Trachurus japonicus igf3 gene and application thereof

Abstract

The application discloses an elagatis bipinnulata IGF3 gene and application thereof, a nucleotide sequence of the elagatis bipinnulata IGF3 gene is shown as SEQ ID NO:1. An amino acid sequence of the encoded protein is shown as SEQ ID NO:2. A prokaryotic expression vector containing the elagatis bipinnulata IGF3 gene and a corresponding recombinant strain are successfully constructed, and a recombinant IGF3 protein is obtained through a high-efficiency expression system. The elagatis bipinnulata recombinant IGF3 protein can be applied to preparation of products for regulating expression of immune-related genes of elagatis bipinnulata and enhancing the antibacterial infection ability of fish. After the elagatis bipinnulata IGF3 recombinant protein is injected into a fish body, expression of immune-related genes such as il8, itgb8, mrc2 and sparc can be significantly promoted, and the bacterial load in the fish body after bacterial infection can be significantly reduced, so that the elagatis bipinnulata IGF3 recombinant protein can be applied to disease prevention and treatment of aquatic animals such as fish.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biotechnology and aquaculture, and particularly relates to a Trachinotus ovatus IGF3 gene and application thereof, in particular to an application of an insulin-like growth factor 3 (IGF3) gene of Trachinotus ovatus and a recombinant protein expressed by the IGF3 gene in enhancing the bacterial resistance of fish. BACKGROUND

[0002] Trachinotus ovatus is an important economic fish and plays an important role in global aquaculture. With the decrease of marine resources and the growth of market demand, mariculture has become one of the main ways to meet the demand for fish supply. In China, the scale of Trachinotus ovatus culture has been continuously expanding, and it has become one of the main mariculture varieties. However, the high-density culture environment leads to frequent diseases, causing huge economic losses to the aquaculture industry and threatening the sustainable development of fisheries.

[0003] Among various diseases, bacterial infection is particularly prominent, especially acute septicemia and other systemic diseases caused by Vibrio harveyi, which significantly reduces the health level and survival rate of fish. Traditionally, chemical drugs (such as antibiotics) are used to control the occurrence and spread of diseases, but long-term use leads to drug resistance problems, drug residues, and environmental pollution, which are not conducive to ecological protection and human health in the long run. Therefore, it is urgent to develop environmentally friendly and efficient disease control methods. Under this background, it is particularly important to screen fish disease-resistant immune genes and develop recombinant disease-resistant proteins through genetic engineering technology.

[0004] The insulin-like growth factor (IGF) gene family is composed of three major ligands (IGF1, IGF2, and IGF3), at least two receptors (IGF1R and IGF2R), and six high-affinity binding proteins (IGFBP1-6), which together form a complex regulatory network involved in regulating growth, reproduction, and metabolism, and other physiological processes.

[0005] Insulin-like Growth Factor 3 (IGF3) is a unique member of the IGF family in teleosts, mainly expressed in gonadal tissue, and plays an important role in regulating cell proliferation, differentiation, and growth related to reproduction. Existing studies have shown that IGF3 is closely related to gonadal maturation, oocyte growth, and ovulation process. However, the potential function of IGF3 in fish disease resistance has not been clearly reported, which provides a new direction for research. SUMMARY

[0006] The present application aims to provide an IGF3 gene of Trachinotus ovatus and a protein encoded by the IGF3 gene, and an expression vector and a recombinant strain comprising the IGF3 gene.

[0007] The present application also aims to provide a recombinant IGF3 protein of Trachinotus ovatus.

[0008] The present application also aims to provide an application of the recombinant IGF3 protein of Trachinotus ovatus in the preparation of a product for regulating the expression of an immune-related gene of Trachinotus ovatus and in the preparation of a product with enhanced fish antibacterial infection ability.

[0009] The first object of the present application can be achieved by the following technical solution: an IGF3 gene of Trachinotus ovatus, wherein the nucleotide sequence of the gene is shown as SEQ ID NO: 1.

[0010] The present application also provides a protein encoded by the IGF3 gene, wherein the amino acid sequence of the protein is shown as SEQ ID NO: 2.

[0011] The present application also provides an expression vector comprising the IGF3 gene of Trachinotus ovatus. The expression vector can efficiently express the IGF3 gene to obtain a target recombinant protein.

[0012] The present application also provides a recombinant strain comprising the expression vector.

[0013] The second object of the present application can be achieved by the following technical solution: a recombinant IGF3 protein of Trachinotus ovatus, which is prepared by the following method: transforming the expression vector into a host cell, culturing the transformed host cell, inducing the expression of the recombinant protein, and separating, purifying and collecting the recombinant IGF3 protein of Trachinotus ovatus from the culture.

[0014] The present application successfully clones the IGF3 gene by reverse transcription PCR technology and constructs a corresponding expression vector, thereby realizing efficient expression and large-scale preparation of the recombinant protein.

[0015] The last object of the present application can be achieved by the following technical solution: the application of the recombinant IGF3 protein of Trachinotus ovatus in the preparation of a product for regulating the expression of an immune-related gene of Trachinotus ovatus.

[0016] Preferably, the immune-related gene comprises il8, itgb8, mrc2 and sparc genes.

[0017] The present application also provides an application of the recombinant IGF3 protein of Trachinotus ovatus in the preparation of a product with enhanced fish antibacterial infection ability.

[0018] Preferably, the fish is Trachurus japonicus.

[0019] Preferably, the bacteria is Vibrio harveyi.

[0020] The present application verifies the potential of the Trachurus japonicus recombinant IGF3 protein in preparing an anti-bacterial infection preparation, especially the application value in preventing and treating Vibrio harveyi infection through experiments.

[0021] The present application has the following advantages:

[0022] (1) The present application focuses on the role of the Trachurus japonicus IGF3 gene in the fish immune system for the first time, a recombinant expression vector of the gene is constructed through cloning and expression analysis, a preparation method of the recombinant protein and an anti-bacterial activity analysis method are established, and the application prospect of the recombinant protein as a potential anti-disease drug is explored, and the preliminary research results show that the Trachurus japonicus recombinant IGF3 protein can effectively enhance the anti-disease immune response of the fish body, and a new gene material and technical means are provided for the research on the anti-disease mechanism of the fish and the disease prevention and control;

[0023] (2) The Trachurus japonicus IGF3 gene is successfully cloned in the present application, the key role of the gene in the anti-disease immune regulation is determined, a recombinant IGF3 protein with significant anti-bacterial activity is developed through experiments, a new technical solution is provided for the disease prevention and control, the recombinant protein has broad application potential, can be used for developing an anti-bacterial infection preparation, and is suitable for coping with the frequent disease problems in the high-density aquaculture environment; in addition, the use of chemical drugs can be significantly reduced, the ecological environment of the water body is protected, and strong support is provided for the sustainable and green development of the aquaculture industry. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 is the expression and purification of the Trachurus japonicus recombinant IGF3 protein in Example 1, 1: supernatant after pGEX-4T-1 induced lysis, 2: flow-through liquid, 3: eluate sample-1, 4: eluate sample-2, M: marker;

[0025] Figure 2 is the SDS-PAGE detection of the Trachurus japonicus recombinant IGF3 protein after affinity purification in Example 1, M: marker, 1: recombinant IGF3 protein;

[0026] Figure 3 is the regulation of the recombinant IGF3 protein on the mRNA transcription level of the immune-related genes in the liver of Trachurus japonicus in Example 2, the numerical value is represented by mean value ± standard error (n=5), compared with the corresponding control group, *P<0.05; **P<0.01;

[0027] Figure 4The values are expressed as mean ± standard error (n = 5). Compared with the corresponding control group, *P < 0.05; **P < 0.01. DETAILED DESCRIPTION

[0028] The present application is further described in conjunction with the following examples. It should be understood that the following examples are intended to illustrate the present application and are not intended to limit the scope of the present application.

[0029] Unless otherwise indicated, the materials, reagents and equipment employed in the present application are commercially available and are used according to the manufacturer's instructions.

[0030] Example 1 Preparation of recombinant IGF3 protein

[0031] 1. Extraction of Trachinotus ovatus RNA and synthesis of cDNA

[0032] Trachinotus ovatus samples were purchased from Xia Shan aquatic product wholesale market in Zhanjiang, Guangdong, China. The fish were anesthetized with a solution containing 50 mg / L tricaine methane sulfonate (MS-222, Sigma, USA) and sacrificed immediately after which the liver tissue was isolated. Total RNA was extracted from the isolated tissue using Trizol reagent (Takara, Japan). The quality of the RNA was assessed by electrophoresis on a 1% agarose gel for its integrity; while its purity and concentration were determined using a NanoDrop 2000C spectrophotometer (Thermo Scientific, USA). The qualified RNA samples were reverse transcribed into cDNA using PrimeScript TM RT Reagent Kit with gDNA Eraser (RR047A; Takara, Japan), which was used as the template for the subsequent PCR reaction.

[0033] 2. Cloning of IGF3 gene

[0034] The cDNA sequence of Trachinotus ovatus IGF3 was obtained based on previous cloning experiments in the laboratory. The sequence encoding the 35-amino acid signal peptide was determined by bioinformatics analysis, and specific primers were designed to amplify the remaining sequence after removing the signal peptide; the primers used are as follows:

[0035] IGF3-RT-F: 5'-tgggatccggtaccaagcttGCCCGCCTCCGCTGTGGCTCTGA-3' (SEQ ID NO: 3);

[0036] IGF3-RT-R: 5'-tggtggtggtggtgctcgagTCACGATTTGAACCTCATTGTTCTC-3' (SEQ ID NO: 4).

[0037] The first strand cDNA synthesized above was used as a template for PCR amplification using primers IGF3-RT-F and IGF3-RT-R to obtain an IGF3 gene fragment without signal peptide, 480 bp in length, 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 the target fragment was recovered by cutting the gel. The purified DNA fragment was ligated to the pGEX-4T-1 vector and transformed into DH5a E. coli, with a GST tag fused to the N terminus. After selecting positive clones, they were sent to Genechem (Shanghai, China) for sequencing verification. The sequencing results were analyzed by BLAST comparison, confirming that the obtained fragment was indeed the IGF3 gene.

[0038] The nucleotide sequence of the IGF3 gene of Trachinotus ovatus is as follows:

[0039] GCCCGCCTCCGCTGTGGCTCTGATCTCCTCAGTGACCTCATATTTGTGTGCGGGGATCGAGGAATCTATTTAGGTAAAGGTTCCTGGTCGGGGTACGGTGCTCGGCCCAGAGGGAAGGGGATAGTGGATCAGTGTTGCCGACCAGCCGGCTGTGAACTTCAGCATCTGGAGATGTACTGTGCCAAACCAAAGAGCCAGCAGCGCACCACAGCTTACCCAACCACAACAACAGCAGCTGCACACACCACCACACAGCTAGACGTGGCCCAGCAGTTCCAAACAGTATTTCACAAGAGACTTGCAGAGCAGCTGGGGGCTCCCAGCAGCCCGAAGAGGGACGCTTACAGGAGGAGAACACAGCCTTCAGGACAGCGGAAACGCAAATCGTCAAACAGGAGGATTAACGGAGTGAACACATCCAGCAGCCCCCCCTCGGCCTCTGGGAGCCCCCTTCAGAGAACAATGAGGTTCAAATCG TGA (SEQ ID NO: 1, the stop codon is underlined).

[0040] The protein encoded by the IGF3 gene is as follows:

[0041] ARLRCGSDLLSDLIFVCGDRGIYLGKGSWSGYGARPRGKGIVDQCCRPAGCELQHLEMYCAKPKSQQRTTAYPTTTTAAAHTTTQLDVAQQFQTVFHKRLAEQLGAPSSPKRDAYRRRTQPSGQRKRKSSNRRINGVNTSSSPPSASGSPLQRTMRFKS (as set forth in SEQ ID NO: 2).

[0042] 3. Construction of the prokaryotic expression vector of IGF3 gene

[0043] The plasmid was extracted from the selected positive clone of E. coli DH5a using a plasmid mini-extraction kit (Tiangen Biotech, Beijing), and the purified plasmid was transformed into Rosetta (DE3) competent cells. The positive single clone was verified by PCR, and the size of the amplified fragment was about 480 bp, which was consistent with the expected size. Then, the positive single colony was sent to a sequencing company for sequencing verification. The sequencing results showed that the obtained sequence was completely matched with the target IGF3 gene sequence, confirming that the prokaryotic recombinant expression vector of IGF3 gene was successfully constructed.

[0044] 4. Large-scale expression and detection of IGF3 protein

[0045] 4.1. Large-scale expression and preliminary detection of IGF3 protein:

[0046] The E. coli single colony containing the recombinant plasmid was inoculated into 5 mL of LB liquid medium containing kanamycin (final concentration 50 μg / mL) and cultured at 37°C overnight. 100 μL of the overnight culture was inoculated into 100 mL of fresh LB liquid medium (containing kanamycin) and cultured at 37°C for 16 hours. Then, the bacterial suspension was inoculated into 2000 mL of LB liquid medium (containing kanamycin) and cultured at 37°C until the OD600 reached 0.6. Then, the culture temperature was reduced to 30°C, IPTG was added to a final concentration of 0.1 mM, and expression was induced for 8 hours.

[0047] After the culture, the bacterial cells were collected by centrifugation at 8000 rpm for 3 minutes, resuspended in 50 mL of pre-cooled lysis buffer (PBS containing lysozyme at a final concentration of 0.1 mg / mL), and incubated on ice for 30 minutes. After ultrasonic disruption of the bacterial cells, the supernatant and precipitate were separated, and 10 μL of the supernatant and precipitate were subjected to SDS-PAGE detection, respectively. The remaining samples were stored at 4°C.

[0048] 4.2. Purification of inclusion body protein

[0049] The precipitate was resuspended in 50 mL of STET buffer (containing DTT at a final concentration of 1 mM), and sonicated for 10 minutes to facilitate the solubilization of the impurities, followed by centrifugation to remove the supernatant. This step was repeated until the supernatant became transparent. Finally, the precipitate was resuspended in PBS, and sonicated for another 10 minutes, and the supernatant was discarded after centrifugation. The precipitate was resuspended in 3 mL of a solution containing 6 M guanidine hydrochloride and 0.5% Triton X-100, and DTT was added to a final concentration of 5 mM. The solution was shaken at 37°C and 220 rpm for 4 hours to ensure complete solubilization of the inclusion bodies. The supernatant was collected after centrifugation at 10,000 rpm and 4°C for 10 minutes, and subjected to SDS-PAGE to determine the solubilization of the protein.

[0050] 4.3, Refolding of the inclusion body protein

[0051] The protein solution was diluted with twice the volume of 3 M guanidine hydrochloride, and added dropwise to 200 mL of refolding buffer at pH 8.0 while stirring at high speed for 24 hours at 4°C. Subsequently, the refolded protein solution was directly placed in a dialysis bag, and dialyzed against refolding buffer at 4°C for 24 hours to gradually remove guanidine hydrochloride. The dialysis buffer was replaced twice during the process (fresh refolding buffer was added after 8 hours of the first dialysis and after 16 hours of the second dialysis). After dialysis, the dialysis buffer was concentrated to the target volume (10-20 mL) using an ultrafiltration device (Amicon Ultra centrifugal concentrator) at 4°C to prevent protein denaturation.

[0052] 4.4, Affinity purification of the refolded protein

[0053] The Glutathione Sepharose resin was ensured to be fully equilibrated, and the affinity column was pre-equilibrated with binding buffer (PBS, pH 7.4, containing 0.5% Triton X-100). The refolded protein solution after dialysis was filtered through a 0.45 μm filter and directly loaded onto the affinity column at a flow rate of 1 mL / min. The column was washed with binding buffer until there was no protein in the effluent (no color change in G250 detection solution). Then, the target protein was eluted in sections using 3 column volumes of elution buffer (50 mM Tris-HCl, pH 8.0, containing 10 mM reduced glutathione (GSH)), and the eluate was detected by SDS-PAGE. The eluate containing the high-purity target protein was collected.

[0054] The collected eluate was directly subjected to SDS-PAGE detection (10 μL was taken), and the target gene was confirmed to form a soluble expression supernatant ( Figure 1 ). The SDS PAGE protein control result showed that the recombinant protein contained a GST tag (-26 kDa), and the molecular weight of the recombinant protein was about 48.0 kDa, which was close to the expected molecular weight of the target protein ( Figure 2 ).

[0055] 4.5 Concentration and preservation of recombinant protein

[0056] The collected high-purity protein solution was concentrated to the target volume (about 1-5 mg / mL) using an ultrafiltration device (Amicon Ultra centrifugal concentrator). During the concentration process, the storage buffer (PBS, pH 7.4, containing 10% glycerol) was simultaneously replaced. The operation was carried out under ice bath conditions to prevent protein denaturation. After the concentration was completed, the purified protein solution was aliquoted and immediately frozen in a -80°C refrigerator for preservation to prevent protein inactivation caused by repeated freezing and thawing.

[0057] Example 2 Regulation of immune-related gene expression by recombinant IGF3 protein

[0058] Healthy Trachurus ovatus purchased from Zhanjiang market were selected as the research object. After the fish were anesthetized using a solution containing 50 mg / L MS-222 (Sigma, USA), the liver tissues were quickly dissected. The obtained liver tissues were cut into small pieces, 5 samples per group, and divided into experimental and control groups. The liver pieces were incubated with L-15 medium (Gibco, USA) containing 5 nM recombinant IGF3 protein (prepared by Example 1) as the experimental group; the control group was incubated with L-15 medium without recombinant IGF3 protein. All incubations were carried out at 25°C for 6 hours. After incubation, the expression of immune-related genes (il8, il10, itgb8, mrc2 and sparc) at the mRNA transcription level was detected by real-time fluorescent quantitative PCR. The primers used were designed based on the corresponding gene sequences in the transcriptome database and optimized to ensure their specificity and amplification efficiency.

[0059] The primers used for real-time fluorescent quantitative PCR to detect immune-related genes il8, il10, itgb8, mrc2 and sparc are as follows:

[0060] il8-F: TCATTGTCATTGCTGTGG (SEQ ID NO: 5);

[0061] il8-R: ATGGGTTTGCTCTCTGTC (SEQ ID NO: 6);

[0062] il10-F: AGTCAGTCTCCACCCCCATCTT (SEQ ID NO: 7);

[0063] il10-R: GCCCACTGGAGTTCAGATGCT (SEQ ID NO: 8);

[0064] itgb8-F: TCCCCTCTCTGTCCTGATGG (SEQ ID NO: 9);

[0065] itgb8-R: AGAGTGCATGCCGATTGTGA (SEQ ID NO: 10);

[0066] mrc2-F: CCTCAGAAGAACACCGGCAT (SEQ ID NO: 11);

[0067] mrc2-R: GTGGCCAGAGTCCCATTCAA (SEQ ID NO: 12);

[0068] sparc-F: AGTTTGACGAGGCCATCGAG (SEQ ID NO: 13);

[0069] sparc-R: AGAAGTGGCAAGAGGTGTCG (SEQ ID NO: 14).

[0070] from Figure 3 The test results showed that recombinant IGF3 protein significantly promoted the mRNA expression levels of il8, itgb8, mrc2 and sparc genes in the liver tissue of pomfret; in contrast, the expression of il10 gene did not show significant changes; this indicates that recombinant IGF3 protein can effectively regulate the expression of multiple immune-related genes and play a role in the immune regulation of pomfret.

[0071] Example 3: Application of recombinant IGF3 protein from oval pomfret in antibacterial immunity

[0072] 1. Injection of recombinant IGF3 protein

[0073] Twenty uniformly sized and healthy oval pomfret (average weight 500±20g) were selected and randomly divided into an experimental group and a control group, with 10 fish in each group. Recombinant IGF3 protein was dissolved in physiological saline and administered to the experimental group fish via intraperitoneal injection at a dose of 10 ng / g (recombinant protein / fish body weight). The control group received an equal volume of physiological saline (without recombinant protein) via injection.

[0074] 2. Preparation of bacterial suspension

[0075] Vibrio harveyi was cultured in LB medium at 37°C with shaking until the OD600 reached 0.8, and then the bacterial cells were collected by centrifugation at 5000g for 10 minutes. The obtained bacterial cells were resuspended in PBS and the final concentration was adjusted to 5 × 10⁻⁶. 6 CFU / mL available for use.

[0076] 3. Virus challenge experiment

[0077] On day 5 after injection of recombinant IGF3 protein, each fish in the experimental and control groups was challenged by injecting 100 μL of the bacterial suspension prepared above. At 6 and 12 hours post-infection, samples were collected from 5 fish in each group, and liver, spleen, and head kidney tissues were taken and homogenized in 1 mL of sterile PBS. Subsequently, 100 μL of the homogenate was spread on LB agar plates and incubated at 30°C for 24 hours to count the colonies.

[0078] from Figure 4 The test results showed that the bacterial counts in the liver, spleen, and head kidney of oval pomfret injected with recombinant IGF3 protein were significantly lower than those in the control group. This indicates that recombinant IGF3 protein can significantly enhance the ability of oval pomfret to resist Vibrio harveyi infection, demonstrating the important role of recombinant IGF3 protein in the antibacterial immune response of fish. This study provides important theoretical basis and technical support for the application of recombinant IGF3 protein in fish disease resistance and immunity.

[0079] The above-described specific embodiments are provided to further illustrate the present invention. It should be noted that the above embodiments are only used to further illustrate the present invention and do not represent the scope of protection of the present invention. Non-essential modifications and adjustments made by others based on the present invention are still within the scope of protection of the present invention.

Claims

1. Use of a Trachurus ovatus recombinant IGF3 protein in the preparation of a product having the ability to enhance the resistance of fish to bacterial infection, said Trachurus ovatus recombinant IGF3 protein being obtained by a method comprising transforming an expression vector containing the Trachurus ovatus IGF3 gene into a host cell, culturing the transformed host cell, inducing the expression of the recombinant protein, isolating, purifying and collecting the Trachurus ovatus recombinant IGF3 protein from the culture, said Trachurus ovatus IGF3 gene having the nucleotide sequence shown in SEQ ID NO: 1, said fish being Trachurus ovatus, and said bacteria being Vibrio harveyi.

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