Trachurus japonicus igf2 gene and application thereof

Abstract

The application discloses an elagatis bipinnulatus IGF2 gene and application thereof, wherein the nucleotide sequence of the elagatis bipinnulatus IGF2 gene is shown as SEQ ID NO:1, the amino acid sequence of the protein coded by the IGF2 gene is shown as SEQ ID NO:2. A prokaryotic expression vector containing the gene and a corresponding recombinant strain are successfully constructed, a recombinant IGF2 protein is prepared through a high-efficiency expression system, and the recombinant IGF2 protein is applied to preparation of a product for regulating fish feeding, preparation of an additive with the function of promoting fish feeding and preparation of artificial feed with the function of promoting fish feeding. The recombinant IGF2 protein can significantly enhance the expression of feeding-related genes of the elagatis bipinnulatus and significantly improve the feeding amount.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biotechnology and aquaculture, and particularly relates to a Trachinotus ovatus IGF2 gene and application thereof, in particular to application of a recombinant protein related to an insulin-like growth factor 2 (IGF2) gene and expression of the Trachinotus ovatus in regulating fish feeding products. BACKGROUND

[0002] Trachinotus ovatus is one of the important marine aquaculture fish in China, which makes a significant contribution to the fishery economy of China. However, in the high-density breeding environment, factors such as water quality deterioration, oxygen deficiency and crowding stress inhibit the appetite of Trachinotus ovatus, leading to reduced feeding and health decline, which seriously affects the growth rate and brings economic losses to the breeders.

[0003] Traditional methods for promoting feeding mainly include improving feed formula (such as adding attractants) or using chemical synthetic appetite stimulants. However, these methods are unstable in effect, and long-term application may cause water pollution and drug residues, and even cause drug resistance problems. Therefore, it is urgent to develop an environmentally friendly, efficient and safe technology to optimize and promote the feeding behavior of farmed fish, so as to improve the growth rate and feed conversion rate.

[0004] In recent years, the insulin-like growth factor (IGF) family has attracted much attention due to its key regulatory role in various physiological processes in organisms, including growth, reproduction and metabolism. In particular, IGF2 has been shown to be involved in the growth regulation and nutritional response of teleosts. However, the role of IGF2 in fish feeding regulation is not clear, and the specific mechanism needs further study. SUMMARY

[0005] The present application aims to provide a Trachinotus ovatus IGF2 gene and the protein encoded by the gene, as well as an expression vector and a recombinant strain containing the IGF2 gene.

[0006] The present application also aims to provide a Trachinotus ovatus recombinant IGF2 protein.

[0007] The last purpose of the present application is to provide the application of the Trachinotus ovatus recombinant IGF2 protein in preparing products for regulating fish feeding, as well as its application in preparing additives with the function of promoting fish feeding and its application in preparing artificial feed with the function of promoting fish feeding.

[0008] The first purpose of the present application can be achieved by the following technical solution: a Trachinotus ovatus IGF2 gene, the nucleotide sequence of which is shown in SEQ ID NO: 1.

[0009] The application further provides a protein encoded by the IGF2 gene, and an amino acid sequence of the protein is shown as SEQ ID NO: 2.

[0010] The application further provides an expression vector comprising the Trachinotus ovatus IGF2 gene.

[0011] The application further provides a recombinant strain comprising the expression vector.

[0012] The application successfully clones the Trachinotus ovatus IGF2 gene through reverse transcription PCR technology, constructs a prokaryotic expression vector and a recombinant strain of the gene, and further realizes large-scale expression of the recombinant protein.

[0013] The second purpose of the application can be realized by the following technical scheme: a Trachinotus ovatus recombinant IGF2 protein is prepared by the following method: the expression vector is transformed into a host cell, the transformed host cell is cultured, expression of the recombinant protein is realized, and the Trachinotus ovatus recombinant IGF2 protein is purified and collected from the culture.

[0014] The application successfully constructs a recombinant expression vector of the gene through gene cloning and expression analysis, and develops a set of efficient recombinant protein preparation method.

[0015] The last purpose of the application can be realized by the following technical scheme: the Trachinotus ovatus recombinant IGF2 protein is applied to preparation of a product for regulating fish feeding, preparation of an additive with the function of promoting fish feeding, and preparation of an artificial feed with the function of promoting fish feeding.

[0016] The application has the following advantages:

[0017] (1) The application successfully clones the Trachinotus ovatus IGF2 gene, and verifies the important role of the Trachinotus ovatus recombinant IGF2 protein in promoting fish feeding through experiments, thereby providing a new solution for improving aquaculture efficiency.

[0018] (2) The application focuses on the research of the Trachinotus ovatus IGF2 gene and the recombinant protein thereof, and explores the potential of the Trachinotus ovatus IGF2 gene in promoting fish feeding; experiments show that the Trachinotus ovatus recombinant IGF2 protein significantly improves the feeding amount of the Trachinotus ovatus, which not only provides a new perspective for understanding the fish feeding regulation mechanism, but also provides a promising technical solution for the aquaculture industry.

[0019] (3) The recombinant IGF2 protein developed by the present application has great potential as a low-cost and high-efficiency feeding attractant, and can be directly used as a feed additive in artificial feed, which not only helps to increase the feeding amount of fish, but also reduces the environmental burden and drug residue problems caused by traditional chemical feeding attractants, thereby promoting the development of aquaculture towards a more environmentally friendly and sustainable direction;

[0020] (4) Compared with traditional methods, the present application uses biotechnological means of fish endogenous proteins to ensure higher safety and environmental friendliness, and does not constitute an additional burden to the ecological system; at the same time, the technology is easy to operate and suitable for actual breeding environment; in the future, this innovative achievement is expected to improve the efficiency of aquaculture, reduce costs and promote the sustainable development of fisheries. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 is the expression and purification of the Trachurus ovatus recombinant IGF2 protein in Example 1, 1: supernatant after induction lysis, 2: flow-through, 3: eluate 1, 4: eluate 2, 5: eluate 3, M: marker;

[0022] Figure 2 is the SDS-PAGE detection of the Trachurus ovatus recombinant IGF2 protein after affinity purification in Example 1, M: marker, 1: recombinant IGF2 protein;

[0023] Figure 3 is the effect of in vivo injection of recombinant IGF2 protein on the expression of agrp and npy genes in the hypothalamus of Trachurus ovatus in Example 2 of the present application, different doses of recombinant IGF2 protein were injected in vivo for 3 hours and 6 hours respectively. The values are expressed as mean ± standard error (n = 5), where ** indicates a very significant difference compared with the control group (P < 0.01);

[0024] Figure 4 is the effect of in vivo injection of recombinant IGF2 protein on the feeding amount of Trachurus ovatus in Example 3 of the present application, the values are expressed as mean ± standard error (n = 10), where ** indicates a very significant difference compared with the control group (P < 0.01). DETAILED DESCRIPTION

[0025] The present application will be further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present application and not to limit the scope of the present application.

[0026] Unless otherwise specified, the materials, reagents and equipment used in the present application can be obtained from commercial channels.

[0027] Example 1 Preparation of recombinant IGF2 protein

[0028] 1. Extraction of Trachinotus ovatus RNA and cDNA synthesis

[0029] Trachinotus ovatus used in the experiment were purchased from the wholesale market of seafood in Xia Shan district, Zhanjiang city, Guangdong province, China. Fish were anesthetized with tricaine methanesulfonate (Sigma, USA) solution and sacrificed immediately after which the hypothalamus tissues were isolated.

[0030] Total RNA was extracted from these tissues using Trizol reagent (Takara, Japan), and the integrity of the RNA was assessed by 1% agarose gel electrophoresis, and the purity and concentration of the RNA were determined using a NanoDrop 2000C spectrophotometer (Thermo Scientific, USA). The qualified RNA samples were then reverse transcribed into cDNA using PrimeScript TM RT Reagent Kit with gDNA Eraser (RR047A; Takara, Japan) as templates for subsequent PCR reactions.

[0031] 2. Cloning of IGF2 gene

[0032] The cDNA sequence of Trachinotus ovatus IGF2 gene was derived from the previous cloning experiment in the laboratory. Through bioinformatics analysis, the region encoding the 52 amino acid signal peptide was determined, and primers were designed to amplify the remaining sequence after removing the signal peptide. The primers used are as follows:

[0033] IGF2-RT-F 5’-tgggatccggtaccaagcttGAGACGCTGTGTGGGGGAGA-3’ (SEQ ID NO: 3);

[0034] IGF2-RT-R 5’-tggtggtggtggtgctcgagTCATTTGTGGTTGACATAGTTG-3’ (SEQ ID NO: 4).

[0035] Using the synthesized first strand cDNA as a template, primers IGF2-RT-F and IGF2-RT-R were used for PCR amplification, and a signal peptide-free IGF2 gene fragment (492 bp) was obtained, corresponding to the nucleotide sequence shown in SEQ ID NO: 1, and the encoded protein sequence is shown in SEQ ID NO: 2. The amplification product was separated by agarose gel electrophoresis and gel recovery purification. The purified DNA fragment was ligated to the pGEX-4T-1 vector and transformed into DH5a E. coli, with a GST tag fused at the N terminus. Positive clones were selected and sent to GenScript Biotech Co., Ltd. (Shanghai, China) for sequencing verification.

[0036] The nucleotide sequence of the Trachinotus ovatus IGF2 gene is as follows:

[0037] GAGACGCTGTGTGGGGGAGAGCTGGTGGATGCGCTGCAGTTTGTCTG

[0038] CGAAGACAGAGGCTTCTATTTCAGTAGGCCAACCAGCAGGGGTAGCAACC

[0039] GGCGCCCCCAGAACCGTGGGATCGTAGAGGAGTGTTGTTTCCGTAGCTGTG

[0040] ACCTCAACCTGCTGGAGCAGTACTGTGCCAAACCCGCCAAGTCCGAAAGG

[0041] GACGTCTCGGCCACCTCTCTACAGGTCATTCCCGTGATGCCTGCACTAAAA

[0042] CAGGAAGTCCCAAGGAAGCAGCATGTGACCATGAAGTATTCCAAATACGA

[0043] GGAGTGGCAGAGGAAGGCGGCCCAGCGGCTCCGGAGGGGTGTCCCCGCC

[0044] ATCCTGAGGGCCAAAAAGTTTCGGAGGCAGGCGGAGAAGATCAAAGCAC

[0045] AGGAGCAGGCAATCTTCCACAGGCCCCTGATCAGCCTGCCTAGCAAACTG

[0046] CCTCCCGTCTTGCTCGCCACGGACAACTATGTCAACCACAAA TGA (As shown in SEQ ID NO: 1, the stop codon is underlined).

[0047] The amino acid sequence of the protein encoded by the IGF2 gene is as follows:

[0048] ETLCGGELVDALQFVCEDRGFYFSRPTSRGSNRRPQNRGIVEECCFRSCDLNLLEQYCAKPAKSERDVSATSLQVIPVMPALKQEVPRKQHVTMKYSKYEEWQRKAAQRLRRGVPAILRAKKFRRQAEKIKAQEQAIFHRPLISLPSKLPPVLLATDNYVNHK (as shown in SEQ ID NO: 2).

[0049] 3. Construction of IGF2 gene prokaryotic expression vector

[0050] The plasmid of the positive clone E. coli was extracted using a plasmid mini extraction kit (Tiangen, Beijing), and the purified plasmid was transformed into Rosetta (DE3) competent cells; the positive single clone was verified by bacterial liquid PCR, and the size of the amplified fragment was confirmed to be consistent with the expected size; then, the positive single colony was sent to a sequencing company for sequencing verification; the sequencing result showed that the obtained sequence was completely matched with the target IGF2 gene sequence, confirming that the prokaryotic recombinant expression vector of the IGF2 gene was successfully constructed.

[0051] 4. Large-scale expression and detection of IGF2 protein

[0052] 4.1. Large-scale expression and preliminary detection of IGF2 protein:

[0053] The E. coli single colony containing the recombinant plasmid was inoculated into LB liquid medium containing kanamycin (50 μg / mL) and cultured overnight at 37°C on a shaker; the next day, 100 μL of the culture was inoculated into fresh LB medium and cultured until the OD600 reached 0.6. The temperature was reduced to 30°C, IPTG (final concentration 0.1 mM) was added, and expression was induced for 8 hours; after expression, the bacterial cells were collected by centrifugation at 8000 rpm, resuspended in pre-cooled lysis buffer (PBS containing lysozyme 0.1 mg / mL), and treated on ice before ultrasonic disruption; the supernatant and precipitate were separated, and SDS-PAGE detection was performed, and the remaining sample was stored for later use.

[0054] 4.2. Purification of inclusion body protein

[0055] The precipitate was resuspended in STET buffer (containing DTT 1 mM) and ultrasonically treated to remove impurities until the supernatant was clear; the final precipitate was resuspended in a solution containing 6M guanidine hydrochloride and 0.5% Triton X-100, DTT (final concentration 5 mM) was added, and the mixture was shaken at 37°C for 4 hours to ensure dissolution of the inclusion bodies; the supernatant was collected by centrifugation, and SDS-PAGE was performed to detect the protein dissolution.

[0056] 4.3. Renaturation of inclusion body protein

[0057] The protein solution was diluted with twice volume of 3M guanidine hydrochloride and then added dropwise into the refolding solution at pH 8.0, and stirred at high speed for 24 hours; then, the protein solution was placed in a dialysis bag for dialysis for 24 hours to gradually remove guanidine hydrochloride; the dialysate was replaced twice during the process; after dialysis, the ultrafiltration device was used to concentrate to the target volume (10-20 mL).

[0058] 4.4, affinity purification of refolded protein

[0059] After the glutathione Sepharose resin was pre-equilibrated, the refolded protein solution was loaded into the affinity column at a flow rate of 1 mL / min; the column was washed with binding buffer until no protein flowed out (no color change in G250 detection solution); the target protein was eluted in sections using elution buffer (50 mM Tris-HCl, pH 8.0, containing 10 mM GSH), and high-purity target protein was confirmed by SDS-PAGE detection; 10 μL of the collected eluate was subjected to SDS-PAGE detection to confirm that the target gene formed a soluble expression supernatant Figure 1 ); the SDS PAGE protein control result showed that the molecular weight of the recombinant protein was about 51.3 kDa (containing a ~ 26 kDa GST tag), which was similar to the expected molecular weight of the target protein Figure 2 ).

[0060] 4.5 Concentration and preservation of recombinant protein

[0061] The purified protein solution was concentrated to the target concentration (about 1-5 mg / mL) using an ultrafiltration device, and was replaced with storage buffer (PBS, pH 7.4, containing 10% glycerol); low temperature was maintained during the operation to prevent protein denaturation; after concentration, the small portions were immediately frozen and stored at -80°C to avoid inactivation caused by repeated freezing and thawing.

[0062] Example 2 Effect of recombinant IGF2 protein on the expression of feeding regulation genes in Trachinotus ovatus

[0063] 1. In vivo injection treatment of recombinant IGF2 protein

[0064] The experimental Trachurus japonicus were purchased from Zhanjiang Xiashan Water Products Wholesale Market, with uniform body length and body weight between 500 and 600 grams to ensure the consistency of the experiment. The experimental fish were raised in indoor water buckets, and the water temperature was maintained at 27-29°C with continuous seawater flow. The recombinant IGF2 protein was dissolved in physiological saline, and the fish were injected intraperitoneally with 1, 5 and 10 ng / g body weight (ng / g BW) of recombinant IGF2 protein after being anesthetized with MS-222 (Sigma, USA); the negative control group was only injected with an equal amount of physiological saline (n = 5); 3 hours and 6 hours after injection, the Trachurus japonicus were anesthetized and sacrificed, and the hypothalamic tissue was quickly removed, frozen in liquid nitrogen and stored in a -80°C refrigerator for subsequent analysis.

[0065] 2. RNA extraction and cDNA synthesis

[0066] Total RNA was extracted from hypothalamic tissue samples according to the method of Example 1 and reverse transcribed into cDNA for subsequent gene expression analysis.

[0067] 3. Quantitative analysis of gene expression

[0068] Real-time fluorescent quantitative PCR (RT-qPCR) was used to evaluate the relative expression levels of feeding-related genes such as appetite stimulating hormone (agrp) and neuropeptide Y (npy) in the hypothalamus. The primers were designed based on the transcriptome database and optimized to ensure their specificity and amplification efficiency; the qPCR reaction conditions were set as follows: denaturation at 95°C for 1 minute; followed by 35 cycles, each cycle including 95°C for 20 seconds, 60°C for 20 seconds and 72°C for 20 seconds; β-actin was used as an internal reference gene to standardize the expression of each target gene.

[0069] The primers used for amplifying the relative expression levels of appetite stimulating hormone (agrp) and neuropeptide Y (npy) by real-time fluorescent quantitative PCR are as follows:

[0070] agrp-F: TCGTCGTTGCATCCCTCAC (as shown in SEQ ID NO: 5);

[0071] agrp-R: GACAGTAGCAGATGGCGTTG (as shown in SEQ ID NO: 6);

[0072] npy-F: GCGGAGGAACTGGCCAAATA (as shown in SEQ ID NO: 7);

[0073] npy-R: GCAGCATCACCACAATGACG (as shown in SEQ ID NO: 8).

[0074] 4. Experimental results

[0075] According toFigure 3 Data showed that, 3 hours after in vivo injection, a dose of 5 ng / g BW of recombinant IGF2 protein significantly increased the expression levels of agrp and npy genes in the hypothalamus of oval pomfret; when the treatment time was extended to 6 hours, doses of 1 ng / g BW and 5 ng / g BW of recombinant IGF2 protein also significantly promoted the expression of these two genes; however, higher doses (10 ng / g BW) of recombinant IGF2 protein did not show the same promoting effect, but instead led to a weakening or disappearance of this promoting effect.

[0076] These results indicate that the effect of recombinant IGF2 protein on the expression of feeding-related genes is dose-dependent. Within a certain range, appropriate doses of recombinant IGF2 protein can affect the regulation of feeding behavior by enhancing the expression of agrp and npy genes in the hypothalamus of the oval pomfret. This finding provides important clues for further understanding the role of recombinant IGF2 protein in the feeding regulation mechanism of fish.

[0077] Example 3: Effect of recombinant IGF2 protein on feed intake of oval pomfret

[0078] 1. Experimental Design

[0079] Oval pomfret of uniform length and weight between 500 and 600 grams were randomly assigned to indoor tanks, with one fish per tank. The experimental fish were fed once a day at 9:00 AM, with a satiated diet of approximately 2% of their body weight. After two weeks of acclimatization, the experiment began. Under MS-222 anesthesia, the weight of each fish was measured, and 0.8% fish physiological saline (control group) or 5 ng / g BW dose of recombinant IGF2 protein (experimental group) was injected intraperitoneally according to weight. Three hours after injection, a fixed weight of food was fed to each group of fish, allowing them free access to feed for one hour. The remaining food was collected and dried, and the amount of food consumed per unit body weight of fish in each group was calculated based on the weight of the remaining food.

[0080] 2. Experimental Results

[0081] like Figure 4 As shown, the average feed intake of the control group of oval pomfret was 0.0205 g / gbw, while the average feed intake of the experimental group was 0.0253 g / gbw. The feed intake of the experimental group of oval pomfret increased by 23.41%, indicating that the recombinant IGF2 protein has a significant feeding-promoting effect.

[0082] These results indicate that in vivo injection of recombinant IGF2 protein at a dose of 5 ng / g BW can significantly increase the feed intake of oval pompano; from both a molecular and practical application perspective, recombinant IGF2 protein has a physiological function of promoting feed intake.

[0083] 3. Application Prospects

[0084] Based on the above research results, the present application first identifies the important role of the recombinant IGF2 protein encoded by the Trachurus ovatus IGF2 gene in regulating fish feeding behavior; the study shows that the recombinant IGF2 protein can synergistically act with the key factors of feeding regulation npy and agrp, and significantly increase the feeding amount of Trachurus ovatus. This discovery provides a new idea for the development of low-cost and high-efficiency attractants, which can be added to fish feed as a feed additive to help improve breeding efficiency and economic benefits.

[0085] Overall, the present application discloses the important function of the recombinant IGF2 protein in the regulation of fish feeding, opens up a new research and development path for a new type of aquatic feed additive, and has wide application prospect and important economic value.

[0086] The above specific embodiments are intended to further illustrate the content of the present application, but should not be regarded as limiting the scope of protection of the present application. Any non-substantial modification or adjustment made by any person on the basis of the present application according to the provided technical inspiration shall be covered within the scope of protection of the present application.

Claims

1. Application of recombinant IGF2 protein from oval pomfret in the preparation of additives that promote fish feeding, wherein the recombinant IGF2 protein from oval pomfret is prepared by the following method: [The method involves] preparing a mixture of oval pomfret... IGF2 The gene expression vector was transformed into host cells, and the transformed host cells were cultured to achieve the expression of recombinant protein. The recombinant IGF2 protein from *Siniperca ovoidea* was purified and collected from the culture. IGF2 The gene, whose nucleotide sequence is shown in SEQ ID NO: 1, is described as the oval pomfret.

2. Application of recombinant IGF2 protein from oval pomfret in the preparation of artificial feed that promotes fish feeding, wherein the recombinant IGF2 protein from oval pomfret is prepared by the following method: [The method involves] preparing feed containing oval pomfret... IGF2 The gene expression vector was transformed into host cells, and the transformed host cells were cultured to achieve the expression of recombinant protein. The recombinant IGF2 protein from *Siniperca ovoidea* was purified and collected from the culture. IGF2 The gene, whose nucleotide sequence is shown in SEQ ID NO: 1, is described as the oval pomfret.

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