CN111471687A

CN111471687A - Synthetic crucian Ferritin L gene, recombinant protein, preparation method and application thereof, and primer

Info

Publication number: CN111471687A
Application number: CN202010213556.4A
Authority: CN
Inventors: 罗盛伟; 刘少军; 罗紫烨; 罗凯坤; 覃钦博; 张纯; 陶敏; 刘庆峰; 胡方舟; 王石
Original assignee: Hunan Normal University
Current assignee: Hunan Normal University
Priority date: 2020-03-24
Filing date: 2020-03-24
Publication date: 2020-07-31

Abstract

The invention discloses a synthetic crucian carp Ferritin L gene and a recombinant protein thereof, wherein the nucleotide sequence of the synthetic crucian carp Ferritin L gene 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 synthetic crucian Ferritin L gene not only enriches the gene bank of the synthetic crucian carp, but also can be applied to the preparation of the recombinant protein, experiments prove that the recombinant protein can inhibit the growth activity of aeromonas hydrophila, and provides a new practical basis for the physiological immune research of the synthetic crucian carp.

Description

Synthetic crucian Ferritin L gene, recombinant protein, preparation method and application thereof, and primer

Technical Field

The invention belongs to the technical field of genetic engineering research and development, and particularly relates to a synthetic crucian carp Ferritin L gene, a synthetic crucian carp Ferritin L recombinant protein, a preparation method of a synthetic crucian carp Ferritin L recombinant protein, application of the synthetic crucian carp Ferritin L recombinant protein, and a primer for amplifying the synthetic crucian carp Ferritin L gene.

Background

Iron ions are one of the essential trace elements in organisms, and have direct connection with ontogenesis, oxidative stress and disease occurrence. In addition, both biotic stress (infection of germs, viruses and the like) and abiotic stress (heavy metals) can cause imbalance of dynamic regulation and control of iron ions in animals and accumulation in cells, so that the level of active oxygen is increased rapidly, and finally macromolecular substances such as intracellular DNA, protein, lipid and the like are damaged.

In higher mammals, mainly H-type and L-type ferritins are mainly found, while related studies indicate that lower vertebrate ferritins mainly contain H-type and M-type subunits, functionally, higher mammalian ferritins H have a Ferritin center and are mainly responsible for the oxidation of iron ions, while ferritins L coat has negative residues capable of forming an intrachain salt bridge structure and promoting the nucleation of iron hydride compounds, while lower vertebrate ferritins L still retain a portion of the Ferritin reaction center, thus have an iron ion oxidation effect, and also have an iron ion oxidation effect, while lower vertebrate Ferritin has a role in forming a nuclear structure and promoting the nucleation of iron hydride compounds, and thus, the lower vertebrate Ferritin family has a role in regulating the activity of iron oxide, and thus has a role in resisting the metabolism of Ferritin H, and also has a role in regulating the metabolism of iron ions, and is found in the family of fishes with no toxicity, and is a family of higher vertebrate animals, and the family of animals which is known to have no regulatory activity of iron oxide, and thus has a role in regulating the metabolism of Ferritin, and is related to the family of animals, and is found that the family of Ferritin, and is related to the family of animals, and is related to the family of animals, which is related to which the family of animals Ferritin, which is known Ferritin, which is related to which, the family of animals Ferritin, which, the family of animals Ferritin, and is found, the family of animals, the family of animals Ferritin, the family of animals Ferritin, and of animals Ferritin, the family of animals, the family of.

The synthetic crucian carp (WR,2n is 100) is a freshwater fish developed by a national key laboratory of the science of freshwater fish development biology of province and ministry of Master university in Hunan province, and is also a new species of national-grade aquatic products. The resultant crucian carp (WR,2n ═ 100) is the first generation of the hybridization between japanese white crucian carp (Carassius cuviaeri, WCC,;, 2n ═ 100) and red crucian carp (Carassius auratus red var, RCC,;, 2n ═ 100). The appearance of the synthetic crucian carp is similar to that of a wild crucian carp, the reproductive capacity is strong, the growth speed is high, and meanwhile, the synthetic crucian carp has high fertility rate and hatchability and is suitable for large-scale production. In addition, the flavor amino acid in the muscles of the crucian carps is obviously higher than that of the parents, the meat is fresh and tender, the nutritional value is high, and the crucian carps are deeply popular with culturists and consumers. However, the serious attack to the healthy culture of the fishes is caused by the problems of rapid climate change and serious pollution of water, and the problem is also a problem which is difficult to solve. Among them, aeromonas hydrophila is one of the main pathogenic microorganisms of bacterial diseases of freshwater fishes, which can cause septicemia of aquatic animals and often cause serious economic loss to freshwater aquaculture industry. The development of high-quality fish-derived feed additive protein is an effective way for solving the problems, is favorable for the development and application of improved fish feed, and can reduce environmental pollution and increase efficiency and income.

Disclosure of Invention

The invention aims to solve the technical problems, overcome the defects and defects in the background technology and provide a newly cloned synthetic crucian Ferritin L gene, a synthetic crucian Ferritin L recombinant protein, a preparation method of a synthetic crucian Ferritin L recombinant protein, application of a synthetic crucian Ferritin L recombinant protein and a primer for amplifying the synthetic crucian Ferritin L gene.

In order to solve the technical problems, the technical scheme provided by the invention is as follows:

the discovery of the synthetic crucian Ferritin L gene not only enriches the gene library of the synthetic crucian, but also can be applied to preparation of recombinant protein, further applied to inhibition of growth of aeromonas hydrophila and used as an immune preparation or feed additive of aquatic animals, and provides a new practical basis for the physiological immune research of the synthetic crucian.

The synthetic crucian carp Ferritin L recombinant protein has an amino acid sequence shown in SEQ ID NO. 2, or is a protein with the same or higher activity obtained by substituting, deleting and/or adding one or more amino acids and/or modifying the tail end of the amino acid sequence shown in SEQ ID NO. 2, wherein the synthetic crucian carp Ferritin L recombinant protein is obtained by encoding a synthetic crucian carp Ferritin L gene, and the nucleotide sequence of the synthetic crucian carp Ferritin L gene is shown in SEQ ID NO. 1.

Based on a general technical concept, the invention also provides a preparation method of the synthetic crucian Ferritin L recombinant proteinThe method comprises the following steps of amplifying a crucian Ferritin L gene sequence of a synergestin by a PCR method, adding enzyme cutting sites, purifying and carrying out double enzyme cutting on a PCR product, connecting the PCR product to an expression vector pET32a, and then connecting the expression vector to a CaCl₂The transformation method comprises the step of transferring an expression vector pET32a containing the synthetic crucian Ferritin L gene into escherichia coli B L21 (E. coli B L21) for induction expression, and finally obtaining the synthetic crucian Ferritin L recombinant protein.

The preparation method preferably comprises the specific operation of amplifying the synthetic crucian carp Ferritin L gene by a PCR method and adding enzyme cutting sites, and comprises the following steps of amplifying the synthetic crucian carp Ferritin L gene with the enzyme cutting sites by the PCR method by taking synthetic crucian liver cDNA obtained by reverse transcription as a template, carrying out sequencing confirmation on the synthetic crucian Ferritin L sequence by directly cutting gel after electrophoretic separation, wherein the nucleotide sequence of the synthetic crucian Ferritin L gene is shown as SEQ ID NO: 1.

Preferably, the reaction conditions of the PCR amplification method are 94 ℃ pre-denaturation for 5min, 94 ℃ denaturation for 30s, 65 ℃ annealing for 30s and 72 ℃ extension for 35s, 5 cycles of 94 ℃ denaturation for 30s, 60 ℃ annealing for 30s and 72 ℃ extension for 35s, 5 cycles of 94 ℃ denaturation for 30s, 56 ℃ annealing for 30s and 72 ℃ extension for 35s, 30 cycles of 94 ℃ denaturation for 30s and 72 ℃ extension for 10min, the primers adopted by the PCR amplification method are an upstream primer Ferritin L-F1 and a downstream primer Ferritin L-R1, the nucleotide sequences of the upstream primer Ferritin L-F1 are shown in SEQ ID NO: 3, and the nucleotide sequences of the downstream primer Ferritin L-R1 are shown in SEQ ID NO: 4.

Preferably, the specific operation of purifying and carrying out double enzyme digestion on the PCR product and connecting the PCR product to an expression vector pET32a comprises the following steps of directly purifying and recovering the PCR product (namely, a synthetic crucian Ferritin L sequence with enzyme digestion sites after electrophoresis) by using a DNA agarose gel recovery kit, then carrying out double enzyme digestion by using restriction enzymes EcoRI and XhoI, after the double enzyme digestion is finished, carrying out product recovery on the synthetic crucian L sequence which is successfully digested, carrying out double enzyme digestion on an expression vector pET32a by using restriction enzymes EcoRI and XhoI, mixing the obtained large vector fragment with the synthetic crucian Ferritin L sequence which is successfully digested, and carrying out connection for 12-18h at 15-17 ℃ by using T4 ligase, namely successfully connecting the synthetic crucian L gene sequence to the expression vector pET32 a.

With the gradual and deep research on the fish Ferritin family members, the Ferritin L as a newly found member in the fish Ferritin family has certain difference with other fish Ferritin family members in amino acid composition, but also contains a conserved Ferritin-like diiron structure, and the preliminary research also indicates that the fish Ferritin L directly participates in the immune regulation and control process caused by fish viruses or pathogenic bacteria, so the development and utilization of the fish-derived Ferritin L recombinant protein are beneficial to research on the immune regulation and control and antibacterial mechanism of fish and the development of a feed immune preparation or an additive of economic aquatic animals.

Based on a general technical concept, the invention also correspondingly provides an application of the synthetic crucian Ferritin L recombinant protein in inhibiting the growth of Aeromonas hydrophila (Aeromonas hydrophylla).

The application method preferably comprises the following steps of mixing and contacting the synthetic crucian Ferritin L recombinant protein and the aeromonas hydrophila in a buffer environment with the pH value of 7.2-8.2, and finishing the inhibition of the aeromonas hydrophila after the reaction is terminated.

More preferably, the concentration of the synthetic crucian Ferritin L recombinant protein is 10.0-80.0 μ g/200 μ L buffer, wherein the optimal concentration is 80.0 μ g/200 μ L buffer.

Based on a general technical concept, the invention also correspondingly provides an application of the synthetic crucian carp Ferritin L recombinant protein in the field of immune preparations or feed additives of aquatic animals, the synthetic crucian carp Ferritin L recombinant protein is immune protein, and the solution containing the synthetic crucian carp Ferritin L recombinant protein obtained by the preparation method can be directly used as an immune preparation or a feed additive to be directly added into daily feeds of the aquatic animals for use.

Preferably, when the synthetic crucian carp Ferritin L recombinant protein is used as an immune preparation, the synthetic crucian carp Ferritin L recombinant protein dissolved in PBS (pH7.9) is injected into the abdominal cavity of a fish body at a dose of 3.5 mu g/g of fish weight, so that the proliferation capacity of aeromonas hydrophila in each organ in the synthetic crucian carp can be effectively inhibited, and the inflammatory reaction caused by the aeromonas hydrophila is reduced.

Based on a general technical concept, the invention also correspondingly provides a primer for amplifying the Ferritin L gene sequence, which comprises an upstream primer Ferritin L-F1 and a downstream primer Ferritin L-R1:

an upstream primer Ferritin L-F1: 5'-CCGGAATTCATGTCTCTAGTCAAGCAGAA-3' (shown as SEQ ID NO: 3);

the downstream primer Ferritin L-R1: 5'-CCGCTCGAGTTAATGATGATGATGATGATGGAGCGTGTGCTTGTCA-3' (shown as SEQ ID NO: 4).

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

1. experiments prove that the synthetic crucian Ferritin L recombinant protein can inhibit the growth activity of aeromonas hydrophila, can be applied to inhibiting the growth of the aeromonas hydrophila, can be used as an immune preparation or a feed additive of aquatic animals, and has wide application potential.

2. According to the preparation method, the nucleotide sequence of the synthetic crucian carp Ferritin L gene is expressed by the recombinant strain, so that the synthetic crucian carp Ferritin L recombinant protein can be produced in a large scale, the production cost is greatly reduced, and the preparation method has high economic value.

3. The invention obtains the Ferritin L gene sequence of the crucian carp, not only enriches the gene library of the crucian carp, but also can be applied to the preparation of recombinant protein, and further applied to the inhibition of the growth of aeromonas hydrophila and the preparation of immune preparations or feed additives of aquatic animals, thereby providing a new practical basis for the physiological immune research of the crucian carp.

4. The synthetic crucian Ferritin L recombinant protein is used as an immune preparation or a feed additive of aquatic animals, can reduce the use of antibiotics, reduce the occurrence of drug-resistant bacteria and environmental pollution, and can also enhance the efficiency and the income.

5. The primer for amplifying the crucian carp Ferritin L gene can quickly and accurately amplify the crucian carp Ferritin L gene, and promotes the production efficiency of the crucian carp Ferritin L recombinant protein.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.

FIG. 1 is an electrophoretic identification chart of the PCR amplification product of the synthetic crucian Ferritin L gene ORF sequence with specific restriction enzyme cutting sites (EcoRI, XhoI) in example 1 (M: DNA molecular weight standard; 1: synthetic crucian Ferritin L gene ORF full-length sequence PCR product with restriction enzyme cutting sites);

FIG. 2 shows the electrophoretic patterns of the expression vector pET32a-WR-Ferritin L double-digested (EcoRI, XhoI) in example 2 (M: DNA molecular weight standard; 1: pET32a-WR-Ferritin L plasmid; 2: pET32a-WR-Ferritin L plasmid);

FIG. 3 is a SDS-PAGE analysis and Western blot identification of the recombinant protein WR-Ferritin L0-B L21 expression product of the recombinant strain pET32a-WR-Ferritin L-B L in example 2 (M: protein molecular weight standard; 1: pET32a-B L121 induction product; 2: pET32a-WR-Ferritin L-B L21 induction product; 3: supernatant of pET32a-WR-Ferritin L-B L21 induction product after disruption; 4: precipitation of pET32a-WR-Ferritin L-B L21 induction product after disruption; 5: WR-Ferritin L after purification; 6: Western blot validation of the recombinant protein WR-Ferritin L after purification);

FIG. 4 is a diagram showing the construction of pET32a-WR-Ferritin L expression vector in example 2;

FIG. 5 is a comparison of the amino acid sequences of the synthetic crucian carp Ferritin L and the human Ferritin L in example 2;

FIG. 6 shows the result of predictive analysis of the secondary structure of the recombinant protein of the synthetic crucian carp Ferritin L in example 2;

FIG. 7 is the combination experiment of different concentrations of the recombinant protein of the synthetic crucian Ferritin L and the E L ISA of Aeromonas hydrophila in example 2 (different letters indicate significant difference, P < 0.05);

FIG. 8 is the effect of treatment of the synthetic Carassius auratus Ferritin L recombinant protein on Aeromonas hydrophila activity in example 2 (different letters indicate significant differences, P < 0.05);

FIG. 9 is the load effect on Aeromonas hydrophila in fish after the treatment of synthetic crucian Ferritin L recombinant protein in example 2;

FIG. 10 is the change in expression of the aerolysin hlyA gene in the liver, kidney and spleen of fish treated with pET32a tag and the synthetic crucian Ferritin L recombinant protein after infection with Aeromonas hydrophila in example 2 (different letters indicate significant difference compared to the control group, P < 0.05);

FIG. 11 is the gene level changes of the liver, kidney and spleen I L-1 β -1 of the fish treated with pET32a tag and the synthetic crucian Ferritin L recombinant protein after Aeromonas hydrophila infection in example 2 (different letters indicate significant difference compared with the control group, P < 0.05);

FIG. 12 is the gene level changes of the liver, kidney and spleen I L-1 β -2 of the fish treated with pET32a tag and the synthetic crucian Ferritin L recombinant protein after Aeromonas hydrophila infection in example 2 (different letters indicate significant difference compared with the control group, P < 0.05);

FIG. 13 shows the level changes of TNF α -1 gene in liver, kidney and spleen of fish treated with pET32a tag and synthetic crucian Ferritin L recombinant protein after Aeromonas hydrophila infection in example 2 (different letters indicate significant differences compared with the control group, P < 0.05);

FIG. 14 shows the change in the liver, kidney and spleen TNF α -2 gene levels of fish treated with pET32a tag and the synthetic crucian Ferritin L recombinant protein after Aeromonas hydrophila infection in example 2 (different letters indicate significant difference compared to the control group, P < 0.05).

Detailed Description

In order to facilitate understanding of the invention, the invention will be described more fully and in detail with reference to the accompanying drawings and preferred embodiments, but the scope of the invention is not limited to the specific embodiments below.

Unless otherwise defined, all terms of art used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.

Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.

Example 1:

a synthetic crucian carp Ferritin L gene has a nucleotide sequence shown in SEQ ID NO. 1, and the synthetic crucian carp Ferritin L gene ORF sequence with specific enzyme cutting sites is obtained by a PCR method, and the specific operation steps are as follows:

specific primers are designed according to the conservation of the gene sequence of Ferritin L, the first primer is an upstream primer Ferritin L-F1: 5'-CCGGAATTCATGTCTCTAGTCAAGCAGAA-3' (the nucleotide sequence of the upstream primer is shown as SEQ ID NO: 3), the second primer is a downstream primer Ferritin L-R1: 5'-CCGCTCGAGTTAATGATGATGATGATGATGGAGCGTGTGCTTGTCA-3' (the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 4), a synthetic crucian Ferritin L gene with an enzyme cutting site is amplified by using an M-M L V reverse transcriptase kit (Promega, Madison, Wis., USA) reverse transcription as a template through a touchdown PCR (touchdown PCR) technology, the obtained PCR amplification product is subjected to electrophoretic separation and direct gel cutting to sequence confirmation on the synthetic crucian Ferritin L sequence, the synthetic crucian Ferritin L gene is determined, the length of 522bp (the nucleotide sequence of the PCR amplification product is shown as SEQ ID NO: 1), the PCR amplification conditions are 94 min, 72 s denaturation extension is carried out at the temperature of 30 ℃, 5s annealing is carried out at the temperature of 30 ℃, 5 ℃ and 30s, 5s annealing is carried out through a cycle of denaturation cycle, and 5s, and 5 ℃ and 5s annealing at the temperature of 30 ℃, and 5 ℃ of 60 ℃ and 30 ℃ of 5 and 30, and 5s, and 5 and 30 ℃ are carried out a cycle of the annealing at the total cycle of the temperature of the PCR amplification product, and 30.

Example 2:

the synthetic crucian carp Ferritin L recombinant protein is obtained by encoding the synthetic crucian carp Ferritin L gene of example 1, and the amino acid sequence of the recombinant protein is shown as SEQ ID NO. 2.

The preparation method of the synthetic crucian Ferritin L recombinant protein comprises the following steps:

(1) obtaining a synthetic crucian Ferritin L gene ORF sequence with a specific enzyme cutting site by a PCR method:

the specific operation method is the same as that in example 1, and the amino acid sequence (shown as SEQ ID NO: 2) of the synthetic crucian Ferritin L gene is deduced according to the ORF nucleotide sequence.

The amino acid sequence of the synthetic crucian carp Ferritin L (WR-Ferritin L shown as SEQ ID NO: 2) and the amino acid sequence of the human Ferritin L (HsFerritin L) are compared by using two sequence analysis software of ClustalX and GeneDoc, the amino acid sequence of the synthetic crucian carp Ferritin L and the amino acid sequence of the human Ferritin L are found to have higher homology (figure 5), and the secondary structure of the synthetic crucian carp L protein is analyzed by the software (figure 6).

(2) Constructing an expression vector pET32a-WR-Ferritin L containing the synthetic crucian carp Ferritin L gene sequence:

directly purifying and recycling the electrophoretic synthetic crucian Ferritin L sequence with the restriction enzyme sites by using a DNA agarose gel recycling kit (Tiangen Biotech) to obtain about 522bp synthetic crucian Ferritin L sequence containing the restriction enzyme sites, separating and recycling the cut Ferritin L sequence fragment and a carrier large fragment by agarose gel electrophoresis after double digestion of restriction enzymes EcoRI and XhoI of a carrier plasmid pET32a (purchased from Novagen), mixing the cut Ferritin L sequence fragment and the cut carrier pET32a large fragment in a volume ratio of 4:1, connecting the cut Ferritin L sequence fragment and the cut carrier pET32a large fragment at 16 ℃ by using T4 ligase (purchased from TAKARA), and then using CaCl₂The method comprises transferring the plasmid obtained after ligation into Escherichia coli DH5a, and screening Amp on L B plate⁺Extracting plasmid by standard method, sending to Invitrogen company for sequencing, comparing the sequencing result, confirming the gene sequence of the synthetic crucian Ferritin L, correctly inserting into expression vector pET32a, naming the recombinant plasmid containing the synthetic crucian Ferritin L gene sequence as pET32a-WR-Ferritin L, using EcoRI and XhoI to carry out double digestion on the expression vector pET32a-WR-Ferritin L, and the enzyme digestion analysis chart is shown in figure 2. the plasmid construction flow is shown in figure 4。

(3) Constructing an escherichia coli recombinant strain pET32a-WR-Ferritin L-B L21 capable of efficiently expressing a synthetic crucian Ferritin L recombinant protein:

according to CaCl₂Transformation method, the recombinant plasmid pET32a-WR-Ferritin L obtained by the T4 ligase ligation was transformed into E.coli B L21 (E.coli B L21) capable of efficiently expressing pET series expression vector cloned in phage T7 promoter, and the plasmid with Amp was screened using L B plate⁺The resistant transformant, the recombinant bacterium is pET32a-WR-Ferritin L-B L21.

(4) The Escherichia coli recombinant bacterium pET32a-WR-Ferritin L-B L21 is used for producing a synthetic crucian Ferritin L recombinant protein:

respectively selecting recombinant B L21 monoclonal engineering bacteria, inoculating to Amp⁺Culturing in resistant L B medium at 37 deg.C and 200rpm, enlarging strain, and replacing fresh strain with Amp⁺The preparation method comprises the steps of stopping a resistant L B culture medium when OD600 is 0.6, adding about 1mM IPTG (isopropyl-beta-thiogalactoside) for induction for 3 hours to enable a synthetic crucian Ferritin L gene to be expressed, collecting thalli at 7500rpm after induction at 4 ℃, adding TEB Buffer and a proper amount of lysozyme to suspend the thalli, standing at 4 ℃ for 6 hours, carrying out ultrasonic crushing in an ice bath to obtain synthetic crucian L recombinant protein (the amino acid sequence of the synthetic crucian L recombinant protein is shown in SEQ ID NO: 2), then using Buffer A containing 30% Trition X-100 to carry out heavy suspension, standing on ice for 30 minutes, adding Buffer B containing urea to dissolve the expression product synthetic crucian Ferritin L recombinant protein, purifying the dissolved protein according to a His-tag bind protein (His-tag library) method, and carrying out SDS-electrophoresis analysis on the expression product synthetic crucian L recombinant protein at room temperature, wherein the SDS-PAGE analysis result is shown in figure 3.

In order to further identify various action indexes of the synthetic crucian Ferritin L recombinant protein, the following tests are carried out:

1. the activity identification experiment of the synthetic crucian carp Ferritin L recombinant protein antigen:

the result is shown in fig. 3, and shows that the mouse-derived His monoclonal antibody can identify the synthetic crucian Ferritin L recombinant protein expressed by escherichia coli, and the obtained protein is proved to be the synthetic crucian Ferritin L recombinant protein.

2. Aeromonas hydrophila E L ISA experiment:

inoculating Aeromonas hydrophila (Aeromonas hydrophylla) into L B medium, continuously culturing at 30 deg.C and 200rpm until OD600 is 0.6, centrifuging, resuspending with PBS buffer solution, and adjusting the concentration to 1 × 10⁷CFUmL^-1And inoculating the above bacteria into a 96-well E L ISA plate, blocking the E L ISA plate with 5% skimmed milk for 2h after overnight at 4 ℃, then washing with 0.5% Tween-20/PBS, respectively adding 2.5. mu.g, 5.0. mu.g, 10.0. mu.g, 20.0. mu.g, 40.0. mu.g and 80.0. mu.g of pET32a tag protein/200. mu. L PBS (pH7.9) and conjunctive crucian Ferritin L recombinant protein (WR-Ferritin L protein)/200. mu. L PBS (pH7.9), incubating for 1.5h at room temperature, then washing with 0.5% Tween-20/PBS, blocking the E L ISA plate with mouse-derived His-tag primary antibody and horse-anti-mouse secondary antibody HRP, respectively, finally adding 200. mu. L TMB solution in dark environment, adding 2M sulfuric acid after about 30min, and performing reaction reading under 450 OD reading, and calculating the result of the OD 450.

The results show that, compared with the pET32a tag control group, the concentration of the synthetic crucian Ferritin L recombinant protein (WR-Ferritin L protein) is gradually increased, the binding ratio of the synthetic crucian Ferritin L recombinant protein to the aeromonas hydrophila is also gradually increased, and the highest binding ratio is reached at 80.0 mu g, which indicates that the synthetic crucian Ferritin L recombinant protein can be bound to the aeromonas hydrophila under the buffer environment with the pH value of 7.9, and the dosage relationship exists.

3. Growth activity test of Aeromonas hydrophila:

inoculating Aeromonas hydrophila (Aeromonas hydrophylla) into L B culture medium, continuously culturing at 30 deg.C and 200rpm until OD600 is 0.6, centrifuging, resuspending with PBS solution, and adjusting bacteria concentration to 1 × 10⁷CFU mL^-12.5. mu.g, 5.0. mu.g, 10.0. mu.g, 20.0. mu.g, 40.0. mu.g and 80.0. mu.g of pET32a tag protein/200. mu. L PBS (pH7.9) and synthetic crucian Ferritin L were each weighedHistone (WR-Ferritin L protein)/200 μ L PBS (pH7.9) were mixed with Aeromonas hydrophila solution at a volume ratio of 4:1, added to a 96-well plate, and mixed with 10 μ L reagent CCK-8(BestBio, Shanghai) for bacterial activity detection, and after further incubation at 30 ℃ for 2.5h, the readings were analyzed at OD450, and Trx-treated group was used as control group for experiment.A calculation formula (OD 450 reading of experiment group/OD 450 reading of control group) × 100% was shown in FIG. 8.

The result shows that under the buffer environment with the pH value of 7.9, the activity of the co-cultured aeromonas hydrophila is gradually reduced along with the gradual increase of the concentration of the added syntype crucian Ferritin L recombinant protein (WR-Ferritin L protein), and the lowest activity value is reached at 80.0 mu g, which indicates that the syntype crucian Ferritin L recombinant protein has the activity of inhibiting the growth of the aeromonas hydrophila under the buffer environment with the pH value of 7.9.

4. Animal experiments:

1) animal treatment and organ bacterial load test

Selecting the same batch of synthetic crucian (about 23.17 +/-0.57 g) with healthy constitution and similar specification, temporarily culturing in 1.0m × 0.65.65 m × 0.65.65 m storage tanks, filling each storage tank with pre-aerated fresh water at the temperature of about 25 +/-1 ℃, respectively suspending the activated aeromonas hydrophila in PBS solution, and adjusting to 1 × 10⁷CFU mL^-1After 50 μ L of the above-mentioned resuspended suspension was intraperitoneally injected for about 25min, and then 3.5 μ g/g of fish weight of pET32a tag protein and synthetic crucian Ferritin L recombinant protein (WR-Ferritin L protein) dissolved in PBS (pH7.9) were intraperitoneally injected for about 24h, then liver, kidney and spleen of fish were separated and about 0.1g of each tissue was weighed out and ground in PBS, and then spread on L B plates and cultured at 30 ℃ for about 8h, and the results are shown in fig. 9.

2) Proliferation test of in-vivo pathogenic bacteria of synthetic crucian carp

The isolated liver, kidney and spleen DNA was isolated using Tissue DNAkit (Omega) and the concentration of the isolated DNA was adjusted to about 100 ng/. mu. L the total volume of the real-time quantitative fluorescence qPCR system was 20. mu. L for upstream and downstream primers diluted 0.8. mu. L each, 5.0. mu. L ultrapure water, 3. mu. L of DNA solution, 0.4. mu. L of ROX reference dye and 10. mu. L of SYBR premix ExTaq^TMII (perfect read time) (TaKaRa, Dalian, China). The reaction procedure comprises the following steps: pre-denaturation at 95 ℃ for 2 min; denaturation at 95 ℃ for 30s, annealing at 58 ℃ for 35s, and total 40 cycles, the amplified product was subjected to dissolution curve analysis. Each qRT-PCR experiment was repeated 3 times. The results of the experiment were analyzed by 7500SDS software (Applied Biosystems, USA), and are shown in FIG. 10.

3) Detection of inflammatory cytokine expression change in crucian carp body caused by germ infection

Total RNA extraction was performed on the above samples using Trizol reagent (Invitrogen), and the quality and purity of the extracted RNA were judged by the values of 260nm and 260/280 nm. According to reverse Aid^TMM-Mu L V Reverse transcriptase Transcriptase kit (MBI Fermentas, USA) method, using 1000ng total mRNA Reverse transcription experiment, the Reverse transcription cDNA was subjected to real-time fluorescence quantitative qRT-PCR experiment, the total system was 20 u L: 0.8 u L diluted upstream and downstream primers, 6.0 u L ultrapure water, 2 u L Reverse transcription cDNA template, 0.4 u L ROX reference dye and 10 u L SYBR premix Ex Taq^TMII (PerfectReal time) (TaKaRa, Dalian, China) reaction program contains 95 ℃ pre-denaturation for 2min, 95 ℃ denaturation for 30s, 59 ℃ annealing for 35s, 40 cycles, and the amplified product is analyzed by dissolution curve analysis, each qRT-PCR experiment is repeated 3 times, the experiment result is analyzed by 7500SDS software (Applied Biosystems, USA), the I L-1 β -1 gene level change is shown in FIG. 11, the I L-1 β -2 gene level change is shown in FIG. 12, the TNF α -1 gene level change is shown in FIG. 13, and the TNF α -2 gene level change is shown in FIG. 14.

The experiments show that after the aeromonas hydrophila is infected for 25min, the synthetic crucian Ferritin L recombinant protein (WR-Ferritin L protein) dissolved in PBS (pH7.9) is injected at the dose of 3.5 mu g/g fish weight, so that the multiplication capacity of the aeromonas hydrophila in each organ in the synthetic crucian can be effectively inhibited, and the inflammatory reaction caused by the aeromonas hydrophila is reduced.

Sequence listing

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aacctcaaac tgacggcctc atatgtgtat ctctcactgg gaatgtattt tgatagagat 120

gatgtggctc tgccaaactt ctcaaagttt ttcttggagc gttcactgaa ggagcgggat 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> 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 (Artificial Sequence)

<400>3

ccggaattca tgtctctagt caagcagaa 29

<210>4

<211>46

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

ccgctcgagt taatgatgat gatgatgatg gagcgtgtgc ttgtca 46

Claims

1. A synthetic crucian carp Ferritin L gene is characterized in that the nucleotide sequence is shown in SEQ ID NO. 1.

2. The synthetic crucian carp Ferritin L recombinant protein is characterized in that the amino acid sequence is shown in SEQ ID NO. 2, or the protein with the same or higher activity is obtained by substituting, deleting and/or adding one or more amino acids and/or modifying the tail end of the amino acid sequence shown in SEQ ID NO. 2.

3. A method for preparing synthetic crucian carp Ferritin L recombinant protein is characterized by comprising the following steps of amplifying a synthetic crucian Ferritin L gene sequence by a PCR method, adding enzyme cutting sites, purifying a PCR product, carrying out double enzyme cutting, connecting the PCR product to an expression vector pET32a, and then passing through CaCl₂The transformation method comprises the step of transferring an expression vector pET32a containing the synthetic crucian Ferritin L gene into escherichia coli B L21 (E. coli B L21) for induction expression, and finally obtaining the synthetic crucian Ferritin L recombinant protein.

4. The preparation method of claim 3, wherein the specific operation of amplifying the synthetic crucian Ferritin L gene by a PCR method and adding enzyme cutting sites comprises the following steps of amplifying the synthetic crucian Ferritin L gene with the enzyme cutting sites by the PCR method by taking synthetic crucian liver cDNA obtained by reverse transcription as a template, and directly cutting gel after electrophoretic separation to sequence and confirm the synthetic crucian Ferritin L sequence, wherein the nucleotide sequence of the synthetic crucian Ferritin L gene is shown in SEQ ID NO. 1.

5. The preparation method of claim 3, wherein the PCR amplification reaction conditions comprise pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 65 ℃ for 30s, and extension at 72 ℃ for 35s for 5 cycles, denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 35s for 5 cycles, denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 35s for 30 cycles, and finally extension at 72 ℃ for 10min, wherein the primers used in the PCR amplification reaction comprise an upstream primer Ferritin L-F1 and a downstream primer Ferritin L-R1, the nucleotide sequence of the upstream primer Ferritin L-F1 is shown as SEQ ID NO: 3, and the nucleotide sequence of the downstream primer Ferritin L-R1 is shown as SEQ ID NO: 4.

6. The preparation method of any one of claims 3 to 5, wherein the specific operation of purifying and double-digesting the PCR product and connecting the PCR product to the expression vector pET32a comprises the following steps of directly purifying and recovering the PCR product by using a DNA agarose gel recovery kit, then carrying out double digestion by using restriction enzymes EcoRI and XhoI, after the double digestion is finished, carrying out product recovery on the successfully digested hybrid crucian Ferritin L sequence, carrying out double digestion on the expression vector pET32a by using the restriction enzymes EcoRI and XhoI, mixing the obtained large vector fragment with the successfully digested hybrid crucian Ferritin L sequence, and carrying out connection for 12-18h at 15-17 ℃ by using T4 ligase, thereby successfully connecting the hybrid crucian Ferritin L gene sequence to the expression vector pET32 a.

7. The use of the synthetic crucian Ferritin L recombinant protein as defined in claim 2 or prepared by the preparation method defined in any one of claims 3-6 for inhibiting the growth of Aeromonas hydrophila (Aeromonas hydrophila).

8. The application of the recombinant protein Ferritin L as the synthetic ingredient is characterized in that the application method comprises the following steps of mixing and contacting the synthetic crucian Ferritin L recombinant protein with the aeromonas hydrophila in a buffer environment with the pH value of 7.2-8.2, and finishing the inhibition of the aeromonas hydrophila after the reaction is terminated.

9. The application of the synthetic crucian Ferritin L recombinant protein as defined in claim 2 or prepared by the preparation method of any one of claims 3-6 in the field of immune preparations or feed additives of aquatic animals.

10. A primer for amplifying Ferritin L gene, which comprises an upstream primer Ferritin L-F1 and a downstream primer Ferritin L-R1:

an upstream primer Ferritin L-F1: 5'-CCGGAATTCATGTCTCTAGTCAAGCAGAA-3';

the downstream primer Ferritin L-R1: 5'-CCGCTCGAGTTAATGATGATGATGATGATGGAGCGTGTGCTTGTCA-3'.