阅读说明：本技术 一种黄颡鱼醛还原酶蛋白及其制备方法和应用 (Pelteobagrus fulvidraco aldehyde reductase protein and preparation method and application thereof ) 是由 李伟 孙文秀 杨龙 于 2021-08-02 设计创作，主要内容包括：本发明公开了一种黄颡鱼醛还原酶重组蛋白及其制备方法和应用,属于基因工程技术领域,其中所述黄颡鱼醛还原酶蛋白的氨基酸序列如序列表SEQ IDNO.1所示。本发明通过从黄颡鱼中克隆AKR1A1基因并进行体外表达和分离纯化,获得黄颡鱼醛还原酶重组蛋白,为研究黄颡鱼醛还原酶的性质和功能奠定基础；同时本发明还分析了黄颡鱼醛还原酶AKR1A1在重金属镉诱导的氧化应激中的调控作用和对免疫相关基因表达的影响,首次验证了所述黄颡鱼醛还原酶AKR1A1在抵抗重金属镉引起的组织损伤中的作用,研究发现经腹腔注射所述黄颡鱼醛还原酶AKR1A1后,可显著提高黄颡鱼在重金属镉诱导的氧化应激条件下肝脏组织的抗氧化能力,减少氧化损伤。(The invention discloses a pelteobagrus fulvidraco aldehyde reductase recombinant protein, a preparation method and application thereof, belonging to the technical field of genetic engineering, wherein the amino acid sequence of the pelteobagrus fulvidraco aldehyde reductase protein is shown as a sequence table SEQ ID NO. 1. The invention clones the AKR1A1 gene from the pelteobagrus fulvidraco and carries out in vitro expression, separation and purification to obtain the pelteobagrus fulvidraco aldehyde reductase recombinant protein, thereby laying a foundation for researching the property and function of the pelteobagrus fulvidraco aldehyde reductase; meanwhile, the regulation and control effect of the yellow catfish aldehyde reductase AKR1A1 in the oxidative stress induced by the heavy metal cadmium and the influence on the immune related gene expression are also analyzed, the effect of the yellow catfish aldehyde reductase AKR1A1 in resisting the tissue damage caused by the heavy metal cadmium is verified for the first time, and researches show that the oxidation resistance of the liver tissue of the yellow catfish under the oxidative stress condition induced by the heavy metal cadmium can be obviously improved and the oxidative damage is reduced after the yellow catfish aldehyde reductase AKR1A1 is injected into the abdominal cavity.)

1. The pelteobagrus fulvidraco aldehyde reductase protein is characterized in that the amino acid sequence of the pelteobagrus fulvidraco aldehyde reductase protein is shown as a sequence table SEQ ID NO. 1.

2. A gene for encoding the pelteobagrus fulvidraco aldehyde reductase protein of claim 1, wherein the nucleotide sequence of the gene is shown as a sequence table SEQ ID No. 2.

3. A vector comprising the gene of claim 2.

4. A cell comprising the vector of claim 3.

5. The preparation method of the yellow catfish aldehyde reductase protein of claim 1, which comprises the following steps: extracting total RNA of liver tissues of the pelteobagrus fulvidraco, performing reverse transcription, performing PCR amplification by using a primer shown in SEQ ID No.3-4, connecting an amplification product with an expression vector, converting the amplification product into a receptor cell, and then inducing the receptor cell to express and purify to obtain the pelteobagrus fulvidraco aldehyde reductase protein.

6. The method according to claim 5, wherein the method comprises:

step 1, extracting total RNA of liver tissues of the pelteobagrus fulvidraco, synthesizing cDNA by using M-MLV reverse transcriptase, performing PCR amplification by using primers shown in SEQ ID NO.3-4, and recovering and purifying amplification products to obtain a coding gene fragment of the pelteobagrus fulvidraco aldehyde reductase protein;

step 2, carrying out EcoRI and Hind III double enzyme digestion on the gene fragment obtained in the step 1 and a vector pET-28a (+) at the same time, and constructing a prokaryotic expression vector pET-AKR1A1 after recovery, purification and connection;

step 3, transforming the prokaryotic expression vector pET-AKR1A1 into E.coli BL21(DE3) receptor cells, coating the E.coli BL21 receptor cells on a kanamycin-containing plate for resistance screening, and obtaining a positive expression strain through colony PCR verification and SDS-PAGE electrophoresis detection;

step 4, inoculating the positive expression strain into a culture medium containing kanamycin for culture, and performing induced expression by using IPTG (isopropyl thiogalactoside);

and 5, centrifugally collecting thalli from the bacteria liquid after induction expression, centrifugally collecting precipitates after carrying out ultrasonic crushing on the thalli, dissolving the precipitates by using a balanced buffer solution, collecting supernatant, adding the supernatant into a Ni ion affinity column, and eluting after combination to obtain the purified recombinant protein of the yellow catfish aldehyde reductase.

7. The pelteobagrus fulvidraco aldehyde reductase protein of claim 1, or the vector of claim 3, or the cell of claim 4 for use in reducing aldehydes and/or ketones.

8. Use according to claim 7, characterized in that the aldehydes are selected from: benzaldehyde, o-phthalaldehyde, formaldehyde, formic acid, glutaraldehyde, glyceraldehyde, and pyruvaldehyde; the ketones are selected from: 2, 3-butanedione and 2, 3-pentanedione.

9. The pelteobagrus fulvidraco aldehyde reductase protein of claim 1, or the vector of claim 3, or the cell of claim 4, for use in preparing a medicament for preventing or treating tissue damage caused by heavy metal cadmium.

10. The use of claim 9, wherein the pelteobagrus fulvidraco aldehyde reductase protein has the effects of inhibiting the expression of IL-1 beta and a cell apoptosis factor caspase3a, promoting the expression of a cell factor IL-10, improving the activities of catalase and glutathione peroxidase, and improving the antioxidant capacity.

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a yellow catfish aldehyde reductase protein, and a preparation method and application thereof.

Background

Aldoketoreductases (AKR) are a class of monomeric proteins with a molecular weight of 37kDa, with NAD (P) H-dependent activity. AKR1a1 is one of the members of the AKR superfamily, also known as aldehyde reductases, with a broad substrate spectrum and is a potential body carbonyl detoxification enzyme. HNE is the most bioactive aldehyde antioxidant produced by lipid peroxidation, and can form a complex GSH-HNE by combining with Glutathione S Transferases (GSTs) or Glutathione (GSH), while AKR1a1 is an important oxidoreductase involved in the reduction reaction of HNE and GSH-HNE. The current research on the function of AKR1A1 mainly focuses on mammals, and related reports of the gene in fishes are few. In mammals, AKR1A1 has been shown to be a novel mammalian S-nitrosoglutathione reductase, which can promote vitamin C biosynthesis and is also a major doxorubicin reductase in human liver. In fishes, AKR1A1 plays an important role in maintaining physiological blood sugar concentration of zebra fishes and relieving organ injury induced by glucose; tilapia AKR1A1 can be induced to express by benzopyrene.

Cadmium pollution is a common water body pollution, fish affected by acute cadmium pollution can cause severe organ oxidation injury and inflammatory reaction, and the fish contaminated by cadmium can enter a human body along with a food chain to affect human health.

The yellow catfish (Pelteobagrus fulvidraco) belongs to the family of the Pseudobagaceae, belongs to common small-sized fresh water economic fishes, is distributed in various large water systems in China, has rich amino acid content, tender meat quality, delicious taste, high nutritional value and no muscle thorns, has nourishing effect and medicinal value, is quickly universally accepted by people, and has wide economic value. At present, the related functions of the aldehyde reductase of the pelteobagrus fulvidraco and the potential role of the aldehyde reductase in cadmium stress are not clear, and further research and research are needed.

Disclosure of Invention

The invention provides a yellow catfish aldehyde reductase protein and a preparation method and application thereof aiming at the blank of the prior art, and the yellow catfish aldehyde reductase recombinant protein is obtained by cloning AKR1A1 gene from yellow catfish and carrying out in vitro expression, separation and purification; the regulation and control function of the recombinant protein in heavy metal cadmium-induced oxidative stress and the influence on immune-related gene expression are further analyzed, and the method has important significance for further exploring the functions of the fish AKR1A1 gene.

One of the purposes of the invention is to provide a pelteobagrus fulvidraco aldehyde reductase protein, wherein the amino acid sequence of the pelteobagrus fulvidraco aldehyde reductase protein is shown as a sequence table SEQ ID NO. 1.

The second purpose of the invention is to provide a gene for coding the yellow catfish aldehyde reductase protein, wherein the nucleotide sequence of the gene is shown as a sequence table SEQ ID NO. 2.

The invention also aims to provide a vector, which comprises the gene for encoding the yellow catfish aldehyde reductase protein.

The fourth object of the present invention is to provide a cell comprising the vector.

The fifth purpose of the invention is to provide a preparation method of the yellow catfish aldehyde reductase protein, which comprises the following steps: extracting total RNA of liver tissues of the pelteobagrus fulvidraco, performing reverse transcription, performing PCR amplification by using a primer shown in SEQ ID No.3-4, connecting an amplification product with an expression vector, converting the amplification product into a receptor cell, and then inducing the receptor cell to express and purify to obtain the pelteobagrus fulvidraco aldehyde reductase protein.

Further, the method specifically comprises:

step 1, extracting total RNA of liver tissues of the pelteobagrus fulvidraco, synthesizing cDNA by using M-MLV reverse transcriptase, performing PCR amplification by using primers shown in SEQ ID NO.3-4, and recovering and purifying amplification products to obtain a coding gene fragment of the pelteobagrus fulvidraco aldehyde reductase protein;

step 2, carrying out EcoRI and Hind III double enzyme digestion on the gene fragment obtained in the step 1 and a vector pET-28a (+) at the same time, and constructing a prokaryotic expression vector pET-AKR1A1 after recovery, purification and connection;

step 3, transforming the prokaryotic expression vector pET-AKR1A1 into E.coli BL21(DE3) receptor cells, coating the E.coli BL21 receptor cells on a kanamycin-containing plate for resistance screening, and obtaining a positive expression strain through colony PCR verification and SDS-PAGE electrophoresis detection;

step 4, inoculating the positive expression strain into a culture medium containing kanamycin for culture, and performing induced expression by using IPTG (isopropyl thiogalactoside);

and 5, centrifugally collecting thalli from the bacteria liquid after induction expression, centrifugally collecting precipitates after carrying out ultrasonic crushing on the thalli, dissolving the precipitates by using a balanced buffer solution, collecting supernatant, adding the supernatant into a Ni ion affinity column, and eluting after combination to obtain the purified recombinant protein of the yellow catfish aldehyde reductase.

The invention further aims to provide the application of the yellow catfish aldehyde reductase protein, the vector or the cell in reducing aldehydes and/or ketones.

Further, the aldehydes are selected from: benzaldehyde, o-phthalaldehyde, formaldehyde, formic acid, glutaraldehyde, glyceraldehyde, and pyruvaldehyde; the ketones are selected from: 2, 3-butanedione and 2, 3-pentanedione.

The seventh purpose of the invention is to provide the application of the yellow catfish aldehyde reductase protein, the carrier or the cell in preparing a medicament for preventing or treating tissue damage caused by heavy metal cadmium.

Furthermore, the pelteobagrus fulvidraco aldehyde reductase protein has the effects of inhibiting the expression of IL-1 beta and a cell apoptosis factor caspase3a, promoting the expression of a cell factor IL-10, improving the activities of catalase and glutathione peroxidase, and improving the antioxidant capacity.

Further, the injection amount of the pelteobagrus fulvidraco aldehyde reductase protein is 1-100 mu g.

Compared with the prior art, the invention has the beneficial effects that:

(1) the invention constructs a prokaryotic expression vector of the yellow catfish aldehyde reductase AKR1A1 gene by using a gene engineering technology, obtains recombinant protein thereof by induced expression and purification, and lays a foundation for further researching the properties and functions of the yellow catfish aldehyde reductase;

(2) the invention verifies the reduction of the pelteobagrus fulvidraco aldehyde reductase AKR1A1 recombinant protein on aldehydes and ketones, analyzes the substrate spectrum of the recombinant protein, and has theoretical guiding significance and reference value on the industrial application of the enzyme;

(3) the invention firstly verifies the effect of the yellow catfish aldehyde reductase AKR1A1 in resisting tissue damage caused by heavy metal cadmium, and researches show that the oxidation resistance of liver tissues of the yellow catfish under the oxidative stress condition induced by the heavy metal cadmium can be obviously improved and the oxidative damage can be reduced after the yellow catfish aldehyde reductase AKR1A1 is injected into the abdominal cavity.

Drawings

Fig. 1 is an SDS-PAGE electrophoresis diagram of the pelteobagrus fulvidraco aldehyde reductase gene AKR1a1 gene after expression and purification in example 1 of the present invention, wherein the lane 1: a blank plasmid strain; lane 2: pET-28-AKR1A 1-containing strain before induction; lane 3: the induced strain containing pET-28-AKR1A 1; lane 4: purified AKR1a1 protein; m: marker;

FIG. 2 is a graph showing the effect of injecting different doses of AKR1A1 recombinant protein into the abdominal cavity of example 3 of the present invention on the expression of IL-1 β gene, a liver inflammatory factor, under cadmium stress;

FIG. 3 shows the effect of abdominal cavity injection of different doses of AKR1A1 recombinant protein on the expression of caspase3a gene under cadmium stress in example 3 of the present invention;

FIG. 4 shows the effect of injecting different doses of AKR1A1 recombinant protein into the abdominal cavity of example 3 of the present invention on the expression of the liver anti-inflammatory factor IL-10 gene under cadmium stress;

FIG. 5 is a graph showing the effect of abdominal cavity injection of different doses of AKR1A1 recombinant protein on the level of liver catalase CAT under cadmium stress in example 3 of the present invention;

FIG. 6 shows the effect of injecting different doses of AKR1A1 recombinant protein into the abdominal cavity of example 3 of the present invention on the content of hepatic glutathione peroxidase GSH-Px under cadmium stress;

FIG. 7 shows the effect of injecting different doses of AKR1A1 recombinant protein into the abdominal cavity of example 3 of the present invention on the total antioxidant capacity T-AOC of liver under cadmium stress.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 Gene cloning, expression vector construction and recombinant protein expression purification of yellow catfish aldehyde reductase

1. Cloning of yellow catfish AKR1A1 gene

Extracting total RNA of the pelteobagrus fulvidraco liver according to the specification of the Trizol kit, synthesizing cDNA by using M-MLV reverse transcriptase, and storing at the low temperature of-80 ℃.

Design primer AKR1A 1-F: 5' -ATTGAATTCATGAATGACTATGCAGTGCT-3' (shown in SEQ ID NO. 3);

AKR1A1-R：CGAAAGCTTATAAGGATCATTAAATGGAT (shown in SEQ ID NO. 4),

wherein, the upstream primer sequence is added with EcoR I enzyme cutting site, and the downstream primer is added with Hind III enzyme cutting site.

Performing conventional PCR amplification, and obtaining an open reading frame sequence of the gene after the amplification product is recovered, purified, connected and sequenced, wherein the nucleotide sequence of the Pseudobagrus fulvidraco AKR1A1 gene is shown as SEQ ID NO.2, and the amino acid sequence of the Pseudobagrus fulvidraco aldehyde reductase coded by the gene is shown as SEQ ID NO. 1.

2. Construction of expression vectors

And carrying out EcoR I and Hind III double enzyme digestion on the PCR amplification product and a prokaryotic expression vector pET-28a (+) at the same time, purifying the enzyme digestion product by using a DNA gel recovery kit respectively, connecting the amplification product and the vector by using T4 DNA ligase, and successfully constructing to obtain a prokaryotic expression vector pET-AKR1A1 after sequencing verification.

3. Expression and purification of yellow catfish AKR1A1 recombinant protein

The prokaryotic expression vector pET-AKR1A1 is transformed into E.coli BL21(DE3), spread on a kanamycin plate containing 50 mug/mL for resistance screening, and a positive expression strain is obtained through colony PCR verification and SDS-PAGE electrophoresis detection.

Respectively inoculating the positive expression strains into 200mL of fresh LB liquid culture medium containing kanamycin according to the ratio of 1:50, and culturing at 200 r.min^-1Shaking culture to OD₆₀₀At about 0.6, 1mM IPTG was added and the culture was continued for 4h to induce recombinant protein expression. Centrifuging the induced bacteria liquid at 4 deg.C and 10000 r.min^-1Centrifuging for 10min to collect thallus, resuspending thallus precipitate with PBS buffer (50mmol/L, pH 8.0), ultrasonically lysing bacteria on ice for 5s at an interval of 10s for 20min, and then performing ultrasonic treatment at 4 deg.C and 10000 r.min^-1After centrifugation for 10min, the precipitate was collected, washed three times with lysis buffer (8mol/L urea, 10mmol/L imidazole, 50mmol/L PBS, pH 8.0), dissolved by adding an appropriate amount of lysis buffer, filtered through a 0.45 μm filter, and the supernatant was applied to a Ni-NTA His Bind Resin purification column, eluted with PBS containing imidazole (100mmol/L), and subjected to SDS-PAGE to detect the purification of the recombinant protein.

The results of SDS-PAGE are shown in FIG. 1, in which lane 1: a blank plasmid strain; lane 2: pET-28-AKR1A 1-containing strain before induction; lane 3: the induced strain containing pET-28-AKR1A 1; lane 4: purified AKR1a1 protein; m: and (5) Marker. The results show that the in vitro expression and purification of the recombinant protein AKR1A1 are successfully realized in the embodiment.

Example 2 detection of enzymatic Activity and analysis of substrate specificity of AKR1A1 recombinant protein

The change of the absorbance value of reduced coenzyme NADPH at 340nm was measured by a general analysis TU-1900 UV spectrophotometer to evaluate the activity of the recombinant protein. The enzyme activity unit is defined as: the aldehyde reductase consumed 1. mu. mol NADPH per minute as one enzyme activity unit (U) under optimum conditions.

Adding 1900 μ L of 50mmol/L in sequence into the cuvette^-1PBS (pH 5.0), 90. mu.L of enzyme solution, 1000. mu.L of substrate (about 40 mmol. multidot.L)^-1) Keeping the temperature at 37 ℃ for 10min, adding 10 mu L of 10mmol/L^-1NADPH, total 3 mL; the cuvette was placed in an ultraviolet spectrophotometer and the change in absorbance at 340nm was measured and recorded, 3 replicates per substrate. Statistical analysis was performed using SPSS20.0, specific enzyme activities and relative enzyme activities were expressed as x + -s, and variance analysis was used for comparison between groups, with differences being very significant when P < 0.01 and significant when P < 0.05.

According to the method, the activities of the AKR1A1 recombinant protein on substrates such as benzaldehyde, o-phthalaldehyde, formaldehyde, formic acid, glutaraldehyde, glyceraldehyde, 2, 3-butanedione, methylglyoxal, 2, 3-pentanedione, acetylacetone, glucose and the like are respectively measured. The results of the activity measurement are shown in Table 1.

TABLE 1 reaction constants of AKR1A1 recombinant protein for different substrates

ND: indicating no detectable activity

The result shows that the AKR1A1 recombinant protein has good reduction activity on the tested aldehydes and medium-long chain ketones, but has no obvious activity on saccharides and acetylacetone.

Example 3 Regulation of oxidative stress on the liver under cadmium stress by AKR1A1 recombinant protein

1. Influence of AKR1A1 recombinant protein on liver inflammatory factor IL-1 beta gene expression under cadmium stress

Taking 60 pelteobagrus fulvidraco (with the same growth environment and similar body type, the body mass of 76 +/-5.2 g), carrying out acclimation culture, randomly and equally dividing into 4 groups, respectively carrying out intraperitoneal injection on 1, 10 and 100 mu g of AKR1A1 recombinant protein, and taking the injected PBS buffer solution as a negative control. Feeding the fish into dechlorinated water with the final concentration of 1.69mg/L cadmium chloride, taking 3 fish livers respectively in 0 hour, 6 hours, 12 hours and 24 hours, taking a proper amount of liver tissues to be filled into a 1.5mL centrifuge tube, adding 500mL Trizol to be stored at the temperature of minus 80 ℃ for RNA extraction and related enzyme index determination. The above experiment was repeated three times.

Designing a real-time fluorescent quantitative PCR primer IL-rt-F: 5'-GCATGGTTCAATCTCTCAGCCTAC-3' and a primer IL-rt-R according to the Pelteobagrus fulvidraco IL-1 beta gene sequence reported in a GenBank database: 5'-TGTTCAGCGAGTCACCGGGTCCG-3', analyzing the expression level of IL-1 beta in the liver of the pelteobagrus fulvidraco induced by heavy metal cadmium. The real-time fluorescent quantitative PCR reaction system is as follows: 20 μ l reaction: 10 μ l SYBR Taq, 0.4 μ l IL-rt-F, 0.4 μ l Rox RD II, 1 μ l cDNA (100 ng/. mu.l), 7.8 μ l H₂And O. The reaction conditions are as follows: 10s at 95 ℃; 95 ℃ for 5s,60 ℃ for 34s, 40 cycles. The relative expression level of the fluorescent quantitative PCR is shown in the formula 2^-ΔΔctCalculated and subjected to one-way analysis of variance by SPSS 19. The difference is significant when P is less than 0.05, and the difference is significant when P is less than 0.01. The results of the detection are shown in FIG. 2.

The result shows that the expression level of the IL-1 beta in the liver tissue of the pelteobagrus fulvidraco is obviously improved in the early period of cadmium stimulation, the peak value is reached after 6 hours, the expression level is improved by about 1.8 times (P is less than 0.05), and then the expression level of the IL-1 beta begins to decline. After 6h of AKR1A1 protein injection, the IL-1 beta expression of the low-concentration dose group (1 mug) and the medium-concentration dose group (10 mug) is obviously lower than that of the control group, and the high-concentration dose group (100 mug) and the control group have no obvious difference; after the AKR1A1 recombinant protein groups are injected for 12h and 24h, the IL-1 beta expression levels of the high-concentration, medium-concentration and low-concentration dose groups are obviously lower than those of the control group, and the results show that the AKR1A1 recombinant protein has the effect of inhibiting the heavy metal cadmium-induced IL-1 beta expression.

2. Influence of AKR1A1 recombinant protein on expression of liver apoptosis factor caspase3a gene under cadmium stress

Grouping of the pelteobagrus fulvidraco and the treatment method of each treatment group are the same as the determination of IL-1 beta, and a real-time fluorescent quantitative PCR primer rt-caspase3a-F is designed according to the gene sequence of the pelteobagrus fulvidraco caspase3a reported in a GenBank database: 5'-GGAGTTCGGTTGTAGAGTAATATGGT-3', respectively; rt-caspase3 a-R: 5'-ATGTTACATGGTGCAGGATATGAGT-3', analyzing the expression level of caspase3a in the liver of the pelteobagrus fulvidraco induced by heavy metal cadmium. The real-time fluorescence quantitative PCR reaction system, reaction conditions, calculation method and data analysis are the same as above. The results of the detection are shown in FIG. 3.

The result shows that the expression level of caspase3a gene in liver is obviously increased in the early stage of heavy metal cadmium stress, after the low, medium and high dose of AKR1A1 recombinant protein is injected for 6h, the expression of apoptosis factor caspase3a is obviously reduced, and no obvious difference exists among three dose groups. And in the liver tissues of the pelteobagrus fulvidraco treated groups at 12h and 24h after injection and at three different concentrations, the gene expression level of the apoptosis factor caspase3a is obviously lower than that of the control group, and the gene expression level of the caspase3a in the not-too-high dose group is slightly higher than that of the low and medium dose groups, which shows that the AKR1A1 recombinant protein can inhibit the expression of the heavy metal cadmium induced apoptosis factor caspase3a, but the too-high dose can also induce the expression of the apoptosis factor caspase3 a.

3. Influence of AKR1A1 recombinant protein on expression of liver anti-inflammatory factor IL-10 gene under cadmium stress

Grouping of the pelteobagrus fulvidraco and the processing method of each processing group are the same as the determination of IL-1 beta, and a real-time fluorescent quantitative PCR primer rt-IL-10-F is designed according to the IL-10 gene sequence of the pelteobagrus fulvidraco reported in a GenBank database: 5'-GTTCTTCATACGCCGTCATCCG-3', respectively; and (2) 5'-GATCCTACATTTAAACAGCTCCCTC-3', analyzing the expression of IL-10 in the liver of the pelteobagrus fulvidraco under the stimulation of heavy metal cadmium. The real-time fluorescence quantitative PCR reaction system, reaction conditions, calculation method and data analysis are the same as above. The results of the detection are shown in FIG. 4.

The result shows that the expression level of the IL-10 gene in livers stressed by heavy metal cadmium for 12h and 24h is obviously reduced compared with that in the early stage of the heavy metal cadmium stress. The low-dose AKR1A1 recombinant protein has no obvious effect of promoting IL-10 expression, and the expression of IL-10 gene is obviously improved 6h, 12h and 24h after the intermediate-dose and high-dose AKR1A1 recombinant protein is injected, which shows that the AKR1A1 recombinant protein has the effect of promoting the expression of anti-inflammatory factor IL-10 in liver cells, thereby protecting the liver.

4. Influence of AKR1A1 recombinant protein on CAT activity of liver catalase under cadmium stress

Grouping of pelteobagrus fulvidraco and the processing method of each processing group are the same as the determination of IL-1 beta, the total protein of liver tissue is extracted by RIPA lysate (Shanghai Biyuntian biological company), and the total protein concentration is determined by a BCA kit (Biyuntian biological, Shanghai, China). Catalase CAT activity was detected using ELISA kit (shanghai enzyme linked). The specific operation is carried out according to the kit instructions. The results of the detection are shown in FIG. 5.

The result shows that compared with the control group, the CAT activity in the liver tissue of the pelteobagrus fulvidraco injected with the high, medium and low dose of AKR1A1 recombinant protein is obviously increased after the treatment of cadmium for 6 hours, and the CAT activity in the low dose group is the highest; the CAT activity of the medium and high dose groups is still obviously higher than that of the control group 12 hours after the treatment; no CAT activity difference was shown between each dose injection group and the control group 24h after the treatment, which indicates that the AKR1A1 recombinant protein has the effect of increasing CAT activity in the early stage of cadmium stress.

5. Influence of AKR1A1 recombinant protein on activity of liver glutathione peroxidase under cadmium stress

Grouping of pelteobagrus fulvidraco and the processing method of each processing group are the same as the determination of IL-1 beta, the total protein of liver tissue is extracted by RIPA lysate (Shanghai Biyuntian biological company), and the total protein concentration is determined by a BCA kit (Biyuntian biological, Shanghai, China). Glutathione peroxidase (GSH-Px) activity was detected using an ELISA kit (Shanghai enzyme-Linked). The specific operation is carried out according to the kit instructions. The results of the detection are shown in FIG. 6.

The results show that the activity of GSH-Px in the liver tissues of the pelteobagrus fulvidraco injected with the high and medium dose of AKR1A1 recombinant protein is obviously improved at 6h, 12h and 24h after cadmium treatment compared with the control group, and the activity of GSH-Px in the low dose group is not obviously different from that in the control group at 24h after cadmium treatment. This indicates that the injected high and medium concentration AKR1A1 recombinant protein has the function of improving GSH-Px activity in the cadmium stress process.

6. Influence of AKR1A1 recombinant protein on total liver antioxidant capacity T-AOC under cadmium stress

Grouping of pelteobagrus fulvidraco and the processing method of each processing group are the same as the determination of IL-1 beta, the total protein of liver tissue is extracted by RIPA lysate (Shanghai Biyuntian biological company), and the total protein concentration is determined by a BCA kit (Biyuntian biological, Shanghai, China). And (3) detecting the content of the total antioxidant capacity T-AOC by using an ELISA kit (Shanghai enzyme-linked immunosorbent assay). The specific operation is carried out according to the kit instructions. The results of the detection are shown in FIG. 7.

The results show that compared with the control group, the T-AOC content in the liver tissue of the pelteobagrus fulvidraco injected with the low-concentration and medium-concentration AKR1A1 recombinant protein is obviously increased in the cadmium treatment of 6h and 12h, and the T-AOC content in all the dose treatment groups is not obviously different from that in the control group 24h after the treatment. The result shows that the recombinant protein with medium and low concentration doses can improve the early oxidation resistance of liver tissue under cadmium stress, so that the recombinant protein has a protective effect on the liver of the pelteobagrus fulvidraco, and effectively reduces the oxidation damage under heavy metal cadmium stress.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention.

Sequence listing

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Gly Leu Gly Thr Trp Lys Ser Glu Ser Gly Lys Val Lys Gln Ala Val

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Ile Trp Ala Leu Gln Ala Gly Tyr Arg His Ile Asp Cys Ala Ser Ile

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Trp Pro Tyr Ala Phe Lys Gln Gly Asp Ser Thr Phe Pro Arg Lys Glu

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Ser Asn Phe Asn Ser Arg Gln Ile Asp Asp Ile Leu Ser Val Ala Asn

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Ile Lys Pro Thr Val Leu Gln Val Glu Gly His Pro Tyr Leu Ala Gln

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Tyr Ser Pro Leu Gly Ser Pro Asp Arg Ala Trp Lys Arg Pro Asp Glu

210 215 220

Pro Val Ile Leu Glu Glu Pro Val Ile Ala Ala Leu Ala Lys Lys Tyr

225 230 235 240

Asn Lys Ser Pro Ala Gln Ile Ile Ile Arg Trp Gln Thr Gln Arg Ser

245 250 255

Val Val Thr Ile Pro Lys Ser Val Thr Glu Ser Arg Ile Lys Glu Asn

260 265 270

Ile Gln Val Phe Asp Phe Thr Leu Glu Ser Ala Glu Met Asp Ser Val

275 280 285

Thr Ala Leu Asn Lys Gly Trp Arg Tyr Ile Val Pro Thr Ile Thr Val

290 295 300

Asp Gly Lys Phe Val Pro Arg Asp Ala Gly His Pro Tyr Tyr Pro Phe

305 310 315 320

Asn Asp Pro Tyr

<210> 2

<211> 975

<212> DNA

<213> Pelteobagrus fulvidraco (Pelteobagrus fulvidraco)

<400> 2

atgaatgact atgcagtgct gaacaccggg cgaaagatgc cacttgttgg actggggacg 60

tggaagagtg agtccggaaa ggtgaaacag gcggttatat gggctcttca ggctggatat 120

cggcacatcg actgcgcttc gatttatgga aatgaatcag aaattggtga agcctttcag 180

gagatgttgg ggcctgataa agccttgaaa cgagaagacg tgtttgtgac ctccaaattg 240

tggaacacga agcaccatcc tgaggatgtg gagccatcac tgctaaacag cctgaaagag 300

ctgaagctgg agtacctgga cctctacctc attcactggc cttatgcctt taagcagggg 360

gatagcactt tccccagaaa ggaagatggg accttactgt atgatgacat tgattacaag 420

gtgacgtggg ccgccatgga gaaactcgtc gaaaagggac ttgtgagggc tatcgggctc 480

tctaacttca acagcagaca gattgatgat atcttgtccg tggccaacat aaagcctact 540

gttttacagg tggagggtca tccttatctt gcccaggtgg agcttcttgc tcactgcagg 600

gagaggggct tagtgatgac tgcctacagt ccacttggct ctccagatcg tgcttggaag 660

agaccagatg agccagttat cttagaggag ccagtcattg cagctctagc caagaagtac 720

aacaaatctc ctgcacaaat tataatcagg tggcagactc agcgaagtgt tgtgacgatc 780

ccaaagagtg ttacagagtc tcgcattaaa gaaaatattc aggtttttga tttcactctt 840

gagtctgccg agatggacag tgtgacagca ttgaacaaag gctggagata cattgtgcca 900

actattactg ttgatggcaa gtttgtacca agggatgcag gacatcccta ctatccattt 960

aatgatcctt attaa 975

<210> 3

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

attgaattca tgaatgacta tgcagtgct 29

<210> 4

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

cgaaagctta taaggatcat taaatggat 29