Application and preparation method of yellow eel aldehyde reductase protein

Abstract

The present invention discloses an application of a yellow eel aldehyde reductase protein and a preparation method thereof. The amino acid sequence of the yellow eel aldehyde reductase protein is shown in SEQ ID NO.1. The present invention shows for the first time that the yellow eel aldehyde reductase protein has reducing activity on S-nitrosoglutathione (GSNO), and is a new GSNO reductase. Intraperitoneal injection of the yellow eel aldehyde reductase protein into fish can significantly reduce inflammatory reactions and oxidative stress damage induced by pathogens, and therefore has very high application value in the prevention and treatment of fish pathogenic bacterial infections.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and particularly relates to an aldehyde reductase protein derived from rice field eel, and a recombinant preparation method and application of the aldehyde reductase protein. Background Art

[0002] Aldehyde reductase (ALR) is a member of the AKR superfamily of aldehyde-keto reductases. ALRs have a very broad substrate spectrum and are capable of reducing a wide range of carbonyl-containing compounds with reduced nitropropene (NADPH), making them potential carbonyl detoxifying enzymes (Pemming, 2017). Mammalian ALRs have a broad substrate spectrum, capable of reducing numerous aldehydes, ketones, and HNE. By converting these carbonyl-containing compounds into less toxic polyols, they reduce the toxic effects of aldehydes on cells. A growing body of research suggests that ALRs may also be involved in physiological processes such as vitamin C synthesis, doxorubicin metabolism, and tumor suppression (Kassner et al., 2008; Lai et al., 2017; Fujii et al., 2021).

[0003] Recent studies have revealed that human ALR, after genetic reprogramming, has the activity to catalyze the conversion of S-nitrosoglutathione (GSNO), identifying it as a novel mammalian S-nitrosoglutathione reductase (GSNOR) (Stomberski et al., 2019). This suggests that it may be involved in NO synthesis and the inhibition of protein S-nitrosylation, but its function has not been verified (Fujii et al., 2021). GSNORs are important enzymes involved in denitrosylation and reductive activities in cells. They can significantly reduce S-nitrosylation of certain proteins, thereby protecting cells from nitrosylation stress (Corpas et al., 2011). Known GSNOR proteins include GSH / GSH-dependent formaldehyde dehydrogenase, type III alcohol dehydrogenase (ADH5), and thioredoxin reductase (Hess et al., 2005). The substrates of GSNOR proteins are primarily alcohols, HMGSH, and GSNO (Chatterji et al., 2021). Recombinant human rhALR can reduce GSNO and coenzyme A in an NADPH-dependent manner, with a substrate preference for SNO-CoA over GSNO (Stomberski et al., 2019).

[0004] Research on the ALR in fish is limited. It has been found that the ALR plays an important role in maintaining physiological blood glucose levels and mitigating glucose-induced organ damage in zebrafish. Furthermore, the ALR in tilapia may be involved in the detoxification of benzopyrene. Yang Long (2022) found that the ALR in yellow catfish may have a protective role against heavy metal-induced oxidative stress. However, there are no reports of GSNO reductase activity in fish ALR. Summary of the Invention

[0005] The present invention provides an aldehyde reductase protein derived from fish and constructs a recombinant preparation system of the enzyme protein using genetic engineering technology. Experiments show that the aldehyde reductase protein provided by the present invention not only has good in vitro GSNO reduction activity, but also can improve the disease resistance of fish to pathogen infection.

[0006] The technical method of the present invention is specifically as follows:

[0007] In a first aspect, the present invention provides an aldehyde reductase protein derived from the rice field eel, the amino acid sequence of which is shown in SEQ ID NO.1, and the nucleotide sequence encoding the protein is shown in SEQ ID NO.2.

[0008] The yellow eel (Monopterus albus) is a common small freshwater economic fish with a wide distribution and high nutritional and medicinal value. The present invention isolates and identifies the yellow eel ALR gene and its open reading frame sequence based on the published yellow eel full genome sequence.

[0009] The second aspect of the present invention provides a method for preparing yellow eel aldehyde reductase protein, which is specifically: first, the target fragment (i.e., the sequence shown in SEQ ID NO.2) is connected to an expression vector to obtain a recombinant plasmid, then the recombinant plasmid is transformed into a recipient cell, the recipient cell is induced to express, and the recombinant protein is obtained by purification.

[0010] In the above method, the target fragment can be obtained by extracting total RNA from the liver tissue of the eel, reverse transcribing and synthesizing cDNA, performing PCR amplification using the primer pairs shown in SEQ ID NO.3-4, and recovering and purifying the amplified product.

[0011] In the above method, the expression vector is a prokaryotic expression vector, and the recipient cell is Escherichia coli; in one embodiment of the present invention, the expression vector is pET-28a(+), and the recipient cell is E. coli BL21(DE3).

[0012] In the above method, IPTG was used to induce expression.

[0013] In the above method, the recombinant protein is purified by centrifuging the bacterial solution after induced expression, then breaking the collected bacteria, centrifuging to obtain the precipitate, dissolving the precipitate with equilibrium buffer and centrifuging to collect the supernatant, adding the supernatant to a Ni ion affinity column, and eluting after binding.

[0014] The third aspect of the present invention provides the use of the eel aldehyde reductase protein with an amino acid sequence as shown in SEQ ID NO.1, which is A1) or A2):

[0015] A1) acts as GSNOR, reducing S-nitrosoglutathione;

[0016] A2) Improve fish's disease resistance.

[0017] It is understandable that the yellow eel aldehyde reductase protein in the above application can be either a natural enzyme protein or a recombinant protein obtained through genetic engineering technology.

[0018] In the above application A1), the reducing activity of the yellow eel aldehyde reductase protein on GSNO is dependent on NADPH.

[0019] In the above application A2), injecting yellow eel aldehyde reductase protein into the fish body can effectively reduce the inflammatory response and oxidative stress damage induced by pathogens.

[0020] In the above application A2), further studies have shown that yellow eel aldehyde reductase protein has the ability to inhibit the expression of IL-1β and nitric oxide synthase 1 (NOS1), promote the expression of antioxidant transcription factor Nrf2, increase the content of small molecule antioxidant GSH in the liver, and reduce oxidative stress indicators ROS and RNS.

[0021] In the above application A2), the pathogenic bacteria include Aeromonas hydrophila, which is a pathogenic pathogen of many species in freshwater aquaculture. For example, it can cause hemorrhagic disease in the eel, seriously affecting the healthy breeding and quality of the eel, resulting in huge economic losses.

[0022] A fourth aspect of the present invention provides a fish medicine for treating pathogenic bacteria infection, wherein the active ingredient is the yellow eel aldehyde reductase protein having an amino acid sequence as shown in SEQ ID NO.1.

[0023] After administering the above-mentioned drugs to individual fish at an effective dose, the antioxidant capacity of the liver tissue of fish under conditions of pathogenic bacteria infection can be significantly improved and the expression of pro-inflammatory factors can be reduced.

[0024] Preferably, the above-mentioned drug is administered by intraperitoneal injection.

[0025] Preferably, the fish is eel.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] (1) The present invention uses genetic engineering technology to construct a prokaryotic expression vector of the yellow eel aldehyde reductase ALR gene, and obtains its recombinant protein by inducing expression and purification, laying the foundation for further research and utilization of yellow eel aldehyde reductase;

[0028] (2) Experiments show that the yellow eel aldehyde reductase recombinant protein prepared by the present invention has the reducing activity for GSNO and is a new teleost GSNOR;

[0029] (3) The present invention demonstrates for the first time the anti-inflammatory effect of yellow eel aldehyde reductase protein in resisting pathogen infection and reducing oxidative stress damage. After intraperitoneal injection of yellow eel aldehyde reductase protein, the antioxidant capacity of the liver tissue of yellow eel under pathogenic bacterial infection conditions can be significantly improved, and the expression of pro-inflammatory factors is reduced. It is expected to be used for the prevention and treatment of bacterial infection in fish. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The SDS-PAGE electrophoresis diagram of the yellow eel aldehyde reductase gene ALR before and after expression in Example 1, wherein lane 1 is a blank plasmid strain, lane 2 is a pET-28-ALR-containing strain before induction, lane 3 is a pET-28-ALR-containing strain after induction, lane 4 is the purified ALR protein, and M is a marker.

[0031] Figure 2 This is a graph showing the effect of intraperitoneal injection of ALR recombinant protein on the gene expression of the liver inflammatory factor IL-1β induced by pathogens in Example 3.

[0032] Figure 3 This is a graph showing the effect of intraperitoneal injection of ALR recombinant protein on the expression of liver nitric oxide synthase 1 gene NOS1 induced by pathogens in Example 3.

[0033] Figure 4 This is a graph showing the effect of intraperitoneal injection of ALR recombinant protein on pathogen-induced liver antioxidant transcription factor Nrf2 gene expression in Example 3.

[0034] Figure 5 This is a graph showing the effect of intraperitoneal injection of ALR recombinant protein on liver GSH content induced by pathogens in Example 3.

[0035] Figure 6 This is a graph showing the effect of intraperitoneal injection of ALR recombinant protein on liver NO content induced by pathogens in Example 3.

[0036] Figure 7This is a graph showing the effect of intraperitoneal injection of ALR recombinant protein on the content of reactive oxygen species (ROS) in the liver induced by pathogens in Example 3.

[0037] Figure 8 This is a graph showing the effect of intraperitoneal injection of ALR recombinant protein on the content of reactive nitrogen species (RNS) in the liver induced by pathogens in Example 3. DETAILED DESCRIPTION

[0038] The technical solutions of the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments.

[0039] In the following examples, unless otherwise specified, all methods are conventional methods; the reagents and materials described, unless otherwise specified, can be obtained from commercial sources.

[0040] Example 1 Gene cloning, expression vector construction and recombinant protein expression and purification of yellow eel aldehyde reductase

[0041] The experiment in this case includes the following aspects:

[0042] (1) Cloning of the ALR gene of the yellow eel.

[0043] Total RNA from eel liver was extracted according to the Trizol kit instructions, and cDNA was synthesized using M-MLV reverse transcriptase and stored at -80°C. Based on the precisely located ALR gene, specific amplification primers were designed:

[0044] ALR-F:5'-CTAA GGATCC ATGAATGACTTTGCAGTTCT-3'(SEQ ID NO.3),

[0045] ALR-R:5'-TACC AAGCTT TCAGTAGGGGTCATTAAAAG-3' (SEQ ID NO. 4);

[0046] A BamH I restriction enzyme site (underlined) was added to the upstream primer sequence, and a Hind III restriction enzyme site (underlined) was added to the downstream primer sequence.

[0047] Using cDNA as a template, conventional PCR amplification was performed using ALR-F / R. The amplified products were recovered, purified, ligated, and sequenced to obtain the open reading frame sequence of the gene. The nucleotide sequence of the yellow eel ALR gene is shown in SEQ ID NO. 2, and the amino acid sequence of the yellow eel aldehyde reductase protein encoded by the gene is shown in SEQ ID NO. 1.

[0048] (2) Construction of expression vector.

[0049] The PCR amplification product obtained in step (1) and the prokaryotic expression vector pET-28a(+) were simultaneously subjected to BamH I and Hind III double enzyme digestion, and the digestion products were purified using a DNA gel recovery kit, and the amplification product and the vector were ligated using T4 DNA ligase. After sequencing verification, the recombinant prokaryotic expression vector pET-28-ALR was successfully constructed.

[0050] (3) Expression and purification of recombinant eel ALR protein.

[0051] The recombinant prokaryotic expression vector pET-28-ALR obtained in step (2) was transformed into E. coli BL21 (DE3), spread on a plate containing 50 μg / mL kanamycin for resistance screening, and positive expression strains were obtained by colony PCR verification and SDS-PAGE electrophoresis detection.

[0052] The positive expression strain was inoculated into 100 mL of fresh LB liquid medium containing kanamycin at a ratio of 1:100 and incubated at 220 r·min. -1 Incubate on a shaker until OD 600 At about 0.6, 0.1 mM IPTG was added and cultured for 4 h to induce the expression of the recombinant protein.

[0053] The induced bacterial solution was centrifuged at 4 °C and 10000 r min -1 The cells were collected by centrifugation for 10 min, and the cell pellet was resuspended in a buffer solution (20 mM Tris, 250 mM NaCl, pH = 7.4). The bacteria were lysed by ultrasonication on ice (ultrasonication for 3 s, intervals of 6 s, total time 20 min), and then spun at 4 ° C, 10000 r min -1 After centrifugation for 10 min, the precipitate was collected and washed three times with loading buffer (20 mMTris, 250 mM NaCl, 8 M urea, pH = 7.4), and then dissolved in an appropriate amount of lysis buffer (20 mMTris, 250 mM NaCl, 8 M urea, 20 mM imidazole, pH = 7.4). After filtering with a 0.45 μm filter membrane, the supernatant was added to a Ni-NTA HisBindResin purification column and eluted with elution buffer (20 mMTris, 250 mM NaCl, 8 M urea, 200 mM imidazole, pH = 7.4). The protein was dialyzed against dialysate (20 mMTris, 250 mM NaCl, 20 mL / L glycerol, pH = 7.4) to remove urea from the recombinant protein, and the purification of the recombinant protein was detected by SDS-PAGE.

[0054] The results of SDS-PAGE electrophoresis are as follows Figure 1As shown, lane 1 is a blank plasmid strain, lane 2 is a pET-28-ALR-containing strain before induction, lane 3 is a pET-28-ALR-containing strain after induction, and lane 4 is the purified ALR recombinant protein; M is a marker. The results show that this example successfully achieved the in vitro expression and purification of the recombinant protein ALR.

[0055] Example 2 Enzyme activity detection and substrate specificity analysis of ALR recombinant protein

[0056] The activity of the recombinant protein was evaluated by measuring the absorbance of reduced coenzyme II (NADPH) at 340 nm using a TU-1900 UV spectrophotometer from Pulse. One unit of activity (U) is defined as the amount of NADPH consumed per minute by aldehyde reductase under optimal conditions.

[0057] To determine GSNO reductase activity, the reaction system consisted of 2.4 mL of 100.0 mmol / L potassium phosphate buffer, 0.4 mL of 3.0 mmol / L GSNO, 60.0 μL of 10.0 mmol / L NADPH, and 140.0 μL of enzyme solution. The cuvette was placed in a UV spectrophotometer, and the absorbance at 340 nm was measured and recorded. This was repeated three times. Statistical analysis was performed using SPSS 20.0. Specific and relative enzyme activities are expressed as x ± s.

[0058] Table 1 Response constants of ALR recombinant protein to GSNO

[0059]

[0060] The results showed that the ALR recombinant protein had good reducing activity for GSNO and was a new type of GSNO reductase that depended on NADPH.

[0061] Example 3 Regulatory Effects of ALR Recombinant Protein on Inflammatory Response and Liver Oxidative Stress During Pathogen Infection

[0062] This case explored the mechanism of action of ALR recombinant protein in regulating inflammatory response through the following aspects:

[0063] (1) Effect of ALR recombinant protein on the gene expression of the liver inflammatory factor IL-1β during pathogen infection.

[0064] 108 healthy and similar-sized eels (40±2.5 g) from the same growing environment were randomly divided into three groups after acclimation and culture, with 36 eels in each group. They were injected with 1 mL of 0.9% saline (control group) and 1 mL of 1×10 6cfu / mL Aeromonas hydrophila (pathogen infection group), 1 mL of 1×10 6 A mixture of 100 cfu / mL of Aeromonas hydrophila and 10 μg / mL of active rALR protein (pathogen infection + recombinant protein group) was used. Liver tissue was collected from six fish at 0, 6, 12, 24, 48, and 72 hours and stored at -80°C for subsequent experiments. Each treatment was repeated three times. An appropriate amount of liver tissue was placed in a 1.5 mL centrifuge tube, added to 500 mL of Trizol, and stored at -80°C for RNA extraction and related parameter determination. The above experiment was repeated three times.

[0065] According to the IL-1B gene sequence of the yellow eel reported in the GENBANK database, real-time fluorescence quantitative PCR primers were designed:

[0066] IL-RT-F:5'-CAACACTGCATCTTGAGACAG-3' (SEQ ID NO.5);

[0067] IL-RT-R: 5'-TAGGTTTTGAAGAGCATGTAGAGG-3' (SEQ ID NO. 6).

[0068] The extracted RNA was reverse transcribed into cDNA, and the above primers were used to analyze the expression of IL-1β in the liver of the eel during the infection with pathogens. The real-time fluorescence quantitative PCR reaction system was: 5μL SYBR Taq, 0.2μL IL-rt-F, 0.2μL IL-rt-F, 1μL cDNA (100ng / μL), 3.6μL H2O, a total of 10μL. The reaction conditions were: 95℃10s; 95℃5s, 56℃45s, 40 cycles. The relative expression level of fluorescence quantitative PCR was calculated by formula 2 -ΔΔct Calculation and one-way ANOVA were performed using SPSS19. The difference was significant when P < 0.05 and extremely significant when P < 0.01. Figure 2 shown.

[0069] The results showed that IL-1β levels in the liver increased significantly 6, 12, and 72 hours after infection with the pathogen, but showed no significant difference between the two groups at 24 and 48 hours after infection. Six hours after ALR protein injection, IL-1β expression in the protein-treated group was significantly lower than in both the bacterial infection and control groups, indicating that ALR recombinant protein can inhibit the expression of the inflammatory factor IL-1β gene during infection.

[0070] (2) Effect of ALR recombinant protein on NOS1 gene expression in the liver during pathogen infection.

[0071] The grouping of rice field eels and the treatment methods of each treatment group were the same as those in step (1). According to the rice field eel NOS1 gene sequence reported in the GenBank database, real-time fluorescence quantitative PCR primers were designed:

[0072] rt-nos1-F:5'-TGGCTTAACAGAATAGAGAAGGGG-3' (SEQ ID NO.7);

[0073] rt-nos1-R: 5'-CTGGAGTAGGCCGCGTAGAG-3' (SEQ ID NO. 8).

[0074] The extracted RNA was reverse transcribed into cDNA, and the above primers were used to analyze the expression of NOS1 (nitric oxide synthase) in the liver of eels infected with pathogens. The real-time fluorescence quantitative PCR reaction system, reaction conditions, calculation method and data analysis were the same as step (1). The test results are shown in Figure 2. Figure 3 shown.

[0075] The results showed that during pathogen infection, NOS1 gene expression in the liver increased significantly from 12 to 72 hours after infection, but decreased slightly 6 hours after infection. After injection of the recombinant ALR protein, NOS1 gene expression in the liver was significantly lower than that in the pathogen-only injection group. This suggests that the ALR recombinant protein can inhibit the increase in NOS1 caused by pathogen infection and suppress the synthesis of NO.

[0076] (3) Effect of ALR recombinant protein on the expression of liver antioxidant transcription factor Nrf2 gene during pathogen infection.

[0077] The grouping of rice field eels and the treatment methods of each treatment group were the same as step (1). According to the rice field eel Nrf2 gene sequence reported in the GenBank database, real-time fluorescence quantitative PCR primers were designed:

[0078] rt-nrf-F:5'-CGAGCTGGATTCACTGAAGGA-3' (SEQ ID NO.9);

[0079] rt-nrf-R:5'-TAATGCGAGGAACAAGGAAGATGGT-3' (SEQ ID NO. 10).

[0080] The extracted RNA was reverse transcribed into cDNA, and the above primers were used to analyze the expression of Nrf2 in the liver of the eel during the infection of pathogens. The real-time fluorescence quantitative PCR reaction system, reaction conditions, calculation method and data analysis were the same as step (1). The test results are shown in Figure 2. Figure 4 shown.

[0081] The results showed that during pathogen infection, Nrf2 gene expression in the liver of eels was significantly suppressed compared to the control group. However, Nrf2 gene expression in the protein-injected group was significantly higher than in the bacterial-treated group at 12, 24, and 72 hours. At 6 and 48 hours after infection, no significant difference was observed between the protein-treated and pathogen-treated groups. This suggests that ALR recombinant protein promotes the expression of the antioxidant transcription factor Nrf2 in the liver, thereby enhancing the liver's antioxidant capacity.

[0082] (4) Effect of ALR recombinant protein on liver GSH content during pathogen infection.

[0083] The grouping of eels and the treatment methods of each treatment group were the same as step (1). The total protein of liver tissue was extracted using RIPA lysis buffer (Shanghai Biyuntian Biological Co., Ltd.), and the total protein concentration was determined using a BCA kit (Biyuntian Biological, Shanghai, China). The GSH content in the eel liver samples was detected using a reduced glutathione (GSH) content detection kit (Solebo, Beijing, China) according to the kit instructions. The liver tissues at each time point of the three treatments were added with reagent 1 in proportion, quickly ground on ice, centrifuged at 8000r for 10 minutes, and the supernatant was taken to measure the absorbance value at a wavelength of 412nm according to the instructions, and then the GSH content (μg / g) was calculated. The specific operations were carried out according to the kit instructions. The test results are as follows. Figure 5 shown.

[0084] The results showed that, compared with the control group, pathogen infection increased liver GSH levels at 48 hours, with no significant difference at other time points. However, the recombinant protein injection group significantly increased liver GSH levels from 12 to 72 hours after infection, indicating that ALR recombinant protein can increase the production of the small molecule antioxidant GSH and enhance the liver's antioxidant capacity.

[0085] (5) Effect of ALR recombinant protein on liver NO content during pathogen infection.

[0086] The grouping of eels and the treatment methods of each treatment group were the same as step (1). The total protein of liver tissue was extracted using RIPA lysis buffer (Shanghai Biyuntian Biological Co., Ltd.), and the total protein concentration was determined using a BCA kit (Biyuntian Biological, Shanghai, China). The NO content in the eel liver samples was detected using a nitric oxide (NO) determination kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the kit instructions. The liver tissues at each time point of the three treatments were prepared into 10% tissue homogenates using 0.9% pre-cooled physiological saline, centrifuged at 2500r for 10 minutes, and the protein concentration was measured using a BCA kit. The supernatant was taken and the absorbance value was measured at a wavelength of 550nm according to the instructions, and then the NO content (μmol / gprot) was calculated. The specific operations were carried out according to the kit instructions. The test results are as follows. Figure 6 shown.

[0087] The results showed that compared with the control group, pathogen infection significantly increased the NO content in the liver tissue of the eel. Compared with the pathogen infection group, the recombinant protein injection group significantly reduced the NO content in the liver at 6h, 12h, and 72h after treatment, but there was no significant difference between the two groups at 24h and 48h after treatment. This shows that the ALR recombinant protein has the effect of reducing NO production during pathogen infection.

[0088] (6) Effect of ALR recombinant protein on the content of reactive oxygen species (ROS) in the liver during pathogen infection.

[0089] The grouping of eels and the treatment methods of each treatment group were the same as step (1). The total protein of liver tissue was extracted using RIPA lysis buffer (Shanghai Biyuntian Biological Co., Ltd.), and the total protein concentration was determined using a BCA kit (Biyuntian Biological, Shanghai, China). The ROS content in the eel liver samples was detected using a fish reactive oxygen species (ROS) enzyme-linked immunosorbent assay kit (Mlbio, Shanghai, China) according to the kit instructions. The liver tissues at each time point of the three treatments were prepared into 10% tissue homogenates using 0.9% pre-cooled physiological saline, centrifuged at 3000r for 20min, and the supernatant was taken to measure the absorbance value at a wavelength of 450nm according to the instructions, and then the ROS content (IU / mL) was calculated. The specific operations were carried out according to the kit instructions. The test results are as follows. Figure 7 shown.

[0090] The results showed that compared with the control group, the ROS content in the liver was significantly increased 12, 48, and 72 hours after pathogen infection. However, after injection of the recombinant protein ALR, the ROS content was significantly decreased 12, 24, 48, and 72 hours after the pathogen infection group. Only after 6 hours after protein injection did the ROS content increase significantly compared with the pathogen infection group. This indicates that the recombinant protein can reduce the content of reactive oxygen species during pathogen infection, thereby protecting the eel liver and effectively reducing oxidative damage. However, in the early stages of treatment, excessive protein content may cause some oxidative damage.

[0091] (7) Effect of ALR recombinant protein on the content of reactive nitrogen (RNS) in the liver during pathogen infection.

[0092] The grouping of eels and the treatment methods of each treatment group were the same as step (1). The total protein of liver tissue was extracted using RIPA lysis buffer (Shanghai Biyuntian Biological Co., Ltd.), and the total protein concentration was determined using a BCA kit (Biyuntian Biological, Shanghai, China). The fish reactive nitrogen (RNS) enzyme-linked immunosorbent assay kit (Mlbio, Shanghai, China) was used to detect the RNS content in the eel liver samples according to the kit instructions. The liver tissues at each time point of the three treatments were prepared into 10% tissue homogenates using 0.9% pre-cooled physiological saline, centrifuged at 3000r for 20min, and the supernatant was taken to measure the absorbance value at a wavelength of 450nm according to the instructions, and then the RNS content (IU / mL) was calculated. The specific operations were carried out according to the kit instructions. The test results are as follows. Figure 8 shown.

[0093] The results showed that compared with the control group, the RNS content in the liver was significantly increased 12, 48, and 72 hours after pathogen infection. However, after injection of the recombinant protein ALR, the RNS content was significantly decreased 12, 24, 48, and 72 hours after the pathogen infection group. Only 6 hours after protein injection did the liver RNS level increase significantly compared with the pathogen infection group. This indicates that the recombinant protein can reduce the content of reactive nitrogen during pathogen infection, thereby protecting the eel liver and effectively reducing oxidative damage. However, in the early stages of treatment, excessive protein content may cause some oxidative damage.

[0094] In summary, the yellow eel aldehyde reductase protein provided by the present invention is a new type of GSNOR that can rely on NADPH to reduce GSNO; moreover, the yellow eel aldehyde reductase protein provided by the present invention can significantly improve the antioxidant capacity of the liver tissue of the yellow eel under conditions of pathogenic bacterial infection and reduce the expression of pro-inflammatory factors after intraperitoneal injection, and is expected to be used for the prevention and treatment of bacterial infections in fish.

[0095] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by any technician familiar with this technical field within the technical scope disclosed by the present invention should be covered by the scope of protection of the present invention.

Claims

1. Application of yellow eel aldehyde reductase protein in the preparation of drugs for preventing and treating fish infections, wherein the amino acid sequence of the yellow eel aldehyde reductase is shown in SEQ ID NO.1, and the pathogen of the infection is Aeromonas hydrophila.

2. The use according to claim 1, characterized in that The nucleotide sequence encoding the yellow eel aldehyde reductase protein is shown in SEQ ID NO.

2.

3. The use according to claim 1, characterized in that The reducing activity of the yellow eel aldehyde reductase protein on GSNO depends on reducing coenzyme II.

4. The use according to claim 1, characterized in that Injecting yellow eel aldehyde reductase protein into fish can effectively reduce liver inflammation and oxidative stress damage induced by pathogens.

5. The use according to claim 4, characterized in that The yellow eel aldehyde reductase protein specifically inhibits the expression of IL-1β and nitric oxide synthase 1, promotes the expression of antioxidant transcription factor Nrf2, reduces liver NO content, ROS and RNS levels, and improves the fish's anti-infection ability.

6. The use according to claim 1, characterized in that The yellow eel aldehyde reductase protein is a recombinant protein, and its preparation method is: the sequence shown in SEQ ID NO.2 is connected to the expression vector and then transformed into the receptor cell, the receptor cell is induced to express, and the protein is purified.

7. The use according to claim 6, characterized in that The process of obtaining the sequence shown in SEQ ID NO.2 is as follows: using the total RNA of the liver tissue of the yellow eel as a template, reverse transcription is performed to synthesize cDNA, and the primers shown in SEQ ID NOs.3-4 are used for amplification.

8. The use according to claim 6, characterized in that The expression vector is a prokaryotic expression vector, and the recipient cell is Escherichia coli.

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