Carp antiviral protein against spring viremia of carp and application thereof

Abstract

The present application relates to aquatic animal antiviral technology field, especially to a kind of anti-carp edema virus protein of brocade carp and its application.The amino acid sequence of the anti-carp edema virus protein provided in the present application is shown as SEQ ID NO.1, and the 1-27th amino acid of the amino acid sequence is signal peptide.The nucleotide sequence of the brocade carp Viperin gene for encoding the anti-carp edema virus protein is shown as SEQ ID NO.2.Injection of the Viperin recombinant protein obtained in the present application can improve the immune gene expression of brocade carp infected with carp edema virus.Moreover, after injection of the Viperin recombinant protein obtained in the present application, the content of carp edema virus in gill tissue of brocade carp is significantly lower than that of the control group, and the anti-carp edema virus protein provided in the present application has a significant inhibitory effect on brocade carp infected with carp edema virus.

Description

Technical Field

[0001] This invention relates to the field of antiviral technology for aquatic animals, and in particular to a koi anti-carp edema virus protein and its application. Background Technology

[0002] Koi, with their robust bodies, vibrant colors, and exquisite patterns, are known as swimming works of art and are among the most valuable ornamental fish species in my country and the world. In recent years, with the rapid development of the koi industry in my country, disease problems have become increasingly serious. Koi sleepy disease (KSD) is a viral disease caused by carp edema virus (CEV), leading to mortality in koi and common carp. CEV is an enveloped DNA virus approximately 200 nm in size, belonging to the Poxviridae family. CEV is a newly emerging and prevalent virus in koi farming in my country in recent years, seriously endangering the health of young koi fry and causing significant losses to producers. Currently, the main methods for preventing and controlling viral diseases in freshwater aquaculture animals are drug treatment and vaccination. However, drug treatment is not very effective against viral diseases and can cause side effects, drug residues, and water pollution. Furthermore, this virus is a newly emerging virus in China in recent years, and vaccine development has been hampered by a lack of sensitive cell lines and inconvenient immunization methods. Therefore, it is necessary to continuously seek ways to control viral diseases.

[0003] Ultimately, the prevention and control of viral diseases boils down to controlling the number of pathogens and enhancing the fish's immunity. This involves relying on the fish's immune system to clear the pathogens and restore health before they cause serious pathological damage. In the early stages of viral infection, the fish primarily relies on its natural immune response. Natural immune factors such as interferon and interferon-stimulated gene products help the body inhibit viral proliferation, adhesion, and release, thereby resisting viral infection. Since CEV is a newly emerging virus in koi farming in recent years, there is currently a lack of research on the CEV infection mechanism and host immune defense.

[0004] The anti-CEV protein of this invention can induce the transcriptional expression of antiviral immune factors and inflammatory factors, inhibit the proliferation of CEV in koi, and clear the virus carried by low-titer CEV in koi, thereby improving the survival rate of koi infected with CEV. It has good prospects in the development of antiviral immunizing agents for koi. Summary of the Invention

[0005] This invention provides a koi anti-carp edema virus protein and its application.

[0006] In a first aspect, the present invention provides an anti-carp edema virus protein, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] The amino acid sequence of the anti-carp edema virus protein provided by this invention has amino acids 1-27 as a signal peptide.

[0008] Secondly, the present invention provides a koi viperin gene encoding the aforementioned anti-koi edema virus protein.

[0009] The nucleotide sequence of the Viperin gene for koi carp provided by this invention is shown in SEQ ID NO.2.

[0010] Thirdly, the present invention provides a method for preparing the above-mentioned anti-carp edema virus protein, wherein the above-mentioned koi Viperin gene is inserted into an expression vector, transformed into competent cells, and then screened for kanamycin resistance, and the correct recombinant plasmid is screened by colony PCR and sequencing.

[0011] The recombinant plasmid was transferred into competent E. coli and cultured with shaking until OD600 ≥ 0.5-0.65. IPTG was added as an inducer and cultured for another 5-6 hours. The bacterial cells were collected by centrifugation, and the cells were suspended and lysed by sonication under ice bath conditions. The lysate and precipitate were collected by centrifugation. The precipitate was suspended in a buffer containing guanidine hydrochloride and the lysate was purified into Viperin protein by affinity chromatography.

[0012] Fourthly, the present invention provides a fluorescent quantitative PCR primer, the sequence of which is shown in SEQ ID NO.3-4.

[0013] The present invention also provides the application of the aforementioned fluorescent quantitative PCR primers in the detection of anti-carp edema virus protein.

[0014] This invention provides the application of the above-mentioned anti-carp edema virus protein or the above-mentioned koi viperin gene in improving the survival rate of koi infected with carp edema virus, preparing anti-carp edema virus drugs and / or immune-activating drugs.

[0015] Fifthly, the present invention provides a drug for treating carp edema virus, comprising the aforementioned anti-carp edema virus protein.

[0016] The beneficial effects of this invention are as follows:

[0017] The anti-carp edema virus protein provided by this invention has a significant inhibitory effect on koi infected with carp edema virus. Injection of the recombinant Viperin protein obtained by this invention can increase the expression of immune genes in koi infected with carp edema virus. Furthermore, after injection of the recombinant Viperin protein obtained by this invention, the carp edema virus content in the gill tissue of koi was significantly lower than that in the control group. Injection of the recombinant Viperin protein also improved the survival rate of koi infected with carp edema virus. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0019] Figure 1 This study analyzes the expression of the Viperin gene in different tissues of koi carp.

[0020] Figure 2 This study analyzed the expression of Viperin genes in the gill and spleen tissues of naturally infected CEV-positive koi carp.

[0021] Figure 3 This represents the changes in the expression of the Viperin gene in the gill and spleen tissues of koi carp at different time points after artificial infection with CEV.

[0022] Figure 4 This study describes the changes in the expression of the Viperin gene in primary spleen cells of koi carp under different immunostimulants.

[0023] Figure 5 This is the result of Western blot analysis of purified recombinant Viperin protein.

[0024] Figure 6 This describes the expression of immune genes in the head kidney tissue after in vivo injection of recombinant Viperin protein.

[0025] Figure 7 This study describes the expression of immune genes in the gill tissue of koi carp infected with CEV after in vivo injection of recombinant Viperin protein.

[0026] Figure 8 This study describes the effect of in vivo injection of recombinant Viperin protein on the expression of immune genes in the spleen tissue of koi carp infected with CEV.

[0027] Figure 9 This study describes the effect of in vivo injection of recombinant Viperin protein on the expression of immune genes in the head kidney tissue of koi carp infected with CEV.

[0028] Figure 10 The effect of in vivo injection of recombinant Viperin protein on the survival rate of koi carp infected with CEV. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0030] Example 1: Obtaining the Viperin gene from koi carp

[0031] To study the characteristics of the immune molecular response in koi after CEV infection, the inventors collected spleen tissue from koi with sleep disorders and healthy koi for RNA-seq and found a differentially expressed interferon-stimulated gene, Viperin, from the results.

[0032] To verify and obtain the ORF sequence of this gene, using koi spleen cDNA as a template and transcriptome sequence as a reference, the open reading frame of the Viperin gene was amplified by PCR. The open reading frame consists of 1047 nucleotides and encodes a protein composed of 348 amino acids with a predicted molecular weight (MW) of 40.37 kDa and a theoretical isoelectric point (pI) of 7.7. The first 27 amino acids are the signal peptide.

[0033] The amino acid sequence is shown in SEQ ID NO.1:

[0034] MLMQFCFKNVHSFLAALLRWIYMLVSGTQVQQTPVGHISLLKTRTKEQKEGSSTQLTTPSSVNYHFTRQCNYKCGFCFHTAKTSFVLPIEEAKRGLRLLKEAGMEKVNFSGGEPFLHERGNFLGELVRYCKQELQLPSVSIVSNGSLITENWFQKYGDYLDILAVSCDSFNEDT NKVIGRGQGRKSHLDKLHQVHNWCRDYKVAFKINSVINTFNVDEDMTEQITVLNPVRWKVFQCLIIEGENAGENSLREAEKFVISDQQFQDFLDRHQSVKCLVPESNQKMRDSYLILDEYMRFLDCREGRKDPSKSILDVGVEEAIKFSGFDEKMFLKRGGKYMWSKEDMKLEW.

[0035] Example 2: Tissue expression distribution of Viperin in koi carp

[0036] The relative expression levels of the Viperin gene in different tissues of healthy koi carp were detected by real-time quantitative PCR to clarify the tissue distribution of Viperin mRNA.

[0037] Specific procedures: Six healthy koi carp (Nine-Striped Dragon) (20.5±1.4g) were randomly selected, and 12 tissues were dissected and collected: gills, eyes, head kidneys, spleen, kidneys, heart, muscle, skin, liver, blood, brain, and intestines. Total RNA was extracted from the tissues using the Trizol method and reverse transcribed into cDNA using the Takara reverse transcription kit.

[0038] Primers for quantitative real-time PCR were designed based on the open reading frame sequence of Viperin. The primer sequences are as follows:

[0039] qVipF:5'-CCTGTTGAGATGGATTTATATGCTT-3'(SEQ ID

[0040] NO.3);

[0041] qVipR: 5'-CCTCTTTCTGCTCTTTGGTGC-3' (SEQ ID NO. 4);

[0042] The primer sequences for the internal reference gene, 40S ribosomal protein S11, are as follows:

[0043] qS11F: 5'-CCGTGGGTGACATCGTTACA-3' (SEQ ID NO. 5);

[0044] qS11R: 5'-TCAGGACATTGAACCTCACTGTCT-3' (SEQ ID NO. 6).

[0045] The relative expression levels of the Viperin gene in the 12 tissues were detected using an ABI 7500 real-time quantitative PCR instrument. The reaction conditions were: 95℃ for 15s; 95℃ for 5s, 59.6℃ for 30s, 72℃ for 30s, for 40 cycles.

[0046] like Figure 1 The results show that the Viperin gene is expressed in all of the above tissues, with the highest expression level in the skin, followed by the spleen. There was no significant difference between the two, but there were significant differences compared with the other 10 tissues. The spleen is one of the most important immune organs in fish, and the skin is also a major mucosal immune organ, indicating that this gene is closely related to the immune function of koi.

[0047] Example 3: Relative expression levels of the Viperin gene in naturally occurring and artificially infected CEV koi carp.

[0048] Six koi fish (10.5±1.2g) were randomly collected from a koi pond in a koi farm exhibiting sleep disorder symptoms. All fish displayed typical symptoms of sleep disorder (listlessness, swollen skin, and a "sleeping" posture). Six healthy koi fish were collected from other ponds as controls. Gill and spleen tissues were collected, total RNA was extracted, reverse transcribed into cDNA, and the relative expression level of the Viperin gene was detected using qPCR.

[0049] Figure 2 The results showed that the relative expression level of Viperin gene in the gill tissue of dying fry was significantly lower than that in the control group of koi, while the expression level of Viperin gene in the spleen tissue was significantly higher than that in the control group of healthy koi (approximately 27 times).

[0050] Sixty healthy koi (Nine-striped Dragon, weighing approximately 20.5 ± 1.4 g) were randomly selected and divided into a control group and an infection group. The 30 koi in the infection group were immersed in an 80 L tank containing 5 mL of crude CEV extract (final virus concentration 1.8 × 10⁻⁶). 7 One group of koi carp was placed in an 80L tank with the same volume of PBS (copies / L), while another 30 carp were placed in the same tank as a control group. On days 0, 1, and 5 of infection, three koi carp were randomly selected from both the control and infected groups, and gill and spleen tissues were collected. The relative expression level of the Viperin gene was detected by qPCR.

[0051] Figure 3 The results showed that the levels of Viperin mRNA in the gill and spleen tissues of koi carp were upregulated after CEV immersion infection, especially the upregulation response in the gill tissue was more rapid and the upregulation fold was higher.

[0052] Example 4: Effect of immune stimulation on the relative expression of Viperin gene in primary spleen cells of koi carp

[0053] Disinfect koi carp (approximately 25g) by immersing them in a 0.01% (w / v) potassium permanganate solution for 30 minutes, followed by disinfection of the fish's body surface with 70% alcohol. Remove the spleen and place it in a sterile culture dish, washing 3-4 times in 1×PBS solution containing penicillin (500U / mL) and streptomycin (500U / mL). Pass the spleen tissue through a 100-mesh sieve, grind it using a 2mL syringe liner, and wash the sieve with serum-free 1640 medium. Centrifuge the cells at 840×g for 5 minutes, and wash the cells once with serum-free medium. Resuspend the centrifuged cell pellet in cell culture medium, count the cells, and adjust the concentration to 1×10⁻⁶. 6Cells were cultured at a density of 1 cell / mL, with 1 mL of cell suspension added to each well of a 24-well cell culture plate. Primary culture was performed at 25°C. On the second day, the culture medium was aspirated, and 1 mL of fresh 1640 medium was added to each well, along with different concentrations of stimulants: LPS (100 μg / mL), poly I:C (50 μg / mL), poly I:C / Lyo Vec (2 μg / mL), poly dA:dT (10 μg / mL), and poly dA:dT / Lyo Vec (2 μg / mL). PBS was added as a control. Cells were harvested at 6 h, 12 h, 24 h, and 48 h after stimulation, and total RNA was extracted and reverse transcribed into cDNA. The relative expression levels of Viperin mRNA at different stimulation times under different immunostimulant treatments were detected using real-time quantitative PCR.

[0054] Figure 4 The results showed that the relative expression level of Viperin in spleen cells of the LPS and poly I:C groups was significantly increased 6 h after stimulation. Until 12 h after stimulation, the relative expression level of Viperin in cells of each stimulation group was higher than that of the control group, indicating that the Viperin gene can respond to the stimulation of bacterial and viral mimics.

[0055] Example 5: Prokaryotic expression and purification of recombinant Viperin protein (rViperin) from koi carp

[0056] 1. Construction of recombinant plasmids

[0057] A Viperin mature region fragment containing restriction enzyme sites NdeI and HindIII was synthesized using a whole-genome synthesis method. The open reading frame of the Viperin gene, positions 82-1047 (positions 1-81 being the signal peptide) (nucleotide sequence shown in SEQ ID NO.2), was inserted into the expression vector pET30a using a double enzyme digestion method. The vector was then transformed into competent E. coli DH5α cells. After kanamycin resistance selection, the correct recombinant plasmid pET30a-Viperin was selected by colony PCR and sequencing.

[0058] 2. Induction and purification of recombinant Viperin protein in koi carp

[0059] The expression plasmid pET30a-Viperin was transformed into BL21(DE3) competent cells. Colonies containing the recombinant plasmid were selected and inoculated into LB medium containing kanamycin (50 μg / μL) for expansion culture. The cells were cultured at 37°C with shaking until the OD600 reached approximately 0.6. 1M IPTG was added as an inducer, and the cells were cultured for another 6 hours. The cells were collected by centrifugation, resuspended in 1 / 10 volume of PBS, and lysed by sonication at 100 W (20 s working, 20 s resting) under ice bath conditions. The lysate was collected by centrifugation, resuspended in buffer containing guanidine hydrochloride, and then subjected to affinity chromatography (Co). 2+ The rViperin protein was purified using resin, following the method described in (X. Wang, et al. Interleukin-6 in Siberian sturgeon (Acipenser baeri): Molecular characterization and immune functional activity. Fish and shellfish immunology).

[0060] 1 μg of purified rViperin was loaded onto an SDS-PAGE gel. After electrophoresis, the protein on the gel was transferred to a PVDF membrane. The membrane was washed three times with PBST for 10 min each time, then blocked overnight at 4°C in PBST solution containing 1% BSA. After PBST washing, the monoclonal antibody Anti-His-HRP (1:10000) was added, and the membrane was incubated at 37°C for 2 h. The membrane was then washed three times with PBST for 10 min each time, followed by DAB staining. The results are shown below. Figure 5 As shown, the band at the target size shows color development, indicating that rViperin with high purity was successfully obtained after induction expression and purification.

[0061] Example 6: Changes in the expression of immune-related genes in koi carp after in vivo injection of rViperin

[0062] Twelve koi carp (approximately 20g each) were randomly selected and divided into a control group and an experimental group (6 carp in each group). Each koi in the experimental group was intraperitoneally injected with 100μL of purified rViperin (0.2mg / mL), while the koi in the control group were injected with the same volume of PBS. Twenty-four hours after injection, three koi carp were randomly selected from each group, and head and kidney tissues were collected. Total RNA was extracted and reverse transcribed into cDNA. The relative expression level of the Viperin gene was detected using real-time quantitative PCR.

[0063] Figure 6The results showed that rViperin injection significantly upregulated the expression of antiviral pathway-related genes STING, MyD88, and IRF-9, as well as inflammatory factors TNF-α and IL-1β, in the head kidney tissue of koi carp.

[0064] Example 7: The effect of in vivo injection of rViperin on the clearance of virus in CEV-carrying koi.

[0065] To determine the clearance effect of rViperin on CEV virus in koi carrying low titers, this invention selected 10 koi carrying CEV virus but without symptoms. Five koi were injected intraperitoneally with 100 μL of rViperin protein (0.2 mg / mL), while the other five were injected with 100 μL of PBS as a control. Twenty-four hours after injection, gill tissue was collected from three koi from each group. After DNA extraction, the viral load in the gill tissue was detected using TaqMan probe-based real-time quantitative PCR.

[0066] As shown in Table 1, compared to the low CEV content in the control group, no virus was detected in the gill tissue of the rViperin injection group. This indicates that rViperin can clear CEV virus carried by low-titer CEV virus in koi.

[0067] Table 1. Virus content (copy / ngDNA) in gill tissue of koi carp injected with recombinant Viperin protein 24h CEV.

[0068] Fish 1 Fish 2 Fish 3 CON 17.5 91.0 347.6 rVip 0 0 0

[0069] Example 8: Effects of in vivo injection of rViperin on anti-CEV proliferation and immune gene expression in koi carp

[0070] Twelve koi carp (approximately 20g each) were randomly selected and divided into two groups of six fish each. One group served as a control group injected with 100μL PBS, while the other group served as an experimental group injected with 100μL recombinant protein rViperin (0.2mg / mL). Five hours after injection of the recombinant protein, crude CEV virus extract was added to the tanks of both groups of fish until the final concentration reached 7.25×10⁻⁶. 6 Copies / L. 72 hours after infection, six fish from each of the two groups were harvested, and gill, spleen, and head kidney tissues were collected. To minimize variability among different fish, equal amounts of tissue from every two fish were pooled into one sample. DNA was extracted from the gill tissue, and the viral load was detected using TaqMan probe-based real-time quantitative PCR. RNA was extracted from the gill, spleen, and head kidney tissues, reverse transcribed into cDNA, and the expression levels of immune-related genes were detected using real-time quantitative PCR.

[0071] See results Figure 7 , Figure 8 and Figure 9Injection of rViperin protein can enhance the expression of immune genes in koi carp infected with CEV. Specifically, it upregulates the expression of antiviral signaling pathway-related genes cGAS, STING, and MyD88 in gill tissue, interferon system-related immune factors IFN-α, IFN-γ, IRF-3, and IRF-9, as well as inflammatory factors IL-6, IL-1β, and TNF-α. Significant upregulation of cGAS, IRF-9, MyD88, and IL-1β genes in spleen tissue, and STING, IL-6, and IL-10 in head kidney tissue compared to the control group was also observed.

[0072] The virus content results are shown in Table 2. The virus content in the gill tissue of koi carp injected with rViperin was significantly lower than that in the control group. This indicates that rViperin has a significant inhibitory effect on CEV virus infection in koi carp.

[0073] Table 2. Virus content (copy / ngDNA) of recombinant Viperin protein in gill tissue of artificially infected CEV fish in koi carp.

[0074] Sample 1 Sample 2 Sample 3 CON 219.7 364.7 215.3 rVip 35.7 17.2 66.6

[0075] Example 9: Effect of in vivo injection of rViperin on the survival rate of koi carp infected with CEV

[0076] Fifty-two koi carp (approximately 20g each) were randomly selected and divided into two groups of 26 fish each. One group served as the control group, injected with 100μL PBS (13 fish per tank). The other group served as the experimental group, injected with 100μL recombinant protein rViperin (0.2mg / mL), with 13 fish per tank. The water temperature was 16±2℃. Five hours after the recombinant protein injection, the fish were anesthetized with MS222. Then, 200μL of crude rViperin virus extract (concentration 2.9×10⁻⁶) was dripped onto both gills of each fish in both groups. 5 (copy / μL). Dead fish should be removed promptly, and the cumulative mortality over 18 days should be recorded.

[0077] Survival status as follows Figure 10 As shown, the survival rate within 18 days in the rViperin injection group was 76.9%, which was much higher than the survival rate in the control PBS injection group (11.5%).

[0078] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of an anti-sacrovirus protein for the preparation of a medicament against the sacro virus, characterized in that, The amino acid sequence of the anti-CVH protein is shown as SEQ ID NO.

1.

2. Use according to claim 1, characterized in that, The amino acid sequence of the anti-CVH protein is shown as SEQ ID NO.

1.

3. Use according to claim 1 or 2, characterized in that, The amino acid sequence of the anti-CVH protein is shown as SEQ ID NO.

1.

4. Use according to claim 3, characterized in that, The amino acid sequence of the anti-CVH protein is shown as SEQ ID NO.

1.

5. A medicament against the spring viremia of carp virus, characterized in that, The amino acid sequence of the anti-CVH protein is shown as SEQ ID NO. 1.

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