Ferritin L gene, recombinant protein, preparation method, application and primers of Hefang crucian carp

Abstract

The present invention discloses a Ferritin L gene of Hefang Crucian Carp and its recombinant protein. The nucleotide sequence of the Ferritin L gene of Hefang Crucian Carp is shown as SEQ ID NO: 1, and the amino acid sequence of the recombinant protein is shown as SEQ ID NO: 2. The discovery of the Ferritin L gene of Hefang Crucian Carp not only enriches the gene library of Hefang Crucian Carp, but also can be used to prepare recombinant proteins. Experiments have shown that the recombinant protein can inhibit the growth activity of Aeromonas hydrophila, providing a new practical basis for the physiological immunity research of Hefang Crucian Carp. The present invention also discloses a preparation method of the Ferritin L recombinant protein of Hefang Crucian Carp, the application of the Ferritin L recombinant protein of Hefang Crucian Carp in the fields of inhibiting the growth of Aeromonas hydrophila, immune preparations or feed additives for aquatic animals, and primers for amplifying the Ferritin L gene of Hefang Crucian Carp.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering research and development, and in particular relates to a Ferritin L gene of crucian carp, a Ferritin L recombinant protein of crucian carp, a preparation method of the Ferritin L recombinant protein of crucian carp, an application of the Ferritin L recombinant protein of crucian carp, and a primer for amplifying the Ferritin L gene of crucian carp. Background Art

[0002] Iron is an essential trace element in living organisms, directly linked to individual development, oxidative stress, and disease. Furthermore, both biotic stress (infection by pathogens or viruses) and abiotic stress (heavy metals) can cause an imbalance in the dynamic regulation of iron ions in animals, leading to accumulation within cells. This can cause a sharp increase in reactive oxygen species (ROS), ultimately damaging intracellular macromolecules such as DNA, proteins, and lipids.

[0003] Ferritin is a globular protein composed of 24 subunits. Its internal shell-shaped structure stores iron ions, directly participating in and maintaining iron homeostasis in the body. Ferritin subunits are primarily classified as ferritin H, ferritin M, and ferritin L, but the subunits present vary among species. In higher mammals, the H and L ferritin subunits are predominant. Related research indicates that lower vertebrate ferritins are primarily composed of the H and M subunits. Functionally, ferritin H in higher mammals possesses a ferroxidase center and is primarily responsible for iron oxidation, while ferritin L has negatively charged residues on its shell that form intrachain salt bridges and promote the nucleation of iron-hydrogen compounds. However, ferritin L in lower vertebrates still retains a partial ferroxidase reaction center and thus also has iron oxidation potential. Ferritin M, on the other hand, possesses both the ferroxidase activity of Ferritin H and the iron nucleation capacity of Ferritin L. Compared to members of the Ferritin family in higher mammals, current research on immune regulation reveals that members of the Ferritin family in lower vertebrates and invertebrates are multifunctional metal-binding proteins. In addition to directly participating in iron metabolism, invertebrate type I and type II Ferritins, as well as fish Ferritin H and Ferritin M, regulate immune function and exhibit broad-spectrum antibacterial, antiviral, and antioxidant properties. Ferritin L is a homolog of the Ferritin family, but because only a few fish species possess all three subunits, the immune regulatory capabilities of fish Ferritin L are rarely reported.

[0004] The Hefang Crucian Carp (WR, 2n = 100) is a freshwater fish developed by the State Key Laboratory of Freshwater Fisheries Developmental Biology, a joint venture between Hunan Normal University and the Hunan Provincial Ministry of Fisheries. It is a nationally recognized new aquatic species. Hefang Crucian Carp (WR, 2n = 100) is the first generation of a hybrid between Japanese white crucian carp (Carassius cuvieri, WCC, ♀, 2n = 100) and red crucian carp (Carassius auratus red var, RCC, ♂, 2n = 100). Hefang Crucian Carp resembles wild crucian carp in appearance, exhibits strong reproductive capacity and rapid growth, and possesses high fertilization and hatching rates, making it suitable for large-scale production. Furthermore, the muscle content of Hefang Crucian Carp is significantly higher than that of its parents, resulting in a tender and nutritious meat, making it highly sought after by fish farmers and consumers. However, rapid climate change and severely polluted water quality have severely impacted the health of fish farming and remain a difficult problem to address. Aeromonas hydrophila is one of the main pathogens of bacterial diseases in freshwater fish, causing septicemia in aquatic animals and often resulting in severe economic losses for the freshwater aquaculture industry. Developing high-quality fish-derived protein supplements in feed is an effective way to address this issue. This will facilitate the development and application of improved fish feeds, reducing environmental pollution while increasing efficiency and revenue. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the deficiencies and defects mentioned in the above background technology and provide a newly cloned Ferritin L gene of Carassius auratus, a recombinant Ferritin L protein of Carassius auratus, a preparation method of the recombinant Ferritin L protein of Carassius auratus, the application of the recombinant Ferritin L protein of Carassius auratus and primers for amplifying the Ferritin L gene of Carassius auratus.

[0006] In order to solve the above technical problems, the technical solution proposed by the present invention is:

[0007] A Ferritin L gene of Hefang Crucian Carp has a nucleotide sequence as shown in SEQ ID NO: 1. The discovery of the Ferritin L gene of Hefang Crucian Carp not only enriches the gene library of Hefang Crucian Carp, but can also be used to prepare recombinant proteins, further used to inhibit the growth of Aeromonas hydrophila, and used as an immune preparation or feed additive for aquatic animals, providing a new practical basis for the physiological immunity research of Hefang Crucian Carp.

[0008] A recombinant protein of Hefang crucian carp ferritin L, whose amino acid sequence is shown in SEQ ID NO: 2, or a protein with equivalent or higher activity obtained by substituting, deleting and / or adding one or more amino acids and / or terminally modifying the amino acid sequence shown in SEQ ID NO: 2; the recombinant protein of Hefang crucian carp ferritin L is encoded by a Hefang crucian carp ferritin L gene, whose nucleotide sequence is shown in SEQ ID NO: 1.

[0009] Based on a general technical concept, the present invention also provides a method for preparing a recombinant protein of Ferritin L from crucian carp, comprising the following steps: amplifying the Ferritin L gene sequence of Ferritin L from crucian carp by PCR and adding enzyme cleavage sites, purifying the PCR product and performing double enzyme digestion, and then ligating it to an expression vector pET32a, and then transferring the expression vector pET32a containing the Ferritin L gene of Ferritin L from crucian carp into Escherichia coli BL21 (E. coli BL21) by CaCl2 transformation method for inducing expression, thereby finally obtaining the Ferritin L recombinant protein of Ferritin L from crucian carp.

[0010] The above-mentioned preparation method, preferably, the specific operation of amplifying the Hefang crucian carp Ferritin L gene by PCR and adding restriction enzyme cleavage sites comprises the following steps: using the Hefang crucian carp liver cDNA obtained by reverse transcription as a template, amplifying the Hefang crucian carp Ferritin L gene with restriction enzyme cleavage sites by PCR, and sequencing and confirming the Hefang crucian carp Ferritin L sequence by direct gel tapping after electrophoresis separation; the nucleotide sequence of the Hefang crucian carp Ferritin L gene is shown in SEQ ID NO: 1.

[0011] Preferably, the reaction conditions for the PCR amplification are: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 65°C for 30 s, and extension at 72°C for 35 s, for a total of 5 cycles; denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 35 s, for a total of 5 cycles; denaturation at 94°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 35 s, for a total of 30 cycles; and finally extension at 72°C for 10 min. The primers used in the PCR amplification are the upstream primer Ferritin L-F1 and the downstream primer Ferritin L-R1. The nucleotide sequence of the upstream primer Ferritin L-F1 is shown in SEQ ID NO: 3, and the nucleotide sequence of the downstream primer Ferritin L-R1 is shown in SEQ ID NO: 4.

[0012] Preferably, the specific operation of purifying the PCR product and performing double enzyme digestion, and then connecting it to the expression vector pET32a, includes the following steps: using a DNA agarose gel recovery kit to directly purify and recover the PCR product (i.e., the combined crucian carp Ferritin L sequence with enzyme cutting sites after electrophoresis), and then performing double enzyme digestion with restriction endonucleases EcoRI and XhoI; after the double enzyme digestion is completed, the combined crucian carp Ferritin L sequence that is successfully digested is recovered as a product; taking the expression vector pET32a and performing double enzyme digestion with restriction endonucleases EcoRI and XhoI, mixing the obtained large vector fragment with the combined crucian carp Ferritin L sequence that is successfully digested, and connecting with T4 ligase at 15-17°C for 12-18h, that is, successfully connecting the combined crucian carp Ferritin L gene sequence to the expression vector pET32a.

[0013] As research on members of the fish ferritin family deepens, Ferritin L, a newly discovered member of the fish ferritin family, while differing from other fish ferritin family members in amino acid composition, shares a conserved ferritin-like diiron structure. Preliminary studies also indicate that fish ferritin L is directly involved in immune regulation induced by fish viruses or pathogens. Therefore, the development and utilization of recombinant fish-derived Ferritin L proteins will facilitate research into immune regulation and antibacterial mechanisms in fish, as well as contribute to the healthy breeding of commercial aquatic animals and the development of feed immune preparations or additives.

[0014] Based on a general technical concept, the present invention also provides a use of the above-mentioned combined crucian carp Ferritin L recombinant protein in inhibiting the growth of Aeromonas hydrophila.

[0015] The above application preferably comprises the following steps: mixing the recombinant protein of Carassius auratus Ferritin L with the Aeromonas hydrophila in a buffer environment with a pH of 7.2-8.2, and inhibiting Aeromonas hydrophila after the reaction is terminated.

[0016] More preferably, the concentration of the recombinant protein of Carassius auratus Ferritin L is 10.0-80.0 μg / 200 μL buffer solution, wherein the optimal concentration is 80.0 μg / 200 μL buffer solution.

[0017] Based on a general technical concept, the present invention also provides an application of the above-mentioned combined crucian carp Ferritin L recombinant protein in the field of immune preparations or feed additives for aquatic animals. The combined crucian carp Ferritin L recombinant protein itself is an immune protein. The solution containing the combined crucian carp Ferritin L recombinant protein obtained by the above-mentioned preparation method can be directly used as an immune preparation, or can be directly added to the daily feed of aquatic animals as a feed additive.

[0018] Preferably, when the Ferritin L recombinant protein of the crucian carp is used as an immune preparation, the Ferritin L recombinant protein of the crucian carp dissolved in PBS (pH = 7.9) is injected into the fish intraperitoneally at a dose of 3.5 μg / g fish weight, which can effectively inhibit the proliferation ability of Aeromonas hydrophila in various organs of the crucian carp and reduce the inflammatory response caused by Aeromonas hydrophila.

[0019] Based on a general technical concept, the present invention also provides a primer for amplifying the Ferritin L gene sequence, including an upstream primer Ferritin L-F1 and a downstream primer Ferritin L-R1:

[0020] Upstream primer Ferritin L-F1: 5'-CCGGAATTCATGTCTCTAGTCAAGCAGAA-3' (as shown in SEQ ID NO: 3);

[0021] Downstream primer Ferritin L-R1: 5′-CCGCTCGAGTTAATGATGATGATGATGATGGAGCGTGTGCTTGTCA-3′ (shown in SEQ ID NO: 4).

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. The recombinant protein of the present invention, Ferritin L, has been shown to inhibit the growth of Aeromonas hydrophila and can be used to inhibit the growth of Aeromonas hydrophila and as an immune preparation or feed additive for aquatic animals, and has broad application potential.

[0024] 2. The preparation method of the present invention expresses the nucleotide sequence of the Ferritin L gene of the crucian carp through a recombinant strain, which can produce a large amount of the Ferritin L recombinant protein of the crucian carp, greatly reducing the production cost and having a high economic value.

[0025] 3. The present invention obtains the Ferritin L gene sequence of Hefang crucian carp for the first time, which not only enriches the gene library of Hefang crucian carp, but also can be used to prepare recombinant proteins, and further used to inhibit the growth of Aeromonas hydrophila, prepare immune preparations or feed additives for aquatic animals, and provide a new practical basis for the physiological immunity research of Hefang crucian carp.

[0026] 4. The combined crucian carp Ferritin L recombinant protein of the present invention is used as an immune preparation or feed additive for aquatic animals, which can reduce the use of antibiotics, reduce the occurrence of drug-resistant bacteria and reduce environmental pollution, and can also increase efficiency and income.

[0027] 5. The primers for amplifying the Ferritin L gene of the present invention can quickly and accurately amplify the Ferritin L gene of the Crucian Carp, thereby promoting the production efficiency of the Ferritin L recombinant protein of the Crucian Carp. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0029] Figure 1 This is an electrophoresis identification diagram of the PCR amplification product of the ORF sequence of the Hefang crucian carp ferritin L gene with specific restriction sites (EcoRI, XhoI) in Example 1 (M: DNA molecular weight standard; I: PCR product of the full-length sequence of the Hefang crucian carp ferritin L gene ORF with restriction sites);

[0030] Figure 2 This is an electrophoresis identification diagram of the expression vector pET32a-WR-Ferritin L double-enzyme digestion (EcoRI, XhoI) in Example 2 (M: DNA molecular weight standard; 1: pET32a-WR-Ferritin L plasmid; 2: double-enzyme digestion pET32a-WR-Ferritin L plasmid);

[0031] Figure 3The SDS-PAGE electrophoresis analysis and Western blot identification of the WR-Ferritin L recombinant protein expressed by the recombinant strain pET32a-WR-Ferritin L-BL21 in Example 2 are shown (M: protein molecular weight standard; 1: pET32a-BL21 induced product; 2: pET32a-WR-Ferritin L-BL21 induced product; 3: supernatant of the pET32a-WR-Ferritin L-BL21 induced product after fragmentation; 4: precipitate of the pET32a-WR-Ferritin L-BL21 induced product after fragmentation; 5: purified recombinant protein WR-Ferritin L; 6: Western blot verification of the purified recombinant protein WR-Ferritin L);

[0032] Figure 4 is a schematic diagram of the construction of the pET32a-WR-Ferritin L expression vector in Example 2;

[0033] Figure 5 This is a comparison diagram of the amino acid sequence of the crucian carp ferritin L in the combined formula in Example 2 and the amino acid sequence of human ferritin L;

[0034] Figure 6 This is the result of the secondary structure prediction analysis of the recombinant protein of Carassius auratus Ferritin L in Example 2;

[0035] Figure 7 This is the ELISA binding experiment between the recombinant protein of Ferritin L from Carassius auratus of Hefang at different concentrations and Aeromonas hydrophila in Example 2 (different letters indicate significant differences, P<0.05);

[0036] Figure 8 This is the effect of the treatment with the recombinant protein of Carassius auratus Ferritin L on the activity of Aeromonas hydrophila in Example 2 (different letters indicate significant differences, P < 0.05);

[0037] Figure 9 The effect of the Ferritin L recombinant protein on the load of Aeromonas hydrophila in fish after treatment in Example 2;

[0038] Figure 10 The expression changes of the Aeromonas hydrophila hemolysin hlyA gene in the liver, kidney, and spleen of fish treated with pET32a tag and Hefang crucian carp Ferritin L recombinant protein after infection with Aeromonas hydrophila in Example 2 (different letters indicate significant differences compared with the control group, P < 0.05);

[0039] Figure 11Figure 2 shows the changes in IL-1β-1 gene levels in the liver, kidney, and spleen of fish infected with Aeromonas hydrophila and treated with pET32a tag and Ferritin L recombinant protein from Hefang Crucian Carp in Example 2 (different letters indicate significant differences compared with the control group, P < 0.05);

[0040] Figure 12 Figure 2 shows the changes in IL-1β-2 gene levels in the liver, kidney, and spleen of fish infected with Aeromonas hydrophila and treated with pET32a tag and Ferritin L recombinant protein from Hefang Crucian Carp in Example 2 (different letters indicate significant differences compared with the control group, P < 0.05);

[0041] Figure 13 The changes in TNFα-1 gene levels in the liver, kidney, and spleen of fish infected with Aeromonas hydrophila in Example 2 and treated with pET32a tag and Ferritin L recombinant protein from Hefang Crucian Carp (different letters indicate significant differences compared with the control group, P < 0.05);

[0042] Figure 14 Figure 2 shows the changes in TNFα-2 gene levels in the liver, kidney, and spleen of fish infected with Aeromonas hydrophila and treated with pET32a tag and Ferritin L recombinant protein from Hefang Crucian Carp in Example 2 (different letters indicate significant differences compared with the control group, P < 0.05). DETAILED DESCRIPTION

[0043] To facilitate understanding of the present invention, the present invention will be described in more comprehensive and detailed form below in conjunction with the accompanying drawings and preferred embodiments. However, the protection scope of the present invention is not limited to the following specific embodiments.

[0044] Unless otherwise defined, all technical terms used hereinafter have the same meanings as those generally understood by those skilled in the art. The technical terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0045] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0046] Example 1:

[0047] A Hefang Crucian Carp Ferritin L gene, the nucleotide sequence of which is shown in SEQ ID NO: 1. The Hefang Crucian Carp Ferritin L gene ORF sequence with a specific enzyme cleavage site is obtained by PCR, and the specific steps are as follows:

[0048] Based on the conservation of the ferritin L gene sequence, specific primers were designed. The first primer was the upstream primer Ferritin L-F1: 5'-CCGGAATTCATGTCTCTAGTCAAGCAGAA-3' (the nucleotide sequence of which is shown in SEQ ID NO: 3), and the second primer was the downstream primer Ferritin L-R1: 5'-CCGCTCGAGTTAATGATGATGATGATGATGGAGCGTGTGCTTGTCA-3' (the nucleotide sequence of which is shown in SEQ ID NO: 4). The cDNA of the liver of the crucian carp (Carassius auratus) obtained by reverse transcription using the M-MLV reverse transcriptase kit (Promega, Madison, WI, USA) was used as a template. The ferritin L gene of the crucian carp (Carassius auratus) with restriction enzyme cleavage sites was amplified by touchdown PCR. The PCR amplification product was separated by electrophoresis and then directly cut into the gel. The ferritin L sequence of the crucian carp (Carassius auratus) was sequenced and confirmed to be the ferritin L gene of the crucian carp (Carassius auratus) with a length of 522 bp (the nucleotide sequence of which is shown in SEQ ID The PCR reaction conditions were as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 65°C for 30 s, and extension at 72°C for 35 s, for a total of 5 cycles; denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 35 s, for a total of 5 cycles; denaturation at 94°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 35 s, for a total of 30 cycles; and finally extension at 72°C for 10 min. The electrophoresis of the PCR amplification products is shown in Figure 1. Figure 1 .

[0049] Example 2:

[0050] The Hefang Crucian Carp Ferritin L recombinant protein was obtained by encoding the Hefang Crucian Carp Ferritin L gene of Example 1, and its amino acid sequence is shown in SEQ ID NO: 2.

[0051] The method for preparing the recombinant protein of Carassius auratus Ferritin L of the present invention comprises the following steps:

[0052] (1) The ORF sequence of the Ferritin L gene of Hefang Crucian Carp with specific enzyme cleavage sites was obtained by PCR:

[0053] The specific operation method is the same as that of Example 1, and the amino acid sequence of the Ferritin L gene of Hefang Crucian Carp is deduced based on the ORF nucleotide sequence (as shown in SEQ ID NO: 2).

[0054] The amino acid sequence of Ferritin L from Hefang Crucian Carp (WR-Ferritin L, as shown in SEQ ID NO: 2) and the amino acid sequence of Ferritin L from human (HsFerritin L) were aligned using ClustalX and GeneDoc sequence analysis software. It was found that the amino acid sequence of Ferritin L from Hefang Crucian Carp and the amino acid sequence of Ferritin L from human had a high homology ( Figure 5 ) and analyzed the secondary structure of Ferritin L protein in Hefang crucian carp by software ( Figure 6 ).

[0055] (2) Construction of the expression vector pET32a-WR-Ferritin L containing the gene sequence of Hefang crucian carp Ferritin L:

[0056] The DNA agarose gel recovery kit (Tiangen Biotech) was used to directly purify and recover the Ferritin L sequence of the crucian carp with enzyme cleavage sites after electrophoresis, and a Ferritin L sequence of about 522bp containing enzyme cleavage sites was obtained. The purified fragment and the vector plasmid pET32a (purchased from Novagen) were double-digested with restriction endonucleases EcoRI and XhoI, and the above-mentioned Ferritin L sequence fragment and the large vector fragment were separated and recovered by agarose gel electrophoresis. Then, the Ferritin L sequence fragment and the large vector pET32a fragment were mixed in a volume ratio of 4:1, and connected with T4 ligase (purchased from TAKARA) at 16°C for about 15 hours. Then, the plasmid obtained after connection was transferred into Escherichia coli DH5a using the CaCl2 method, and the plasmid with Amp was screened on the LB plate. + Resistant transformants. The plasmid was extracted using standard methods and sent to Invitrogen for sequencing. The sequencing results were compared and confirmed to be the gene sequence of Ferritin L from Hefang Crucian Carp. It was correctly inserted into the expression vector pET32a. The recombinant plasmid containing the Ferritin L gene sequence from Hefang Crucian Carp was named pET32a-WR-Ferritin L. The expression vector pET32a-WR-Ferritin L was double-digested with EcoRI and XhoI. The enzyme digestion analysis is shown in the figure. Figure 2 The plasmid construction process is shown in Figure 4 .

[0057] (3) Construction of the recombinant E. coli strain pET32a-WR-Ferritin L-BL21 capable of efficiently expressing the recombinant protein of Ferritin L from Hefang crucian carp:

[0058] According to the CaCl2 transformation method, the recombinant plasmid pET32a-WR-FerritinL obtained by T4 ligase ligation was transferred into Escherichia coli BL21 (E. coli BL21) which can efficiently express the pET series expression vector containing phage T7 promoter. The Amp + The resistant transformant, the recombinant bacteria is pET32a-WR-FerritinL-BL21.

[0059] (4) Production of Ferritin L recombinant protein from Carassius auratus using the recombinant Escherichia coli pET32a-WR-Ferritin L-BL21:

[0060] Recombinant BL21 monoclonal engineering bacteria were picked and inoculated into the medium containing Amp + Culture in LB medium with resistance at 37°C and 200 rpm, expand the strain and replace with fresh medium containing Amp + The resistant LB medium was stopped when OD600 reached 0.6, and about 1mM IPTG was added for induction for 3 hours to express the Ferritin L gene of the crucian carp. After induction, the cells were collected at 7500rpm and 4℃; TEB buffer and an appropriate amount of lysozyme were added to suspend the cells, and the cells were allowed to stand at 4℃ for 6 hours, and then ultrasonically disrupted in an ice bath until clear, thereby obtaining the Ferritin L recombinant protein of the crucian carp (its amino acid sequence is shown in SEQ ID NO: 2). The cells were resuspended in Buffer A containing 30% Trition X-100, and allowed to stand on ice for 30 minutes. Buffer B containing urea was added to dissolve the expressed product, the Ferritin L recombinant protein of the crucian carp at room temperature. The dissolved protein was purified according to the His-tag bind resin (Millipore) method. The results of SDS-PAGE electrophoresis analysis of the expressed product, the Ferritin L recombinant protein of the crucian carp are shown in FIG. Figure 3 .

[0061] In order to further identify the various indicators of the recombinant protein of Hefang Crucian Carp Ferritin L, the following experiments were conducted:

[0062] 1. Antigen activity identification experiment of Ferritin L recombinant protein of Hefang Crucian Carp:

[0063] Western blotting was used to immunoidentify the recombinant protein of crucian carp Ferritin L. The primary antibody used was mouse His monoclonal antibody (Novagen), and the secondary antibody used was horse anti-mouse IgG-AP (Beijing Dingguo Biological Company). Figure 3, showing that the mouse His monoclonal antibody can recognize the recombinant protein of Ferritin L expressed by Escherichia coli, proving that the obtained protein is the recombinant protein of Ferritin L of Crucian carp.

[0064] 2. Aeromonas hydrophila ELISA test:

[0065] Aeromonas hydrophila was inoculated into LB medium and cultured continuously at 30°C and 200 rpm until OD600 = 0.6. The bacteria were then resuspended in PBS buffer after centrifugation and the bacterial concentration was adjusted to 1 × 10 7 CFUmL -1 The above bacteria were inoculated into a 96-well ELISA plate; after overnight at 4°C, the ELISA plate was blocked with 5% skim milk for 2 hours, then washed with 0.5% Tween-20 / PBS, and then 2.5 μg, 5.0 μg, 10.0 μg, 20.0 μg, 40.0 μg and 80.0 μg of pET32a tag protein / 200 μL PBS (pH 7.9) and combined crucian carp Ferritin L recombinant protein (WR-FerritinL protein) / 200 μL PBS (pH 7.9) were added, respectively, and incubated at room temperature for 1.5 hours; then washed with 0.5% Tween-20 / PBS, and the ELISA plate was blocked with mouse His-tag primary antibody and horse anti-mouse HRP secondary antibody, respectively; finally, 200 μL TMB solution was added for color development in a dark environment, and 2 M sulfuric acid was added after about 30 minutes to terminate the display reaction, and the reading analysis was performed at OD450. Calculation formula: OD450 reading of experimental group / OD450 reading of negative control group, the results are shown in Figure 7 .

[0066] The results showed that compared with the pET32a tag control group, as the concentration of the combined crucian carp Ferritin L recombinant protein (WR-Ferritin L protein) gradually increased, its binding ratio with Aeromonas hydrophila also gradually increased, and reached the highest binding ratio at 80.0 μg, indicating that the combined crucian carp Ferritin L recombinant protein can bind to Aeromonas hydrophila in a pH = 7.9 buffer environment, and there is a dose relationship.

[0067] 3. Growth activity test of Aeromonas hydrophila:

[0068] Aeromonas hydrophila was inoculated into LB medium and cultured continuously at 30°C and 200 rpm until OD600 = 0.6. The bacteria were then resuspended in PBS solution after centrifugation and the bacterial concentration was adjusted to 1×10 7CFU mL -1 . 2.5μg, 5.0μg, 10.0μg, 20.0μg, 40.0μg and 80.0μg of pET32a tag protein / 200μL PBS (pH7.9) and Ferritin L recombinant protein from Carassius auratus (WR-Ferritin L protein) / 200μL PBS (pH 7.9) were mixed with Aeromonas hydrophila solution at a volume ratio of 4:1, and added to a 96-well culture plate, mixed with 10μL of bacterial activity detection reagent CCK-8 (BestBio, Shanghai). After continuing to culture at 30℃ for 2.5h, the readings were analyzed at OD450, and the Trx-treated group was used as the control group of the experiment. Calculation formula: (OD450 reading of the experimental group / OD450 reading of the control group) × 100%, the results are shown in Figure 8 .

[0069] The results showed that in a buffer environment of pH = 7.9, as the concentration of the added recombinant Ferritin L protein (WR-Ferritin L protein) gradually increased, the activity of the co-cultured Aeromonas hydrophila gradually decreased and reached the lowest activity value at 80.0 μg. This indicates that the recombinant Ferritin L protein of WR-Ferritin L has the activity to inhibit the growth of Aeromonas hydrophila in a buffer environment of pH = 7.9.

[0070] 4. Animal experiments:

[0071] 1) Animal handling and organ bacterial load test

[0072] Healthy crucian carp of similar size and from the same batch (approximately 23.17 ± 0.57 g) were selected and temporarily housed in 1.0 m × 0.65 m × 0.65 m storage boxes. Each box was filled with pre-aerated fresh water at a temperature of approximately 25 ± 1 °C. Activated Aeromonas hydrophila was resuspended in PBS solution and adjusted to 1 × 10 7 CFU mL -1 . After 50 μL of the above-mentioned resuspended bacterial solution was intraperitoneally injected for about 25 minutes, the fish were intraperitoneally injected with pET32a tag protein and combined crucian carp Ferritin L recombinant protein (WR-Ferritin L protein) dissolved in PBS (pH=7.9) at a dose of 3.5 μg / g fish weight. About 24 hours after the injection, the liver, kidney and spleen of the fish were separated and about 0.1 g of tissue was weighed from each of them and ground in PBS. Then, the tissues were spread on LB plates and cultured at 30°C for about 8 hours. The results are shown in Table 1. Figure 9 .

[0073] 2) Pathogen proliferation test in Hefang crucian carp

[0074] The DNA from the liver, kidney, and spleen was isolated using the Tissue DNA kit (Omega), and the concentration of the isolated DNA was adjusted to approximately 100 ng / μL. The total volume of the real-time fluorescence quantitative qPCR system was 20 μL: 0.8 μL of diluted upstream and downstream primers, 5.0 μL of ultrapure water, 3 μL of DNA solution, 0.4 μL of ROX reference dye, and 10 μL of SYBR premix ExTaq TM II (Perfect Real Time) (TaKaRa, Dalian, China). The reaction procedure included: 95°C pre-denaturation for 2 min; 95°C denaturation for 30 s; 58°C annealing for 35 s, for a total of 40 cycles. The amplified products were subjected to melting curve analysis. Each qRT-PCR experiment was repeated three times. The experimental results were analyzed using 7500SDS software (Applied Biosystems, USA). Figure 10 .

[0075] 3) Detection of changes in inflammatory cytokine expression in Hefang crucian carp caused by bacterial infection

[0076] Total RNA was extracted from the samples using Trizol reagent (Invitrogen), and the quality and purity of the extracted RNA were determined by the values ​​at 260 nm and 260 / 280 nm. TM The reverse transcription experiment was performed using 1000 ng of total mRNA using the M-MuLV Reverse Transcriptase Kit (MBI Fermentas, USA). The reversed cDNA was subjected to real-time fluorescence quantitative qRT-PCR experiments. The total system was 20 μL: 0.8 μL of diluted upstream and downstream primers, 6.0 μL of ultrapure water, 2 μL of reverse transcribed cDNA template, 0.4 μL of ROX reference dye and 10 μL of SYBR premix Ex Taq TM II (Perfect Real Time) (TaKaRa, Dalian, China). The reaction procedure included: 95°C pre-denaturation for 2 min; 95°C denaturation for 30 s; 59°C annealing for 35 s, for a total of 40 cycles. The amplified products were subjected to melting curve analysis. Each qRT-PCR experiment was repeated three times. The experimental results were analyzed using 7500SDS software (Applied Biosystems, USA). Changes in IL-1β-1 gene levels are shown in Figure 11 , changes in IL-1β-2 gene levels are shown in Figure 12 , changes in TNFα-1 gene levels are shown in Figure 13 , changes in TNFα-2 gene levels are shown in Figure 14 .

[0077] The above experiments show that 25 minutes after infection with Aeromonas hydrophila, injection of 3.5 μg / g fish weight of the recombinant Ferritin L protein (WR-Ferritin L protein) dissolved in PBS (pH = 7.9) effectively inhibited the proliferation of Aeromonas hydrophila in various organs of the crucian carp and reduced the inflammatory response caused by Aeromonas hydrophila. This indicates that the recombinant Ferritin L protein has potential for development in antibacterial and antimicrobial applications and can also be used directly as an immune preparation or feed additive for aquatic animals. Sequence Listing <110> Hunan Normal University <120> Ferritin L gene, recombinant protein, preparation method, application and primers of Hefang crucian carp <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 522 <212> DNA <213> Hefang Crucian Carp (WR) <400> 1 atgtctctag tcaagcagaa tcttcacccg aataatgagg caaacatcaa caaactggtc 60 aacctcaaac tgacggcctc atatgtgtat ctctcactgg gaatgtattt tgatagagat 120 gatgtggctc tgccaaactt ctcaaagttt ttcttggagc gttcactgaa ggagcggggat 180 caggcggagc acctgctgga gtatcaaaac acaagaggag gacgaatcgt tctgcagacc 240 gttgcgaagc ccagtcgtga tgattggaaa ggaggtatgg aggctctcac tttttctctg 300 gaccatcaaa agtctcttaa ccaatccctg ctggaggtcc ataaagcagc tggagaaaac 360 tctgaccctc atctgtgtga tttcctagag agcaacttct ttactgacag tcatgacacc 420 attaagacgc tgggtgacta cgctggcagc ctgagtcgcc tcatctcttc tgacccgcat 480 ggaaaaatgg gagagtacct gtttgacaag cacacgctct ga 522 <210> 2 <211> 173 <212> PRT <213> Hybrid crucian carp (WR) <400> 2 Met Ser Leu Val Lys Gln Asn Leu His Pro Asn Asn Glu Ala Asn Ile 1 5 10 15 Asn Lys Leu Val Asn Leu Lys Leu Thr Ala Ser Tyr Val Tyr Leu Ser 20 25 30 Leu Gly Met Tyr Phe Asp Arg Asp Asp Val Ala Leu Pro Asn Phe Ser 35 40 45 Lys Phe Phe Leu Glu Arg Ser Leu Lys Glu Arg Asp Gln Ala Glu His 50 55 60 Leu Leu Glu Tyr Gln Asn Thr Arg Gly Gly Arg Ile Val Leu Gln Thr 65 70 75 80 Val Ala Lys Pro Ser Arg Asp Asp Trp Lys Gly Gly Met Glu Ala Leu 85 90 95 Thr Phe Ser Leu Asp His Gln Lys Ser Leu Asn Gln Ser Leu Leu Glu 100 105 110 Val His Lys Ala Ala Gly Glu Asn Ser Asp Pro His Leu Cys Asp Phe 115 120 125 Leu Glu Ser Asn Phe Phe Thr Asp Ser His Asp Thr Ile Lys Thr Leu 130 135 140 Gly Asp Tyr Ala Gly Ser Leu Ser Arg Leu Ile Ser Ser Asp Pro His 145 150 155 160 Gly Lys Met Gly Glu Tyr Leu Phe Asp Lys His Thr Leu 165 170 <210> 3 <211> 29 <212> DNA <213> Artificial Sequence <400> 3 ccggaattca tgtctctagt caagcagaa 29 <210> 4 <211> 46 <212> DNA <213> Artificial Sequence <400> 4 ccgctcgagt taatgatgat gatgatgatg gagcgtgtgc ttgtca 46

Claims

1. A use of a recombinant protein of Carassius auratus Ferritin L in the preparation of an inhibitory agent against Aeromonas hydrophila, characterized in that: The amino acid sequence of the recombinant protein of Carassius auratus Ferritin L is shown in SEQ ID NO:

2.

2. The use according to claim 1, characterized in that The nucleotide sequence of the gene encoding the recombinant protein of Carassius auratus Ferritin L is shown in SEQ ID NO: 1.

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