Trachinotus ovatus IGF3 gene and application thereof

Abstract

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

Description

Technical Field

[0001] The invention belongs to the technical fields of biotechnology and aquaculture, and specifically relates to an IGF3 gene of silver pomfret and an application thereof, in particular to an insulin-like growth factor 3 (IGF3) gene of silver pomfret and an application of the recombinant protein expressed by the gene in enhancing the anti-bacterial ability of fish. Background Art

[0002] Trachinotus ovatus is an important economic fish species and plays an important role in the global aquaculture industry. With the reduction of marine resources and the growth of market demand, marine aquaculture has become one of the main ways to meet the demand for fish supply. In China, the scale of aquaculture of Trachinotus ovatus continues to expand and has become one of the main marine aquaculture species. However, the high-density aquaculture 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 infections are particularly prominent, especially acute sepsis and other systemic diseases caused by Vibrio harveyi, which significantly reduce 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 is not conducive to ecological protection and human health in the long run. Therefore, the development of environmentally friendly and efficient disease prevention and control methods has become an urgent problem to be solved. In this context, it is particularly important to screen fish disease resistance genes and develop recombinant disease resistance proteins through genetic engineering technology.

[0004] The insulin-like growth factor (IGF) gene family consists 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 construct a complex regulatory network and are involved in regulating various physiological processes such as growth, reproduction and metabolism.

[0005] Insulin-like Growth Factor 3 (IGF3), as a member of the IGF family unique to teleosts, is mainly expressed in gonadal tissue and plays an important role in regulating reproduction-related cell proliferation, differentiation and growth. Existing studies have shown that IGF3 is closely related to gonadal maturation, oocyte growth and ovulation. However, there has been no clear report on the potential function of IGF3 in fish disease resistance, and this gap provides a new direction for research. Summary of the invention

[0006] The purpose of the present invention is to provide an IGF3 gene of silver pomfret and a protein encoded by it, and an expression vector and a recombinant strain comprising the IGF3 gene.

[0007] The present invention also aims to provide a recombinant IGF3 protein of silver pomfret.

[0008] The last object of the present invention is to provide the use of the above-mentioned recombinant IGF3 protein of silver pomfret in the preparation of products for regulating the expression of immune-related genes of silver pomfret and in the preparation of products for enhancing the ability of fish to resist bacterial infection.

[0009] The first object of the present invention can be achieved by the following technical solution: an IGF3 gene of silver pomfret, the nucleotide sequence of which is shown in SEQ ID NO:1.

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

[0011] The present invention also provides an expression vector, which contains the IGF3 gene of the ovate pomfret, and can efficiently express the IGF3 gene to obtain the target recombinant protein.

[0012] The present invention also provides a recombinant strain, which contains the expression vector.

[0013] The above-mentioned second purpose of the present invention can be achieved by the following technical scheme: a recombinant IGF3 protein of silver pomfret is prepared by the following method: comprising transforming the expression vector into host cells, culturing the transformed host cells, inducing the expression of the recombinant protein, and isolating, purifying and collecting the recombinant IGF3 protein of silver pomfret from the culture.

[0014] The present invention successfully cloned the IGF3 gene through reverse transcription PCR technology and constructed a corresponding expression vector, thereby achieving efficient expression and large-scale preparation of recombinant protein.

[0015] The last object of the present invention can be achieved by the following technical solution: the above-mentioned recombinant IGF3 protein of silver pomfret is used in the preparation of products for regulating the expression of immune-related genes of silver pomfret.

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

[0017] The present invention also provides the use of the above-mentioned recombinant IGF3 protein of silver pomfret in preparing a product capable of enhancing the ability of fish to resist bacterial infection.

[0018] Preferably, the fish is oval pomfret.

[0019] Preferably, the bacterium is Vibrio harveyi.

[0020] The present invention verifies the potential of the recombinant IGF3 protein of Pieris ovalis in the preparation of anti-bacterial infection preparations through experiments, especially its application value in preventing and treating Vibrio harveyi infection.

[0021] The present invention has the following advantages:

[0022] (1) The present invention focuses on the role of the IGF3 gene of silver pomfret in the immune system of fish for the first time. Through cloning and expression analysis, a recombinant expression vector of the gene was constructed, a method for preparing the recombinant protein and analyzing the antibacterial activity was established, and its application prospect as a potential anti-disease drug was explored. Preliminary research results show that the recombinant IGF3 protein of silver pomfret can effectively enhance the anti-disease immune response of fish, providing new genetic materials and technical means for the study of fish disease resistance mechanism and disease prevention and control;

[0023] (2) The present invention successfully cloned the IGF3 gene of silver pomfret, clarified its key role in disease resistance and immune regulation, and developed a recombinant IGF3 protein with significant antibacterial activity through experiments, providing a new technical solution for disease prevention and control. The recombinant protein has broad application potential and can be used to develop preparations against bacterial infections, which is suitable for dealing with frequent disease problems in high-density breeding environments. In addition, the present invention can significantly reduce the use of chemical drugs, protect the ecological environment of water bodies, and provide strong support for the sustainable and green development of the aquaculture industry. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0025] Figure 2 It is the SDS-PAGE detection of the affinity-purified recombinant IGF3 protein of the oval pomfret in Example 1, M: marker, 1: recombinant IGF3 protein;

[0026] Figure 3 The recombinant IGF3 protein in Example 2 regulates the mRNA transcription level of immune-related genes in the liver of the oval pomfret. The values ​​are expressed as mean ± standard error (n = 5). Compared with the corresponding control group, *P < 0.05; **P < 0.01;

[0027] Figure 4The bacterial loads in the liver (A), spleen (B) and head kidney (C) tissues after the recombinant IGF3 protein was intraperitoneally injected to inhibit Vibrio harveyi infection in vivo in Example 3. The 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 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.

[0029] Unless otherwise specified, the materials, reagents and equipment used in the present invention can be obtained from commercial sources.

[0030] Example 1 Preparation of recombinant IGF3 protein

[0031] 1. RNA extraction and cDNA synthesis of Oval Pomfret

[0032] The oval pomfret samples were purchased from Xiashan Aquatic Products Wholesale Market, Zhanjiang City, Guangdong Province, China. The oval pomfret was anesthetized with a solution containing 50 mg / L tricaine methane sulfonate (MS-222, Sigma, USA) and then killed, and the liver tissue was immediately sampled and isolated. Total RNA was extracted from the isolated tissue using Trizol reagent (Takara, Japan). The quality of RNA was evaluated by electrophoresis on 1% agarose gel; its purity and concentration were determined using a NanoDrop 2000C spectrophotometer (Thermo Scientific, USA), and qualified RNA samples were analyzed using PrimeScript TM The DNA was reverse transcribed into cDNA using RT Reagent Kit with gDNA Eraser (RR047A; Takara, Japan), and the cDNA was used as a template for the subsequent PCR reaction.

[0033] 2. Cloning of IGF3 gene

[0034] The cDNA sequence of IGF3 of the oval pomfret was obtained based on the previous cloning experiment in the laboratory. The sequence encoding the 35 amino acid signal peptide was determined through 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 IDNO: 3);

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

[0037] Using the above-mentioned first-strand cDNA synthesized as a template, PCR amplification was performed using primers IGF3-RT-F and IGF3-RT-R to obtain an IGF3 gene fragment without a signal peptide, with a length of 480 bp, 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 product was separated by agarose gel electrophoresis and the target fragment was recovered by gel cutting. The purified DNA fragment was connected to the pGEX-4T-1 vector and transformed into DH5α Escherichia coli, and a GST tag was fused to the N-terminus. After selecting the positive clones, they were sent to Sangon Biotech Co., Ltd. (Shanghai, China) for sequencing verification. The sequencing results were analyzed by BLAST comparison to confirm that the obtained fragment was indeed the IGF3 gene.

[0038] The nucleotide sequence of the IGF3 gene of the oval pomfret is as follows:

[0039] GCCCGCCTCCGCTGTGGCTCTGATCTCCTCAGTGACCTCATATTTGTGTGCGGGGATCGAGGAATCTATTTAGGTAAAGGTTCCTGGTCGGGGTACGGTGCTCGGCCCAGAGGGAAGGGGATAGTGGATCAGTGTTGCCGACCAGCCGGCTGTGAACTTCAGCATCTGGAGATGTACTGTGCCAAACCAAAGAGCCAGCAGCGCACCACAGCTTACCCAACCACAACAACAGCAGCTG CACACACCACCACACAGCTAGACGTGGCCCAGCAGTTCCAAACAGTATTTCACAAGAGACTTGCAGAGCAGCTGGGGGCTCCCAGCAGCCCGAAGAGGGACGCTTACAGGAGGAGAACACAGCCTTCAGGACAGCGGAAACGCAAATCGTCAAACAGGAGGATTAACGGAGTGAACACATCCAGCAGCCCCTCGGCCTCTGGGAGCCCCCTTCAGAGAACAATGAGGTTCAAATCG TGA (shown in SEQ ID NO: 1, the underline indicates the stop codon).

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

[0041] ARLRCGSDLLSDLIFVCGDRGIYLGKGSWSGYGARPRGKGIVDQCCRPAGCELQHLEMYCAKPKSQQRTTAYPTTTTAAAHTTTQLDVAQQFQTVFHKRLAEQLGAPSSPKRDAYRRRTQPSGQRKRKSSNRRINGVNTSSSPPSASGSPLQRTMRFKS (shown in SEQ ID NO: 2).

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

[0043] From the selected positive clone Escherichia coli DH5α, the plasmid was extracted using a plasmid extraction kit (Tiangen Biochemical Technology, Beijing), and the purified plasmid was transformed into Rosetta (DE3) competent cells. The positive single clone colony was verified by bacterial liquid PCR, confirming that the amplified fragment size was about 480bp, which was consistent with expectations. Subsequently, the positive single colony was selected and sent to a sequencing company for sequencing verification. The sequencing results showed that the obtained sequence completely matched the target IGF3 gene sequence, confirming that the prokaryotic recombinant expression vector of the IGF3 gene was successfully constructed.

[0044] 4. IGF3 protein mass expression and detection

[0045] 4.1. Massive expression and preliminary detection of IGF3 protein:

[0046] Inoculate a single colony of E. coli containing the recombinant plasmid into 5 mL of LB liquid medium containing kanamycin (final concentration 50 μg / mL) and culture overnight at 37°C in a shaking incubator. Take 100 μL of the overnight cultured bacterial solution and inoculate it into 100 mL of fresh LB liquid medium (containing kanamycin) and culture it at 37°C for 16 hours. Subsequently, transfer the bacterial solution to 2000 mL of LB liquid medium (containing kanamycin) and continue to culture it at 37°C until the OD600 reaches 0.6. Then lower the culture temperature to 30°C, add IPTG to a final concentration of 0.1 mM, and induce expression for 8 hours.

[0047] After the culture was completed, the 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 placed on ice for 30 minutes. After the cells were disrupted by ultrasound, the supernatant and the precipitate were separated, and 10 μL of the supernatant and the precipitate were taken for SDS-PAGE detection, and the remaining samples were stored at 4°C for later use.

[0048] 4.2. Inclusion body protein purification

[0049] The above precipitate was resuspended in 50mL STET buffer (containing DTT final concentration of 1mM), ultrasonically treated for 10 minutes to promote the dissolution of impurities, and then centrifuged to remove the supernatant. Repeat this step until the supernatant becomes transparent. Finally, the precipitate was resuspended in PBS, ultrasonically treated again for 10 minutes, and the supernatant was discarded after centrifugation. The precipitate was resuspended in 3mL of a solution containing 6M guanidine hydrochloride and 0.5% TritonX-100, DTT was added to a final concentration of 5mM, and shaken at 37°C and 220rpm for 4 hours to ensure that the inclusion bodies were completely dissolved. After centrifugation at 10000rpm and 4°C for 10 minutes, the supernatant was taken and the protein dissolution was detected by SDS-PAGE.

[0050] 4.3. Inclusion body protein renaturation

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

[0052] 4.4. Refolded protein affinity purification

[0053] Ensure that the Glutathione Sepharose resin is fully equilibrated and pre-equilibrate the affinity column with binding buffer (PBS, pH 7.4, containing 0.5% Triton X-100). Filter the dialyzed refolded protein solution through a 0.45μm filter membrane and load it directly onto the affinity column, injecting at a flow rate of 1mL / min. Wash the column with binding buffer until there is no protein in the effluent (no color change in the G250 detection solution). Then use 3 column volumes of elution buffer (50mM Tris-HCl, pH 8.0, containing 10mM reduced glutathione (GSH)) to elute the target protein in sections, and detect the eluate by SDS-PAGE. Collect the eluate of the high-purity target protein.

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

[0055] 4.5 Recombinant protein concentration and storage

[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 replaced at the same time. The operation was carried out under ice bath conditions to prevent protein denaturation. After the concentration was completed, the purified protein solution was divided into small portions and immediately placed in a -80°C refrigerator for freezing and storage to prevent repeated freezing and thawing from causing protein inactivation.

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

[0058] Healthy oval pomfret purchased from the Zhanjiang market was selected as the research object. After the fish was anesthetized with a solution containing 50 mg / LMS-222 (Sigma, USA), the liver tissue was quickly dissected. The obtained liver tissue was cut into small fragments, 5 samples per group, and divided into an experimental group and a control group. The liver fragments were incubated in L-15 medium (Gibco, USA) containing 5nM recombinant IGF3 protein (prepared by Example 1) as the experimental group; the control group was incubated in L-15 medium without recombinant IGF3 protein. All incubations lasted for 6 hours at 25°C. After the incubation, the expression of immune-related genes (il8, il10, itgb8, mrc2 and sparc) at the mRNA transcription level was detected using real-time fluorescence quantitative PCR technology. 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 fluorescence 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 show that recombinant IGF3 protein significantly promoted the mRNA expression levels of il8, itgb8, mrc2 and sparc genes in the liver tissue of oval pomfret; in contrast, there was no significant change in the expression of il10 gene; 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 oval pomfret.

[0071] Example 3 Application of recombinant IGF3 protein of Agaricus ovatus in antibacterial immunity

[0072] 1. Injection of Recombinant IGF3 Protein

[0073] Twenty healthy oval pomfret (average weight 500±20g) of uniform size 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 saline and injected intraperitoneally into the experimental group fish at a dose of 10ng / g (recombinant protein / fish weight). The control group received an equal amount of saline (without recombinant protein) injection.

[0074] 2. Preparation of Bacterial Suspension

[0075] Vibrio harveyi was cultured in LB medium at 37°C with shaking until OD600 reached 0.8, and then centrifuged at 5000g for 10 minutes to collect the cells. The resulting cells were suspended in PBS and the final concentration was adjusted to 5×10 6 CFU / mL is reserved.

[0076] 3. Virus challenge experiment

[0077] On the 5th day after the injection of recombinant IGF3 protein, each fish in the experimental group and the control group was injected with 100 μL of the bacterial suspension prepared above for infection. At 6 hours and 12 hours after infection, 5 fish were randomly selected from each group for sampling, and the liver, spleen and head kidney tissues were taken and homogenized in 1 mL of sterile PBS; then 100 μL of the homogenate was spread on the LB plate and the number of colonies was counted after incubation at 30°C for 24 hours.

[0078] from Figure 4 The test results show that the number of bacteria in the liver, spleen and head kidney of the oval pomfret injected with recombinant IGF3 protein was significantly lower than that in the control group. This shows that recombinant IGF3 protein can significantly enhance the ability of oval pomfret to resist Vibrio harveyi infection, proving the important role of recombinant IGF3 protein in the antibacterial immune response of fish. This study provides an important theoretical basis and technical support for the application of recombinant IGF3 protein in fish disease resistance.

[0079] The specific embodiments listed above are used to supplement the description of the present invention. It should be pointed out 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 suggestions of the present invention still fall within the scope of protection of the present invention.

Claims

1. An IGF3 gene of silver pomfret, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

2. The protein encoded by the IGF3 gene according to claim 1, characterized in that The amino acid sequence of the protein is shown in SEQ ID NO:

2.

3. An expression vector, characterized in that: The expression vector contains the IGF3 gene of the silver pomfret according to claim 1.

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

5. A recombinant IGF3 protein of silver pomfret, characterized in that: The method is prepared by the following method: comprising transforming the expression vector described in claim 3 into host cells, culturing the transformed host cells, inducing the expression of the recombinant protein, and isolating, purifying and collecting the recombinant IGF3 protein of silver pomfret from the culture.

6. Use of the recombinant IGF3 protein of Agaricus ovatus according to claim 5 in the preparation of a product for regulating the expression of immune-related genes of Agaricus ovatus.

7. The use according to claim 6, characterized in that: The immune-related genes include il8, itgb8, mrc2 and sparc genes.

8. Use of the recombinant IGF3 protein of Pisces ovalis according to claim 5 in the preparation of a product capable of enhancing the ability of fish to resist bacterial infection.

9. The use according to claim 8, characterized in that: The fish is an oval pomfret.

10. The use according to claim 8, characterized in that: The bacterium is Vibrio harveyi.

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