Acipenser baerii appetite promoting factor angiopoietin-like protein and application thereof

Abstract

The invention relates to the technical field of feeding of acipenser sibiricum, in particular to an acipenser sibiricum appetite promoting factor angiopoietin-like protein and application thereof, in-vitro prokaryotic recombinant expression of ANGPTL4 mature peptide is achieved for the first time, and acipenser sibiricum ANGPTL4 / pET-32a protein with biological activity is obtained through purification. The provided Acipenser sibiricum ANGPTL4 / pET-32a recombinant protein can lay a foundation for subsequent research on biological functions of ingestion regulation and control.

Description

Technical Field

[0001] The present invention relates to the technical field of the feeding of Acipenser baerii, and specifically relates to an appetite-promoting factor angiopoietin-like protein of Acipenser baerii and its application. Background Art

[0002] Feeding is a basic physiological process for animals to maintain life activities and is also the basis for the growth and development of animals. It is closely related to appetite, hunger, and satiety. Appetite is a physiological and psychological factor that dominates feeding and food selection. The formation and regulation mechanism of animal appetite are mainly controlled by the "appetite regulation network" composed of the central neuroendocrine system and peripheral organs. Among them, the central neuroendocrine system includes neuropeptides and monoamines, and the peripheral feeding system includes gastrointestinal peptides and hormones.

[0003] These in-vivo appetite factors either play an anorectic role or play an orexigenic role. Orexigenic factors include orexin, agouti-related protein (AgRP), and neuropeptide (NPY), etc.; anorectic factors include proopiomelanocortin (POMC), cocaine- and amphetamine-regulated transcript (CART), leptin, cholecystokinin (CCK), and angiopoietin-like 4 (ANGPTL4), etc.

[0004] ANGPTL4 was successively discovered by three independent research teams in 2000. It is also known as hepatic fibrinogen / angiopoietin-related protein (HFARP), peroxisome proliferator-activated receptor gamma (PPARγ)-angiopoietin related (PGAR), and fasting-induced adipose factor (FIAF). The structure of ANGPTL4 is conserved among species, and its distribution is similar. ANGPTL4 has multiple functions, including energy balance (food intake, glucose metabolism, lipid metabolism), wound healing, tumorigenesis, angiogenesis and vascular permeability, cell differentiation, and redox regulation (fatty acid oxidation). However, there are very few reports on the role of ANGPTL4 in regulating food intake. Currently, it is only reported that it may play an anorectic role in mice, and there is no report in fish. Summary of the Invention

[0005] In view of this, the present invention provides an appetite-promoting factor angiopoietin-like protein of Acipenser baerii and its application method in the feeding of Acipenser baerii.

[0006] To solve the above technical problems, the technical solution provided by the present invention is: an appetite-promoting factor angiopoietin-like protein of Acipenser baerii, and the amino acid sequence of the angiopoietin-like protein is shown as SEQ ID NO: 1.

[0007] An in vitro encoded recombinant protein as claimed in claim 1, and the amino acid sequence of the recombinant protein sequence is shown as SEQ ID NO: 2.

[0008] The second aspect of the present invention discloses the application of an in vitro encoded recombinant protein in the feeding regulation of Acipenser baerii.

[0009] The third aspect of the present invention discloses the application of an in vitro encoded recombinant protein in the feeding regulation of Acipenser baerii.

[0010] The advantages of the present invention compared with the prior art are as follows: The present invention takes Acipenser baerii as the research object, clones the cDNA sequence of the Acipenser baerii ANGPTL4 gene by Reverse Transcription polymerase Chain Reaction (RT-PCR); uses Real-time quantitative reverse transcription-PCR (qRT-PCR) to detect the tissue distribution of ANGPTL4, laying a foundation for further studying its physiological functions and action mechanisms; uses qRT-PCR to detect the effects of different feeding strategies (before and after feeding, fasting, and refeeding) on the expression of ANGPTL4; and by intraperitoneal injection of the functional fragment of ANGPTL4, detects the changes in indicators such as the food intake of Acipenser baerii and the expression levels of related appetite regulatory factors, providing a theoretical reference for the feeding regulation of Acipenser baerii. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 It is a diagram of the CDS sequence of Acipenser baerii ANGPTL4 and the deduced amino acid sequence.

[0012] Figure 2 It is a multiple alignment diagram of the ANGPTL4 amino acid sequence.

[0013] Figure 3 It is a phylogenetic tree analysis diagram of the ANGPTL4 amino acid sequence.

[0014] Figure 4 It is a predicted structural diagram of the ANGPTL4 protein.

[0015] Figure 5 It is the identification result of the recombinant pET-32a-ANGPTL4 nucleic acid fragment by colony PCR gel electrophoresis.

[0016] Figure 6 It is the SDS-PAGE map of the pET-32a-ANGPTL4 transformed bacteria induced by different IPTG concentrations.

[0017] Figure 7 It is a schematic diagram of the SDS-PAGE map of the pET-32a-ANGPTL4 transformed bacteria expressed at different induction times.

[0018] Figure 8 It is the identification diagram of the soluble expression of the recombinant pET-32a-ANGPTL4.

[0019] Figure 9 It is the affinity purification electrophoresis analysis diagram of the recombinant pET-32a-ANGPTL4.

[0020] Figure 10 It is the electrophoretic analysis diagram of ANGPTL4 affinity purification.

[0021] Figure 11 It is the western-blotting verification diagram of the ANGPTL4 recombinant protein.

[0022] Figure 12 It is the diagram of the changes in glucose and triglyceride levels in serum after injecting ANGPTL4.

[0023] Figure 13 It is the schematic diagram of the effect of intraperitoneal injection of ANGPTL4 on the expression of appetite factors in the hypothalamus (A), liver (B), stomach (C) and valvular intestine (D) of Acipenser baerii.

[0024] Figure 14 It is the schematic diagram of the effect of intraperitoneal injection of ANGPTL4 on the expression of factors in the hypothalamic feeding-related pathway. Detailed implementation mode

[0025] The present invention will be further described in detail below with reference to the accompanying drawings.

[0026] An appetite-promoting factor angiopoietin-like protein of Acipenser baerii, characterized in that the amino acid sequence of the angiopoietin-like protein is as shown in SEQ ID NO: 1.

[0027] The amino acid sequence of the recombinant protein sequence is as shown in SEQ ID NO: 2.

[0028] The present invention also provides an application of an in vitro encoded recombinant protein in the feeding regulation of Acipenser baerii.

[0029] On the other hand, the present invention provides an application of an in vitro encoded recombinant protein in the feeding regulation of Acipenser baerii.

[0030] During specific implementation: Gene cloning, tissue distribution sample collection, RNA extraction and cDNA synthesis:

[0031] Six healthy juvenile Acipenser baerii (215.20 ± 29.63 g) were randomly selected 6 h before feeding. After anesthesia with 0.01% MS-222, five brain regions (forebrain, midbrain, hypothalamus, cerebellum and medulla oblongata), as well as 16 peripheral tissues (esophagus, stomach, pyloric caeca, duodenum, valvular intestine, rectum, heart, liver, pancreas, spleen, kidney, eye, gill, swim bladder, white muscle and skin) were frozen rapidly in liquid nitrogen and ground into powder, then stored at -80 °C for RNA extraction.

[0032] The isolated tissues were used to extract total RNA with an RNA extraction kit. The integrity of the RNA was detected by 1.0% agarose gel electrophoresis, and the purity and concentration of the RNA were detected by a nucleic acid protein analyzer to screen out RNA with qualified quality.

[0033] The obtained total RNA was reverse transcribed using a 047 reverse transcription kit to prepare a cDNA template for gene cloning and fluorescence quantitative analysis.

[0034] Cloning and sequence analysis of angiopoietin-like protein 4:

[0035] Based on the genome of Acipenser ruthenus published by NCBI, the sequence information of the ANGPTL4 gene was retrieved. The sequence was subjected to multiple sequence alignment with the ANGPTL4 genes of other species. According to the retrieved conserved regions, a pair of specific primers was designed to amplify a partial sequence.

[0036] The specificity and brightness of the PCR product bands were detected by 1.5% agarose gel electrophoresis. Subsequently, the target fragment band was excised, and DNA recovery was performed using a gel extraction kit.

[0037] The obtained target fragment was ligated to the cloning vector pMD19-T and transformed into DH5α competent cells. After recovery and overnight culture on plates, colonies were picked and cultured in LB liquid medium containing 100 μg / μl ampicillin. After PCR identification, the bacterial solution was sent for inspection.

[0038] Finally, the software DNAman was used to analyze the partial sequence obtained by cloning to obtain the full-length coding region of the target gene cDNA sequence.

[0039] DNAman was used to predict the amino acid sequence and isoelectric point. SignalP 5.0 was used to predict the signal peptide of ANGPTL4. MEGA was used for amino acid multiple sequence alignment and phylogenetic tree construction. SWISS-MODEL was used to predict the tertiary structure of ANGPTL4, and TMpred and TMHMM were used to analyze the transmembrane region of ANGPTL4.

[0040] In vitro recombinant expression and activity analysis of angiopoietin-like protein 4:

[0041] Using the cloned ANGPTL4 from Acipenser baerii as a template, ANGPTL4-yf and ANGPTL4-yr as primers. The PCR reaction system was 10 μL, containing 5 μL of 2×Taq PCR MasterMix (TianGen Biotech, Beijing, China), 3 μL of ultrapure water, 1 μL of bacterial solution, and 0.5 μL of each upstream and downstream primer. The specificity and brightness of the PCR product bands were detected by 1.5% agarose gel electrophoresis. Subsequently, the target fragment band was excised, and a gel extraction kit was used for DNA recovery.

[0042] After that, the obtained target fragment was ligated to the cloning vector pMD19-T, transformed into DH5α competent cells. After resuscitation and overnight culture on the plate, colonies were picked into LB liquid medium containing 100 μg / μL ampicillin and cultured for several hours. After PCR identification, the bacterial solution was sent to a biological company for sequencing.

[0043] Construction of recombinant plasmid: After resuscitating the pMD19-T-ANGPTL4 bacteria and the expression vector pET-32a bacterial solution, they were cultured overnight at 37 °C. Plasmids were extracted according to the instructions of the plasmid extraction kit, and the purity and concentration were determined.

[0044] The target gene fragment amplified by PCR was double digested with restriction endonucleases BamHⅠ and EcoRⅠ at a constant temperature of 37 °C for 5 - 15 min. 5.5 μL of 10×loading buffer was added to terminate the reaction. After the reaction, the digested products were separated by 1.5% agarose gel electrophoresis. The target-sized fragment was cut off, and a gel extraction kit was used for recovery and purification. The expression vector pET-32a was also double digested with BamHⅠ and EcoRⅠ, and the large fragment was recovered by gel extraction. The purity and concentration of the recovered products were determined.

[0045] Under the condition of 4 °C, the digested vector and target fragment were mixed with T4 DNA Ligase and ligated overnight at 4 °C. Subsequently, the ligation products were transformed into DH5α competent cells, and after resuscitation, they were spread on LB solid medium containing ampicillin and cultured overnight.

[0046] Expression of recombinant plasmid in expression strains: The extracted correct recombinant expression plasmid was transformed into Transetta(DE3) competent cells, spread on a solid plate containing 1‰ ampicillin. The next day, single colonies were selected for colony PCR. The PCR products were subjected to 1.5% agarose gel electrophoresis to judge the product size, and positive clones were screened. Subsequently, they were sent to a biological company for sequencing.

[0047] Inoculate the engineered bacteria identified as PCR positive and with correct sequencing into LB liquid medium containing 1‰ ampicillin, place it in a shaker at 37°C, and culture it overnight with shaking at a frequency of 180 r / min for activation. The next day, transfer it to LB medium containing 1‰ ampicillin at a ratio of 1:100 and culture it at 37°C for expansion, and detect the bacterial growth density. When the OD600 value of the bacterial solution reaches 0.5, explore the optimal IPTG induction concentration (set at 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, and 1.4 mmol / L) and induction time (0, 2, 4, 6, 8, 10, 12, and 14 h). Centrifuge at 6000 r / min for 5 min to collect the induced bacteria, resuspend and wash them 2-3 times with 25 mM Tris-HCl, then add lysozyme to break the bacteria until it becomes viscous. Centrifuge at 12000 r / min for 10 min, collect the supernatant and precipitate, and then perform 12% SDS-PAGE electrophoresis for identification to analyze the expression form of recombinant pET-32a-ANGPTL4.

[0048] Purification and refolding of the recombinant protein: Take the centrifuged supernatant after disruption and bind it to a nickel ion chelating affinity chromatography column ( Ni-NTA Resin Kit, TransGen Biotech) at a rate of 1 mL / min, and refer to the column loading operation instructions of TransGen Biotech Ni-NTA Resin for purification.

[0049] Slowly equilibrate the chromatography column with Tris-HCl buffer for 5-10 column volumes until the baseline is level. Wash with the equilibration buffer to remove bacterial impurities, elute with 20 mmol / L imidazole to collect the target protein. Add 5× protein loading buffer to the collected eluate, boil for 10 min to denature the protein, and analyze the purity of recombinant pET-32a-ANGPTL4 by 12% SDS-PAGE.

[0050] Take the frozen pET-32a-ANGPTL4 protein solution, measure the concentration using a BCA protein concentration detection kit, add an appropriate amount of enterokinase, and the ratio of protein to enterokinase is 50 μg:1 U. Mix well. To maximize the biological activity of ANGPTL4, select the enzymatic digestion conditions as a 25 mM Tris-HCl (pH 8.0) buffer system, digest overnight at 4°C for 16 h. After enzymatic digestion, take the mixed sample for 15% SDS-PAGE electrophoresis to analyze the enzymatic digestion efficiency, enzymatic digestion specificity, and the relative molecular mass of the target protein ANGPTL4.

[0051] Western blot verification of recombinant protein: The overnight cultured recombinant bacterium pET-32a-ANGPTL4 was diluted and expanded in LB liquid medium at a ratio of 1:50. The culture was continued until the OD600 reached approximately 0.5. Then, 0.8 mM IPTG was added to induce expression for 6 h. After ultrasonic disruption, the supernatant was collected. After obtaining the purified fusion protein through an affinity chromatography column, the ANGPTL4 protein was obtained after cleavage with enterokinase. Subsequently, it was separated by SDS-PAGE electrophoresis and electrotransferred onto a PVDF membrane.

[0052] After blocking with the membrane blocking solution (5% skim milk powder) for 1 h, TBST was added and the membrane was washed on a shaker at 120 r / min for 10 min, and this was repeated 3 times. The primary antibody (Anti-His Mouse Monoclonal Antibody) diluted with 1:2000 TBST was added and incubated overnight at 4°C. Then, it was washed 3 times with TBST, 10 min each time. The secondary antibody (Goat Anti-MouseIgG(H+L), HRP Conjugate) diluted with 1:2000 TBST was added and incubated at room temperature for 1 h, and then washed 3 times with TBST. The PVDF membrane was placed in a gel imaging system, ECL developer was added and developed for 2 minutes, and then photographed and observed.

[0053] Secondary affinity purification of recombinant protein: The molecular chaperone protein Trx-Tag thioredoxin carries a 6×His tag. After cleavage, the Trx-Tag thioredoxin was removed by Ni ion chelating affinity column chromatography to separate and purify the ANGPTL4 protein mixture after cleavage. The mixture of the fusion protein after cleavage was taken and passed through the Ni ion chelating affinity chromatography column at a speed of 1 mL / min 3 times. The flow-through was carefully collected. The flow-through was taken for 15% SDS-PAGE analysis of the purity and relative molecular mass of the purified ANGPTL4 protein, and the purification yield of the target protein ANGPTL4 after cleavage was calculated. The purified ANGPTL4 protein was fully concentrated by centrifugation using an ultrafiltration tube. The concentrated target protein was also subjected to 15% SDS-PAGE electrophoresis for identification. The purified and concentrated target protein was stored at -80°C for later use.

[0054] Intraperitoneal injection experiment:

[0055] Forty-eight healthy juvenile Amur sturgeons (120.91 ± 19.76 g) were selected and randomly divided into 4 groups, with 3 replicates in each group and 4 fish in each replicate.

[0056] Among them, 3 groups were intraperitoneally injected with ANGPTL4 functional fragments at concentrations of 100, 200, and 400 ng / g BW respectively; 1 group was intraperitoneally injected with PBS buffer. Before the protein injection experiment started, each group was intraperitoneally injected with PBS buffer for 2 days to adapt to the acupuncture stimulation. Intraperitoneal injection started at 13:30 every day, and the fish were anesthetized with 0.01% MS-222 before injection. Feed was provided at 14:00, and the remaining baits of each group were collected at 15:00, 17:00, and 20:00 and dried and weighed. The food intake was calculated based on the remaining bait amount, and the lowest effective concentration affecting the food intake was used as the optimal concentration for sample collection after injection.

[0057] Twenty-four healthy juvenile Amur sturgeons (119.55±20.63 g) were selected and randomly divided into 2 groups, which were intraperitoneally injected with PBS buffer and the lowest effective dose of ANGPTL4 respectively. Each group had 3 parallels, and each parallel had 4 fish. Before the protein injection started, each group was intraperitoneally injected with PBS buffer for 2 days to adapt to the acupuncture stimulation. Intraperitoneal injection started at 13:30 every day, and the fish were anesthetized with 0.01% MS-222 before injection. Feed was provided at 14:00. In the formal experiment, 6 fish were randomly selected from each treatment group at 1 h after injection and blood was collected via the caudal vein to detect the serum glucose and triglyceride levels. Then, they were decapitated and the hypothalamus, liver, stomach, and valvular intestine tissues were quickly dissected on an ice plate. Among them, the collected blood was allowed to stand overnight at 4°C and then centrifuged at 4000 r / min for 10 min at 4°C, and the upper serum was aspirated and stored at -20°C for subsequent physiological and biochemical index detection; after the hypothalamus was taken out, it was placed in RNAlater and permeated overnight at 4°C, and then stored in a -80°C refrigerator; after the liver, stomach, and valvular intestine tissues were taken out, they were rinsed with ice-cold physiological saline, dried, and immediately frozen in liquid nitrogen, and then stored in a -80°C refrigerator to detect the changes in the expression levels of other appetite factors and signaling pathway factors after the injection of ANGPTL4 functional fragments.

[0058] Data were expressed as Mean±SEM, and the data of the experimental results were statistically analyzed using IBM SPSS Statistics 20. After the data were tested for normal distribution, one-way analysis of variance (ANOVA) and Duncan's method were used for multiple comparisons to test the significance of differences among multiple groups of data, and an independent-samples T-test was used to analyze the differences between two groups of data. Different letters indicate significant differences between different experimental groups, *P<0.05 indicates significant differences, and **P<0.01 indicates extremely significant differences.

[0059] The coding region of the Amur sturgeon ANGPTL4 gene is 1404 bp long and encodes 467 amino acids as Figure 1 shown.

[0060] In vitro recombinant expression of angiopoietin-like protein 4:

[0061] The expression vector pET-32a and the ANGPTL4 nucleotide fragment were double-digested with the restriction endonucleases BamHⅠ and EcoRⅠ. The recovered ANGPTL4 nucleic acid fragment after double digestion was ligated to the expression vector pET-32a and transformed into competent DH5α cells, which were then spread on LB solid medium. Positive clones were screened by colony PCR using the synthesized pET-32a identification primers.

[0062] After extracting the recombinant plasmid with correct sequencing, it was transferred into Escherichia coli Transetta(DE3). The verified engineering strain was taken for enlarged culture, and 0.8 mmol / L IPTG ( Figure 6 ) was added for induction at 37 °C for 6 h ( Figure 7 ). The bacterial cells were collected, lysed by repeated freezing and thawing, and after centrifugation at high speed for 10 min, the supernatant and precipitate were subjected to 12% SDS-PAGE ( Figure 8 ). The results showed that the relative molecular mass of the fusion protein was about 29×10 3 .

[0063] The supernatant after fragmentation and lysis was passed through a Ni ion affinity chromatography column at a slow rate, and then the chromatography column was equilibrated with Tris-HCl buffer. Subsequently, the fusion protein was eluted and collected with the equilibrium buffer (containing 10 mmol / L imidazole) to elute the impurity proteins and 20 mmol / L imidazole, and was identified by 12% SDS-PAGE electrophoresis ( Figure 9 ). After affinity chromatography, the purity of the pET-32a-ANGPTL4 fusion protein was above 90%, and the purified fusion protein was stored at -80 °C for standby.

[0064] Enterokinase digestion was carried out at 4 °C for 16 h, and then the functional fragment was recovered again. The digested mixed sample and the secondary affinity purification sample were analyzed by 16.5% SDS-PAGE ( Figure 10 ). The results showed that the digestion efficiency under this condition was 100%, and no non-specific digestion phenomenon occurred. The relative molecular mass of the ANGPTL4 functional fragment after digestion was about 11×10 3 .

[0065] Western-blotting was used to verify the pET-32a-ANGPTL4 expression bacteria after induced expression and the products after digestion. The results showed that bands of about 31 kDa (pET-32a-ANGPTL4) and about 11 kDa (6×His tag) appeared on the PVDF membrane ( Figure 11 ) and Figure 3-7It was consistent with the specific band in [[]], and the target protein expressed by the recombinant expression bacteria could be specifically recognized by the His-tag antibody, showing antigen activity, indicating that the recombinant protein of Acipenser baerii ANGPTL4 was successfully expressed and successfully digested.

[0066] Intraperitoneal injection:

[0067] Use the relevant kits to detect the changes of glucose and triglyceride in serum after injecting ANGPTL4.

[0068] Injecting ANGPTL did not affect the serum glucose level of Acipenser baerii ( Figure 12 A), but the triglyceride level increased significantly ( Figure 12 B).

[0069] Changes in the expression levels of appetite factors in key tissues for appetite regulation after 1 hour of intraperitoneal injection of the effective dose of ANGPTL4 (200 ng / g BW). Compared with the PBS control group, intraperitoneal injection of ANGPTL4 could significantly inhibit the expression of agrp and pomc mRNA in the hypothalamus, and up-regulate the expression of npy and cart mRNA ( Figure 13 A). Compared with the control group, intraperitoneal injection of ANGPTL4 could significantly reduce the expression of the liver satiety factors leptin and nucb2 mRNA ( Figure 13 B), down-regulate the expression of the gastric hunger factor ghrelin ( Figure 13 C), increase the expression of pyy mRNA in the valvular intestine and reduce the expression of apela ( Figure 13 D). It can be seen from the above that peripheral ANGPTL4 can inhibit the feeding of Acipenser baerii by reducing the expression of the gastric orexigenic factor ghrelin mRNA, promoting the expression of the anorexigenic factor pyy mRNA in the valvular intestine, inhibiting the expression of the orexigenic factor agrp mRNA in the hypothalamus, and increasing the expression of cart mRNA in the hypothalamus.

[0070] Effect of intraperitoneal injection of ANGPTL4 on the expression levels of appetite regulation-related receptors, kinases and transcription factors in the hypothalamus ( Figure 14 ).

[0071] One hour after intraperitoneal injection of 200 ng / g BW ANGPTL4, compared with the PBS control group, ANGPTL4 significantly promoted the mRNA expression of hypothalamic ampkα1, ampkα2, ampkβ1, ampkβ2 and ampkγ2, and inhibited the mRNA expression of ampkγ1. After intraperitoneal injection of ANGPTL4, the expression level of cpt1 mRNA increased significantly, while the expression level of cpt1 mRNA did not change significantly. At the same time, peripheral ANGPTL4 treatment could significantly up-regulate the mRNA expression of pi3k, akt and mtor. In addition, the mRNA expression levels of hypothalamic foxo1 and bsx decreased significantly after peripheral ANGPTL4 treatment, and socs3 increased significantly, but the mRNA expression levels of jak2 and stat3 were not significantly affected by peripheral ANGPTL4 treatment.

[0072] In the above implementation process, the present invention realized the in vitro prokaryotic recombinant expression of ANGPTL4 mature peptide for the first time, and purified and obtained the bioactive Acipenser baerii ANGPTL4 / pET-32a protein.

[0073] The prokaryotic expression was used to express the ANGPTL4 mature peptide in vitro, and the in vitro recombinant ANGPTL4 / pET-32a protein contained the pET-32a empty protein. Therefore, the pET-32a empty protein and PBS were set as the control group:

[0074] Intraperitoneal injection of ANGPTL4 could inhibit the feeding of Acipenser baerii, and the minimum effective dose was 200 ng / g BW. After ANGPTL4 treatment, the expression of orexigenic factors in the hypothalamus and stomach decreased, and the expression of anorexigenic factors in the hypothalamus and ileocecum increased.

[0075] Appendix 1 Primers related to Acipenser baerii angptl4 cloning and fluorescence quantification

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[0081] The content not described in detail in this specification belongs to the prior art well-known to those skilled in the art.

[0082] The above description is made on the present invention and its embodiments, such description is not restrictive, and what is shown in the drawings is only one of the embodiments of the present invention, and the actual structure is not limited thereto. Generally speaking, if those of ordinary skill in the art are inspired by it and design similar structural modes and embodiments to this technical solution without creative work under the premise of not departing from the gist of the present invention, they shall fall within the protection scope of the present invention.

Claims

1. An appetite-stimulating factor angiopoietin-like protein of Acipenser baerii, characterized in that: The amino acid sequence of the angiopoietin-like protein is shown in SEQ ID NO:

1.

2. An in vitro encoded recombinant protein of claim 1, characterized in that: The amino acid sequence of the recombinant protein sequence is shown in SEQ ID NO:

2.

3. A recombinant protein comprising the recombinant protein according to claim 2, characterized in that, The construction vector of the recombinant plasmid includes pET-32a, and the original bacterium of the recombinant bacterium includes Escherichia coli DH5α.

4. Application of an in vitro encoded recombinant protein according to claim 2 in the feeding regulation of Acipenser baerii.

5. Application of an in vitro encoded recombinant protein according to claim 2 in the expression level of appetite regulatory factors of Acipenser baerii.

6. An in vitro encoded recombinant protein according to claim 3 or 4 can reduce the expression of appetite-stimulating factors in the hypothalamus and stomach of Acipenser baerii and increase the expression of appetite-suppressing factors in the hypothalamus and valvular intestine.

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