Application of RBP4 protein in increasing food intake of fishes

Abstract

The invention discloses application of RBP4 protein in increasing the food intake of fishes, and belongs to the technical field of genetic engineering. The amino acid sequence of the protein is as shown in SEQ ID NO.1, and the gene sequence for coding the protein is as shown in SEQ ID NO.2. The novel protein capable of improving the food intake of the acipenser sibirica is obtained, does not influence the expression of stomach Ghrelin, but can strongly activate the expression of hypothalamus appetite increasing signals NPY and AgRP, has the potential of inducing appetite enhancement of the acipenser sibirica, and is beneficial to rapid growth of juvenile fish.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and particularly relates to a use of RBP4 protein in increasing the food intake of fish. Background Art

[0002] Feeding is the only way for animals to obtain nutrients. How to regulate animal growth, development and reproduction by intervening in feeding behavior has become a crucial issue. Some genes with feeding regulatory functions have been identified in the brain and peripheral tissues of animals, but these genes mainly encode proteins that inhibit feeding, including leptin, cholecystokinin (CCK) and peptide YY (PYY). Therefore, it is urgent to discover genes and proteins with appetite-stimulating effects and potential application value.

[0003] Retinol binding protein 4 (RBP4) is not only produced and secreted by adipose tissue, but also abundantly expressed in mammalian liver. More and more studies have shown that the expression of RBP4 is regulated by nutritional status. For example, in rodents, refeeding after an overnight fast can significantly increase the expression level of RBP4 in serum. Similarly, the expression level of RBP4 in HEIIE cells can be upregulated after the culture medium is renewed. In contrast, when mice face energy-deficient states such as starvation, the expression of RBP4 in the liver is very low. From the above, it can be seen that RBP4 can sensitively sense changes in the body's energy status and has characteristics similar to potential feeding regulatory genes. However, there have been no reports on research on RBP4 regulating animal feeding behavior.

[0004] Acipenser siberianus Acipenser baerii Brandt is one of the main farmed species of sturgeon in the world. Due to the frequent occurrence of temperature changes, high ammonia nitrogen, low dissolved oxygen and other problems during the breeding process, the seedlings often have problems such as low feeding rate, metabolic abnormalities and high mortality, which restrict the green and efficient development of sturgeon breeding. Therefore, it is of great significance to explore the feeding regulation mechanism and develop feeding regulatory proteins. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the present invention provides a use of RBP4 protein in increasing the food intake of fish, which can not only increase the food intake of Siberian sturgeon, but also activate the appetite-stimulating signals in the hypothalamus, the feeding regulatory center, and is expected to serve as a potential attractant to enhance and strengthen its feeding behavior, thereby facilitating the rapid growth of young fish.

[0006] To achieve the above purpose, the technical solution adopted by the present invention to solve the technical problem is: The purpose of the present invention is to provide a use of RBP4 protein in increasing the food intake of fish, and its amino acid sequence is shown in SEQ ID NO.1.

[0007] Another object of the present invention is to provide a use of RBP4 protein in the preparation of a fish anorectic agent having the effect of promoting feeding.

[0008] Another object of the present invention is to provide a use of RBP4 protein in the preparation of fish feed additives.

[0009] Furthermore, the fish is a Siberian sturgeon.

[0010] Furthermore, the effective dose of RBP4 protein is 100~300ng / gBW.

[0011] Furthermore, the effective dose of RBP4 protein is 100, 300 ng / g BW.

[0012] Another object of the present invention is to provide a gene encoding the above RBP4 protein, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0013] Another object of the present invention is to provide a vector comprising the above gene.

[0014] Another object of the present invention is to provide a cell line comprising the above gene or vector.

[0015] Another object of the present invention is to provide a recombinant bacterium comprising the above gene or vector.

[0016] Another object of the present invention is to provide a preparation for promoting fish feeding, which contains a substance that promotes the expression of the protein encoding the above RBP4 as an active ingredient.

[0017] Beneficial effects of the present invention: The present invention obtains a protein that can promote the feeding of Siberian sturgeon. The protein can not only increase the feeding amount of Siberian sturgeon, but also activate the appetite-promoting signal in the hypothalamus, the feeding regulation center, and is expected to serve as a potential attractant to enhance and strengthen its feeding behavior, thereby facilitating the rapid growth of young fish. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the electrophoresis pattern of the PCR product of the target gene containing the mature peptide region; Figure 2 The results of IPTG concentration screening for inducing recombinant protein expression are shown in Figure 1. M: protein marker; 1: sample without recombinant vector induction; 2-8: IPTG concentrations of 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 and 1.4 mmol / L, respectively, induce recombinant vector expression; Figure 3The result diagram of the time screening for inducing recombinant protein expression; where 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 solubility test result of the recombinant mature protein is shown in the figure; wherein, M: Marker; 1: mixed solution after the recombinant protein is crushed; 2: precipitate after the recombinant protein is crushed; 3: supernatant after the recombinant protein is crushed; Figure 5 This is a diagram of recombinant protein purification; where M: Marker; 1: stock solution before purification; 2: column effluent; 3: 10mM Urea washing solution; 4: 20mM Urea eluent; 5: 50mM Urea eluent; 6: 100mM Urea eluent; 7: 150mM Urea eluent; 8: 200mM Urea eluent; 9: 300mM Urea eluent Figure 6 Dialysis refolding and concentration diagram of recombinant protein: M: marker; 1: recombinant protein Figure 7 This is a diagram of purification after recombinant proteinase cleavage. M: Marker; 1: enzyme cleavage mixture; 2: column effluent; 3: 50mM imidazole elution; 4: target protein concentrated solution; Figure 8 This is a Western-blotting diagram of protease cleavage purification; where M: Marker; 1: recombinant protein; 2: enzyme cleavage mixture; 3: purified protein after enzyme cleavage; 4: 50mM imidazole elution solution; 5: target protein concentrate Fig. 9 To investigate the effect of injection of target protein on the food intake of Siberian sturgeon; Fig.10 Effects of injected protein on the expression of appetite genes in the hypothalamus and stomach of Siberian sturgeon. DETAILED DESCRIPTION

[0019] 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.

[0020] Example 1 1. Cloning of target gene According to the instructions of the Animal Tissue Total RNA Extraction Kit (FOREGENE), the total RNA of the Siberian sturgeon liver was extracted, and then the first-strand cDNA was synthesized by reverse transcription according to the 047A (TaKaRa) kit. Then, the prokaryotic expression primers with restriction sites were designed: Upstream primer F1: 5'-C GGATCCGACGACGACGACAAA CAAGATAATTGCTT AC-3'; Downstream primer R1: 5'-GGGGGG AAGCTT TTAATACTGTCCTCTGTACTTATTGC-3'; Add protective base (C), BamHI restriction site (GGATCC), and enterokinase restriction site (GATGACGATGACAAA) to the 5' end of F1, and add protective base and HindⅢ restriction site (AAGCTT) to the 5' end of R1. PCR amplification conditions: 94℃, 5min denaturation, 35 cycles (94℃, 30s; 65℃, 30s; 72℃, 50s), 72℃ extension for 10 min. After purification and recovery, the PCR product was connected to the pMD19-T vector, and the positive bacterial clone was selected for sequencing verification. The gel electrophoresis results are as follows Figure 1 shown.

[0021] The nucleotide sequence encoding the functional region of Siberian sturgeon RBP4 is as follows: CAAGATAATTGCTTACTAAGCAACATCAACGTGATGCAAAATTTTGATAGACACAGATATGCAGGAACATGGTACGCTGTTGCCAAAAAAGACCCAGTGGGACTTTTTCTTCTAGATAACATAAAAGCTAACTATGTCATCAATGAGGATGGTGTAATGACCGCCACAGCCGAGGGAAGAGTCATCATTTTAAATAACTGGGAGCTGTGTGCAGAAATGTTTGGCACTTTTGAGGATACAGTTGACCCTGCTAAATTCAAGATGAAGTACTGGGGAGCTGCAGCTGTACTTCAACAAGGACTTGACAACCACTGGGTTATAGACACAGACTATGACAACTATGCCATCCACTACTCCTGCCGCGAGCAGAGCTGGGATGGCACCTGCTTGGACAGCTACTCCTTCATCTTCTCCCGTGACCCTAAAGGCCTGACCCCAGAGTCACAGAGGATTGTCAGGAAGAAGCAGGAGGGGCTCTGCTTGGTTGGGAAATACAGGCGTGTGGCTCAGGACGGATTCTGCAATAAGTACAGAGGACAGTATTAA (SEQ ID NO.1).

[0022] The amino acid sequence of the RBP4 (SsRBP4) protein of Acipenser baerii is as follows, with a molecular mass of 20.881 kDa: QDNCLLSNINVMQNFDRHRYAGTWYAVAKKDPVGLFLLDNIKANYVINEDGVMTATAEGRVIILNNWELCAEMFGTFEDTVDPAKFKMKYWGAAAVLQQGLDNHWVIDTDYDNYAIHYSCREQSWDGTCLDSYSFIFSRDPKGLTPESQRIVRKKQEGLCLVGKYRRVAQDGFCNKYRGQY (SEQ ID NO.2).

[0023] 2. Protein expression and purification (1) Construction of recombinant plasmid The plasmid for cloning the coding region and the expression vector pET-32a were used, and the restriction endonucleases BamHI and HindⅢ were used for double digestion. The target fragment and the large vector fragment were recovered, and the recombinant expression plasmid pET32a-SsRBP4 was obtained by ligation with T4 enzyme. 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.

[0024] (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 ampicillin) until OD 600 The value reached 0.4~0.6 (37℃, 180 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 41kD.

[0025] (3) Optimization of induction conditions The bacterial culture solution was cultured to OD 600 When the value was 0.4-0.6, different volumes of IPTG stock solution were added to the culture medium with final concentrations of 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 and 1.4 mmol / L, respectively, and cultured at 37°C for 8 h to explore the induction effect of different concentrations of IPTG on protein expression. Figure 2 As shown, when the IPTG induction concentration was 1.0-1.4 mM, the expression level of the recombinant protein was higher than that in other concentration groups.

[0026] When the bacterial culture reached OD 600 When the value was 0.4-0.6, 1.0 mmol / L IPTG was added and the cells were cultured at 37℃ for 0, 2, 4, 6, 8, 10, 12 and 14 h to explore the effect of different induction times on protein expression. The bacterial precipitate was collected and subjected to SDS-PAGE electrophoresis. The results are shown in Figure 3 As shown, a large amount of recombinant protein can be obtained by inducing for 2-14 hours.

[0027] (4) Recombinant protein solubility test The bacterial culture solution was cultured to OD 600When the value was 0.4-0.6, 1.0 mM IPTG was added to induce expression for 2 h. The precipitate was collected by centrifugation at 12000 rpm for 1 min, washed twice with PBS, and then resuspended with 1 ml PBS. Lysozyme (0.1 mg / ml) was added to digest at room temperature for 30 mins, and the cells were repeatedly frozen and thawed 4-5 times for disruption. The cells were centrifuged at 12000 rpm for 5 min, and the supernatant and precipitate were collected for SDS-PAGE electrophoresis to analyze whether the target protein expression product was mainly present in the precipitate or the supernatant, and then determine whether the expressed protein was an inclusion body protein or a soluble protein. The results are shown in Figure 4 As shown, the recombinant protein was mainly present in the supernatant, indicating that it was an inclusion body protein.

[0028] (5) Purification and renaturation of recombinant proteins The expression bacteria were cultured overnight at 37°C with shaking, and 5 ml was inoculated into 250 ml of fresh culture medium. The bacterial solution was cultured until OD 600 When the value was 0.4-0.6, 1.0 mM IPTG was added to induce expression for 2 h, and the precipitate was collected by centrifugation for 10 min. PBS was added to resuspend and wash the precipitate 3 times. Lysozyme (0.1 mg / ml) was then added to react for 0.5 h. After freezing and thawing 3 times, the precipitate was collected by centrifugation. The precipitate was dissolved in 8 M Urea buffer, impurities were filtered out with a 0.45 μm microporous filter, and Ni 2+ -NTA affinity chromatography column was used to separate and purify the target protein using 8M Urea buffer containing different concentrations of imidazole, and the protein purity was detected by SDS-PAGE electrophoresis. Figure 5 As shown, the optimal elution condition for recombinant protein purification is 8M Urea elution buffer containing 50mM imidazole.

[0029] (6) Recombinant protein dialysis refolding and concentration The purified fusion protein was placed in a dialysis bag, and the external solution was replaced by gradient renaturation to 6M, 4M and 2MUrea, and finally dialyzed in PBS at 4°C overnight. After being concentrated to 2ml using an ultrafiltration tube, the protein purity was detected by SDS-PAGE electrophoresis. Figure 6 As shown, the recombinant protein was dialyzed into PBS solution in a soluble form.

[0030] (7) Enterokinase digestion to obtain mature peptide protein The purified recombinant protein was dialyzed into 25 mM Tris-HCl (pH 8.0) solution, the protein concentration was determined by BCA method, and an appropriate amount of enterokinase (Solebol) was added and digested at 4 °C for 14 h. 2+-NTA affinity chromatography column was used to separate 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 7 As shown, the mature peptide target protein with high purity can be successfully eluted using a 25 mM Tris-HCl (pH 8.0) solution containing 50 mM imidazole.

[0031] (8) Immunoblotting to detect the purity of mature peptides The recombinant protein before enzyme cleavage, the reaction solution of protease cleavage, the purified outflow target protein, the protein eluate containing 50mM imidazole and the purified outflow target protein concentrate were subjected to SDS-PAGE electrophoresis, and then wet-transferred to a 0.22 μm PVDF membrane, blocked with 5% BSA membrane blocking solution for 1 hour, repeatedly rinsed with 1XTBST for 30 minutes, incubated with anti-6×His tag mouse monoclonal antibody (Transgen) primary antibody at room temperature for 1 hour, rinsed with TBST 3 times, incubated with HRP-labeled goat anti-mouse IgG (Transgen) secondary antibody, rinsed with TBST 3 times, and photographed with E-BLOT development in combination with ECL colorimetric solution to further verify the purity of the target protein. The results are as follows Figure 8 As shown, the target protein was purified successfully after enzyme digestion.

[0032] Example 2 Regulatory effect of target protein on feeding of Siberian sturgeon Regulatory effect of target protein on food intake Forty-eight Siberian sturgeons (29.42±3.13 g) were randomly divided into four groups, with three fish tanks in each group and four fish in each tank. At 13:30, the fish were anesthetized by immersion in MS-222 (0.01%), and PBS and SsRBP4 target protein (30, 100 and 300 ng / g BW) were injected intraperitoneally. Feed was given at 14:00, which was marked as 0h. The residual bait was collected at 0-1h (15:00), 1-3h (17:00) and 3-6h (20:00), and dried in an oven to constant weight. The changes in food intake at 0-1h, 1-3h and 3-6h were measured and analyzed to clarify the regulatory effect of SsRBP4 on the feeding of Siberian sturgeons.

[0033] The results are as follows Fig. 9 As shown in the figure, compared with the PBS group, intraperitoneal injection of three doses of protein did not affect the food intake of Siberian sturgeon at 0-1h, while 30, 100 and 300 ng / gBW protein significantly increased the food intake at 1-3h and 3-6h (P<0.05). The above results show that SsRBP4 protein can promote the feeding of Siberian sturgeon, and its effective time is 3h and 6h.

[0034] Regulatory effect of target protein on the expression of appetite factors 24 Siberian sturgeon juveniles (32.12±5.43g) were selected and randomly divided into 2 groups, with 3 fish tanks in each group and 4 fish in each tank. At 13:30, they were anesthetized by immersion in MS-222 (0.01%) and intraperitoneally injected with PBS and an effective dose (100 ng / g BW) of SsRBP4 target protein. Three sturgeons were randomly selected from each tank, and after weighing, hypothalamus and stomach tissues were collected to determine the changes in the expression of appetite factors (NPY, Agrp, Ghrelin and Insulin). The sequences of fluorescent quantitative primers are shown in Table 1.

[0035] Table 1 Fluorescence quantitative primers

[0036] like Fig.10 As shown in the figure, intraperitoneal injection of 100ng / g BW SsRBP4 target protein can significantly increase the expression of NPY and AgRP mRNA in the hypothalamus, but does not affect the expression of hypothalamic Insulin and gastric Ghrelin mRNA. This indicates that SsRBP4 has the effect of promoting NPY and AgRP factors in the hypothalamus, the feeding regulation center of Siberian sturgeon, to increase appetite.

[0037] 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. Use of RBP4 protein in increasing fish food intake, the amino acid sequence of which is shown in SEQ ID NO.

1.

2. Use of RBP4 protein in the preparation of fish anorectic agents with the effect of promoting feeding.

3. Use of RBP4 protein in the preparation of fish feed additives.

4. The use according to any one of claims 1 to 3, characterized in that The fish is a Siberian sturgeon.

5. The use according to any one of claims 1 to 3, characterized in that: The effective amount of the RBP4 protein is 100-300 ng / g BW.

6. A gene encoding the RBP4 protein of claim 1, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

7. A carrier, characterized in that Comprising the gene according to claim 6.

8. A cell line, characterized in that Comprising the gene according to claim 6 or the vector according to claim 7.

9. A recombinant bacterium, characterized in that: Comprising the gene according to claim 6 or the vector according to claim 7.

10. A preparation for promoting fish feeding, characterized in that: The active ingredient of the invention is a substance that promotes the expression of the protein encoding the RBP4 as claimed in claim 1.

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