A protein that mediates probiotic colonization and its application in regulating intestinal homeostasis

Abstract

The present invention discloses a protein that mediates probiotic colonization and its application in regulating intestinal homeostasis, belonging to the field of disease prevention and control and aquaculture technology. The present invention reports for the first time a protein that mediates probiotic colonization and its encoding gene. Through research, the present invention found that the protein functions in the intestine at the expression level of various tissues of shrimp. The protein has the function of helping probiotics to colonize in the intestine and can increase the survival rate of shrimp under Vibrio infection. It shows excellent disease resistance in shrimp farming, especially in the prevention and control of Vibrio disease. The technology of using the protein to increase the content of probiotics in the intestine of shrimp and then prevent Vibrio 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.

Description

Technical Field

[0001] The invention belongs to the technical field of disease prevention and treatment and aquaculture, and specifically relates to a protein mediating probiotic colonization and an application thereof in regulating intestinal homeostasis. 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] 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, shrimp are susceptible to environmental stress and pathogenic microorganisms, especially bacterial diseases such as vibriosis, which has become the main bottleneck restricting the sustainable development of aquaculture.

[0004] Vibrio disease is a highly pathogenic disease caused by pathogenic bacteria of the genus Vibrio (such as Vibrio parahaemolyticus), which can spread rapidly and cause mass mortality of shrimp, causing serious economic losses to farmers. At present, the main means of preventing and controlling vibriosis is the use of antibiotics and chemical drugs. However, the long-term and widespread use of antibiotics not only leads to the emergence of drug-resistant strains, weakening the effectiveness of prevention and control, but also causes the problem of antibiotic residues, posing a threat to the sustainability of aquaculture and food safety. Therefore, there is an urgent need to develop a greener, more effective and risk-free alternative strategy to address these problems.

[0005] Probiotics are widely used in aquaculture because they can regulate the balance of intestinal microecology and inhibit the growth of harmful bacteria. For example, patent CN117487719A discloses a bacillus with anti-vibrio activity, patent CN103405479A discloses a lactobacillus for preventing and controlling vibrio in shrimp ponds in the late stage of aquaculture, and patent CN109880767A discloses a strain of Bacillus BHK201801 for preventing or treating vibrio disease in shrimp. However, due to the short and straight structure of the shrimp intestine and the lack of a mucus layer, it is difficult for probiotics to remain and adhere stably after entering the intestine, which leads to the unstable colonization effect of probiotics in the shrimp intestine, and it is often difficult to play a role for a long time, resulting in limited prevention and control effects on pathogens such as vibrio. Therefore, how to improve the colonization rate, stability and function of probiotics in the shrimp intestine has become a technical problem that needs to be solved urgently. Summary of the invention

[0006] In order to solve the shortcomings of the prior art, the purpose of the present invention is to provide a protein that mediates probiotic colonization and its application in regulating intestinal homeostasis. The protein provided by the present invention has the effect of promoting the colonization of probiotics in the intestine of shrimp, can effectively regulate the intestinal homeostasis of shrimp, and enhance the ability of shrimp to resist foreign pathogens.

[0007] In order to achieve the above object, the technical solution of the present invention is:

[0008] The first aspect of the present invention provides a protein that mediates probiotic colonization, wherein the amino acid sequence of the protein is shown in SEQ ID NO:1.

[0009] The second aspect of the present invention provides a nucleic acid molecule encoding the above-mentioned protein.

[0010] In some embodiments of the present invention, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:2.

[0011] The third aspect of the present invention provides a recombinant expression vector, which contains the above-mentioned nucleic acid molecule.

[0012] A fourth aspect of the present invention provides an engineered bacterium, wherein the engineered bacterium contains the above-mentioned recombinant expression vector.

[0013] A fifth aspect of the present invention provides a use of the above protein in the preparation of a product for regulating intestinal homeostasis of aquatic animals;

[0014] The aquatic animals are crustacean aquatic animals.

[0015] In some embodiments of the present invention, the product has any one or more of the following functions:

[0016] (1) Mediate the colonization of probiotics in the intestines of aquatic animals;

[0017] (2) Increase the number of probiotics in the intestines of aquatic animals;

[0018] (3) Improve the ability of aquatic animals to resist exogenous pathogens;

[0019] (4) Maintaining the homeostasis of the intestinal flora of aquatic animals;

[0020] (5) Improve the survival rate of aquatic animals infected by exogenous pathogens;

[0021] (6) Prevent bacterial diseases of aquatic animals;

[0022] The probiotics are Gram-positive probiotics, the exogenous pathogenic bacteria are Vibrio, and the bacterial disease of aquatic animals is Vibriosis.

[0023] In some embodiments of the present invention, the probiotic is Bacillus subtilis, the exogenous pathogenic bacteria is Vibrio parahaemolyticus, and the bacterial disease of aquatic animals is vibriosis caused by Vibrio parahaemolyticus.

[0024] In some embodiments of the present invention, the aquatic animal is a shrimp.

[0025] In some embodiments of the present invention, the protein is applied to aquatic animals by direct feeding, feed addition or preparation of medicine.

[0026] The beneficial effects of the present invention are:

[0027] The present invention provides a protein that mediates probiotic colonization, which effectively prevents and treats diseases by regulating the intestinal homeostasis of shrimp. Specifically, by analyzing the expression level of the protein in various tissues of shrimp, it is determined that the protein functions in the intestine. The recombinant protein is further purified by prokaryotic expression and verified by in vitro bacterial binding experiments and in vivo colonization experiments to have the function of helping probiotics colonize in the intestine and increase the survival rate of shrimp under Vibrio infection. The protein provided by the present invention shows excellent disease resistance in the shrimp farming industry, especially in the prevention and control of Vibrio disease. The technology of using the protein to increase the content of probiotics in the shrimp intestine and thus prevent Vibrio infection has the characteristics of being green and environmentally friendly, easy to operate, and long-term effective, and provides an efficient and safe solution for shrimp farming, with broad application prospects and economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] 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.

[0029] Figure 1 The protein obtained in Example 1 of the present invention;

[0030] Figure 2A This is a graph of the expression levels of the gene encoding the protein in Example 2 of the present invention in various tissues;

[0031] Figure 2B This is a diagram of the protein domain analysis in Example 2 of the present invention;

[0032] Figure 3 This is a diagram showing the results of in vitro recombinant protein verification combined with probiotics in Example 3 of the present invention;

[0033] Figure 4A This is a diagram showing the location of bacteria in the intestine in Example 4 of the present invention;

[0034] Figure 4B This is a graph showing the number of bacteria on the surface of a portion of the intestine in Example 4 of the present invention;

[0035] Figure 4C This is a graph showing the number of probiotics in the entire intestine in Example 4 of the present invention;

[0036] Figure 5A This is a graph showing the expression level of the protein gene after silencing in Example 5 of the present invention;

[0037] Figure 5B The 16S rDNA sequencing of Example 5 of the present invention reveals the changes in the intestinal flora structure at the phylum level after the protein gene expression is silenced;

[0038] Figure 5C The 16S rDNA sequencing of Example 5 of the present invention reveals the changes in the intestinal flora structure at the genus level after the protein gene expression is silenced;

[0039] Figure 6 This is the survival rate of the shrimp fed with the recombinant protein according to Example 6 of the present invention under Vibrio infection. DETAILED DESCRIPTION

[0040] The present application provides a protein that mediates probiotic colonization and its application in regulating intestinal homeostasis, which is used to solve the technical defects in the prior art that shrimp farming is unstable, the colonization effect of probiotics in the shrimp intestine is unstable, and it is often difficult to exert a lasting effect, resulting in limited prevention and treatment effects on pathogens such as Vibrio.

[0041] A first typical embodiment of the present invention provides a protein that mediates probiotic colonization, wherein the amino acid sequence of the protein is shown in SEQ ID NO:1.

[0042] The specific information of the amino acid sequence of the protein is as follows:

[0043] (a) Sequence characteristics

[0044] Length: 556 amino acids

[0045] Type: Amino Acid

[0046] Chain type: Single chain

[0047] (b) Molecule type: protein

[0048] (c) Original source: Japanese shrimp ( Marsupenaeus japonicus )

[0049] (d) Sequence description: Details are as follows:

[0050] (SEQ ID NO.1)

[0051] The amino acid sequence shown in SEQ ID NO.1 is composed of the abbreviations of the amino acids in the protein. The specific amino acid sequence of the protein can also be represented in the following ways:

[0052] Phe Pro Leu Pro Met Ser Lys Ser Val Cys His Asp Glu Gly Arg Trp GluAsp Thr Glu Asp Cys Gly Ala Tyr Ile Asp Cys Ile Pro Asp Ser Asn Gly Glu LeuIle Val His Arg Gly Ser Cys Asn Gly Gly Val Tyr Asn Pro Val Val Met Ala CysPhe Glu Ala Asp Asp Thr Ser Ser Cys Gln Gln Leu His Ser Arg Leu Trp Lys GlyThr Thr Val Asp Glu Asp Tyr Ser Tyr Ile Cys Lys Gly Val Pro Asn Gly Phe TyrCys Ala Asp Cys Thr Thr Val Val Asn Cys Ile Ser Glu Ala Ala Phe Pro Tyr ThrCys Gln Met Pro Ser Arg Cys Asp Ser Arg Glu Ala Phe Gly Gly Gly Val Cys TyrPro Glu Arg Pro Gly Glu Cys Leu Cys His Lys Arg Asn Arg Phe Gln Val Asp HisTyr Asp Asp Arg Lys Phe Phe Phe Cys Asp Asp Val Met Thr Asp Tyr Thr Leu TyrGln Cys Pro Ser Asn Met Val Phe Ala Glu Lys Glu Ile Gln Cys Lys Thr Arg SerGly Leu Pro Asn Cys Arg Ser Val Gly Met Gly Ile Phe Ser Tyr Ile Pro Asp CysArg Thr Tyr Phe Val Cys Ile Phe Ala Ile Gln Gly Trp Val Gln Lys Ala Phe LysCys Pro Ala Asp Pro Leu Leu Gly Glu Leu Met Phe Asn Glu Ile Thr Gly Gln CysGlu Asp Pro Cys Ser Trp Thr AlaGln Lys Phe Ser Cys Gln Thr Glu Gly Arg PheGly Asp Leu Tyr Leu Cys Asn Ile Tyr His Glu Cys Val Ala Ile Ser Thr Gly PheArg His Ile Lys His Glu Cys Pro Leu Gly Phe Thr Trp Asp Pro Ser Thr Leu LeuGly His Gly Leu Cys Val Pro Ala Ala Thr Asn Thr Arg Cys Pro Lys Lys His ThrVal Asn Lys Cys Val Val Pro Asp Gly Arg Cys Pro Gly Val Gln Val Gly Ile SerSer Ser Gly Val Pro Pro Lys Glu Ser Val His Asn Val Ile Asn Pro Glu Ala LeuAsp Ile Pro Ser Ile Ile Ala Asn Ile Leu Val Ser Thr Glu Pro Pro Val Pro ThrAsp Pro Leu Gln Thr Gly Val Pro Ser Glu Asp Thr Ala Thr Thr Thr Ile Pro IleGlu Thr Pro Thr Thr Ala Gly Ser Val Gly Leu Pro Asn Pro Leu Ser Gln Gly ProLeu Arg Ile Asp Glu Ala Ser Pro Leu Cys Pro Tyr Pro Phe Thr Leu Met Ala AspGlu Cys Ile Met Val Thr Gly Glu Asn Glu Lys Arg Thr Tyr Ser Gly Ala Met GluTyr Cys Ser Gly Leu Gly Ala Gly Leu Ala Ser Pro Thr Asn Leu Met Ala Leu ArgAsn Tyr Val Leu Leu Glu Leu Gly Ile Ile Asn Val Arg Val Gly Ala Thr Arg SerPro Gly Ser Asn Gly Ala Trp Leu Trp Pro Asp Gly Arg ProVal Leu Ala Ser AlaTrp Gly Pro Arg Gln Pro Asp Asn Ala Gly Asn Asp Glu Tyr Cys Ala Met Leu AsnThr Asn Leu Leu Trp Asn Gly Pro Leu Asn Asp Phe Asn Cys Asp Gly Leu Phe ProPhe Val Cys Gln Tyr Asn Leu

[0053] The protein mediating probiotic colonization of the present invention can be artificially synthesized, or its encoding gene can be synthesized first and then biologically expressed, 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 polyclonal or monoclonal antibodies of the protein to separate and purify it in shrimp.

[0054] The protein mediating probiotic colonization described in the present invention can be in the form of natural protein, recombinant protein or synthetic polypeptide, or chemically or physically modified on the basis of natural or recombinant protein to improve its stability, activity or targeting in the intestine, which is not specifically limited here.

[0055] The invention provides an effective disease prevention and control method 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.

[0056] A second typical embodiment of the present invention provides a nucleic acid molecule encoding the above-mentioned protein.

[0057] The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:2.

[0058] The specific information of the nucleic acid molecule is as follows:

[0059] (a) Sequence characteristics

[0060] Length: 1668 bases

[0061] Type: Nucleic Acid

[0062] Chain type: Single chain

[0063] (b) Molecule type: Nucleic acid

[0064] (c) Original source: Japanese shrimp ( Marsupenaeus japonicus )

[0065] (d) Sequence description: Details are as follows:

[0066]

[0067] 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.

[0068] A third typical embodiment of the present invention provides a recombinant expression vector, wherein the recombinant expression vector contains the above-mentioned nucleic acid molecule.

[0069] A fourth typical embodiment of the present invention provides an engineered bacterium, wherein the engineered bacterium contains the above-mentioned recombinant expression vector, wherein the engineered bacterium may be a eukaryotic bacterium or a prokaryotic bacterium, for example, but not limited to, Agrobacterium, yeast, Escherichia coli, etc.

[0070] A fifth typical embodiment of the present invention provides a use of the above protein in preparing a product for regulating intestinal homeostasis of aquatic animals;

[0071] The aquatic animals are crustacean aquatic animals.

[0072] In some examples of this embodiment, the product has any one or more of the following functions:

[0073] (1) Mediate the colonization of probiotics in the intestines of aquatic animals;

[0074] (2) Increase the number of probiotics in the intestines of aquatic animals;

[0075] (3) Improve the ability of aquatic animals to resist exogenous pathogens;

[0076] (4) Maintaining the homeostasis of the intestinal flora of aquatic animals;

[0077] (5) Improve the survival rate of aquatic animals infected by exogenous pathogens;

[0078] (6) Prevent bacterial diseases of aquatic animals;

[0079] The probiotics are Gram-positive probiotics, the exogenous pathogenic bacteria are Vibrio, and the bacterial disease of aquatic animals is Vibriosis.

[0080] In some examples of this embodiment, the probiotic is Bacillus subtilis.

[0081] In some examples of this embodiment, the Vibrio includes but is not limited to one or more of Vibrio parahaemolyticus, Vibrio anguillarum and Vibrio harveyi, preferably Vibrio parahaemolyticus.

[0082] In some examples of this embodiment, the bacterial diseases of aquatic animals include but are not limited to vibriosis caused by one or more of Vibrio parahaemolyticus, Vibrio anguillarum or Vibrio harveyi, preferably vibriosis caused by Vibrio parahaemolyticus.

[0083] In some examples of this embodiment, the aquatic animal is a prawn. In the specific example of the present invention, Japanese prawn is selected.

[0084] In some examples of this embodiment, the protein is applied to aquatic animals by direct feeding, feed addition, or preparation of medicine.

[0085] A sixth typical embodiment of the present invention provides a product for regulating intestinal homeostasis of aquatic animals, the product comprising the above-mentioned protein, and the product having one or more of the following functions:

[0086] (1) Mediate the colonization of probiotics in the intestines of aquatic animals;

[0087] (2) Increase the number of probiotics in the intestines of aquatic animals;

[0088] (3) Improve the ability of aquatic animals to resist exogenous pathogens;

[0089] (4) Maintaining the homeostasis of the intestinal flora of aquatic animals;

[0090] (5) Improve the survival rate of aquatic animals infected by exogenous pathogens;

[0091] (6) Prevent bacterial diseases of aquatic animals;

[0092] The probiotics are Gram-positive probiotics, the exogenous pathogenic bacteria are Vibrio, and the bacterial disease of aquatic animals is Vibriosis.

[0093] In some examples of this embodiment, the aquatic animal is a crustacean aquatic animal, preferably a prawn. In the specific embodiment of the present invention, the aquatic animal is a Japanese shrimp.

[0094] In some examples of this embodiment, the Vibrio includes but is not limited to one or more of Vibrio parahaemolyticus, Vibrio anguillarum and Vibrio harveyi, preferably Vibrio parahaemolyticus.

[0095] In some examples of this embodiment, the vibriosis includes but is not limited to vibriosis caused by one or more of Vibrio parahaemolyticus, Vibrio anguillarum and Vibrio harveyi, preferably vibriosis caused by Vibrio parahaemolyticus.

[0096] In some examples of this embodiment, the product is a feed additive, a medicine or a water treatment agent.

[0097] Specifically, the feed additive, medicine or water treatment agent can be in the form of powder, soluble powder, granular premix, compound preparation, liquid coating or microcapsule embedding, etc., which is not limited here and can be selected according to actual needs.

[0098] More specifically, the feed additive, agent or water treatment agent can be directly mixed with the feed, added to the feed in combination with probiotics, or prepared into a corresponding agent for use, which is not specifically limited here and can be selected according to actual needs. The specific application method can be direct feeding, which is not specifically limited here and can be selected according to actual needs.

[0099] 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.

[0100] Example 1 Amplification and expression of protein mature peptide sequence.

[0101] 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:

[0102] F1: 5'-GCCATGGCTGATATCGGATCCTTCCCACTCCCCATGAGCA-3' (SEQ ID NO. 3);

[0103] R1: 5'-GTGGTGGTGGTGGTGCTCGAGAAGATTATATTGACACACAAATGGAAAC-3' (SEQ ID NO. 4), containing BamH I and Xho I restriction sites.

[0104] The sequence encoding the mature peptide of the protein was amplified by the above primers and recovered by gel. Subsequently, the PET-30a (+) 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-thiogalactoside (IPTG) was used to induce expression at 28°C for 6 hours. Since the protein is an insoluble protein, it needs to be dissolved in 8 M urea at 28°C and 160 rpm overnight, and the supernatant was taken after centrifugation at 12000 rpm. The target protein was obtained after dialyzed and renatured in PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mMKH2PO4, pH 7.4) (see Figure 1 ).

[0105] Example 2 Tissue distribution of mRNA levels and protein domain analysis

[0106] After extracting RNA from various shrimp tissues and converting it into cDNA, real-time fluorescence quantitative PCR (qPCR) was used to detect the expression level of the gene encoding the protein in various tissues. The primer sequences are:

[0107] F2: 5'-CCAGCATCATCGCAAACAT-3' (SEQ ID NO.5)

[0108] R2: 5'-AAGGTAAACGGGTAGGGACA-3' (SEQ ID NO.6)

[0109] β-actin was used as an internal reference, and the primer sequences were:

[0110] F3: 5'-AGTAGCCGCCCTGGTTGTAGAC-3' (SEQ ID NO.7)

[0111] R3: 5'-TTCTCCATGTCGTCCAGT-3' (SEQ ID NO.8)

[0112] The qPCR 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.

[0113] Test results ( Figure 2A ) showed that the gene encoding this protein was most highly expressed in intestinal tissue, suggesting that this protein may function in the intestine.

[0114] The amino acid sequence of the protein (SEQID NO.1) was predicted by the online SMART tool (https: / / smart.embl.de / ), and it was found that it contains multiple chitin binding domains. Since chitin is abundant in the intestines of shrimp, the protein also has a carbohydrate recognition domain, indicating that it may have the function of binding bacteria and mediating their colonization in the intestines ( Figure 2B ).

[0115] Example 3 In vitro validation of recombinant protein combined with probiotics

[0116] Gram-positive bacteria (such as Bacillus subtilis, Bacillus megaterium, Microbacterium laceae, and Micrococcus plantarum) and Gram-negative bacteria (such as Vibrio harveyi, Vibrio fischeri, Vibrio anguillarum, and Aeromonas hydrophila) were cultured at 37°C. Bacteria were collected by centrifugation at 6000×g for 3 minutes, washed three times with PBS, and then resuspended in PBS. Then, bacteria (1×107 CFU) and 20 μg of recombinant protein were gently shaken at 28°C for 1 h. Bacteria were collected by centrifugation at 6000 × g for 5 min and washed five times with PBS. Subsequently, bound proteins were eluted with 7% SDS and protein levels were detected by western blotting. The primary antibody was mouse anti-6-His monoclonal antibody (Abbkine, Wuhan, China; ABT 2050) at a dilution of 1:1500. The secondary antibody was horseradish peroxidase (HRP)-labeled goat anti-mouse antibody (ZB-2305, Zhongshan Biotechnology) at a dilution of 1:10000. Immunoreactive protein bands were visualized using High-sig enhanced chemiluminescence (ECL) western blotting substrate (180-5001, Tanon, Shanghai, China) and the Tanon 5200 chemiluminescence imaging system.

[0117] result( Figure 3 ) showed that the protein had a strong binding ability to Gram-positive probiotics (such as Bacillus subtilis), but a weaker binding ability to Vibrio harveyi and other Vibrio, indicating that the protein has the property of selectively binding to probiotics.

[0118] Example 4 In vivo verification that the recombinant protein helps probiotics colonize in the intestine

[0119] The experimental animal treatment process was as follows: first, the shrimp were orally fed with 50 μL of an antibiotic mixture (ampicillin, 25 mg / mL; kanamycin, 25 mg / mL; oxytetracycline, 25 mg / mL, Sangon Biotech (Shanghai)). After 24 h, the antibiotics were repeated. After 48 h, the shrimp were fed with 20 μg of recombinant protein and 1×10 7 CFU Bacillus subtilis mixture, and the localization of bacteria in the intestine was detected 24 hours after feeding ( Figure 4A ), the number of bacteria in some areas of the intestine ( Figure 4B ), the total number of bacteria in the intestine ( Figure 4C ).in, Figure 4A The cells were fed with FITC (fluorescein isothiocyanate) labeled bacteria.

[0120] After the experimental animals were treated, the intestines of the two groups of shrimp were fixed in Davidson's fixative (30% ethanol, 22% formalin and 11.5% acetic acid) for 24 hours, and then immersed in 15% and 30% sucrose solutions for 24 hours for dehydration. After that, they were placed in the embedding medium and immersed in 4°C overnight and then transferred to -20°C for half an hour. The wax blocks of the embedded tissue were cut into wax slices with a thickness of 10 μm using a slicer (Leica CM 1860). The wax slices were attached to the slides and dried at 60°C for half an hour, and then the protein was labeled with His primary antibody (mouse) and Dylight 649-labeled fluorescent secondary antibody (goat anti-mouse, 1:500 dilution). The localization of the protein and FITC-labeled Bacillus subtilis in the intestine was observed under a confocal microscope (LeicaSTELLARIS 5, Germany). Results ( Figure 4A ) found that the protein can bind to the intestinal chitin layer and help probiotics colonize on it.

[0121] After the experimental animals were treated, intestinal samples of about 1 mm 3 The samples were washed with PBS for 3 times, each time for 15 min. Dehydrated with 30%, 50%, 70%, 90%, and 100% ethanol, each time for 15 min. The samples were then dried in a critical point dryer and coated with gold using a sputtering coater (HITACHI MC 1000, Hitachi, Tokyo, Japan) (with the inner surface of the intestine facing up), and images were taken using a field emission scanning electron microscope (HITACHI Regulus 8100, Hitachi). Results ( Figure 4B ) showed that after feeding the protein and probiotic mixture, the number of probiotics on the intestinal surface increased, indicating that the protein can help probiotics colonize on the intestinal surface.

[0122] After the experimental animals were treated, the intestinal tissues of 6 shrimps in each group were homogenized in PBS, and then the number of bacteria was determined by plate counting method. It was found that after feeding the protein and probiotics, the number of probiotics in the intestine increased ( Figure 4C ).

[0123] Experimental Example 5 16S rDNA sequencing revealed changes in the intestinal flora structure after silencing of the protein gene expression.

[0124] Gene expression silencing (RNAi): using specific primers linked to a T7 promoter:

[0125] F4:5'-GCGTAATACGACTCACTATAGGGCTGGATTCCGTCACATAAAGC-3' (SEQ ID NO.9)

[0126] R4:5'-GCGTAATACGACTCACTATAGGGTGGGTATTGTCGTGGTTGC-3' (SEQ ID NO.10)

[0127] Partial cDNA fragments of the target gene were amplified and used as templates for dsRNA synthesis using an in vitro T7 transcription kit (TR102; Vazyme, Nanjing, China). dsRNA specific for the green fluorescent protein (GFP) gene was used as a control. dsRNA was injected into the hemocoel of shrimp at a dose of 50 μg / shrimp. The RNAi efficiency was determined 24 h later (using SEQ ID NO.5; SEQ ID NO.6 as primers) ( Figure 5A ).

[0128] Two days after the protein gene expression was silenced, the shrimp intestinal tissue was collected, frozen in liquid nitrogen, and sent to BioMarker (Beijing, China) for commercial sequencing. The results showed that after gene silencing, the relative abundance of two types of positive bacteria (Firmicutes and Proteobacteria) at the phylum level was significantly reduced ( Figure 5B ). At the genus level, the relative abundance of Gram-negative pathogens (Vibrio, Vibrio fischeri) increased significantly ( Figure 5C ). This indicates that the absence of the protein in the intestine will lead to intestinal flora disorder, and combined with the previous examples (Example 3 and Example 4), it is further concluded that the protein helps maintain intestinal homeostasis by mediating probiotic colonization.

[0129] Experimental Example 6 Effect of feeding recombinant protein on survival rate under Vibrio infection

[0130] In this example, the shrimps were kept in a normal temperature circulating oxygenated seawater environment, and the stocking density was set at 40 shrimps / mm 3 After oral feeding of the recombinant protein (20 μg / prawn) for 24 h, 1×10 7 CFU of Vibrio parahaemolyticus were orally fed into the shrimp intestine. Figure 6 ) found that feeding the recombinant protein in advance could increase the survival rate of shrimp infected with Vibrio parahaemolyticus.

[0131] 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. A protein that mediates probiotic colonization, characterized in that The amino acid sequence of the protein is shown in SEQ ID NO:

1.

2. A nucleic acid molecule encoding the protein of claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:

2.

4. A recombinant expression vector, characterized in that: The recombinant expression vector contains the nucleic acid molecule according to claim 2 or 3.

5. An engineered bacterium, characterized in that: The engineered bacteria contains the recombinant expression vector according to claim 4.

6. Use of the protein according to claim 1 in preparing a product for regulating intestinal homeostasis of aquatic animals; The aquatic animals are crustacean aquatic animals.

7. The use according to claim 6, characterized in that The product has any one or more of the following functions: (1) Mediate the colonization of probiotics in the intestines of aquatic animals; (2) Increase the number of probiotics in the intestines of aquatic animals; (3) Improve the ability of aquatic animals to resist exogenous pathogens; (4) Maintaining the homeostasis of the intestinal flora of aquatic animals; (5) Improve the survival rate of aquatic animals infected by exogenous pathogens; (6) Prevent bacterial diseases of aquatic animals; The probiotics are Bacillus subtilis, the exogenous pathogenic bacteria are Vibrio parahaemolyticus, and the bacterial disease of aquatic animals is vibriosis caused by Vibrio parahaemolyticus.

8. The use according to any one of claims 6 to 7, characterized in that: The aquatic animal is a prawn.

9. The use according to any one of claims 6 to 7, characterized in that: The protein is applied to aquatic animals by direct feeding, feed addition or preparation into medicine.

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