Trachinotus ovatus IGF2 gene and application thereof

Abstract

According to the trachinotus ovatus IGF2 gene and the application of the trachinotus ovatus IGF2 gene, the nucleotide sequence of the trachinotus ovatus IGF2 gene is shown as SEQ ID NO: 1, and the amino acid sequence of protein coded by the trachinotus ovatus IGF2 gene is shown as SEQ ID NO: 2. A prokaryotic expression vector containing the gene and a corresponding recombinant strain are successfully constructed, and the recombinant IGF2 protein is prepared through a high-efficiency expression system. The invention also discloses an application of the recombinant IGF2 protein in preparation of a product for regulating fish ingestion, an application in preparation of an additive with a function of promoting fish ingestion and an application in preparation of an artificial feed with a function of promoting fish ingestion. The recombinant IGF2 protein provided by the invention can significantly enhance the expression of the ingestion-related genes of trachinotus ovatus and significantly improve the food intake of trachinotus ovatus.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and aquaculture technology, and specifically relates to an IGF2 gene of silver pomfret and an application thereof, in particular to an insulin-like growth factor 2 (IGF2) gene of silver pomfret and an application of the recombinant protein expressed by the gene in regulating fish food intake. Background Art

[0002] The ovate pomfret (Trachinotus ovatus) is one of the important marine aquaculture fish in China, and has made significant contributions to my country's fishery economy. However, in high-density aquaculture environments, factors such as deteriorating water quality, lack of oxygen, and crowding stress inhibit the appetite of the ovate pomfret, leading to reduced food intake and decreased health, which seriously affects growth rate and causes economic losses to farmers.

[0003] Traditional methods of promoting feeding mainly include improving feed formula (such as adding attractants) or using chemically synthesized appetite stimulants. However, the effects of these methods are unstable, and long-term application may cause water pollution and drug residues, and even lead to 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, thereby improving 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 growth regulation and nutritional response in teleosts. However, the role of IGF2 in fish feeding regulation is still unclear, and its specific mechanism of action needs further study. Summary of the invention

[0005] The object of the present invention is to provide an IGF2 gene of silver pomfret and a protein encoded by the gene, as well as an expression vector and a recombinant strain containing the IGF2 gene.

[0006] The present invention also aims to provide a recombinant IGF2 protein of silver pomfret.

[0007] The last object of the present invention is to provide the use of the recombinant IGF2 protein of silver pomfret in the preparation of products for regulating fish feeding, in the preparation of additives with the function of promoting fish feeding, and in the preparation of artificial feed with the function of promoting fish feeding.

[0008] The first object of the present invention can be achieved by the following technical solution: an IGF2 gene of silver pomfret, the nucleotide sequence of which is shown in SEQ ID NO:1.

[0009] The present invention also provides a protein encoded by the IGF2 gene, and its amino acid sequence is shown in SEQ ID NO:2.

[0010] The present invention also provides an expression vector, comprising the above-mentioned silver pomfret IGF2 gene. The vector can efficiently express the silver pomfret IGF2 gene, thereby obtaining a recombinant protein.

[0011] The present invention further provides a recombinant strain, comprising the expression vector.

[0012] The invention successfully cloned the IGF2 gene of silver pomfret by reverse transcription PCR technology, constructed its prokaryotic expression vector and recombinant strain, and then realized the large-scale expression of recombinant protein.

[0013] The above-mentioned second purpose of the present invention can be achieved by the following technical scheme: a recombinant IGF2 protein of silver pomfret is prepared by the following method: comprising transforming the expression vector into host cells, culturing the transformed host cells to achieve the expression of the recombinant protein, purifying and collecting the recombinant IGF2 protein of silver pomfret from the culture.

[0014] The present invention successfully constructed a recombinant expression vector of the gene through gene cloning and expression analysis, and developed a set of efficient recombinant protein preparation methods.

[0015] The last object of the present invention can be achieved by the following technical scheme: the use of the above-mentioned recombinant IGF2 protein of silver pomfret in the preparation of products for regulating fish feeding, in the preparation of additives with the function of promoting fish feeding, and in the preparation of artificial feed with the function of promoting fish feeding.

[0016] The present invention has the following advantages:

[0017] (1) The present invention successfully cloned the IGF2 gene of the oval pomfret, and experimentally confirmed the important role of the oval pomfret recombinant IGF2 protein in promoting fish feeding, providing a new solution for improving aquaculture efficiency;

[0018] (2) The present invention focuses on the study of the IGF2 gene and its recombinant protein of the oval pomfret, exploring its potential in promoting fish feeding; experiments show that the oval pomfret recombinant IGF2 protein significantly increases the oval pomfret's food intake, which not only provides a new perspective for understanding the mechanism of fish feeding regulation, but also provides a promising technical solution for the aquaculture industry;

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

[0020] (4) Compared with traditional methods, the present invention adopts biotechnology methods of endogenous fish proteins, which ensures higher safety and environmental protection and will not impose additional burden on the ecosystem; at the same time, the technology is easy to operate and suitable for actual breeding environments; in the future, this innovative achievement is expected to improve aquaculture efficiency, reduce costs and promote the sustainable development of fisheries. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The recombinant IGF2 protein expression and purification of the oval pomfret in Example 1, 1: supernatant after induced lysis, 2: flow-through, 3: eluent 1, 4: eluent 2, 5: eluent 3, M: marker;

[0022] Figure 2 It is the SDS-PAGE detection of the affinity-purified recombinant IGF2 protein of the oval pomfret in Example 1, M: marker, 1: recombinant IGF2 protein;

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

[0024] Figure 4 The figure shows the effect of in vivo injection of the recombinant IGF2 protein in Example 3 of the present invention on the food intake of the oval pomfret. The values ​​are expressed as mean±standard error (n=10), wherein the ** mark indicates a very significant difference compared with the control group (P<0.01). DETAILED DESCRIPTION

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

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

[0027] Example 1 Preparation of recombinant IGF2 protein

[0028] 1. RNA extraction and cDNA synthesis of Oval Pomfret

[0029] The oval pomfret used in the experiment was purchased from the aquatic product wholesale market in Xiashan District, Zhanjiang City, Guangdong Province, China. The fish were anesthetized with tricaine methane sulfonate (Sigma, USA) solution and quickly killed, and the hypothalamic tissue was immediately isolated.

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

[0031] 2. Cloning of IGF2 gene

[0032] The cDNA sequence of the IGF2 gene of the oval pomfret 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, PCR amplification was performed using primers IGF2-RT-F and IGF2-RT-R to obtain an IGF2 gene fragment (492 bp) without a signal peptide, corresponding to the nucleotide sequence shown in SEQ ID NO:1, and the encoded protein sequence is shown in SEQ ID NO:2; the amplified products were separated by agarose gel electrophoresis and purified by gel extraction; the purified DNA fragments were ligated to the pGEX-4T-1 vector and transformed into DH5α Escherichia coli, and a GST tag was fused to the N-terminus; positive clones were selected and sent to Sangon Biotechnology Co., Ltd. (Shanghai, China) for sequencing verification.

[0036] The nucleotide sequence of the IGF2 gene of the oval pomfret 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 underline indicates the stop codon).

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

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

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

[0050] A plasmid extraction kit (Tiangen, Beijing) was used to extract the plasmid of the positive cloned Escherichia coli, and the purified plasmid was transformed into Rosetta (DE3) competent cells; the positive single clone colony was verified by bacterial liquid PCR to confirm that the size of the amplified fragment was consistent with the expectation; then, the positive single colony was selected and sent to a sequencing company for sequencing verification; the sequencing results 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. Massive expression and detection of IGF2 protein

[0052] 4.1. Extensive expression and preliminary detection of IGF2 protein:

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

[0054] 4.2. Inclusion body protein purification

[0055] The precipitate was resuspended in STET buffer (containing 1 mM DTT) and sonicated to remove impurities until the supernatant was transparent; the precipitate was finally resuspended in a solution containing 6 M 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 that the inclusion bodies were dissolved; the supernatant was taken by centrifugation and the protein solubility was detected by SDS-PAGE.

[0056] 4.3. Inclusion body protein renaturation

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

[0058] 4.4. Refolded protein affinity purification

[0059] After pre-equilibration of Glutathione Sepharose resin, filter the refolded protein solution and load it onto the affinity column, inject at a flow rate of 1 mL / min; wash the column with binding buffer until no protein flows out (no color change in G250 detection solution); use elution buffer (50 mM Tris-HCl, pH 8.0, containing 10 mM GSH) to elute the target protein in sections, and confirm the high-purity target protein by SDS-PAGE detection; take 10 μL of the collected eluate for SDS-PAGE detection to confirm that the target gene forms a soluble expression supernatant ( Figure 1 ); SDS PAGE protein control results showed that the molecular weight of the recombinant protein was about 51.3kDa (containing a GST tag of 26kDa), which was close to the expected molecular weight of the target protein ( Figure 2 ).

[0060] 4.5 Recombinant protein concentration and storage

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

[0062] Example 2 Effect of recombinant IGF2 protein on the expression of feeding regulatory genes in silver pomfret

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

[0064] The oval pomfret used in the experiment was purchased from Xiashan Aquatic Products Wholesale Market in Zhanjiang. The individual body length was uniform and the weight ranged from 500 to 600 grams to ensure the consistency of the experiment. The experimental fish were kept in indoor buckets, the water temperature was maintained at 27-29℃, and the seawater was continuously flowing. Recombinant IGF2 protein was dissolved in saline, and the fish were anesthetized with MS-222 (Sigma, USA), and then intraperitoneally injected with 1, 5 and 10 ng / g body weight (ng / g BW) of recombinant IGF2 protein; the negative control group was injected with an equal amount of saline only (n=5); 3 hours and 6 hours after injection, the oval pomfret was anesthetized and killed, and the hypothalamic tissue was quickly removed, frozen with liquid nitrogen and stored in a -80℃ refrigerator for subsequent analysis.

[0065] 2. RNA extraction and cDNA synthesis

[0066] According to the method of Example 1, total RNA was extracted from the hypothalamic tissue sample and reverse transcribed into cDNA for subsequent gene expression analysis.

[0067] 3. Quantitative analysis of gene expression

[0068] Real-time 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. Primers were designed based on the transcriptome database and optimized to ensure their specificity and amplification efficiency; 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 real-time fluorescence quantitative PCR amplification of the relative expression levels of appetite stimulating hormone (agrp) and neuropeptide Y (npy) are as follows:

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

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

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

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

[0074] 4. Experimental results

[0075] according to Figure 3 According to the data, 3 hours after in vivo injection, a 5ng / g BW dose 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, 1ng / g BW and 5ng / g BW doses of recombinant IGF2 protein could also significantly promote the expression of these two genes; however, a higher dose (10ng / g BW) of recombinant IGF2 protein did not show the same promoting effect, but instead caused the promoting effect to weaken or disappear.

[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, an appropriate dose of recombinant IGF2 protein can affect the regulation of feeding behavior by enhancing the expression of agrp and npy genes in the hypothalamus of 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 food intake of silver pomfret

[0078] 1. Experimental design

[0079] Egg-shaped silver pomfret with uniform body length and body weight between 500 and 600 g were randomly distributed into indoor water buckets, with one fish in each bucket; the experimental fish were fed once a day at 9:00 a.m., fed to satiation, and the feeding amount was about 2% of body weight; the experiment began after two weeks of acclimation; under MS-222 anesthesia, each fish was weighed and intraperitoneally injected with 0.8% fish saline (control group) or 5 ng / g BW of recombinant IGF2 protein (experimental group) according to body weight; 3 hours after injection, each group of fish was fed with a fixed weight of food and allowed to eat freely for 1 hour; the remaining food was collected and dried, and the food intake per unit body weight of each group of fish was calculated based on the weight of the remaining food.

[0080] 2. Experimental results

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

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

[0083] 3. Application prospects

[0084] Based on the above research results, the present invention identified for the first time the important role of the recombinant IGF2 protein encoded by the IGF2 gene of the oval pomfret in regulating the feeding behavior of fish; the study showed that the recombinant IGF2 protein can synergize with the key factors of feeding regulation, npy and agrp, to significantly increase the feeding amount of the oval pomfret. This discovery provides a new idea for the development of low-cost and efficient attractants, which can be added to fish feed as feed additives to help improve breeding efficiency and economic benefits.

[0085] In general, the present invention reveals the important function of recombinant IGF2 protein in regulating fish feeding, opens up a research and development path for new aquatic feed additives, and has broad application prospects and important economic value.

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

Claims

1. An IGF2 gene of silver pomfret, characterized in that: Its nucleotide sequence is shown in SEQ ID NO:

1.

2. The protein encoded by the IGF2 gene according to claim 1, characterized in that Its amino acid sequence is shown in SEQ ID NO:

2.

3. An expression vector, characterized in that: It includes the IGF2 gene of silver pomfret according to claim 1.

4. A recombinant strain, characterized in that: Comprising the expression vector according to claim 3.

5. A recombinant IGF2 protein of silver pomfret, characterized in that: The method is prepared by the following steps: transforming the expression vector described in claim 3 into host cells, culturing the transformed host cells to achieve expression of the recombinant protein, and purifying and collecting the recombinant IGF2 protein of silver pomfret from the culture.

6. Use of the recombinant IGF2 protein of Pieris ovalis according to claim 5 in the preparation of products for regulating fish feeding.

7. Use of the recombinant IGF2 protein of Pisces ovalis according to claim 5 in the preparation of an additive having the function of promoting fish feeding.

8. Use of the recombinant IGF2 protein of Pieris ovalis according to claim 5 in the preparation of artificial feed having the function of promoting fish feeding.

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