 37°C for 10 h to explore the induction effect of different concentrations of IPTG on the expression of recombinant proteins. Figure 4 As shown in the figure, when the IPTG induction concentration is 0.2-1.4 mM, it can stimulate the expression of recombinant protein in large quantities.

[0026] (5) Purification and renaturation of recombinant proteins The recombinant expression bacteria were cultured overnight at 37°C and 200 rpm, and 5 ml was inoculated into 500 ml of fresh LB medium. The bacterial solution was cultured until OD 600 When the value is 0.4-0.6, add 0.2 mM IPTG to induce expression for 10 hours, centrifuge for 5 minutes to collect the precipitate, add sterile PBS to resuspend and wash the precipitate twice, add lysozyme (0.1 mg / ml) to react for half an hour, freeze and thaw three times at -20℃, and then centrifuge to collect the precipitate. The precipitate was dissolved in 8M Urea, reacted at 4℃ overnight, centrifuged at 12000rpm to collect the supernatant, and the filtrate was filtered out of impurities with a 0.45μm microporous filter membrane. Ni was used to filter the impurities. 2+ -NTA affinity chromatography column was used for purification analysis, 8MUrea buffer with different concentrations of imidazole was used for elution, and protein purity was detected by SDS-PAGE electrophoresis. Figure 5 As shown, 8M Urea elution buffer containing 50mM imidazole can elute a large amount of recombinant protein.

[0027] (6) Recombinant protein dialysis refolding and enzyme digestion purification The purified recombinant protein was placed in a dialysis bag, and the external solution was replaced by gradient renaturation to 6M, 4M and 2MUrea, and dialyzed in a PBS 4°C refrigerator overnight. After being concentrated to 2ml using an ultrafiltration tube, the protein purity was detected by SDS-PAGE electrophoresis. The purified recombinant protein was dialyzed into a 25mM Tris-HCl (pH8.0) solution, and the protein concentration was determined by the BCA method. Enterokinase (Solebol) was used for enzyme digestion at 4°C for 14h. Ni 2+ -NTA purification column was used to purify the target protein (the enzyme cleavage reaction solution was passed through the column three times), and the protein purity was detected by SDS-PAGE electrophoresis. The concentration of the target protein was determined by BCA method, and after sterilization by filtration through a 0.22 μm microporous filter membrane, it was frozen in a -80℃ ultra-low temperature refrigerator for future use. Figure 6 As shown, the target protein with high purity can be successfully eluted using 25 mM Tris-HCl (pH 8.0) containing 50 mM imidazole.

[0028] (8) Immunoblotting to detect the purity of mature peptides The recombinant protein before enzyme cleavage, the protease cleavage reaction solution, the outflow target protein, the protein eluate containing 50mM imidazole and the target protein concentrate samples were subjected to SDS-PAGE electrophoresis and wet-transferred to a 0.22 μm PVDF membrane. The membrane was blocked with 5% BSA membrane blocking solution for 1 hour, and then rinsed with TBST for 3 times, with an interval of 5 minutes each time. The membrane was incubated with the primary antibody of anti-6×His tag mouse monoclonal antibody (Transgen) at room temperature for 1 hour. After rinsing with TBST for 3 times, the membrane was incubated with the secondary antibody of HRP-labeled goat anti-mouse IgG (Transgen). After rinsing with TBST, the membrane was developed with ECL colorimetric solution and photographed with E-BLOT to further verify the purity of the target protein. The results are shown in Figure 2. Figure 7 As shown, the target protein was purified successfully after enzyme digestion.

[0029] Example 2 Regulatory effect of target protein on feeding of Siberian sturgeon Regulatory effect on food intake Siberian sturgeons (48, 102.16±11.57 g) of uniform size were randomly divided into 4 groups, with 3 parallel tanks in each group and 4 sturgeons in each tank. PBS and target protein (50, 100 and 200 ng / g BW) were intraperitoneally injected after MS-222 (0.01%) immersion anesthesia 30 min before the fixed feeding point (14:00), and feed was fed at 14:00, 15:00 and 17:00, respectively. The residual bait was collected at 0-1h (15:00), 1-3h (17:00) and 3-6h (20:00), respectively, and dried in an oven to constant weight. The changes in food intake at 1h, 3h and 6h were measured and analyzed to explore the effect of protein on the food intake of Siberian sturgeons.

[0030] The results are as follows Figure 8 As shown in the figure, compared with the PBS group, intraperitoneal injection of three doses of the target protein did not affect the 1-hour food intake of Siberian sturgeon, while 100 ng / g BW and 200 ng / g BW proteins significantly increased the food intake at 3 and 6 hours (P<0.05). The above results show that the cloned target protein can promote the feeding of Siberian sturgeon, and its effective time is 3 hours and 6 hours.

[0031] Regulatory effect on the expression of appetite factors 24 Siberian sturgeon juveniles (104.78±13.03 g) were selected and randomly divided into two groups, with 3 fish in each tank and 4 fish in each tank. MS-222 (0.01%) was used for immersion anesthesia, and PBS and an effective dose of the target protein (100 ng / g BW) were injected intraperitoneally. Three hours after injection, three sturgeons were randomly selected from each tank, deeply anesthetized and weighed, and quickly decapitated. The hypothalamus and stomach tissues were collected to determine the changes in the expression of appetite factors. The sequences of fluorescent quantitative primers are shown in Table 1.

[0032] Table 1 Fluorescence quantitative primers

[0033] like Fig. 9 As shown, intraperitoneal injection of 100 ng / g BW of the target protein can significantly increase the expression of npy, agrp and ghrelin genes in the hypothalamus, and significantly inhibit the expression of anorexia factors pomc and nucb2, but does not affect the expression of ucn3. This shows that the target protein isolated and obtained by the present invention has an important role in stimulating the appetite of Siberian sturgeon and increasing the food intake.

[0034] Finally, it should be noted that the above specific implementation methods are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A protein for promoting sturgeon feeding, characterized in that: The amino acid sequence of the protein is shown in SEQ ID NO.

2.

2. A gene encoding the protein according to claim 1, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

3. An expression vector, characterized in that: Comprising the gene according to claim 2.

4. Use of the gene according to claim 2 or the expression vector according to claim 3 in regulating fish feeding.

5. Use of the protein according to claim 1 in the preparation of a fish anorectic agent having the effect of promoting feeding.

6. Use of the protein according to claim 1 in the preparation of fish feed additives.

7. A method for promoting sturgeon feeding, characterized in that: Feeding sturgeons with the protein of claim 1 as a feeding promoter, or promoting overexpression of the protein of claim 1 in sturgeons.

8. The method according to claim 7, characterized in that The sturgeon is a Siberian sturgeon.

9. The method according to claim 7, characterized in that: The effective amount of the protein is 100-200 ng / g BW.

Images