Fish interferon regulatory factor 4 gene, protein, expression vector and application thereof

Abstract

This invention relates to a fish interferon regulator IRF4 gene, protein, expression vector, and their applications, belonging to the field of fish molecular immunology technology. The fish interferon regulator IRF4 is derived from the oval pomfret, and the cDNA nucleotide sequence of the oval pomfret IRF4 gene is shown in SEQ ID NO.1. The amino acid sequence of the protein encoded by the oval pomfret IRF4 gene is shown in SEQ ID NO.2. This invention also provides an expression vector containing the oval pomfret IRF4 gene, and an anti-Vibrio harveyi infection preparation containing the protein or a eukaryotic expression vector. The oval pomfret IRF4 protein of this invention can promote the respiratory burst activity and phagocytic capacity of oval pomfret head kidney macrophages. Overexpression in oval pomfret... Believe After IRF4 is activated, the body's antibacterial ability can be significantly improved, and it can be used as an immune enhancer.

Description

Technical Field

[0001] This invention belongs to the field of fish molecular immunology technology, and in particular relates to a fish interferon regulatory factor IRF4 gene, protein, expression vector and its application. Background Technology

[0002] Interferon (IFN) regulatory factors (IRFs) participate in antiviral and antibacterial immune responses by regulating the expression of IFN-related immune genes. In mammals, IRF4 is produced by various immune cells, including T and B cells, macrophages, and dendritic cells, and plays a crucial role in both the innate and adaptive immune systems. Currently, turbot (… Paralichthys olivaceus ), Croaker ( Miichthys miiuy ), spotted catfish ( Ictalurus punctate ), large yellow croaker ( Larimichthys crocea ), semi-smooth tongue sole ( Cynoglossus semilaevis ), eel ( Monopterus albus ) and zebrafish ( Danio rerio The IRF4 gene has been successively identified in fish species such as [list of fish]. Studies have shown that fish IRF4 can be highly upregulated induction by viral, bacterial, or pathogen-associated molecular patterns (PAMPs, such as poly I:C or LPS). However, mammalian IRF4 shows no response to poly I:C stimulation. This indicates that IRF4 in fish and other vertebrates is species-specific. Although the IRF4 gene has been identified in various fish species, most studies have focused on analyzing the structural features and expression patterns of fish IRF4. The functional role of fish IRF4 remains largely unknown.

[0003] Oval pomfret ( Trachinotus ovatus The oval pomfret is a farmed fish of significant economic value in the coastal areas of southern China and is currently the most important species in deep-water cage aquaculture in the south. However, in recent years, Vibrio harveyi has been repeatedly found to have caused outbreaks in farmed pomfret populations. Vibrio harveyi This has caused a variety of diseases and resulted in huge economic losses.

[0004] To date, there are no reports on the involvement of the interferon regulator IRF4 in the body's resistance to Vibrio harveyi infection in pomfret. Therefore, research on the IRF4 of pomfret (…) Believe The methods and approaches for preventing and controlling fish diseases using IRF4 (Immune Reflux 4) and the development of useful immunomodulators are of significant reference value for the prevention and control of fish diseases. Summary of the Invention

[0005] The technical problem this invention aims to solve is to provide an interferon regulatory factor (IRF4) in oval pomfret and its application in the body's resistance to Vibrio harveyi infection. This invention prepares the IRF4 protein by constructing a prokaryotic expression vector and verifies that it can promote the respiratory burst activity and phagocytic capacity of oval pomfret head kidney macrophages. Furthermore, this invention constructs a eukaryotic expression vector to overexpress or interfere with the expression of the IRF4 gene in fish, thereby affecting the body's resistance to Vibrio harveyi infection. Therefore, it has good practical application value.

[0006] To solve the above-mentioned technical problems, the present invention is achieved through the following technical solution: A fish interferon regulatory factor IRF4 gene, wherein the fish interferon regulatory factor IRF4 gene is the oval pomfret IRF4 gene, and the cDNA nucleotide sequence of the oval pomfret IRF4 gene is shown in SEQ ID NO.1:

[0007] The amino acid sequence of the oval pomfret IRF4 is shown in SEQ ID NO.2: MNPEVDYGGSGSSGNGKLRQWLIEQVDCGKYPGLVWENDEKTIFRIPWKHAGKQDYNRDEDAALFKAWALFKGKFREGIDKPDPPTWKTRLRCALNKSNDFEELVDRSQLDI SDPYKVYRIIPEGAKKRPRQEDSPLSPMSYQVHNYPPLQTQMQQYMATPECGWRDYCQEQASLPELPFTQCPCPPSLPWQGPSMENGYQLRASIYSYGPADSQPPPFTLDAS IRSAEALSDFRLHVSVYFRDSLVKEVTTSSPEGCHITPCPPEKHYLPTGGPDVVPLPVDSLSGQRRAEECPPSPPSALERGVYLWMGADGLYARRMCQSRVYWQGGLSPYGDK LNKLEREVNCKLLHTQDYLTEIQSYGLHSRPLPRFQVLLHFGDECLDPQRQRRTLTVQVEPLFARQLLYLAHQQAGGHYFRSYEHPGVTEHINPSEDYQRAITHHHSSSLQE.

[0008] The present invention also provides a eukaryotic expression vector containing the oval pomfret IRF4 gene.

[0009] The present invention also provides an injectable formulation for improving the anti-infection ability of pomfret, wherein the injectable formulation contains the pomfret IRF4 protein or the eukaryotic expression vector, and the anti-infection refers to anti-Vibrio harveyi infection.

[0010] The beneficial effects of this invention compared to the prior art are as follows: The present invention discloses that the interferon regulatory factor IRF4 of the oval pomfret participates in the body's resistance to Vibrio harveyi infection. The IRF4 protein in the oval pomfret can promote the respiratory burst activity and phagocytic capacity of macrophages in the head kidney of the oval pomfret. Furthermore, the present invention achieves overexpression of this protein in the oval pomfret. Believe After IRF4 treatment, the body's antibacterial ability was significantly enhanced. Following Vibrio harveyi infection, the bacterial counts in the liver, spleen, and head kidney were significantly lower than in the control group. Consistent with this, when IRF4 was interfered with in fish... BelieveAfter IRF4 expression, the body's antibacterial ability is significantly reduced, and after Vibrio harveyi infection, the number of bacteria in various tissues increases significantly. The IRF4 of oval pomfret of the present invention can improve the fish's resistance to bacterial infection and can be used as an immune enhancer. It has application value in the preparation of antiviral drugs, antibacterial drugs and anti-inflammatory agents, and the prevention and treatment of fish diseases. Attached Figure Description

[0011] Figure 1 Figure 1 shows the results of the IRF4 protein promoting the respiratory burst activity and phagocytic rate of head kidney macrophages in an embodiment of the present invention; A, respiratory burst activity of head kidney macrophages; B, phagocytic rate of head kidney macrophages. Figure 2 This invention provides an embodiment for overexpression in experimental fish. Believe The graph shows the changes in bacterial counts in the liver, spleen, and head kidney at various time points after IRF4, where A represents the liver, B represents the spleen, and C represents the head kidney. Figure 3 This invention provides an embodiment for interfering with expression in experimental fish. Believe The graph shows the changes in bacterial counts in the liver, spleen, and head kidney at various time points after IRF4, where A represents the liver, B represents the spleen, and C represents the head kidney. Detailed Implementation

[0012] The technical solution of the present invention will be further explained below through embodiments, but the technical solution of the present invention is not limited in any form by the embodiments.

[0013] Example 1 The oval pomfret IRF4, the nucleotide sequence of the oval pomfret IRF4 gene cDNA is as shown in SEQ ID NO.1, and the amino acid sequence of the oval pomfret IRF4 gene is as shown in SEQ ID NO.2.

[0014] The construction methods and protein purification processes of the above-mentioned prokaryotic and eukaryotic expression vectors for the oval pomfret IRF4 were carried out sequentially according to the following steps: 1. Construction of recombinant vectors RNA was extracted from the head and kidney tissue of the oval pomfret, and the RNA was reverse transcribed into cDNA. Then, using the cDNA as a template and the following primer sequence as primers, the nucleotide sequence of the IRF4 gene was obtained by PCR. The primer sequence is as follows: Believe IRF4-F: 5'- gatatcgccacc ATGAACCCTGAGGTGGATTACG-3', (SEQ ID NO. 3); Believe IRF4-R: 5'- streetatcCTCCTGCAGGCTGCTGCTGT-3', (SEQ ID NO. 4).

[0015] The specific steps are as follows: following the standard TA cloning method (reaction conditions: 95 ℃ 5 min; 95 ℃ 30 s; 60 ℃ 30 s; 72 ℃ 100 s; 72 ℃ 10 min), the IRF4 gene, specifically the fragment of SEQ ID NO.1, is ligated into the pMD19-T vector to obtain the recombinant plasmid. Believe IRF4-Tsimple.

[0016] Extract the DNA from the above recombinant plasmid, and use... Eco The 1350 bp fragment was recovered after digestion with RV restriction endonuclease; DNA was extracted from the pET-32a plasmid and processed... Eco After RV digestion, the recovered 1350 bp fragment was ligated to the plasmid using T4 ligase to construct the recombinant plasmid. Gene sequencing confirmed the presence of IRF4 in the recombinant plasmid, and it was named pET32a- Believe IRF4. The 1350 bp fragment was ligated into the eukaryotic expression vector pCN3 using the same method to obtain p. Believe IRF4 recombinant plasmid.

[0017] 2. Recombinant protein r Believe IRF4 induction and purification To express the oval pomfret r Believe IRF4 recombinant protein, we will use pET32a- Believe IRF4 and pET32a expressing rTrx (thioredoxin) were introduced into *E. coli* BL21, and the strains were stored at -80 °C. The strains were then inoculated into LB broth and cultured at 37 °C until OD200. 600 ≈0.6. The bacterial cells were then collected after inducing with 0.5 mM IPTG at 16 °C for 24 h. The cells were then resuspended in 10 mM imidazole buffer, lysed by sonication, and purified using Ni column affinity to obtain the target protein. The purified recombinant protein, after dialysis, met the expected protein size. Thus, the oval pomfret was obtained. Believe The IRF4 recombinant protein has the amino acid sequence shown in SEQ ID NO.2, with a length of 450 amino acids, a type of amino acid, a chain type of single chain, and characteristics of a molecular weight of 51.49 kDa and an isoelectric point of 6.67.

[0018] Experimental Example 2: Recombinant protein r Believe IRF4 can significantly promote the respiratory burst activity and phagocytic capacity of oval pomfret head kidney macrophages.

[0019] We seeded freshly extracted oval pomfret head kidney macrophages into 96-well plates and cultured them at 28 °C until they adhered. Then, we used 200 μg / mL r... Believe Cells were treated with IRF4, 200 μg / mL rTrx, or PBS (as a control group) for 12 or 24 hours. The absorbance at 630 nm was measured in each well using a microplate reader according to the NBT reduction assay. Simultaneously, freshly extracted oval pomfret head kidney macrophages were seeded in 6-well plates and cultured at 28 °C until adherence. Subsequently, cells were treated with 200 μg / mL rTrx. Believe Cells were treated with IRF4, 200 μg / mL rTrx, or PBS (as a control group) for 4 hours, then incubated with fluorescent microspheres, and finally the phagocytosis rate of the cells was detected by flow cytometry.

[0020] The results showed that, compared with the control group, r Believe The respiratory burst effect of IRF4-treated head kidney macrophages was significantly increased by 8.16-fold and 3.17-fold at 12 and 24 hours, respectively (e.g., ...). Figure 1 (A) Furthermore, according to flow cytometry results, compared with the control group and the rTrx group (protein control group), r... Believe The phagocytic efficiency of the IRF4-treated group increased significantly, reaching 31.70% (e.g., Figure 1 B in the text indicates that r Believe IRF4 can significantly enhance the phagocytic capacity of phagocytes. These results indicate that IRF4 in oval pomfret can promote the phagocytic capacity of phagocytes, suggesting that this protein can significantly enhance the efficiency of macrophages in recognizing and clearing pathogens.

[0021] Experimental Example 3: The oval pomfret IRF4 of the present invention can significantly improve the antibacterial infection ability of oval pomfret. To investigate the antibacterial immunity induced by IRF4 overexpression in vivo, we extracted p... Believe IRF4 endotoxin-free plasmids were diluted with PBS to 200 μg / mL. Furthermore, 36 oval pomfret (average weight 14.2 ± 1.3 g) were divided into 3 groups (12 fish / group) and each group was intramuscularly injected with 100 μL of p Believe IRF4, pCN3, or PBS (as control group) were administered. Five days after immunization, three fish were randomly selected from each group and aseptically dissected to collect liver, spleen, and head kidney tissues. RNA was then extracted from the liver, spleen, and head kidney tissues, reverse-engineered into cDNA, and used as a template for PCR detection to confirm plasmid expression. Subsequently, the remaining fish were intraperitoneally injected with 100 μL of Vibrio harveyi (5 × 10⁻⁶). 7(CFU / mL). At 6, 9, and 12 hours post-injection, liver, spleen, and head kidney were aseptically collected from three fish in each group. The extracted tissues were then ground, diluted, and plated onto LB agar plates containing ampicillin. Bacterial load in each group was determined using the plate count method.

[0022] To further determine Believe IRF4 is involved in the antibacterial activity of oval pomfret. We used siRNA (small interfering RNA) technology to interfere with the fish's... Believe We observed the expression of IRF4 to see if the antibacterial effect was weakened. Specifically, we designed and synthesized IRF4 using the T7 RiboMAX™ Express RNAi System (Promega, USA). Believe IRF4 primers, i.e., si Believe IRF4-P1 / si Believe IRF4-P2 and si Believe IRF4-P3 / si Believe IRF4-P4, followed by preparation of si according to the T7 RiboMAX™ Express RNAiSystem kit instructions. Believe IRF4.

[0023] The above si Believe The primer sequences required for IRF4 synthesis are: si Believe IRF4-P1: GGATCCTAATACGACTCACTATAGCTGTTCAAGGGCAAGTTT, (SEQ IDNO.5); si Believe IRF4-P2: AAAAACTTGCCCTTGAACAGCTATAGTGAGTCGTATTAGGAT CC, (SEQ IDNO.6); si Believe IRF4-P3: GGATCCTAATACGACTCACTATAAAACTTGCCCTTGAACAGC, (SEQ IDNO.7); si Believe IRF4-P4: AAGCTGTTCAAGGGCAAGTTTTATAGTGAGTCGTATTAGGAT CC, (SEQ ID NO. 8).

[0024] We randomly divided 36 fish into three groups (12 fish per group) and injected each group with 15 μg of si intramuscularly. Believe IRF4, 15 μg si BelieveIRF4-C (control siRNA) and 100 μL PBS (control group). Twelve hours after siRNA injection, three fish were extracted from each group, and as described above, confirmation was achieved. Believe IRF4 was knocked down. Then, 100 μL of Vibrio harveyi (5 × 10⁻⁶) was injected intraperitoneally into each of the 27 fish. 7 (CFU / mL). Six, 12, and 24 hours after injection, the bacterial load in the liver, spleen, and head kidney of each group of fish was analyzed using plate count as described above.

[0025] The results showed that, compared with the control group, overexpression of p Believe IRF4 can effectively reduce the bacterial load in fish (e.g., Figure 2 Liver: 6, 9, and 12 hours after viral invasion, inject p... Believe The bacterial load in IRF4-treated fish decreased by 42.80%, 75.30%, and 74.91% respectively compared to the control group (e.g., ...). Figure 2 (A) Spleen: 6, 9, and 12 hours after viral invasion, inject p Believe The bacterial load in IRF4 fish decreased by 66.06%, 59.86%, and 50.52% compared to the control group (e.g., ...). Figure 2 (B) Head kidney: 6, 9, and 12 hours after viral invasion, inject p. Believe The bacterial count in IRF4 fish decreased by 66.31%, 68.35%, and 39.46% compared to the control group (e.g., ...). Figure 2 (C) Consistent with this, compared to the control group, the interference expression of si Believe Following IRF4, the bacterial load in the fish significantly increased in three tissues examined at each time point (e.g., Figure 3 In comparison, si Believe The bacterial load in fish treated with TNF-α1-C was almost the same as that in the control group (e.g. Figure 3 These results indicate that IRF4 in oval pomfret can enhance the body's antibacterial function.

Claims

1. A fish interferon regulator IRF4 gene, characterized in that, The fish interferon regulator IRF4 is the oval pomfret IRF4, and the cDNA nucleotide sequence of the oval pomfret IRF4 gene is shown in SEQ ID NO.

1.

2. The protein encoded by the fish interferon regulator IRF4 gene according to claim 1, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. A eukaryotic expression vector, characterized in that, The expression vector contains the IRF4 gene as described in claim 1.

4. An injectable formulation for enhancing the anti-infective ability of oval pomfret, characterized in that, The injectable formulation contains the protein of claim 2 or the eukaryotic expression vector of claim 3. The anti-infection refers to anti-Vibrio harveyi infection, which is achieved by overexpressing the IRF4 gene to promote the respiratory burst activity and phagocytic capacity of the head kidney macrophages of the oval pomfret.

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