Application of a protein in regulating intestinal homeostasis and resisting pathogenic bacteria infection

Abstract

The present invention relates to the field of biotechnology, and specifically to the use of a protein in regulating intestinal homeostasis and resisting pathogenic bacteria infection. The protein regulating intestinal homeostasis in the present invention effectively prevents and treats diseases caused by exogenous pathogenic bacteria by maintaining the dynamic balance of intestinal flora and the integrity of the intestinal epithelial barrier. Through gene silencing, it is clear that the target protein plays a role in the intestine of shrimp, and it is determined that it can affect the abundance of intestinal flora and intestinal tissue morphology, thereby affecting the survival of shrimp. The recombinant protein was further purified by prokaryotic expression and applied to shrimp. It was found that the recombinant protein increased the survival rate of shrimp under Vibrio parahaemolyticus infection and alleviated tissue damage caused by Vibrio parahaemolyticus infection. The technology of using the target protein to enhance intestinal homeostasis and then defend against pathogenic bacteria infection has the characteristics of green environmental protection, simple operation, long-term effectiveness, etc., and provides an efficient and safe solution for shrimp farming, with broad application prospects and economic benefits.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to application of a protein in regulating intestinal homeostasis and resisting pathogenic bacteria infection. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] The intestine is an important barrier for the host to defend against pathogen invasion, and the maintenance of its homeostasis is crucial to the host's health. The maintenance of intestinal homeostasis depends on the coordination of the intestinal microbiota, the host immune system, and physiological and biochemical processes, among which the intestinal microbiota is one of the important driving factors of intestinal homeostasis. The intestinal microbiota can play a positive role in intestinal homeostasis by maintaining intestinal barrier function, participating in nutrient metabolism, and regulating the host immune system. In recent years, with the in-depth study of intestinal function, utilizing and improving intestinal flora to enhance intestinal homeostasis has become an important means to defend against pathogen invasion and treat diseases.

[0004] Shrimp farming is an extremely important branch of aquaculture, and its output value and output occupy an important position in the entire aquaculture industry. At present, shrimp farming is gradually developing towards intensive and high-density farming. However, under such high-density farming conditions, many disease problems have become bottlenecks restricting the healthy development of shrimp farming, among which bacterial diseases (mainly caused by Vibrio bacteria) are particularly prominent. During shrimp farming, exogenous pathogens can easily enter the intestine through the mouth and proliferate in the body to cause disease. Based on the key role of the intestine in defending against pathogen invasion, many studies have currently used prebiotics and probiotics to regulate intestinal flora and enhance intestinal defense function. This has become an effective method for shrimp to defend against pathogen invasion.

[0005] Since there are individual differences in the host's response to probiotics, and some probiotics have poor colonization effects in the body, these problems often lead to the inability of exogenously added substances to function effectively in the host. However, some molecules produced by the host itself often have potential advantages in regulating intestinal homeostasis. Such molecules usually exist naturally in the host and can regulate specific intestinal physiological processes or molecular targets of the host, and have higher accuracy and stability in regulating intestinal homeostasis. Therefore, if the host's own molecules that regulate intestinal homeostasis can be discovered, developed and utilized, it may become a more effective way for shrimp to defend against pathogen infection. Summary of the invention

[0006] In order to overcome the above problems, the present invention provides an application of a protein in regulating intestinal homeostasis and resisting pathogenic bacteria infection. The protein provided by the present invention is derived from the host itself, and regulates intestinal homeostasis by maintaining the dynamic balance of intestinal flora and the integrity of the intestinal epithelial barrier, thereby enhancing the resistance of aquatic animals to pathogenic bacteria infection.

[0007] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0008] The first aspect of the present invention provides an application of a protein in resisting infection by pathogenic bacteria, wherein the application includes any of the following:

[0009] (A1), use of a protein or a substance that regulates the expression of a gene encoding the protein or a substance that regulates the activity or content of the protein in resisting infection by exogenous pathogenic bacteria in aquatic animals;

[0010] (A2) Use of a protein or a substance that regulates the expression of a gene encoding the protein or a substance that regulates the activity or content of the protein in the preparation of a product for resisting infection by exogenous pathogenic bacteria in aquatic animals;

[0011] Wherein, the amino acid sequence of the protein is shown in SEQ ID NO: 1;

[0012] The exogenous pathogenic bacteria are Vibrio.

[0013] In one or more embodiments, the application includes any of the following:

[0014] (B1) Use of a protein or a substance that regulates the expression of a gene encoding the protein or a substance that regulates the activity or content of the protein in improving the ability of aquatic animals to resist exogenous pathogens;

[0015] (B2) Use of a protein or a substance that regulates the expression of a gene encoding the protein or a substance that regulates the activity or content of the protein in the preparation of a product that improves the ability of aquatic animals to resist exogenous pathogens;

[0016] (B3) Use of a protein or a substance that regulates the expression of a gene encoding the protein or a substance that regulates the activity or content of the protein in improving the survival rate of aquatic animals infected by exogenous pathogenic bacteria;

[0017] (B4) Use of a protein or a substance that regulates the expression of a gene encoding the protein or a substance that regulates the activity or content of the protein in the preparation of a product for improving the survival rate of aquatic animals infected by exogenous pathogens;

[0018] (B5) Use of a protein or a substance that regulates the expression of a gene encoding the protein or a substance that regulates the activity or content of the protein in alleviating tissue damage caused by infection with exogenous pathogenic bacteria in aquatic animals;

[0019] (B6) Use of a protein or a substance that regulates the expression of a gene encoding the protein or a substance that regulates the activity or content of the protein in the preparation of a product that alleviates tissue damage caused by infection with exogenous pathogens in aquatic animals.

[0020] In one or more embodiments, the Vibrio includes one or more of Vibrio parahaemolyticus, Vibrio anguillarum and Vibrio harveyi, preferably Vibrio parahaemolyticus.

[0021] The second aspect of the present invention provides an application of a protein in regulating intestinal homeostasis, wherein the application includes any of the following:

[0022] (C1) Use of a protein or a substance that regulates the expression of a gene encoding the protein or a substance that regulates the activity or content of the protein in maintaining the homeostasis of intestinal flora and the integrity of the intestinal epithelial barrier of aquatic animals;

[0023] (C2) Use of a protein or a substance that regulates the expression of a gene encoding the protein or a substance that regulates the activity or content of the protein in the preparation of a product for maintaining the homeostasis of intestinal flora and the integrity of the intestinal epithelial barrier of aquatic animals;

[0024] (C3) Use of a protein or a substance that regulates the expression of a gene encoding the protein or a substance that regulates the activity or content of the protein in enhancing the ability of aquatic animals to control intestinal symbiotic flora;

[0025] (C4) Use of a protein or a substance that regulates the expression of a gene encoding the protein or a substance that regulates the activity or content of the protein in the preparation of a product that enhances the ability of aquatic animals to control intestinal symbiotic flora;

[0026] Wherein, the amino acid sequence of the protein is shown in SEQ ID NO:1.

[0027] Preferably, the intestinal symbiotic flora in (C3) or (C4) is a microbial flora that stably exists in the intestines of aquatic animals.

[0028] In one or more embodiments, the substance that regulates the expression of the protein encoding gene or the substance that regulates the activity or content of the protein is a biological material, and the biological material is any one of the following:

[0029] (D1), a nucleic acid molecule encoding the protein;

[0030] (D2), an expression cassette containing the nucleic acid molecule described in (D1);

[0031] (D3), a recombinant vector containing the nucleic acid molecule described in (D1), or a recombinant vector containing the expression cassette described in (D2);

[0032] (D4), a recombinant microorganism containing the nucleic acid molecule described in (D1), or a recombinant microorganism containing the expression cassette described in (D2), or a recombinant microorganism containing the recombinant vector described in (D3);

[0033] (D5), a transgenic animal cell line containing the nucleic acid molecule described in (D1), or a transgenic animal cell line containing the expression cassette described in (D2), or a transgenic animal cell line containing the recombinant vector described in (D3).

[0034] In one or more embodiments, the aquatic animal includes a crustacean aquatic animal, preferably a shrimp, and more preferably a Japanese shrimp.

[0035] In one or more embodiments, the product includes one or more of a drug and a drug composition, a feed and a feed additive.

[0036] The third aspect of the present invention provides a product for aquatic animals to resist infection by exogenous pathogenic bacteria, comprising a protein, wherein the amino acid sequence of the protein is shown in SEQ ID NO:1.

[0037] In one or more embodiments, the product includes one or more of a drug and a drug composition, a feed and a feed additive.

[0038] The beneficial effects of the present invention are:

[0039] The present invention provides a protein for regulating intestinal homeostasis, which effectively prevents and treats diseases caused by exogenous pathogenic bacteria by maintaining the dynamic balance of intestinal flora and the integrity of the intestinal epithelial barrier. Specifically, gene silencing is used to clarify the function of the target protein in the intestine of shrimp, and it is determined that it can affect the abundance of intestinal flora and intestinal tissue morphology, thereby affecting the survival of shrimp. The recombinant protein was further purified by prokaryotic expression and applied to shrimp. It was found that the recombinant protein increased the survival rate of shrimp under Vibrio parahaemolyticus infection and alleviated tissue damage caused by Vibrio parahaemolyticus infection. The technology of using the target protein to enhance intestinal homeostasis and thus defend against pathogenic bacteria infection has the characteristics of being green and environmentally friendly, easy to operate, and long-term effective. It provides an efficient and safe solution for shrimp farming and has broad application prospects and economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0041] Figure 1 The SDS-PAGE test result of the target protein obtained in Example 1 of the present invention;

[0042] Figure 2 This is a graph showing the expression level of the target protein after the target protein gene expression is silenced in Example 2 of the present invention;

[0043] Figure 3 The graphs are the changes in the survival rate, intestinal tissue morphology and intestinal flora abundance of shrimp after the target protein gene is silenced in Example 3 of the present invention, wherein A is the survival rate graph of shrimp after the target protein gene expression is silenced, B is the intestinal tissue morphology graph of shrimp in the control group and after the target protein gene expression is silenced, the scale bar is 200 μm, C is the total bacterial abundance graph of shrimp intestine after the target protein gene expression is silenced, and D is the abundance graph of culturable bacteria in shrimp intestine after the target protein gene expression is silenced;

[0044] Figure 4 This is a graph showing the survival rate of shrimp injected with recombinant protein under infection with Vibrio parahaemolyticus in Example 4 of the present invention;

[0045] Figure 5 This is a morphological diagram of the hepatopancreas tissue of the shrimp injected with the recombinant protein under infection with Vibrio parahaemolyticus in Example 5 of the present invention. DETAILED DESCRIPTION

[0046] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0047] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0048] A first typical embodiment of the present invention provides an application of a protein in resisting infection by pathogenic bacteria, wherein the application includes any of the following:

[0049] (A1), use of a protein or a substance that regulates the expression of a gene encoding the protein or a substance that regulates the activity or content of the protein in resisting infection by exogenous pathogenic bacteria in aquatic animals;

[0050] (A2) Use of a protein or a substance that regulates the expression of a gene encoding the protein or a substance that regulates the activity or content of the protein in the preparation of a product for resisting infection by exogenous pathogenic bacteria in aquatic animals;

[0051] Wherein, the amino acid sequence of the protein is shown in SEQ ID NO: 1;

[0052] The exogenous pathogenic bacteria are Vibrio.

[0053] The specific information of the amino acid sequence of the protein is as follows:

[0054] (a) Sequence characteristics

[0055] Length: 170 amino acids

[0056] Type: Amino Acid

[0057] Chain type: Single chain

[0058] (b) Molecule type: protein

[0059] (c) Original source: Japanese shrimp ( Marsupenaeus japonicus )

[0060] (d) Sequence description: Details are as follows:

[0061] RRCLTIKQKRAIENTYLNFKCMQPGIKLVELDVPEEYIVFTPSVAAVSRCSMMYCITNGMQCLSVEKRNTTIRVEALSRLQEIKCLEIPVEEDLSCGCQKCQPEICPNNDMVFNSNTCKCECTNRFLSQCNEKVNKGTHTFDRKTCRCMCKETKDCGQRMWNDNTCRCMD (SEQ ID NO. 1).

[0062] The protein of the present invention can be synthesized artificially, or its coding gene can be synthesized first and then expressed biologically, or it can be sent to a biological company for synthesis or the entire sequence can be synthesized using an automatic peptide synthesizer, or it can be obtained by preparing and using a polyclonal antibody or a monoclonal antibody of the protein and isolating and purifying it in shrimp.

[0063] The protein of the present invention may be in the form of a natural protein, a recombinant protein or a synthetic polypeptide, or may be chemically or physically modified on the basis of a natural or recombinant protein to improve its stability, activity or targeting in the intestine, which is not specifically limited here.

[0064] The invention provides an effective disease prevention and control means for the shrimp farming industry by applying the novel protein, while reducing the dependence on antibiotics and chemical drugs, and has good application prospects and promotion value.

[0065] In one or more embodiments, the application includes any of the following:

[0066] (B1) Use of a protein or a substance that regulates the expression of a gene encoding the protein or a substance that regulates the activity or content of the protein in improving the ability of aquatic animals to resist exogenous pathogens;

[0067] (B2) Use of a protein or a substance that regulates the expression of a gene encoding the protein or a substance that regulates the activity or content of the protein in the preparation of a product that improves the ability of aquatic animals to resist exogenous pathogens;

[0068] (B3) Use of a protein or a substance that regulates the expression of a gene encoding the protein or a substance that regulates the activity or content of the protein in improving the survival rate of aquatic animals infected by exogenous pathogenic bacteria;

[0069] (B4) Use of a protein or a substance that regulates the expression of a gene encoding the protein or a substance that regulates the activity or content of the protein in the preparation of a product for improving the survival rate of aquatic animals infected by exogenous pathogens;

[0070] (B5) Use of a protein or a substance that regulates the expression of a gene encoding the protein or a substance that regulates the activity or content of the protein in alleviating tissue damage caused by infection with exogenous pathogenic bacteria in aquatic animals;

[0071] (B6) Use of a protein or a substance that regulates the expression of a gene encoding the protein or a substance that regulates the activity or content of the protein in the preparation of a product that alleviates tissue damage caused by infection with exogenous pathogens in aquatic animals.

[0072] In one or more embodiments, the Vibrio includes one or more of Vibrio parahaemolyticus, Vibrio anguillarum and Vibrio harveyi, preferably Vibrio parahaemolyticus.

[0073] A second typical embodiment of the present invention provides an application of a protein in regulating intestinal homeostasis, wherein the application includes any of the following:

[0074] (C1) Use of a protein or a substance that regulates the expression of a gene encoding the protein or a substance that regulates the activity or content of the protein in maintaining the homeostasis of intestinal flora and the integrity of the intestinal epithelial barrier of aquatic animals;

[0075] (C2) Use of a protein or a substance that regulates the expression of a gene encoding the protein or a substance that regulates the activity or content of the protein in the preparation of a product for maintaining the homeostasis of intestinal flora and the integrity of the intestinal epithelial barrier of aquatic animals;

[0076] (C3) Use of a protein or a substance that regulates the expression of a gene encoding the protein or a substance that regulates the activity or content of the protein in enhancing the ability of aquatic animals to control intestinal symbiotic flora;

[0077] (C4) Use of a protein or a substance that regulates the expression of a gene encoding the protein or a substance that regulates the activity or content of the protein in the preparation of a product that enhances the ability of aquatic animals to control intestinal symbiotic flora;

[0078] Among them, the amino acid sequence of the protein is shown as SEQ ID NO: 1.

[0079] Preferably, the intestinal symbiotic flora in (C3) or (C4) is a microbial flora that stably exists in the intestines of aquatic animals.

[0080] In one or more embodiments, the substance that regulates the expression of the protein encoding gene or the substance that regulates the activity or content of the protein is a biological material, and the biological material is any one of the following:

[0081] (D1), a nucleic acid molecule encoding the protein;

[0082] (D2), an expression cassette containing the nucleic acid molecule described in (D1);

[0083] (D3), a recombinant vector containing the nucleic acid molecule described in (D1), or a recombinant vector containing the expression cassette described in (D2);

[0084] (D4), a recombinant microorganism containing the nucleic acid molecule described in (D1), or a recombinant microorganism containing the expression cassette described in (D2), or a recombinant microorganism containing the recombinant vector described in (D3);

[0085] (D5), a transgenic animal cell line containing the nucleic acid molecule described in (D1), or a transgenic animal cell line containing the expression cassette described in (D2), or a transgenic animal cell line containing the recombinant vector described in (D3).

[0086] The nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

[0087] Specifically, the nucleotide sequence of the nucleic acid molecule in (D1) is shown in SEQ ID NO:2.

[0088] The specific information of the nucleic acid molecule is as follows:

[0089] (a) Sequence characteristics

[0090] Length: 510 bases

[0091] Type: Nucleic Acid

[0092] Chain type: Single chain

[0093] (b) Molecule type: Nucleic acid

[0094] (c) Original source: Japanese shrimp ( Marsupenaeus japonicus )

[0095] (d) Sequence description: Details are as follows:

[0096] AGGCGATGCTTGACCATCAAGCAAAAGAGAGCCATCGAGAACACTTACTTGAATTTCAAGTGCATGCAACCGGGAATCAAATTGGTCGAGCTTGATGTCCCAGAAGAATACATAGTGTTTTACCCCCTC AGTGGCCGCAGTGAGTCGCTGTTCTATGATGTACTGTATAACGAATGGCATGCAGTGTCTGTCCGTGGAAAAGAGGAATACAACAATCAGGGTCGAGGCGCTCAGTCGGTTACAAGAAATTAAGTGCTT AGAAATCCCCGTTGAAGAAGATCTGAGTTGTGGATGCCAAAAATGCCAGCCCGAGATATGCCCTAACAATGATATGGTCTTCAACTCCAACACCTGCAAATGCGAGTGTACCAATCGGTTCCTCAGTC AATGCAACGAGAAAGTCAACAAAGGAACACACACTTTCGATCGTAAGACCTGTCGCTGCATGTGCAAGGAAACAAAGGACTGTGGACAGCGCATGTGGAACGACAATACATGCAGATGTATGGAC (SEQ ID NO.2).

[0097] In one or more embodiments, the aquatic animal includes a crustacean aquatic animal, preferably a shrimp, and more preferably a Japanese shrimp.

[0098] In one or more embodiments, the product includes one or more of a drug and a drug composition, a feed and a feed additive.

[0099] A third typical embodiment of the present invention provides a product for aquatic animals to resist infection by exogenous pathogenic bacteria, comprising a protein, wherein the amino acid sequence of the protein is shown in SEQ ID NO:1.

[0100] In one or more embodiments, the product includes one or more of a drug and a drug composition, a feed and a feed additive.

[0101] Specifically, the product can be a granular premix such as powder or soluble powder. Excipients include solvents (water for injection, saline), stabilizers (to prevent drug decomposition or deformation), buffers (to maintain solution pH stability), etc. There is no specific limitation here and it can be selected according to actual needs.

[0102] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0103] In the following examples, the shrimp is selected from the Japanese Capsulatus.

[0104] Example 1

[0105] Amplification and expression of protein mature peptide sequences:

[0106] RNA from shrimp intestinal tissue was extracted using TRIzol Reagent, and then reverse transcribed into cDNA using a reverse transcription kit (Novozyme, Nanjing, China, R323-01). Expression primers were designed according to SEQ ID NO. 2 provided by the present invention to amplify the sequence encoding the mature peptide of the protein from the cDNA of shrimp intestine.

[0107] F1:

[0108] 5'-GCCATGGCTGATATCGGATCCAGGCGATGCTTGACCATCAA-3' (SEQ ID NO. 3);

[0109] R1:

[0110] 5'-GTGGTGGTGGTGGTGCTCGAGTCAGTCCATACATCTGCATGTATTGT-3' (SEQ ID NO. 4), containing BamH I and Xho I restriction sites.

[0111] The sequence encoding the mature peptide of the protein was amplified by the above primers and recovered by gel. Subsequently, the pET-32a (+) plasmid was double-digested with BamH I and Xho I, and the target sequence was connected to the plasmid by homologous recombination. The recombinant vector was transformed into the Escherichia coli Rosetta (DE3) strain, and 0.2 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was used to induce expression at 28 °C for 6 h. Since the target protein is an insoluble protein, the target protein needs to be dissolved in 8 M urea and then in PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 The target protein was obtained after dialyzing and renaturation in 5% paraformaldehyde (pH 7.4). The His-tagged thioredoxin expressed in the pET-32a (+) plasmid was used as the control protein in this example, example 4 and example 5. The amino acid sequence of the control protein is:

[0112] MIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMADIGSEFELRRQACGRTRAPPPLPLRSGC (SEQ ID NO. 5).

[0113] The nucleic acid sequence encoding the control protein is:

[0114] ATGATCGCCCCGATTCTGGATGAAATCGCTGACGAATATCAGGGCAAACTGACCGTTGCAAAACTGAACATCGATCAAAACCCTGGCACTGCGCCGAAATATGGCATCCGTGGTATCCCGACTCTGCTGCTGTTCAAAAACGGTGAAGTGGCGGCAACCAAAGTGGGTGCACTGTCTAAAGGTCAGTTGAAAGAGTTCCTCGACGCTAACCTGGCCGGTTCTGGTTCTGG CCATATGCACCATCATCATCATTCTTCTGGTCTGGTGCCACGCGGTTCTGGTATGAAAGAAACCGCTGCTGCTAAATTCGAACGCCAGCACATGGACAGCCCAGATCTGGGTACCGACGACGACGACAAGGCCATGGCTGATATCGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACTACCACTGAGATCCGGCTGC (SEQ ID NO.6).

[0115] The control protein was expressed as a soluble protein and purified using the affinity column Ni-NTA His Resin (Merck, Darmstadt, Germany, 770666). The purification of the control protein and the target protein was detected by SDS-PAGE (see Figure 1 ).

[0116] Example 2

[0117] Target protein gene expression silencing efficiency:

[0118] Gene expression silencing (RNAi): using a synthetic nucleic acid sequence linked to a T7 promoter:

[0119] Oligo1:

[0120] 5'-GATCACTAATACGACTCACTATAGGGAGGCGCTCAGTCGGTTACAAGAAATT-3' (SEQ ID NO. 7).

[0121] Oligo2:

[0122] 5'-AATTTCTTGTAACCGACTGAGCGCCTCCCTATAGTGAGTCGTATTAGTGATC-3' (SEQ ID NO. 8).

[0123] Oligo3:

[0124] 5'-AAAGGCGCTCAGTCGGTTACAAGAAATCCCTATAGTGAGTCGTATTAGTGATC-3' (SEQ ID NO. 9).

[0125] Oligo4:

[0126] 5'-GATCACTAATACGACTCACTATAGGGATTTCTTGTAACCGACTGAGCGCCTTT-3' (SEQ ID NO. 10).

[0127] The above sequence was annealed and transcribed into a template for siRNA. The siRNA was synthesized using the T7 RNAi transcription kit (Novozyme, Nanjing, China; TR102) and purified by phenol / chloroform / isoamyl alcohol extraction. After verification by gel electrophoresis and quantification by spectrophotometer, siRNA was injected into the hemolymph of shrimp at a dose of 5 μg / g body weight. The detection primers of the target protein were designed according to SEQ ID NO.2 provided by the present invention, and the RNAi efficiency was determined after 24 hours. The primer sequence is:

[0128] F2: 5'-GCAGTGAGTCGCTGTTCTAT-3' (SEQ ID NO. 11);

[0129] R2: 5'-TTCAACGGGGGATTTCTAAG-3' (SEQ ID NO. 12);

[0130] β-actin was used as an internal reference, and the primer sequences were:

[0131] F3: 5'-CAGCCTTCCTTCCTGGGTATGG-3' (SEQ ID NO. 13);

[0132] R3: 5'-GAGGGAGCGAGGGCAGTGATT-3' (SEQ ID NO. 14);

[0133] The qRT-PCR program was set as follows: 94°C for 5 minutes, 94°C for 10 seconds, 60°C for 1 minute, and 40 cycles. The melting curve was measured from 65°C to 95°C at 1°C intervals.

[0134] Test results such as Figure 2 The results show that siRNA significantly knocked down the expression of the target protein. The knockdown efficiency of the target protein was analyzed using an unpaired Student's t-test. ***, p <0.001.

[0135] Example 3

[0136] Changes in shrimp survival rate, intestinal tissue morphology and intestinal flora abundance after protein gene silencing:

[0137] After siRNA injection, the shrimp were placed in a circulating oxygenated environment at room temperature, and the stocking density was set at 40 shrimp / m 3 The death of shrimp was counted every 12 hours. Figure 3 As shown in A, after gene silencing, the survival rate of shrimp was significantly reduced. The log-rank (Mantel-Cox) test was used for survival analysis.

[0138] At 6 days after siRNA injection, the intestinal tissues of the two groups were fixed in Davidson's fixative (30% ethanol, 22% formalin and 11.5% acetic acid) for 24 h, and then the intestinal tissue morphology was observed by paraffin embedding, sectioning and H&E staining. Figure 3 As shown in B, after gene silencing, the integrity of the shrimp intestine was significantly damaged, with thinner intestinal walls, looser or even falling intestinal epithelial cells, and disappearance of the villi in the intestinal cavity. The scale bar in this figure is 200 μm.

[0139] At 6 days after siRNA injection, the intestines of 12 shrimps in each group were homogenized in PBS, and then the total intestinal DNA was extracted using the QIAamp DNABlood Mini Kit (Qiagen, Hilden, Germany; 51104). qPCR was performed using universal primers designed for the conserved region of bacterial 16S rRNA to detect the abundance of total intestinal bacteria. The total intestinal bacterial abundance was expressed as the ratio of 16S rDNA level to host β-actin. Each sample came from one shrimp. The results are shown in Figure 3 As shown in C, after gene silencing, the homeostasis of shrimp intestinal flora was unbalanced and the total bacterial abundance increased. The difference in total bacterial abundance in shrimp intestine was analyzed using unpaired Student's t test. ***,p <0.001.

[0140] The specific primers for bacterial quantification are:

[0141] F5:ACTCCTACGGGAGGCAGCAGT (SEQ ID NO.15);

[0142] R5: TATTACCGCGGCTGCTGGC (SEQ ID NO.16);

[0143] At 6 days after siRNA injection, the intestines of 12 shrimps in each group were homogenized in sterile PBS, mixed and spread on 2216E solid culture medium and cultured at 30 °C for 24 h to observe the growth of bacterial colonies. Figure 3 As shown in D, the abundance of cultivable bacteria in the shrimp intestine increased after gene silencing.

[0144] The results of Example 3 show that the target protein is a protein that regulates the intestinal homeostasis of shrimp, and has the function of maintaining the dynamic balance of the intestinal flora of shrimp and the integrity of the intestinal epithelial barrier. The target protein maintains the survival of shrimp by effectively controlling the intestinal symbiotic microbial flora.

[0145] Example 4

[0146] Effect of injection of recombinant protein on the survival rate of shrimp infected with Vibrio:

[0147] In this example, the shrimp were injected with recombinant proteins such as the target protein and the control protein (2 μg / shrimp) and placed in a circulating oxygenated seawater environment at room temperature. The stocking density was set at 40 shrimp / m 3 The target protein and control protein were obtained according to the method described in Example 1. After 2 days, the shrimps were immersed in seawater containing Vibrio parahaemolyticus, and the mortality of the shrimps was counted every 12 hours. Figure 4 As shown in the figure, pretreatment with the target protein can improve the survival rate of shrimp infected with Vibrio parahaemolyticus. The log-rank (Mantel-Cox) test was used for survival analysis.

[0148] Example 5

[0149] Effects of injection of recombinant protein on hepatopancreas tissue of shrimp infected with Vibrio:

[0150] In this example, the shrimp were injected with recombinant proteins such as the target protein and the control protein (2 μg / shrimp) and placed in a circulating oxygenated seawater environment at room temperature. The stocking density was set at 40 shrimp / m 3The target protein and control protein were obtained according to the method described in Example 1. After 2 days, the shrimp were immersed in seawater containing Vibrio parahaemolyticus. After 2 days of infection with Vibrio parahaemolyticus, the hepatopancreas tissues of the two groups of shrimp were fixed in Davidson's fixative for 24 hours, and then the tissue morphology was observed by paraffin embedding, sectioning and H&E staining. The results are shown in Figure 5 As shown in the figure, after shrimp were infected with Vibrio parahaemolyticus, the hepatopancreas tissue swelled, increased in size, with massive cell necrosis, cytoplasmic vacuolation, and a large number of blood cell infiltration. After the shrimp were pre-treated with the target protein, the damage to the hepatopancreas tissue was alleviated, as shown by the reduced swelling of the hepatopancreas tissue and the reduction of the cavity.

[0151] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of a protein or a substance that upregulates the expression of a gene encoding the protein or a substance that upregulates the activity or content of the protein in the preparation of a product for resisting infection by exogenous pathogenic bacteria in aquatic animals; in, The amino acid sequence of the protein is shown in SEQ ID NO: 1; The exogenous pathogenic bacteria are Vibrio; The substance that upregulates the expression of the protein encoding gene or the substance that upregulates the activity or content of the protein is a biological material, and the biological material is any one of the following: (A1), a nucleic acid molecule encoding the protein; (A2), an expression cassette containing the nucleic acid molecule described in (A1); (A3), a recombinant vector containing the nucleic acid molecule described in (A1), or a recombinant vector containing the expression cassette described in (A2); (A4), a recombinant microorganism containing the nucleic acid molecule described in (A1), or a recombinant microorganism containing the expression cassette described in (A2), or a recombinant microorganism containing the recombinant vector described in (A3); (A5), a transgenic animal cell line containing the nucleic acid molecule described in (A1), or a transgenic animal cell line containing the expression cassette described in (A2), or a transgenic animal cell line containing the recombinant vector described in (A3).

2. The use according to claim 1, characterized in that The application includes any of the following: (B1) Use of a protein or a substance that upregulates the expression of a gene encoding the protein or a substance that upregulates the activity or content of the protein in the preparation of a product that improves the ability of aquatic animals to resist exogenous pathogens; (B2) Use of a protein or a substance that upregulates the expression of a gene encoding the protein or a substance that upregulates the activity or content of the protein in the preparation of a product for improving the survival rate of aquatic animals under infection by exogenous pathogenic bacteria; (B3) Use of a protein or a substance that upregulates the expression of a gene encoding the protein or a substance that upregulates the activity or content of the protein in the preparation of a product for alleviating tissue damage caused by infection with exogenous pathogens in aquatic animals.

3. The use according to claim 1, characterized in that The Vibrio includes one or more of Vibrio parahaemolyticus, Vibrio anguillarum and Vibrio harveyi.

4. The use according to any one of claims 1 and 2, characterized in that: The aquatic animals include crustacean aquatic animals.

5. The use according to any one of claims 1 and 2, characterized in that: The product includes one or more of a medicine and a medicine composition, a feed and a feed additive.

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