A protein that enhances fish feed intake, its encoding gene, and its uses.

Abstract

The present invention discloses a protein, a coding gene and a use for increasing the food intake of fish, and belongs to the field of genetic engineering technology. The amino acid sequence of the protein is shown in SEQ ID NO.1. The present invention obtains a new protein that can increase the food intake of Siberian sturgeon, which can not only increase the food intake of Siberian sturgeon, but also significantly enhance the antioxidant capacity of Siberian sturgeon, and is expected to reduce the mortality rate of Siberian sturgeon and improve its growth and development level.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a protein for increasing the food intake of fish, a coding gene and a use thereof. Background Art

[0002] Maintaining the body's energy homeostasis is crucial for the growth, development and reproduction of animals, while an imbalance in food intake and consumption will damage the body's energy homeostasis and lead to metabolic abnormalities. In the process of aquaculture, fish often produce adverse reactions such as oxidative stress and inflammation due to inappropriate temperature, disease, water quality and stocking density, causing adverse hazards such as lipid peroxidation, DNA damage and malabsorption, and even causing fish to stop eating, seriously reducing the economic benefits of farmers. Animal appetite is often jointly regulated by appetite regulatory factors secreted by the central feeding system and the peripheral feeding system. Some appetite regulatory factors have been isolated from the central system and peripheral tissues of animals, including appetite-increasing factors: neuropeptide Y (NPY), agouti related protein (AgRP) and ghrelin; appetite-suppressing factors: proopiomelanocortin (POMC), leptin and cholecystokinin (CCK). The central nervous system can regulate fish feeding behavior and energy homeostasis through the signal transduction effects and central signaling pathways of these central and peripheral appetite regulatory factors.

[0003] The Siberian sturgeon (Acipenser baerii Brandt) is known as black gold because of its meat rich in high-quality protein and polyunsaturated fatty acids, and its cartilage rich in sulfur chondroitin. Therefore, it is deeply loved by consumers and farmers, making China the largest producer and exporter of sturgeon caviar. The ultimate goal of aquaculture is to obtain high-quality and high-yield aquatic products with minimal input, and to meet the people's growing material and cultural needs while maximizing economic benefits. However, the low feeding rate, high mortality rate and long breeding cycle of Western sturgeon seedlings are important reasons that restrict the efficient development of sturgeon farming. Metabolic diseases in the breeding process seriously affect the growth and development of Siberian sturgeons, reduce meat quality, and lead to a decrease in caviar production and quality, affecting economic benefits and living standards. Therefore, a deep understanding of the feeding mechanism of Siberian sturgeons is of great production significance to promote the development of Western sturgeon farming. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the present invention provides a protein, encoding gene and use for increasing the food intake of fish, which can effectively increase the food intake of Siberian sturgeon.

[0005] To achieve the above purpose, the technical solution adopted by the present invention to solve the technical problem is:

[0006] A protein for increasing the food intake of fish, the amino acid sequence of the protein is shown in SEQ ID NO.1.

[0007] Furthermore, the protein can upregulate the expression level of the appetite factor Ghrelin.

[0008] A nucleotide encoding the above protein, whose sequence is shown in SEQ ID NO.2.

[0009] A recombinant plasmid comprises the above nucleotides.

[0010] A recombinant bacterium comprises the above-mentioned recombinant plasmid.

[0011] The protein is used in preparing a preparation for increasing the food intake of fish.

[0012] Use of the above protein in improving the antioxidant capacity of fish.

[0013] Furthermore, the fish is a Siberian sturgeon.

[0014] Furthermore, the protein can promote the expression of SOD1 and SOD2.

[0015] Beneficial effects of the present invention:

[0016] The present invention obtains a new protein that can increase the food intake of Siberian sturgeon. The protein can not only increase the food intake of Siberian sturgeon, but also significantly enhance the antioxidant capacity of Siberian sturgeon, and is expected to reduce the mortality rate of Siberian sturgeon and improve its growth and development level. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The electrophoresis pattern of the PCR product of the target gene;

[0018] Figure 2 The result diagram of IPTG induction concentration optimization for recombinant vector expression; where M: protein marker; 1: sample without recombinant vector induction; 2-8: recombinant vector expression induced by IPTG concentrations of 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 and 1.4 mmol / L, respectively; 9: pET-32a (+) induction;

[0019] Figure 3 The result diagram of IPTG induction time optimization for recombinant vector expression; M: protein marker; 1: sample without recombinant vector induction; 2-8: samples of recombinant vector transformed bacteria induced for 2, 4, 6, 8, 10, 12 and 14 hours respectively; 9: pET-32a(+) induction;

[0020] Figure 4 The solubility test result of the recombinant mature protein is shown in the figure; M: Marker; 1: pET32a empty load; 2: mixed solution after the recombinant protein bacterial solution is broken; 3: supernatant after the recombinant protein bacterial solution is broken; 4: precipitate after the recombinant protein bacterial solution is broken;

[0021] Figure 5 The figure shows the purification of recombinant protein. M: Marker; 1: effluent; 2: washing solution; 3: pH 6.0 eluent; 4: pH 5.0 eluent; 5: pH 4.0 eluent; 6: pET32a empty load;

[0022] Figure 6 This is the result diagram of optimization of recombinant protease cleavage conditions; where M: Marker; 1-4: induced at 4°C for 14, 16, 18 and 20 hours respectively; 5-8: induced at 25°C for 14, 16, 18 and 20 hours respectively;

[0023] Figure 7 The diagram of protease cleavage and purification; M: Marker; 1: protein before cleavage; 2: cleavage mixture; 3: purified protein after cleavage; 4: pET32a empty protein;

[0024] Figure 8 The SDS-Page electrophoresis diagram (left) and Western blotting diagram (right) of protease cleavage purification; M: Marker; 1: protein before cleavage; 2: cleavage mixture; 3: purified protein after cleavage; 4: pET32a empty protein;

[0025] Fig. 9 The effect of intraperitoneal protein injection on the food intake of Siberian sturgeon;

[0026] Fig.10 The effect of intraperitoneal protein injection on the expression of appetite-regulating genes in the hypothalamus of Siberian sturgeon;

[0027] Fig.11 Effects of intraperitoneal protein injection on the expression of appetite-regulating genes in the liver of Siberian sturgeon;

[0028] Fig.12 The effect of intraperitoneal protein injection on the expression of SOD gene in the liver of Siberian sturgeon;

[0029] Fig.13 The effect of intraperitoneal protein injection on the expression of SOD gene in the duodenum of Siberian sturgeon;

[0030] Fig.14 This study is the effect of intraperitoneal injection of protein on the expression of SOD gene in the Siberian sturgeon. DETAILED DESCRIPTION

[0031] 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.

[0032] Example 1

[0033] 1. Cloning of target gene

[0034] Total RNA from the liver of Siberian sturgeon was extracted according to the instructions of Animal Total RNA Isolation Kit (FOREGENE), and then the first-strand cDNA was synthesized by reverse transcription using the PrimeScript TM RT Reagent Kit 037A (TaKaRa). Then, the upstream primer P1: 5'-GGAATTCGATGACGATGACAAAATGTCTCAGGCC-3' and the downstream primer P2: 5'-GAACTAAAAGAAAAATACGGCATTTAAAAGCTTGGG-3' were designed for prokaryotic expression. The EcoI restriction site (GAATTC), protective bases and enterokinase cleavage site (GATGACGATGACAAA) were added to the 5' end of P1, and the HindIII restriction site (AAGCTT) and protective bases were added to the 5' end of P2. PCR amplification was performed using the above primers, and the PCR amplification conditions were: 94℃, 5min denaturation, 35 cycles (94℃, 30s; 67℃, 30s; 72℃, 50s), and 72℃ extension for 10min. The target fragment was excised and recovered from the gel and connected to the pMD19-T vector. The positive clones were selected for sequencing verification. The gel electrophoresis after PCR reaction was as shown in Figure 1 shown.

[0035] The nucleotide sequence encoding the Siberian sturgeon appetite stimulating protein is as follows:

[0036] ATGTCTCAGGCCGACTTTGATAAAGCTGCTGAGGAGGTCAAGAATTTGAAATCTCAGCCCTCAGACAAGGAAATGCTGGAGATTTACAGTTTGTTCAAACAAGCAACAGTTGGAGATGTCAACACGGCACGTCC TGGCATGCTGGACTTCAAAGGAAAGGCCAAGTGGGACGCCTGGGATGCTCGTAAAGGAATGGGCAAGGAAGATGCAATGAAGGCCTATATTGCAAAAGTTGAAGAACTAAAAGAAAAATACGGCATTTAA(SEQ ID NO.2).

[0037] The amino acid sequence of the Siberian sturgeon appetite-stimulating protein is as follows, and the molecular mass is 9821.23Da:

[0038] MSQADFDKAAEEVKNLKSQPSDKEMLEIYSLFKQATVGDVNTARPGMLDFKGKAKWDAWDARKGMGKEDAMKAYIAKVEELKEKYGI (SEQ ID NO. 1).

[0039] 2. Protein expression and purification

[0040] (1) Construction of recombinant plasmid

[0041] The recombinant clone plasmid and expression vector pET-32a were extracted, double-digested with EcorI and HindⅢ respectively, recovered by gel excision, and ligated with T4 ligase to obtain the expression recombinant plasmid pET32a, which was transformed into DH5α competent cells. After bacterial culture, PCR detection and double-digestion verification were performed, and the correct recombinant plasmid was sequenced.

[0042] (2) Expression of recombinant plasmid in expression strain

[0043] The expression recombinant plasmid with correct sequencing was transformed into the expression strain E. coli BL21 (DE3) and cultured in liquid LB medium (containing ampicillin) until OD 600 The value reached 0.4-0.6 (37°C, 200rpm / min), and isopropyl-β-D-thiogalactoside (IPTG, 1mmol / L) was added for induction culture to express the recombinant plasmid in the expression strain. The estimated size of the recombinant expression protein was 28.389kD.

[0044] (3) Optimization of induction conditions

[0045] The bacterial solution was cultured at 37°C until OD 600When the value was 0.4-0.6, IPTG was added to the final concentrations of 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 and 1.4 mmol / L, respectively, and cultured with shaking for 8 hours to study the induction effect of different IPTG concentrations on the expression of recombinant protein.

[0046] The results are as follows Figure 2 It shows that when the IPTG induction concentration is 0.2-0.8mM, the expression level of pET32a recombinant protein is higher than that of other IPTG treatment groups.

[0047] According to the concentration optimization method, the bacterial solution was cultured at 37°C until OD 600 When the value was 0.4-0.6, IPTG was added to a final concentration of 0.8 mmol / L, and the time gradient was set to 0, 2, 4, 6, 8, 10, 12 and 14 hours. SDS-PAGE electrophoresis was then performed to study the induction effect of different induction times on the expression of recombinant proteins. The results are shown in Figure 3 It shows that the optimal induction time of pET32a recombinant protein is 8h.

[0048] (4) Solubility detection of recombinant mature protein

[0049] The bacterial solution was cultured at 37°C until OD 600 When the value is 0.4-0.6, add 0.6mM IPTG to induce for 8h. Centrifuge at 10000g for 5min in a centrifuge, collect the precipitate, and wash it repeatedly for 3 times with 30mL of Tris-HCl-NaCl (25mM, pH 8.0) each time. Add 30mL of Tris-HCl-NaCl (25mM, pH8.0) to resuspend the precipitate, add 0.1mg / ml lysozyme, digest at room temperature for 30min, and freeze-thaw repeatedly 4-5 times. After the bacteria are broken, centrifuge at 10000g for 5min in a centrifuge, collect the supernatant and precipitate at the same time, and finally perform SDS-PAGE electrophoresis to observe whether the expression product of the target gene is mainly present in the supernatant or the precipitate, so as to determine whether the expression product is a soluble protein or an inclusion body protein. The results are as follows Figure 4 As shown, the pET-32a recombinant protein was in soluble form and mainly existed in the supernatant.

[0050] (5) Purification of recombinant protein

[0051] The recombinant bacteria induced by IPTG (0.6 mmol / L) for 8 h at 37°C were centrifuged (10000 g / min, 5 min), the precipitate was collected and washed and resuspended with Tris-HCl-NaCl (25 mM, pH 8.0), lysozyme (0.1 mg / ml) was added and the bacteria were repeatedly frozen and thawed to break them, the supernatant was collected, filtered with a 0.45 μm microporous filter membrane, and the fusion protein was separated and purified using a Ni2+-NTA affinity chromatography column (GE), and the protein purity was detected by SDS-PAGE electrophoresis.

[0052] The results are as follows Figure 5 As shown, the optimal elution condition for recombinant protein purification was tris-HCl-NaCl buffer at pH 4.0.

[0053] (6) Obtaining target protein by recombinant protease cleavage

[0054] The purified fusion protein was dialyzed into a pH 8.0 25mM Tris-HCl solution, and the protein concentration was determined by the BCA method. Enterokinase was added to the fusion protein solution and digested at 4°C / 25°C for 14, 16, 18, and 20 hours. The optimal digestion time of enterokinase was screened by SDS-page.

[0055] The results are as follows Figure 6 As shown, the optimal enzyme cleavage temperature for the recombinant fusion protein is 4°C, and the optimal enzyme cleavage time is 14-20h.

[0056] (7) Purification and detection of target protein

[0057] Take the protein solution digested at 4°C for 14 hours, separate and purify the target protein using Ni2+-NTA affinity chromatography column (GE) (repeat the column three times), and detect the protein purity by SDS-PAGE electrophoresis. Determine the concentration of the target protein by BCA method, and then filter and sterilize it with a 0.22μm microporous filter membrane and store it in a -80°C ultra-low temperature refrigerator for later use.

[0058] After SDS-PAGE electrophoresis, the pET-32a fusion protein before enzyme digestion, the enzyme digestion mixture, the target protein recovered by enzyme digestion, and the pET 32a empty protein were transferred to a 0.2μm PVDF membrane (Bio-rad) using a semi-dry electrotransfer instrument, blocked with a membrane blocking solution, and incubated at room temperature for 2h and 1h respectively with an anti-6×His tag mouse monoclonal antibody (Transgen) as the primary antibody and HRP-labeled goat anti-mouse IgG (Transgen) as the secondary antibody. The samples were then developed with an ECL colorimetric solution, and photographed to detect whether the target protein was successfully prepared and its purity. The results are shown in Figure 2. Figure 7 and Figure 8 As shown, the target protein was purified successfully after enzyme digestion.

[0059] Example 2 Effect of protein on feeding of Siberian sturgeon

[0060] 1. Effects of intraperitoneal injection of protein on feeding of Siberian sturgeon

[0061] Forty-eight healthy Siberian sturgeons weighing 35±4.3g were randomly divided into four groups, with three replicates in each group and four fish in each replicate. The four groups were intraperitoneally injected with 1×PBS, 100ng / g BW protein, 300ng / g BW protein and 500ng / g BW protein at 13:30, respectively. The Siberian sturgeons were anesthetized by immersion in 0.01% MS-222 before injection. The feeding point at 14:00 was recorded as 0h, and the feed residues were collected at 1h (15:00), 3h (17:00) and 6h (20:00), respectively. At the same time, a blank control group was set up at each time point to detect the feed loss rate. The feed residues were dried in an oven at 60℃ to constant weight, and the changes in the food intake of Siberian sturgeons at 1h, 3h and 6h were detected. The cumulative food intake was calculated to clarify the regulatory effect of intraperitoneal injection of protein on the appetite of Siberian sturgeons.

[0062] Effects of intraperitoneal protein injection on food intake (A) and cumulative food intake (B) of Siberian sturgeon Fig. 9 As shown, intraperitoneal injection of protein can promote the feeding of Siberian sturgeon. Compared with the PBS control group, intraperitoneal injection of protein at various concentrations did not affect the feeding amount of Siberian sturgeon from 0 to 1 h; 100 ng / g BW protein can significantly increase the feeding amount of Siberian sturgeon from 1 to 3 h and 3 to 6 h (P<0.01) and the cumulative feeding amount of 3 h and 6 h (P<0.05); 300 ng / g BW and 500 ng / g BW protein can also significantly increase the feeding amount of Siberian sturgeon from 1 to 3 h (P<0.01) and 3 to 6 h (P<0.001) and the cumulative feeding amount of 3 h (P<0.01) and 6 h (P<0.001). The above results show that intraperitoneal injection of protein can promote the feeding of Siberian sturgeon.

[0063] 2. Effect of intraperitoneal protein injection on the expression of appetite regulatory factors in Siberian sturgeon

[0064] Twenty-four Siberian sturgeons weighing 35±4.3g and eating normally were selected and divided into two groups, with three replicates in each group and four sturgeons in each replicate. PBS and 300ng / g BW protein were injected intraperitoneally. Nine sturgeons were randomly selected from each group, weighed, and hypothalamic tissues were collected to detect changes in the mRNA expression of appetite regulatory factors (POMC, CART, NPY, Agrp, Ghrelin, CCK, and PYY). The nucleotide sequences of the fluorescence quantitative primers are shown in Table 1, where F represents the forward primer and R represents the reverse primer.

[0065] Table 1 Fluorescence quantitative primers

[0066]

[0067]

[0068] Effects of intraperitoneal protein injection on the expression of appetite-regulating genes in Siberian sturgeon Figure 10-11 In the hypothalamus, intraperitoneal injection of 300 ng / g BW protein significantly downregulated the expression levels of CART, Agrp (p<0.001), NPY and CCK (p<0.01), upregulated the expression level of Ghrelin (p<0.001), and had no significant effect on POMC and PYY ( Fig.10 ); In the liver, the protein significantly downregulated the expression levels of APN and insulin (p<0.001), but had no significant effect on the expression level of leptin ( Fig.11 ).

[0069] 3. Effect of intraperitoneal protein injection on the expression of SOD, a key gene in the antioxidant system of Siberian sturgeon

[0070] Effects of intraperitoneal protein injection on the expression of SOD, a key gene of the antioxidant system in Siberian sturgeon Figure 12-14 In the liver, intraperitoneal injection of protein significantly upregulated the expression levels of SOD1 and SOD2 genes (p<0.01) and significantly downregulated the expression level of SOD3 gene (p<0.001) ( Fig.12 In the duodenum, the protein significantly upregulated the expression levels of SOD1 (p<0.05) and SOD2 (p<0.01), but had no significant effect on the expression level of SOD3 ( Fig.13 In the valvular intestine, the protein significantly upregulated the expression of SOD1 and SOD2 (p<0.01), but had no significant effect on the SOD3 gene ( Fig.14 ). The above results showed that intraperitoneal injection of protein could promote the antioxidant capacity of Siberian sturgeon.

[0071] 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 increasing the food intake of fish, characterized in that The amino acid sequence of the protein is shown in SEQ ID NO.

1.

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

2.

3. A recombinant plasmid, characterized in that: Comprising the nucleic acid of claim 2.

4. A recombinant bacterium, characterized in that: It comprises the nucleic acid according to claim 2 or the recombinant plasmid according to claim 3.

5. Use of the protein according to claim 1 in preparing a preparation for increasing the food intake of Siberian sturgeon.

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