CN114671945A

CN114671945A - Grass carp bacteria small peptide recognition receptor and preparation method and application thereof

Info

Publication number: CN114671945A
Application number: CN202210042932.7A
Authority: CN
Inventors: 瞿符发; 刘臻; 汤杰; 李嘉玲; 房佳美; 郭美杏; 唐建洲; 曾璇; 佘青; 高超然
Original assignee: Changsha University
Current assignee: Changsha University
Priority date: 2022-01-14
Filing date: 2022-01-14
Publication date: 2022-06-28

Abstract

The invention discloses a grass carp bacterial small peptide recognition receptor and a preparation method and application thereof, wherein the grass carp bacterial small peptide recognition receptor is grass carp NOD2-LRR protein, the amino acid sequence is shown as SEQ ID NO.1, and the cDNA sequence of a coding gene is shown as SEQ ID NO. 2. The preparation of the grass carp bacterial small peptide recognition receptor comprises the steps of cloning grass carp NOD2 gene, expressing, purifying and renaturing. According to the invention, an immune recognition model of grass carp NOD2 on bacterial MDP is established for the first time by using a molecular docking method, and NOD2-LRR protein is successfully obtained by using a prokaryotic expression system, and the result shows that the NOD2-LRR protein has the binding activity on the bacterial MDP, can specifically recognize the bacterial MDP, plays an important role in the immune recognition of the bacterial MDP, and has potential application value in the aspects of developing novel antibacterial drugs, immunopotentiators, feed additives and the like.

Description

Grass carp bacteria small peptide recognition receptor and preparation method and application thereof

Technical Field

The invention relates to a grass carp bacteria small peptide recognition receptor and a preparation method and application thereof.

Background

The bacterial enteritis of fishes is emphasized by the characteristics of wide morbidity (almost all cultured fishes are involved), serious harm (high infection rate and mortality rate), complex pathogenesis (influenced by factors such as hosts, pathogens, environment and the like), and the like, and becomes a hotspot and difficulty in the research field of aquatic diseases and immunology. Aiming at pathogenic bacteria of fish intestinal infection, early immunology theory proves that an organism can identify exogenous pathogens and danger early warning signals generated by the exogenous pathogens by means of an inherent immune system (endogenous immune system), induce the organism to generate immune response reaction and finally remove exogenous pathogenic substances so as to keep the intestinal homeostasis. Research shows that innate immunity is the first defense line of body to defend infection, and the mediated immune response process comprises immune recognition of pathogens, transmission of immune signals, induction of effector molecules and the like. Wherein, the recognition of self and non-self is the first step of inducing immune response, and is also the basis for the body to realize effective defense against pathogenic microorganisms. Research finds that the non-self recognition of the innate immunity depends on a corresponding Pattern Recognition Receptor (PRR) encoded by a host gene, and the non-self recognition of the innate immunity can activate a downstream signal path in a cell by recognizing a Pathogen Associated Molecular Pattern (PAMP) which is a conserved structure of pathogenic microorganisms, so as to trigger an immune response reaction of an organism. Because of the central role of PRRs in pathogen recognition, studies of pattern recognition receptors have been the focus of immunology.

Studies from higher animals have found that intestinal bacteria over-propagate or dysbacteriosis can release a large amount of Muramyl Dipeptide (MDP), a metabolite, to induce the body to produce an intestinal inflammatory response by crossing the intestinal mucosal barrier with small peptide transporter 1 (PepT 1) of intestinal epithelial cells. Research shows that intracellular nucleotide binding oligomerization domain receptor protein 2 (NOD 2) of intestinal cells can recognize foreign bacterial oligopeptide product MDP mediated by PepT1, and then expression of inflammatory factors of the intestinal cells is induced through two pathways of nuclear factor kappa B (NF-kappa B) and mitogen-activated protein kinase (MAPK), so that inflammatory reaction is caused. Research shows that NOD2 is a pattern recognition receptor protein capable of specifically recognizing cytoplasmic bacterial MDP, the expression of which is closely related to Inflammatory Bowel Disease (IBD), and plays an important role in innate immune response.

Currently, studies on expression and activity of NOD2 in lower vertebrate fish are relatively poor. Meanwhile, compared with higher animals, because fish NOD2 has larger difference in structure, whether the pathogen pattern recognition receptor protein NOD2 participates in bacterial enteritis reaction of the fish is not clear. Therefore, research on expression and activity of NOD2 in fish can provide theoretical basis for monitoring intestinal bacterial infection and preventing and treating bacterial enteritis in fish, and has potential application value in aspects of developing novel antibacterial drugs and feed additives.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a grass carp bacteria small peptide recognition receptor, a preparation method and application.

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

a grass carp bacterial small peptide recognition receptor, which is a grass carp NOD2-LRR protein; the amino acid sequence of the grass carp NOD2-LRR protein is shown in SEQ ID NO. 1.

As a general technical concept, the invention also provides a gene for coding the grass carp bacterial small peptide recognition receptor, and the cDNA sequence of the gene is shown as SEQ ID NO. 2.

As a general technical concept, the invention also provides a recombinant plasmid of the gene of the grass carp bacterial small peptide recognition receptor.

In the above-mentioned recombinant plasmid, further improvement, said recombinant plasmid is recombinant plasmid pET32a-NOD 2-LRR.

As a general technical concept, the present invention also provides a preparation method of the grass carp bacterial small peptide recognition receptor, comprising the following steps:

s1, cloning a grass carp NOD2 gene;

s2, constructing an NOD2-LRR functional domain expression vector according to the cDNA sequence of the grass carp NOD2 gene, and performing codon optimization to obtain an NOD2-LRR gene;

S3, carrying out double enzyme digestion on NOD2-LRR gene and pET32a plasmid by adopting BamH I and Xho I, and carrying out conversion expression after connection to obtain recombinant plasmid pET32a-NOD 2-LRR;

s4, carrying out IPTG induction expression on the recombinant plasmid pET32a-NOD2-LRR to obtain recombinant protein, and purifying and renaturing to obtain the grass carp bacterial small peptide recognition receptor.

In the step S1, the constructed grass carp cDNA library is used as a template, and CiNOD2-F1 and CiNOD2-R1 are used as primers to perform PCR reaction to obtain a grass carp NOD2 gene; the sequence of the CiNOD2-F1 is shown as SEQ ID NO. 3; the sequence of the CiNOD2-R1 is shown in SEQ ID NO. 4.

In the preparation method of the grass carp bacterial small peptide recognition receptor, the temperature is controlled to be 37 ℃ in the IPTG induction expression process in the step S4; the concentration of IPTG in the control system in the IPTG induction expression process is 0.05 mM.

As a general technical concept, the invention also provides an application of the grass carp bacterial small peptide recognition receptor, and the grass carp bacterial small peptide recognition receptor is used for preparing a medicine for specifically recognizing bacterial enteritis of fishes.

As a general technical concept, the invention also provides an application of the grass carp bacterial small peptide recognition receptor, and the grass carp bacterial small peptide recognition receptor is used for preparing antibacterial drugs or immunopotentiators for treating bacterial enteritis of fishes.

As a general technical concept, the invention also provides an application of the grass carp bacterial small peptide recognition receptor, and the grass carp bacterial small peptide recognition receptor is used for preparing fish health products or feed additives.

Compared with the prior art, the invention has the advantages that:

according to the invention, an immune recognition model of grass carp NOD2 on bacterial MDP is established for the first time by using a molecular docking method, NOD2-LRR protein is successfully obtained by using a prokaryotic expression system, and the NOD2-LRR protein is found to have binding activity on the bacterial MDP through a surface plasmon resonance (SRP) technology and can specifically recognize the bacterial MDP, so that the grass carp NOD2-LRR protein plays an important role in immune recognition of the bacterial MDP, provides a theoretical basis for monitoring the bacterial infection condition of fish intestinal tracts and preventing and treating bacterial enteritis of fish, and has potential application values in the aspects of developing novel antibacterial drugs, immunopotentiators, feed additives and the like.

Drawings

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

FIG. 1 shows the expression level of grass carp NOD2 gene under the stimulation of bacterial MDP in example 1 of the present invention.

FIG. 2 shows the molecular binding pattern of grass carp NOD2 protein and bacterial MDP in example 1 of the present invention.

FIG. 3 shows the SDS-PAGE results of NOD2 protein in fish of example 1.

Detailed Description

The invention is further described below with reference to the drawings of the specification and to specific preferred embodiments, without thereby limiting the scope of protection of the invention.

In the following examples, all the raw materials and instruments used are commercially available unless otherwise specified.

Example 1

A grass carp bacterial small peptide recognition receptor is grass carp NOD2-LRR protein, and the amino acid sequence is shown in SEQ ID NO. 1.

A method for preparing the grass carp bacterial small peptide recognition receptor (grass carp NOD2-LRR protein) in the embodiment comprises the following steps:

(1) collecting samples:

healthy fresh grass carp (average weight about 30 g) is divided into 2 groups on average. The experimental group of grass carp was intraperitoneally injected with 100. mu.L of MDP solution (10. mu.g/mL, Invitrogen), and the control group was injected with an equal volume of PBS buffer per fish. The injected grass carp was immediately returned to the cultivation bucket, while 3 random samples were taken from each group as 0h samples. At 3, 6, 12, 24, 48 and 72 hours post-injection, 3 grass carp from each group were randomly taken, dissected from the fish body with sterile forceps and scissors and intestinal tissue removed, and the samples were placed in liquid nitrogen for total RNA extraction.

(2) Total RNA extraction:

total RNA of the intestinal tract sample is extracted by using a reagent of RNAioso (Takara), and the specific steps are as follows: taking 100mg of grass carp intestinal tissue in a mortar precooled by liquid nitrogen, grinding the tissue into powder, transferring the powder into a centrifugal tube filled with 1mL of RNAioso, blowing, beating and uniformly mixing; adding 200 μ L chloroform, shaking vigorously for 40s, and standing at room temperature for 5 min; centrifuging at 4 deg.C for 15min at 12,000 Xg, sucking supernatant 0.5mL, and transferring to a new tube; adding 0.5mL of isopropanol, and uniformly mixing; centrifuging at 4 deg.C for 10min at 12,000 Xg, and discarding the supernatant; washing the RNA precipitate with 1mL of 75% ethanol; discarding the supernatant, adding 30 μ L DEPC water to dissolve RNA; RNA integrity and concentration were checked using 1.2% agarose gel electrophoresis and a nucleic acid protein quantifier, respectively.

(3) constructing a cDNA library:

construction of grass carp intestinal cDNA library Using PrimeScriptTMRT Reagent Kit with gDNA Eraser (TaKaRa), the specific steps are as follows: removing genome DNA reaction: preparing reaction liquid in a Microtube, wherein the reaction liquid comprises 2 mu L of 5 Xg DNA Eraser Buffer, 1 mu L of gDNA Eraser, 1 mu g of grass carp intestinal total RNA and RNase Free H₂O, supplementing the total volume to 10 mu L; 42 ℃ for 2min, and 4 ℃ is finished. Reverse transcription reaction: the reverse transcription reaction solution was prepared in the Microtube described above, including 10. mu.L of the reaction solution in step 1, 4. mu.L of 5 XPrimeScript Buffer 2(for Real Time), 1. mu.L of PrimeScript RT Enzyme Mix 1, 4. mu.L of RNase Free H ₂O and 1. mu.L RT Primer Mix; the reaction system is flicked and mixed evenly and is slightly eccentric; 15min at 37 ℃; inactivating reverse transcriptase at 85 deg.C for 5 s; cooled on ice and stored at-20 ℃.

(4) Cloning cDNA sequence of grass carp NOD2 gene:

according to sequence information screened in a grass carp intestinal transcriptome database, a pair of specific primers CiNOD2-F1 and CiNOD2-R1 containing an open reading frame of grass carp NOD2 gene are designed, and are shown as follows:

CiNOD2-F1：5'-ATTGTGACTGTTGACATTGAG-3'(SEQ ID NO.3)；

CiNOD2-R1：5'-TACATTTATGACAGCCCCGAT-3'(SEQ ID NO.4)。

and carrying out PCR reaction by taking the constructed cDNA library as a template and taking CiNOD2-F1 and CiNOD2-R1 as primers.

The PCR reaction system is as follows: 37.75 μ L ddH₂O,0.25μL Ex Taq DNA Polymerase(5U/μL,TaKaRa),5μL 10×Ex PCR Buffer(Mg²⁺Plus), 4. mu.L dNTP mix (2.5mM each), 1. mu.L forward and reverse primers (10. mu.M), and 1. mu.L cDNA templateAnd (3) a plate.

The PCR reaction program is: pre-denaturation at 94 ℃ for 3 min; 94 ℃ 30s, 56 ℃ 30s, 72 ℃ 90s (35 cycles); 10min at 72 ℃; storing at 4 ℃.

Detecting the product by using 1.5% agarose gel after the PCR reaction is finished, connecting the purified product with a pMD-19T (TaKaRa) vector, converting the product into an escherichia coli competent cell DH5 alpha, selecting positive clone after the colony PCR detection, and sending the positive clone to a platforming biotechnology (Shanghai) Limited company for sequencing to obtain the sequence information of the grass carp NOD2 gene cDNA.

Detection of transcriptional level of NOD2 gene:

according to the cloned cDNA sequence information of the grass carp NOD2 gene, a pair of specific primers CiNOD2-F2 and CiNOD2-R2 for detecting the expression level of the NOD2 gene are designed, and are shown as follows:

CiNOD2-F2：5'-ACTTTTGATGGGCTTGACGA-3'(SEQ ID NO.5)；

CiNOD2-R2：5'-GCACCTCTTTGCGGAGATAAC-3'(SEQ ID NO.6)。

And (3) carrying out fluorescence quantitative PCR reaction by taking the cDNA constructed in the step (3) as a template and CiNOD2-F2 and CiNOD2-R2 as primers, and taking the transcription level of the grass carp housekeeping gene beta-actin as an internal reference control.

The PCR reaction system is as follows: 5.68 μ L ddH₂O, 8. mu.L of TB Green Premix Ex Taq (Tli RNaseH Plus) (2X, TaKaRa), 0.32. mu.L of ROX Reference Dye II (50X), 4. mu.L of dNTP mix (2.5mM each), 0.5. mu.L of forward and reverse primers (10. mu.M), and 1. mu.L of cDNA template.

The PCR reaction program is: pre-denaturation at 94 ℃ for 4 min; 94 ℃ 10s, 56 ℃ 10s, 72 ℃ 10s (45 cycles).

After the fluorescent quantitative PCR reaction is finished, 2 is applied according to the collected CT values of the target gene and the reference gene^-ΔΔCTThe method calculates the relative expression amount, and the result is shown in FIG. 1.

As can be seen from figure 1, the real-time fluorescent quantitative PCR technology is used for detection, expression level of the NOD2 gene in grass carp intestinal tracts is obviously increased (24.5 times of that of a PBS injection group) after bacteria MDP is injected for 6 hours, and the NOD2 gene is disclosed to be well related to grass carp bacterial enteritis, the NOD2 gene can be used as a fish bacterial enteritis diagnosis molecular marker, and the grass carp bacterial enteritis can be effectively diagnosed by using a detection method of the expression level of the NOD2 gene.

Molecular docking of grass carp NOD2 with bacterial MDP:

in the project, a single template 5irm protein A chain is adopted for modeling, the similarity of the template and the amino acid sequence of the template is 55%, the modeling result is optimized by AMBER16 software, the optimization time is 5000ps, and the final average structure is taken for subsequent analysis. Molecular docking pocket prediction using the software using the modeled crystal protein structure of template 5irm, it can be seen that the protein has a large binding cavity, and the cavity pocket contains the small active molecule, i.e., the substrate binding pocket. The binding region is measured in the region of the LRR protein and can act with small molecules. Flexible docking is carried out on the NOD2 protein and the small molecule MDP by utilizing Autodock software to obtain a primary docking phase structure, and the phase with the best docking energy is selected for structure extraction for subsequent molecular dynamics research.

Based on the docking results in the above steps, molecular dynamics simulations were performed using AMBER16 software. The whole protein system adopts the force fields of gaff and ff14SB, takes protein as the center, adds a 10A cubic water box and Na⁺The system is made electrically neutral, the topology and coordinate structure are preserved, and then simulation is performed. In the molecular dynamics process, all heavy atoms of the protein are restricted (position stress: -2000kcal/mol), i.e. the overall structure of the protein is kept unchanged. If the protein structure is changed greatly under the parameter setting, the amino acid of the part of the protein structure is easy to change, and the influence on the binding of small molecules is large.

As shown in FIG. 2, the binding pocket of grass carp NOD2 protein for MDP is mainly composed of LRR region (composed of 9 LRR structures, each LRR comprises typical beta-sheet and alpha-helix secondary structures), contains more polar amino acids, and can form better polar hydrogen bonding with small molecule MDP; the MDP structure contains more polar chemical groups, and can form better combination effect with hydrophilic and hydrophobic amino acid pockets of NOD2-LRR region (FIG. 2A, B). Through further molecular binding pattern analysis, potential key amino acid sites affecting grass carp NOD2 binding to bacterial MDP include: arg793, Asp768, Phe821, Arg847, Lys817, etc. (FIG. 2C, D). The model for combining the grass carp NOD2-LRR with the bacterial dipeptide MDP, which is established by the research, lays a foundation for subsequently disclosing an immune recognition mechanism of the grass carp NOD2 with the bacterial MDP and carrying out preparation research on NOD2 active protein.

(6) Expression of grass carp NOD2-LRR protein

(6.1) vector construction:

based on the results shown in FIG. 2, the sequence of LRR region (NOD 2-LRR: 731-977aa) of MDP binding pocket in grass carp NOD2 protein was selected, codon optimization was performed in E.coli system to obtain optimized gene fragment sequence, which was synthesized by the company as NOD2-LRR gene sequence, whose nucleotide sequence is shown in SEQ ID NO.2 and contains 741 bases. The BamH I and Xho I are adopted to carry out double enzyme digestion on pET32a plasmid, and the reaction system is as follows: quickcut Xho I1. mu.L, Quickcut BamH I1. mu.L, 10 XQuickcut Buffer 5. mu.L, pET32a plasmid 1. mu.g, ddH ₂O Up to 50. mu.L. Mixing, centrifuging, and keeping the temperature at 37 deg.C for 10 min. After the enzyme digestion reaction is finished, detecting the double enzyme digestion effect by utilizing agar gel electrophoresis, and recovering the double enzyme digestion effect in a tapping mode. Using Clonexpress^TMII (Vazyme) reagent for recombination reaction, the reaction system is as follows: exnase^TMII 2. mu.L, 5 XCE II Buffer 4. mu.L, 50-200 ng of linearized pET32a vector, 200ng of PCR amplification product, ddH₂O Up to 20. mu.L. After the system preparation is completed, the reaction is carried out for 30min at 37 ℃. After the reaction was completed, the reaction tube was immediately placed in an ice-water bath to cool for 5 min. After the recombination reaction is finished, the product is transformed into an escherichia coli competent cell DH5 alpha, and positive clones are selected after colony PCR detection and sent to platforming biotechnology (Shanghai) limited company for sequencing. After the sequencing is successful, the amplification culture is carried out, and HiPure Plasmid EF Micro Kit (magenta) is used for extracting a recombinant Plasmid (pET32a-NOD2-LRR), wherein the recombinant Plasmid (pET32a-NOD2-LRR) contains a gene of grass carp bacteria small peptide recognition receptor NOD2-LRR protein.

(6.2) expression of grass carp NOD2-LRR protein

Transferring the recombinant plasmid pET32a-NOD2-LRR with correct sequencing into escherichia coli BL21 competent cells, and coating the competent cells on a flat plate containing corresponding antibiotics after heat shock for culture; selecting a single clone to be cultured in a liquid culture medium containing ampicillin; when the OD value reaches 0.6, adding 0.5mM IPTG (isopropyl thiogalactoside) of an inducer, continuously culturing for 12h at 37 ℃, and taking the negative control without the inducer; centrifuging, discarding the supernatant, and collecting the thallus; the collected cells were suspended in buffer A (PBS, pH7.4), and the suspension was thoroughly dissolved using an ultrasonication apparatus, centrifuged, and the precipitate after centrifugation was dissolved using buffer B (8M Urea,50mM Tris-HCl,300mM NaCl, pH8.0), and the supernatant and the precipitate were treated separately, and then sampled and examined by SDS-PAGE.

(6.3) purification of grass carp NOD2-LRR protein

Cell thalli is dissolved and ultrasonically broken by buffer solution C (8M Urea,50mM Tris,300mM NaCl, 0.1% Triton X-100, pH8.0), and supernatant crude protein is collected by centrifugation; 5mL of Ni-NTA was taken, and the equilibrated column was washed with 5 bed volumes of Binding buffer (8M Urea,50mM Tris,300mM NaCl, pH8.0) at a flow rate of 5 mL/min; incubating the crude protein with the balanced column packing for 1 h; putting the incubated product on a column, and collecting and flowing out; washing the equilibrium column with a Binding buffer; washing the column with Washing buffer (8M Urea,50mM Tris,300mM NaCl,20/50mM Imidazole, pH8.0) and collecting the effluent; eluting with Elution buffer (8M Urea,50mM Tris,300mM NaCl,500mM Imidazole, pH8.0), and collecting the eluate; respectively processing the crude protein, the elution impurity outflow and the elution outflow, preparing a sample, and preparing SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) for detection; the fractions of better purity were dialyzed into 500mM L-Arginine,2mM DTT, 0.1% SKL, pH8.0 buffer. After the dialysis was completed, the mixture was dialyzed to 10mM Tris-HCl,2mM DTT, 0.1% SKL,1mM EDTA, 10% Glycerol, pH 8.0. Concentrating the protein with PEG20000 after dialysis, filtering with 0.45 μm filter membrane, packaging into 1ml/tube, and storing at-80 deg.C to obtain grass carp NOD2-LRR protein with amino acid sequence shown in SEQ ID NO.1 and 247 amino acids.

Detection of grass carp NOD2-LRR protein:

SDS-PAGE detection: mixing the purified protein with 2 × loading buffer solution at ratio of 1: 1, water bathing at 100 deg.C for 10min, and taking out; SDS-PAGE protein gel was prepared using a protein gel kit (Bio-Rad); and respectively loading the sample and the protein molecule Marker by 10 mu L, wherein the constant voltage is 80V when the electrophoresis is started, and the constant voltage is 120V after the sample enters the separation gel until the bromophenol blue moves to the front edge. After the electrophoresis, the electrophoresis was performed by Coomassie brilliant blue staining and destaining with destaining solution, and the results of the observation experiment are shown in FIG. 3. As can be seen from FIG. 3, compared with the control group without IPTG, the experimental group induced by IPTG addition has obvious bands at the corresponding positions of theoretical molecular weight + -5 kDa, which confirms that the grass carp NOD2-LRR recombinant protein is successfully induced, and the NOD2-LRR protein with higher purity is obtained by purification operation.

The binding activity of grass carp NOD2-LRR protein and bacterial MDP is detected:

installing a COOH chip according to an OpenSPRTM instrument standard operating program; start running PBS buffer (pH 7.4) at maximum flow rate (150. mu.L/min); after the signal baseline was reached, 200 μ L of 80% IPA (isopropyl alcohol) was loaded, 10s of air-bubbling was run, and after the baseline was reached, the sample loop was flushed with buffer and evacuated with air; after the signal reaches the baseline, the flow rate of the buffer is adjusted to 20 mu L/min; load 200 μ L of EDC/NHS solution, buffer wash sample loop, and evacuate with air; load 200 μ L of ligand protein NOD2(6 μ g) diluted in activation buffer for 4 min; loading 200 μ L of Blocking solution, washing the sample loop with buffer, and evacuating with air; baseline was observed for 5 minutes to ensure stability; the analyte was loaded at a gradient concentration of MDP (0. mu.M, 100. mu.M, 200. mu.M, 400. mu.M, 800. mu.M) and the binding time of the polypeptide to the ligand was 240 s; natural dissociation for 160 s; the analysis software used in the experimental result is as follows: TraceDrawer (Ridgeview Instruments ab, Sweden) was analyzed by the One To One analysis model.

The results show that the grass carp NOD2-LRR protein has binding activity to bacterial MDP, K_DThe molecular weight of the strain reaches 1.84e-4M, which indicates that the grass carp NOD2-LRR protein can specifically recognize the bacterial MDP. Therefore, the grass carp NOD2-LRR protein can be used for preparing a medicine for specifically recognizing bacterial enteritis of fishes, can be used for preparing an antibacterial medicine or an immunopotentiator for treating the bacterial enteritis of the fishes, and can be used for preparing health-care products or feed additives of the fishes, so that the immunity and the resistance of the fishes (such as grass carps) to the bacterial enteritis can be enhanced. At the same time, for the NOD2-LRR regionTargeted inhibitors or activators are designed to interfere with the binding activity of NOD2 to bacterial MDP, thereby providing conditions for modulating the levels of inflammation in the grass carp gut induced by MDP.

In the embodiment, the fluorescence quantitative PCR method is used for finding that the expression of grass carp intestinal NOD2 and MDP-induced bacterial intestinal inflammation have good correlation, and specifically comprises the following steps: the in-vivo intraperitoneal injection experiment is carried out on the grass carp by using the bacterial MDP, real-time fluorescent quantitative PCR detection finds that the expression level of NOD2 in the grass carp intestinal tract is obviously increased after 6 hours of injection, so that the NOD2 gene has good correlation with the bacterial enteritis of the grass carp, and the expression detection of the gene can be used for carrying out effective early diagnosis on the bacterial intestinal inflammation of the grass carp.

In this embodiment, a molecular docking model combining a grass carp immune pattern recognition receptor (NOD2) and a bacterial pathogen-associated molecular pattern (MDP) is established, specifically: the 3D structure of the grass carp intestinal NOD2 protein is obtained by using a homologous modeling mode, the structure of the bacterial MDP is obtained from an NCBI database, the MDP is used as a ligand, the NOD2 protein is used as a receptor, and the binding mode analysis is carried out by using docking software such as MOE, AUTODOCK and the like, so that the binding free energy under the optimal binding conformation is obtained, and meanwhile, the binding pocket of NOD2 to the MDP and the key binding site thereof are predicted. The NOD2-MDP binding model established by molecular docking can provide a theoretical basis for further research on grass carp intestinal immune response mechanism and related drug development by taking NOD2 as a target.

In this example, the NOD2-LRR prokaryotic expression system was optimized from the aspects of coding sequence (rare codon) and expression conditions (temperature and IPTG concentration), specifically: a prokaryotic expression vector of grass carp pET32a-NOD2-LRR for identifying MDP is constructed by using a codon optimization technology, and a prokaryotic expression system for efficiently expressing grass carp NOD2-LRR protein fragments is obtained by screening at a proper temperature (37 ℃) and IPTG concentration (0.05mM), so that an expression product with higher purity is finally obtained. The prokaryotic expression system and conditions established by the invention can lay a foundation for the next step of industrial production of NOD2-LRR, and provide conditions for developing NOD2-LRR immune recognition function research and developing NOD2 as an immunopotentiator to be applied to aquaculture industry.

In this example, it was found by the surface plasmon resonance technology that the no 2 protein, an immune pattern recognition receptor, has a good binding activity to bacterial MDP, specifically: the surface plasmon resonance technology is utilized to detect the binding activity of the grass carp NOD2-LRR fragment and the bacterial MDP, and the result shows that the grass carp NOD2-LRR and the bacterial MDP have certain affinity, thereby providing a feasible technical means for developing the recognition mechanism research of a fish immune pattern recognition receptor on pathogen-related molecular patterns, and simultaneously providing an important basis for developing the subsequent research of gene therapy, drug therapy and the like by taking NOD2 as a molecular target for bacterial enteritis treatment of the grass carp.

The foregoing is illustrative of the preferred embodiments of the present invention and is not to be construed as limiting the invention in any way. Although the present invention has been described with reference to the preferred embodiments, it is not intended to be limited thereto. Those skilled in the art can make many variations and modifications to the disclosed solution, or equivalent embodiments, without departing from the spirit and technical scope of the invention, using the method and technical content disclosed above. Therefore, any simple modifications, equivalent substitutions, equivalent changes and modifications made to the above embodiments according to the technical essence of the present invention are within the scope of the technical scheme of the present invention, unless departing from the content of the technical scheme of the present invention.

Sequence listing

<110> Changsha college

<120> grass carp bacteria small peptide recognition receptor, preparation method and application

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 247

<212> PRT

<213> grass carp (Ctenophaggodon idella)

<400> 1

Lys Leu Asp Val Glu His Leu Lys Leu Thr Tyr Cys Ser Ile Gly Pro

1 5 10 15

Val Glu Cys Thr Ala Leu Ala Tyr Val Leu Gln His Leu Arg Asn Pro

20 25 30

Val Gly Leu Gln Leu Asp Asn Asn Ser Val Gly Asp Val Gly Val Glu

35 40 45

Gln Leu Leu Pro Cys Leu His Ile Cys His Ser Leu Tyr Leu Arg Asn

50 55 60

Asn Asn Ile Ser Asp Glu Gly Ile Arg Lys Leu Leu Glu Lys Gly Val

65 70 75 80

Lys Cys Glu Ser Phe Gln Lys Ile Ala Leu Phe Asn Asn Lys Leu Thr

85 90 95

Asp Ala Cys Thr Gln His Phe Ala Cys Leu Leu Lys Thr Lys Gln Asn

100 105 110

Phe Leu Ala Leu Arg Leu Gly Asn Asn Asn Ile Thr Ser Gln Gly Ala

115 120 125

Glu Gln Leu Ala Gly Gly Leu Ser Tyr Asn Gln Ser Leu Gln Phe Leu

130 135 140

Gly Leu Trp Gly Asn Lys Ile Gly Asp Arg Gly Ala Glu Ala Leu Ala

145 150 155 160

Asn Ala Leu Lys Asn Ser Thr Thr Leu Ile Trp Leu Ser Leu Val Asp

165 170 175

Asn Gly Val Gly Ser Ala Gly Ala Cys Ala Leu Ala Lys Leu Ile Ser

180 185 190

Gln Ser Lys Thr Leu Asp Glu Leu Trp Leu Asn Lys Asn Cys Ile Ser

195 200 205

Arg Asp Gly Val Glu Cys Leu Ile Glu Ala Leu Lys Met Asn Ser Ser

210 215 220

Val Arg Glu Val Trp Leu Arg Gly Asn Asn Leu Ser Pro Glu Glu Glu

225 230 235 240

Val Glu Leu Ser Lys Gln Glu

245

<210> 2

<211> 741

<212> DNA

<213> grass carp (Ctenophaggodon idella)

<220>

<221> misc_feature

<222> (1)..(741)

<223> grass carp NOD2-LRR gene cDNA

<400> 2

aagctggacg ttgaacacct gaaactgacc tactgcagca ttggtccggt ggagtgcacc 60

gcgctggcgt atgttctgca gcacctgcgt aacccggtgg gcctgcaact ggacaacaac 120

agcgtgggtg atgtgggcgt tgaacagctg ctgccgtgcc tgcacatctg ccacagcctg 180

tacctgcgta acaacaacat cagcgacgag ggtattcgta agctgctgga aaagggcgtt 240

aaatgcgaga gcttccaaaa gattgcgctg tttaacaaca aactgaccga tgcgtgcacc 300

cagcacttcg cgtgcctgct gaagaccaaa caaaactttc tggcgctgcg tctgggtaac 360

aacaacatta ccagccaagg tgctgaacaa ctggcgggtg gcctgagcta taaccagagc 420

ctgcaattcc tgggtctgtg gggcaacaag atcggtgatc gtggcgcgga ggcgctggcg 480

aacgcgctga aaaacagcac caccctgatt tggctgagcc tggtggataa cggtgtgggc 540

agcgcgggtg cgtgcgcgct ggcgaagctg attagccaga gcaaaaccct ggacgaactg 600

tggctgaaca agaactgcat cagccgtgat ggtgttgaat gcctgattga ggcgctgaaa 660

atgaacagca gcgtgcgtga ggtttggctg cgtggcaaca acctgagccc ggaggaagag 720

gtggaactga gcaaacaaga g 741

<210> 3

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(21)

<223> CiNOD2-F1 as a forward primer for cloning NOD2 gene designed according to experimental requirements

<400> 3

attgtgactg ttgacattga g 21

<210> 4

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(21)

<223> reverse primer CiNOD2-R1 designed according to experimental requirements and used as clone NOD2 gene

<400> 4

tacatttatg acagccccga t 21

<210> 5

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(22)

<223> is designed according to the experimental requirements, and is used as a forward primer CiNOD2-F2 for detecting the expression level of NOD2 gene

<400> 5

cagcagggac ggagttgagt gt 22

<210> 6

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(22)

<223> reverse primer CiNOD2-R2 designed according to experimental requirements and used for detecting expression level of NOD2 gene

<400> 6

ctggatttgg aagtttggtg gc 22

Claims

1. A grass carp bacterial small peptide recognition receptor, which is a grass carp NOD2-LRR protein; the amino acid sequence of the grass carp NOD2-LRR protein is shown in SEQ ID NO. 1.

2. A gene for coding the grass carp bacterial small peptide recognition receptor of claim 1, wherein the cDNA sequence of the gene is shown as SEQ ID No. 2.

3. A recombinant plasmid containing a gene of the grass carp bacterial small peptide recognition receptor of claim 2.

4. The recombinant plasmid of claim 3, wherein the recombinant plasmid is the recombinant plasmid pET32a-NOD 2-LRR.

5. A method for preparing a grass carp bacterial small peptide recognition receptor according to claim 1, which comprises the following steps:

s1, cloning a grass carp NOD2 gene;

s3, carrying out double enzyme digestion on NOD2-LRR gene and pET32a plasmid by using BamH I and Xho I, and carrying out transformation expression after connection to obtain recombinant plasmid pET32a-NOD 2-LRR;

6. The method for preparing a grass carp bacterial small peptide recognition receptor as claimed in claim 5, wherein in step S1, a PCR reaction is performed by taking a constructed grass carp cDNA library as a template and taking CiNOD2-F1 and CiNOD2-R1 as primers to obtain a grass carp NOD2 gene; the sequence of the CiNOD2-F1 is shown as SEQ ID NO. 3; the sequence of the CiNOD2-R1 is shown in SEQ ID NO. 4.

7. The method for preparing a grass carp bacterial small peptide recognition receptor as claimed in claim 5 or 6, wherein in step S4, the temperature is controlled to 37 ℃ during the IPTG induced expression; the concentration of IPTG in the control system is 0.05mM in the IPTG induction expression process.

8. The use of the grass carp bacterial small peptide recognition receptor as defined in claim 1, wherein the grass carp bacterial small peptide recognition receptor is used for preparing a medicament for specifically recognizing fish bacterial enteritis.

9. The use of the grass carp bacterial small peptide recognition receptor according to claim 1, wherein the grass carp bacterial small peptide recognition receptor is used for preparing antibacterial drugs or immunopotentiators for treating bacterial enteritis in fish.

10. The use of the grass carp bacterial small peptide recognition receptor according to claim 1, wherein the grass carp bacterial small peptide recognition receptor is used for preparing fish health products or feed additives.