Application of micropterus salmoides type 2 interferon related factor IFN-gammarel in preparation of medicine for treating and / or preventing iridovirus infection of micropterus salmoides

Abstract

The invention discloses application of a micropterus salmoides type 2 interferon related factor IFN-gamma rel in preparation of a medicine for treating and / or preventing iridovirus infection of micropterus salmoides. Through screening of transcriptome data after LMBV infection of micropterus salmoides and qPCR verification, expression of IFN gamma rel after LMBV infection is significantly up-regulated. The recombinant bacillus subtilis B.s-IFN-gammarel for expressing the protein is obtained by carrying out codon optimization on the gene of IFN gammarel, connecting the gene of IFN gammarel into plasmids to construct recombinant plasmids, and carrying out transformation and screening. Results show that the B.s-IFN-gammarel, as an antiviral oral drug for preventing or treating LMBV infection, can significantly improve the survival rate of micropterus salmoides, and has good safety. Therefore, the invention provides the application of IFN gammarel as an oral antiviral drug for preventing and treating LMBV infection in micropterus salmoides culture.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to the application of interferon-associated factor IFN-γrel (IAFR) in the preparation of drugs for the treatment and / or prevention of largemouth bass iridovirus infection. Background Technology

[0002] Largemouth bass ( Micropterus salmoides Largemouth bass (LMBV) is an important aquaculture species in China, but its cultivation is severely threatened by LMBV. LMBV belongs to the genus *Ranavirus* of the family Iridoviridae and can cause mortality in largemouth bass; it is highly contagious. Currently, biosecurity measures are mainly used to control LMBV, but their effectiveness is limited. LMBV infection leads to severe mortality in largemouth bass, and there is a lack of antiviral drugs, especially easily administered oral antiviral drugs.

[0003] Interferons (IFNs) are key cytokines of the innate immune system, playing a central role in antiviral defense. In mammals, interferons are classified into types I, II, and III, while in bony fish, they are classified into types I, II, and IV. Type I interferons are further divided into two groups based on the number of cysteine ​​residues (two cysteines and four cysteines). Group I is further divided into subgroups a, d, e, and h, while subgroup II is divided into subgroups b, c, and f. Type II interferons include two members: IFN-γ and the IFN-γ-related gene (IFN-γrel). Type IV interferons have only one member.

[0004] Since interferon was discovered to interfere with viral replication, its effects have been continuously studied, and it is increasingly being used to treat viral diseases, including chronic hepatitis C virus (HCV), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Different types of IFNs exhibit varying antiviral effects. Studies on Atlantic salmon infectious salmon anemia virus (ISAV) have shown that IFNc can protect Atlantic salmon from ISAV infection, but the protective effects of IFNa1 and IFNb are very weak. Interferon not only treats viral diseases but also provides protection against bacterial infections. Type I interferon IFN-β has direct antibacterial activity, directly killing Staphylococcus aureus, and the peptides synthesized from IFN-β also possess antibacterial properties. Type III interferon IFNλ is generally considered to be an inducible antiviral cytokine involved in intestinal immunity, while naturally occurring IFNλ4 in the pig intestine has agglutinating and bactericidal effects, significantly reducing bacterial load in mouse models.

[0005] While interferons offer protection against a variety of viral and bacterial infections, studies have shown that they can have negative effects on some viruses and bacteria. In lymphocytic choriomeningovirus (LCMV) infection, blocking type I interferon signaling reduces chronic immune activation and immunosuppression, restores lymphoid tissue structure, and increases immune parameters associated with viral replication control, ultimately promoting LCMV clearance. During influenza virus infection, type I interferon increases morbidity and mortality, rather than providing protection. Type I interferon IFNα / β is also detrimental during Brucella abortus infection; bacterial control in mice is associated with increased IFNγ and nitric oxide production, as well as decreased TRAIL expression and apoptosis. Therefore, interferons are a double-edged sword when used for viral and bacterial infections, and their antiviral function requires validation through animal studies.

[0006] IFN-γrel is a gene unique to bony fishes. Unlike IFN-γ, bony fish IFN-γrel lacks the C-terminal nuclear localization sequence (NLS) motif, which consists of four consecutive cationic residues (lysine and arginine). This motif is conserved in IFN-γ molecules of both bony fish and mammals. (Mandarin fish) Siniperca chuatsi IFN-γ and IFN-γrel from sea bass can induce significant upregulation of Mx, IRF1, and STAT1. Lateolabrax japonicus IFN-γ can activate the JAK-STAT signaling pathway, upregulating the expression of Mx, ISG15, and Viperin, and exhibits significant antiviral activity against red-spotted grouper neuronecrosis virus (RGNNV). However, IFN-γrel does not possess these functions, indicating that the role of IFN-γrel varies among different fish species. In goldfish, IFN-γ has a persistent effect on ROI production in monocytes, while IFN-γrel has a transient ROI initiation potential and ultimately downregulates IFN-γ-induced ROI production. In pufferfish, IFN-γ induces Mx gene expression, while IFN-γrel inhibits its expression.

[0007] Patent CN108264548A discloses that recombinant IFN-γrel protein can induce the upregulation of immune-related genes Mx, IRF1, STAT1, and SOCS1, and can be used as an immune enhancer or adjuvant for fish. Patent CN115093476A discloses that a combination of type II interferon IFN-γrel and type II interferon receptor for largemouth bass can significantly reduce the copy number of LBUSV virus (largemouth bass ulcer syndrome virus) in the spleen, liver, and head kidney tissues of largemouth bass after infection. However, the prior art does not disclose the application of IFN-γrel in largemouth bass iridovirus.

[0008] Interferon is available in various formulations for humans, such as injections, sprays, gels, and effervescent tablets. In fish, current research on interferon mainly focuses on in vivo injection, but this is cumbersome, harmful to the fish, and impossible when the fish are too small. In contrast, oral administration is a promising alternative. Oral administration is the most ideal method of administration in aquaculture due to its ease of operation and minimal damage to the fish. The antiviral and antibacterial efficacy of orally delivered interferon has been verified in various fish species. Oral administration of type I interferon to Atlantic salmon (Salmo salar) significantly reduced the viral load of infectious pancreatic necrosis virus (IPNV) and induced a systemic antiviral immune response. In studies of oral type II interferon delivery, in addition to stimulating serum lysozyme activity, it upregulated the expression of IL-6, IFN-γ, and IL-12 genes in the spleen and kidneys, significantly improving survival rates after bacterial infection. The antiviral effects of orally delivered type I interferon have also been observed in rainbow trout (…). Oncorhynchus mykiss ),carp( Cyprinus carpio ) and grass carp (Ctenopharyngodon idella) It has been verified in studies that it significantly improves the relative survival rate after infection with infectious hematopoietic necrosis virus (IHNV), carp virus spring viremia (SVCV), and grass carp hemorrhagic virus (GCHV).

[0009] Therefore, exploring whether IFN-γrel can prevent and treat LMBV in largemouth bass is of great significance for providing a new anti-LMBV drug, especially an oral anti-LMBV drug. Summary of the Invention

[0010] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide the application of interferon-associated factor IFN-γrel in the preparation of drugs for the treatment and / or prevention of iridovirus infection in largemouth bass.

[0011] A second objective of this invention is to provide the use of interferon-associated factor IFN-γrel (IAFR) in the preparation of a medicament for the treatment and / or prevention of largemouth bass virus disease caused by largemouth bass iridovirus infection.

[0012] A third objective of this invention is to provide the use of a recombinant strain expressing the interferon-associated factor IFN-γrel (IAA) in the preparation of a drug for the treatment and / or prevention of largemouth bass iridovirus infection or largemouth bass virus disease caused by AIA.

[0013] The above-mentioned objective of this invention is achieved through the following technical solution: This invention, through analysis and screening of transcriptome data from largemouth bass infected with largemouth bass virus (LMBV), discovered that IFN in the JAK-STAT signaling pathway is significantly reduced after LMBV infection. Upregulation of genes such as γrel; qPCR was used to verify IFN. γrel was significantly upregulated after LMBV infection, and preliminary analysis showed that IFN was upregulated in fish. γrel is used to combat LMBV virus infection. The IFN-γrel gene of largemouth bass was codon-optimized and ligated into a plasmid to construct the recombinant plasmid pHT43-IFN-γrel. This recombinant plasmid was transformed, cultured, and screened for positive monoclonal antibodies to obtain recombinant Bacillus subtilis Bs-IFN-γrel. After activation, amplification, and induced expression, Bacillus subtilis containing this protein was obtained. Furthermore, Bacillus subtilis containing this protein was used as an oral antiviral drug for the prevention and treatment of LMBV infection. Results showed that the recombinant Bacillus subtilis Bs-IFN-γrel significantly improved the survival rate of largemouth bass and exhibited good biocompatibility.

[0014] Therefore, the present invention provides the use of interferon-associated factor IFN-γrel in the preparation of a medicament for the treatment and / or prevention of iridovirus infection in largemouth bass, wherein the amino acid sequence of the IFN-γrel is shown in SEQ ID No. 2.

[0015] This invention also provides the use of interferon-associated factor IFN-γrel in the preparation of a drug for treating and / or preventing largemouth bass virus disease caused by largemouth bass iridovirus infection, wherein the amino acid sequence of IFN-γrel is shown in SEQ ID No. 2; and the fish is largemouth bass.

[0016] The recombinant Bacillus subtilis Bs-IFN-γrel constructed in this invention can efficiently express the interferon-associated factor IFN-γrel protein in largemouth bass. Oral administration is simple, non-invasive, and suitable for large-scale aquaculture. Results showed that Bacillus subtilis delivery of interferon-associated factor IFN-γrel in largemouth bass not only prevents LMBV infection (survival rate of 78.9% and relative survival rate of 65.1% in the Bs-IFN-γrel group) but also has a therapeutic effect (survival rate of 59.4% and relative survival rate of 31.6% in the Bs-IFN-γrel group), providing a new and effective method for the prevention and control of LMBV infection in largemouth bass, with promising application prospects.

[0017] Therefore, the present invention also provides the use of a recombinant strain expressing interferon-associated factor IFN-γrel in the preparation of a drug for treating and / or preventing largemouth bass iridovirus infection or largemouth bass virus disease caused by largemouth bass iridovirus infection, wherein the amino acid sequence of the IFN-γrel is shown in SEQ ID No. 2; and the fish is largemouth bass.

[0018] Furthermore, the nucleotide sequence encoding the IFN-γrel is shown in SEQ ID No. 1.

[0019] Furthermore, the recombinant strain is Bacillus subtilis. Bacillus subtilis ( Bacillus subtilis Bacillus subtilis is a non-pathogenic Gram-positive bacterium with good biosafety and environmental adaptability. It can be used as an excellent vector for recombinant protein expression and is suitable for the development of oral vaccines or drugs. The present invention shows that Bacillus subtilis can also be used to express and deliver IFN-γrel protein.

[0020] Furthermore, the recombinant strain is prepared by constructing the recombinant plasmid pHT43-IFN-γrel; transforming the plasmid into Bacillus subtilis and screening to obtain recombinant Bacillus subtilis Bs-IFN-γrel.

[0021] Furthermore, the number of recombinant strains in the drug is 1×10⁻⁶. 6 ~1×10 8 CFU.

[0022] Preferably, the number of recombinant strains in the drug is 1×10⁻⁶. 7 CFU.

[0023] Furthermore, the drug is formulated as an oral or injectable preparation.

[0024] Furthermore, the drug also contains other pharmaceutically acceptable excipients.

[0025] Furthermore, the excipients are selected from pharmaceutically acceptable carriers and excipients.

[0026] Compared with the prior art, the present invention has the following beneficial effects: This invention provides the application of interferon-associated factor IFN-γrel (IAFR) in the preparation of drugs for the treatment and / or prevention of largemouth bass iridovirus (LMBV) infection. This invention, through screening transcriptomic data and qPCR validation of largemouth bass infected with LMBV, discovered that IFN-γrel after LMBV infection... γ-rel expression was significantly upregulated. IFN After codon optimization of the γrel gene, it was ligated into a plasmid to construct a recombinant plasmid. Transformation and screening yielded recombinant Bacillus subtilis Bs-IFN-γrel expressing this protein. Results showed that Bs-IFN-γrel, as an oral antiviral drug for the prevention or treatment of LMBV infection, significantly improved the survival rate of largemouth bass and exhibited good safety. Therefore, this invention provides IFN... The application of γrel and Bs-IFN-γrel as oral antiviral drugs for the prevention and treatment of LMBV infection in largemouth bass farming. Attached Figure Description

[0027] Figure 1 KEGG analysis of co-regulated DEGs in the liver and spleen after LMBV infection.

[0028] Figure 2 A heatmap of DEGs enriched in the JAK-STAT signaling pathway of the spleen after LMBV infection.

[0029] Figure 3 This describes the expression pattern of host IFN-γrel after LMBV infection.

[0030] Figure 4 Figure shows the PCR identification results of single-clone colonies obtained from cloning culture of recombinant plasmids. Caption: Lane M: DNA molecular weight standard marker; Lanes 1-2: Empty vector control; Lanes 3-5: Bs-IFN-γrel.

[0031] Figure 5 This figure shows the Western Blot results of Bs-IFN-γrel in Bacillus subtilis at different time points after IPTG induction. Caption: Lane M: Protein molecular weight standard marker; Lane 1: Expression 0h; Lane 2: Expression 3h; Lane 3: Expression 6h; Lane 4: Expression 12h; Lane 5: Expression 24h; Lane 6: Expression 48h; Lane 7: Expression 72h.

[0032] Figure 6 Paraffin sections of largemouth bass tissue after oral administration of Bacillus subtilis Bs-IFN-γrel.

[0033] Figure 7 The mortality curve of artificially infected largemouth bass with LMBV after oral administration of Bacillus subtilis.

[0034] Figure 8 Mortality curve of largemouth bass treated with intraperitoneal injection of LMBV followed by oral administration of Bacillus subtilis one day later. Detailed Implementation

[0035] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0036] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0037] Example 1: LMBV Infection, Transcriptome Sequencing, and Data Analysis I. Experimental Methods 1. Viral infection Sixty fish were randomly divided into two groups: anesthetized and injected with LMBV (LMBV-infected group) and PBS (control group). When more than 50% of the fish in the virus-injected group showed clinical symptoms of disease, liver and spleen tissues from the control and diseased fish were collected for transcriptome sequencing.

[0038] 2. Transcriptome sequencing and analysis Total RNA was extracted from immune tissues (liver and spleen tissues) using the Trizol kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. After RNA integrity was checked using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), mRNA was enriched with oligonucleotide (dT) beads, fragmented into short fragments, and then reverse transcribed into cDNA using the NEBNext Ultra RNA Library Preparation Kit (NEB #7530, New England Biolabs, Ipswich, MA, USA). Finally, the cDNA library was sequenced using an Illumina Novaseq 6000 from Gene Denovo Biotechnology Co. (Guangzhou, China).

[0039] Raw sequencing reads were filtered to obtain high-quality clean reads by removing reads containing adapters, reads containing more than 10% unknown nucleotides (N), and low-quality reads containing more than 50% low-quality (Q value ≤ 20) bases. The clean reads were mapped to the largemouth bass reference genome (NCBI, PRJNA640112) using HISAT 2.2.4. Mapped reads for each sample were assembled using StringTie v1.3.1, and expression abundance and variation levels for each transcribed region were normalized using the FPKM (number of kilobase transcript fragments per million mapped reads) method.

[0040] Differential gene expression analysis was performed on the LMBV group and the control group using DESeq2 software. Genes with a false discovery rate (FDR) below 0.05 and an absolute fold change ≥2 were defined as differentially expressed genes (DEGs). DEG enrichment analysis was performed on the Kyoto Genome and Genome Encyclopedia (KEGG) using KOBAS 2.0 software. Hypergeometric tests were used to identify significantly enriched KEGG entries in the DEGs. Enrichment with a p-value <0.05 was defined as significant enrichment.

[0041] II. Experimental Results like Figure 1 As shown, the DEGs identified in the liver and spleen were significantly enriched in the KEGG pathway, which is associated with infection and immunity (such as JAK-STAT signaling and cytokine-cytokine receptor interactions).

[0042] like Figure 2 As shown in the heatmap, the expression profiles of DEGs enriched in the JAK-STAT signaling pathway are displayed. Analysis revealed that IFN-γrel, IFN-γR1 (interferon type II receptor), IFNa, STAT1, and JAK1 were significantly upregulated in the JAK-STAT signaling pathway.

[0043] Example 2: Expression pattern of IFN-γrel after LMBV infection I. Experimental Methods The experiment consisted of an LMBV infection group and a PBS control group, with 30 fish in each group. Spleen tissue samples were collected from three randomly selected fish at 1, 3, and 7 days after LMBV infection. Total RNA was extracted from the spleen tissue of LMBV-infected largemouth bass using the Animal Total RNA Isolation Kit (FOREGENE). cDNA was synthesized by reverse transcription using the extracted RNA as a template via the ReverTra Ace-a kit (Toyobo). The gene sequence (GenBank accession number: XM_038709291) was downloaded from https: / / www.ncbi.nlm.nih.gov, and specific primers were designed: upstream primer: 5'-CCAGGAGCATCAACACC-3' (SEQ ID NO.3), and downstream primer: 5'-TCGGGATCGTCCACAT-3' (SEQ ID NO.4). qPCR was then used to analyze the expression level of the IFN-γrel gene in the spleen tissue.

[0044] II. Experimental Results The results are as follows Figure 3As shown, LMBV infection activates the expression of IFN-γrel. Compared with the uninfected control, the transcriptional level was significantly upregulated by 8.8-fold after 1 day of infection, reached a peak of 11.5-fold after 3 days of infection, and remained elevated by 11.3-fold after 7 days of infection.

[0045] Example 3 Construction of recombinant Bacillus subtilis I. Experimental Methods 1. Constructing recombinant plasmids PCR amplification was performed using cDNA as a template to obtain the IFN-γrel gene fragment. The upstream primer was 5'-cggtaccATGTCTTCTTGCTGTGGTTCAGTC-3' (containing a Kpn I restriction site) (SEQ ID NO.5), and the downstream primer was 5'-tggatccTCATTCAGCAGAAGAAGAGTGGG-3' (containing a BamHI restriction site) (SEQ ID NO.6).

[0046] The nucleotide sequence was optimized using the IFN-γrel gene fragment as a template (the optimized nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2). The optimized nucleotide sequence was sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis, and then inserted between the BamHI and XbaI restriction sites of the pHT43 vector to construct the recombinant expression vector pHT43-IFN-γrel.

[0047] 2. Constructing recombinant expression strains The recombinant plasmid was transformed into Bacillus subtilis WB600N competent cells using chemical transformation. The competent Bacillus subtilis cells, preserved in glycerol at -80°C, were thawed on ice. After complete thawing, the recombinant plasmid pHT43-IFN-γrel and the empty vector pHT43 were quickly added to a final concentration of 1 µg / mL. The mixture was gently stirred and incubated in a 37°C water bath for 1 hour. Add 700 μL of antibiotic-free LB liquid medium and incubate at 37°C in a shaker for 2 hours. Centrifuge at 5000 rpm for 10 minutes, discard part of the supernatant, and retain approximately 100 μL of bacterial suspension. Gently pipette the suspension evenly and spread it onto LB solid medium containing 60 μg / mL chloramphenicol. Invert the container and incubate overnight at 37°C. The next day, pick single colonies and incubate them on 1 mL of 50 μg / mL chloramphenicol LB liquid medium at 37°C in a shaker for 4 hours. Centrifuge at 5000 rpm for 10 minutes at room temperature, discard the supernatant, resuspend the bacterial cells in 100 μL of PBS, and add lysozyme to a final concentration of 0.5 μg / mL. After incubation for 1 hour, extract recombinant bacterial DNA using a DNA extraction kit (Tiangen Biotech Co., Ltd.) for PCR verification. Identification primers: Upstream primer: 5'-TGTGAGCGGATAACAATTCC-3' (SEQ ID NO.7), Downstream primer: 5'-CAAAGATCTCCATGGACGCG-3' (SEQ ID NO.7). NO.8), deionized water and positive plasmid were added to the negative control and positive control, respectively.

[0048] 3. Expression and identification of recombinant proteins The Bs-IFN-γrel recombinant bacteria were inoculated into 50 mL of 2YT liquid medium containing 50 μg / mL chloramphenicol and cultured at 37°C and 200 rpm in a shaker to allow OD to develop. 600 When the concentration reached 0.6–0.8, IPTG was added to make a final concentration of 1 mM. After induction at 37°C for 0 h, 3 h, 6 h, 12 h, 24 h, 48 h, and 72 h, 1 mL of sample was taken and centrifuged at 4°C at 5000 × g for 10 min. The bacterial cells were resuspended in 100 μL of PBS, lysozyme was added, and the cells were incubated at 37°C for 30 min. The supernatant of the culture medium and the treated bacterial cells were analyzed by Western blot.

[0049] II. Experimental Results The results are as follows Figure 4 As shown, PCR was performed on the recombinant bacterial DNA using specific primers. The electrophoresis results of the PCR products showed the target band appearing in the region of theoretical value. The successfully constructed positive recombinant bacteria was named Bs-IFN-γrel. The recombinant bacteria were added to 50% sterile glycerol to a final concentration of 25% and then stored at -80℃ for later use.

[0050] The results are as follows Figure 5As shown, IFN-γrel expression can be detected within 3 hours, gradually increasing until reaching a peak level at 48 hours. However, the target band is almost undetectable at 72 hours, indicating that it may degrade or have reduced expression stability under prolonged induction.

[0051] Example 4 Safety identification of recombinant bacteria Bs-IFN-γrel I. Experimental Methods Largemouth bass were randomly divided into three groups of 30 individuals each: a PBS group, a Bs group, and a Bs-IFN-γrel group. Bs and Bs-IFN-γrel cells were diluted with PBS to a final concentration of 10⁻⁶. 9 CFU / mL. The Bs and Bs-IFN-γrel groups were orally administered 10... 7 Bacillus subtilis CFU (g / fish / day) was administered to the fish in the PBS group, along with an equal volume of PBS, for 7 consecutive days. Feeding behavior, activity level, and mortality were observed daily. After 7 days of administration, liver, spleen, and intestinal tissues were collected for H&E staining to observe histopathological changes.

[0052] The results are as follows Figure 6 As shown, the Bs-IFN-γrel group showed no significant histopathological damage compared to the PBS group, indicating that recombinant Bacillus subtilis Bs-IFN-γrel has good safety.

[0053] Example 5: Application of type II interferon-associated factor IFN-γrel in antiviral activity in largemouth bass. I. Experimental Methods 1. Antiviral preventive application effect Largemouth bass were randomly divided into three groups of 60 individuals each: a PBS group, a Bs group, and a Bs-IFN-γrel group. Bs and Bs-IFN-γrel cells were diluted with PBS to a final concentration of 10⁻⁶. 9 CFU / mL. The Bs and Bs-IFN-γrel groups received oral administration of 10 CFU / mL for 7 consecutive days. 7 CFU (g / fish / day) of Bacillus subtilis was administered to the PBS group, who received the same amount of PBS. On day 7, LMBV was administered intraperitoneally for challenge. Mortality rates were recorded over 14 days, and relative survival rates (RPS) were calculated.

[0054] 2. Efficacy of antiviral therapy Largemouth bass were intraperitoneally injected with LMBV and randomly divided into three groups of 60 fish each one day later: a PBS group, a Bs group, and a Bs-IFN-γrel group. Bs and Bs-IFN-γrel cells were diluted with PBS to a final concentration of 10⁻⁶. 9CFU / mL. The Bs and Bs-IFN-γrel groups were orally administered 10... 7 Bacillus subtilis CFU (g / fish / day) was administered for 3 consecutive days, while the PBS group received an equal volume of PBS. Mortality was recorded over 14 days, and RPS was calculated.

[0055] II. Experimental Results The results are as follows Figure 7 As shown, the survival rate of the Bs-IFN-γrel group was 78.9%, and the RPS was 65.1%, indicating that recombinant Bacillus subtilis Bs-IFN-γrel has a significant antiviral protective effect and improves the survival rate of largemouth bass.

[0056] The results are as follows Figure 8 As shown, the survival rate of the Bs-IFN-γrel group was 59.4%, and the RPS was 31.6%, indicating that recombinant Bacillus subtilis Bs-IFN-γrel has a therapeutic effect after LMBV infection and improves the survival rate of largemouth bass.

[0057] The embodiments described above are merely preferred embodiments of this application and are not intended to limit the scope of protection of this application. Various modifications and variations can be made to this application by anyone skilled in the art. Any simple equivalent changes and modifications made based on the scope of protection of this application and the content of the specification should be included within the scope of protection of this application.

Claims

1. The application of interferon-associated factor IFN-γrel (IAN-γrel) in the preparation of drugs for the treatment and / or prevention of largemouth bass iridovirus infection, characterized in that, The amino acid sequence of the IFN-γrel is shown in SEQ ID No.

2.

2. The application of interferon-associated factor IFN-γrel (IAN-γrel) in the preparation of drugs for the treatment and / or prevention of largemouth bass virus disease caused by largemouth bass iridovirus infection, characterized in that, The amino acid sequence of the IFN-γrel is shown in SEQ ID No. 2; the fish is the largemouth bass.

3. The use of a recombinant strain expressing the largemouth bass type II interferon-associated factor IFN-γrel in the preparation of a drug for treating and / or preventing largemouth bass iridovirus infection or largemouth bass virus disease caused by largemouth bass iridovirus infection, characterized in that, The amino acid sequence of the IFN-γrel is shown in SEQ ID No. 2; the fish is the largemouth bass.

4. The application according to any one of claims 1 to 3, characterized in that, The nucleotide sequence encoding the IFN-γrel is shown in SEQ ID No.

1.

5. The application according to claim 3, characterized in that, The recombinant strain is Bacillus subtilis.

6. The application according to claim 5, characterized in that, The recombinant strain was prepared by constructing the recombinant plasmid pHT43-IFN-γrel; transforming the plasmid into Bacillus subtilis and screening to obtain recombinant Bacillus subtilis Bs-IFN-γrel.

7. The application according to claim 3, characterized in that, The number of recombinant strains in the drug is 1×10. 6 ~1×10 8 CFU.

8. The application according to claim 7, characterized in that, The number of recombinant strains in the drug is 1×10. 7 CFU.

9. The application according to any one of claims 1 to 3, characterized in that, The drug is prepared as an oral or injectable form.

10. The application according to any one of claims 1 to 3, characterized in that, The drug also contains other pharmaceutically acceptable excipients.

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