Eriocheir sinensis lrr-ig4 recombinant protein and application thereof

Abstract

The application discloses a kind of Eriocheir sinensis LRR-IG4 recombinant proteins and application thereof, belong to molecular biology and immunology technical field.The recombinant protein is the I type transmembrane protein comprising signal peptide, extracellular region, single transmembrane region and intracellular region, the extracellular region comprises the leucine-rich repeat LRR domain shown in SEQ ID NO:2 and the immunoglobulin-like IG domain shown in SEQ ID NO:3, and the protein has the broad-spectrum pathogen-associated molecular pattern PAMPs binding activity of lipopolysaccharide LPS and peptidoglycan PGN, wherein: the LRR domain is located at the N terminal 45th-378th amino acid of the protein, the IG domain is located at the 451th-554th amino acid of the protein, the single transmembrane region is located at the 571th-592th amino acid, and the intracellular region is located at the 593th-1005th amino acid;The protein can activate the nuclear transposition of Dorsal transcription factor downstream of Eriocheir sinensis Toll signal pathway by binding with lipopolysaccharide and peptidoglycan, and up-regulate the expression of anti-lipopolysaccharide factor ALF.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biology and immunology, specifically relating to a recombinant protein of Chinese mitten crab (Eriocheirsinensis) LRR-IG4, its encoded nucleic acid, recombinant expression vector, recombinant host cell, and the application of the recombinant protein in pathogen detection, crustacean immune enhancement, and in vitro evaluation of disease resistance. Background Technology

[0002] The Chinese mitten crab (Eriocheir sinensis), commonly known as the hairy crab, is an extremely important economic aquaculture species in my country. In recent years, with the promotion of high-density intensive farming models, diseases in Chinese mitten crab farming have become increasingly frequent. Various bacterial diseases caused by pathogens such as Vibrio parahaemolyticus, Aeromonas hydrophila, and Staphylococcus aureus have caused huge economic losses to the aquaculture industry. Traditionally, aquaculture often relies on antibiotics for disease control, but this easily leads to drug resistance in pathogens and causes environmental pollution and food safety risks. Therefore, the development of green and safe new aquatic immune enhancers and vaccine adjuvants, as well as the development of molecular marker tools for breeding disease-resistant varieties, has become an urgent need in the field of modern crustacean disease control.

[0003] Unlike vertebrates, crustaceans lack a specific acquired immune system mediated by T and B lymphocytes. Their defense against invading exogenous pathogens relies entirely on their innate immune system. Innate immunity in crustaceans primarily includes cellular immunity (such as phagocytosis, cyst formation, and nodulation by hemocytes) and humoral immunity (such as the synthesis and release of antimicrobial peptides and activation of the prophenoloxidase system). During pathogen recognition, the host's pattern recognition receptors (PRRs) are responsible for specifically recognizing pathogen-associated molecular patterns (PAMPs) on the surface of pathogenic microorganisms, such as lipopolysaccharide (LPS) on the surface of Gram-negative bacteria and peptidoglycan (PGN) on the surface of Gram-positive bacteria, thereby amplifying and initiating downstream defense responses.

[0004] Among numerous pattern recognition receptors, proteins rich in leucine-rich repeats (LRR) and immunoglobulin (IG) domains play crucial roles in host defense. Existing research indicates that the LRR domain is highly variable and primarily responsible for providing protein-protein interaction interfaces for binding PAMPs; while the IG domain typically mediates cell adhesion, receptor molecule dimerization, and signal transduction.

[0005] Although the above studies have preliminarily revealed the potential role of LRR or IG domains in crustacean immune recognition, the following technical shortcomings still exist in the existing technology: First, current technologies have only predicted the existence of LRR or IG domain proteins in some crustaceans based on bioinformatics analysis, but no studies have experimentally verified the specific immune function of **type I transmembrane receptors containing both LRR and IG domains** in the Chinese mitten crab. In particular, current technologies have not disclosed whether such dual-domain receptors possess broad-spectrum PAMP binding activity, nor have they clarified whether their binding activity is sufficient to activate downstream signaling pathways.

[0006] Second, existing research on the Toll / Dorsal signaling pathway in crustaceans largely focuses on the expression analysis of downstream effector molecules, but lacks in-depth research on the molecular structural characteristics of the upstream **specific recognition receptor** and its binding mechanism with ligands. While those skilled in the art are aware that the LRR domain can participate in pattern recognition, they cannot predict whether specific spatial combinations of the LRR and IG domains (such as the specific arrangement of the domains shown in SEQ ID NO:2 and SEQ ID NO:3 within the full-length protein of SEQ ID NO:1) will produce synergistic or antagonistic effects, nor can they predict whether this receptor can effectively trigger the nuclear translocation of Dorsal after binding to LPS and PGN.

[0007] Third, existing technologies do not provide any functional evidence regarding the EsLRR-IG4 gene (GenBank accession number OP921210) in pathogen immune responses in the Chinese mitten crab. Although the gene sequence has been published, the publication of the sequence does not equate to the publication of its function. Those skilled in the art cannot directly infer from the sequence information whether the gene is involved in immune recognition, whether it can serve as an effective component of immune enhancers, or whether its expression level is related to an individual's immune capacity.

[0008] Therefore, there is an urgent need in the field for an experimentally validated recombinant protein of the Chinese mitten crab LRR-IG4 with a clear immune-activating function, as well as pathogen detection tools and molecular marker detection methods based on this protein, to make up for the shortcomings of the above-mentioned existing technologies. Summary of the Invention

[0009] The technical problem to be solved by this invention is to provide a recombinant protein of Chinese mitten crab LRR-IG4 with pattern recognition receptor function, capable of broadly binding to LPS / PGN and activating downstream immune signals intracellularly, and its encoded nucleic acid, and to apply it to pathogen detection, crustacean immune enhancement and in vitro disease resistance assessment, thereby filling the gaps in the existing technology for crustacean broad-spectrum PAMP binding detection reagents, adjuvants for crustacean recombinant protein vaccines and disease resistance molecular markers.

[0010] To solve the above-mentioned technical problems, the present invention provides the following technical solution: This invention provides a recombinant protein, LRR-IG4, from the Chinese mitten crab. The recombinant protein is a type I transmembrane protein comprising a signal peptide, an extracellular region, a single transmembrane region, and an intracellular region. The extracellular region includes a leucine-rich repeat-sequence LRR domain (SEQ ID NO:2) and an immunoglobulin-like IG domain (SEQ ID NO:3). Furthermore, the protein exhibits broad-spectrum pathogen-associated molecular pattern (PAMP) binding activity, specifically lipopolysaccharide (LPS) and peptidoglycan (PGN). The LRR domain is located at amino acids 45 to 378 at the N-terminus of the protein, the IG domain is located at amino acids 451 to 554 at the protein, the single transmembrane region is located at amino acids 571 to 592 at the protein, and the intracellular region is located at amino acids 593 to 1005 at the protein. The protein can activate the nuclear translocation of the Dorsal transcription factor, a downstream component of the Toll signaling pathway in the Chinese mitten crab, by binding to lipopolysaccharide and peptidoglycan, and upregulate the expression of the anti-lipopolysaccharide factor ALF.

[0011] In some embodiments, the amino acid sequence of the recombinant protein is shown in SEQ ID NO:1. The amino acid sequence shown in SEQ ID NO:1 is codon-optimized and can be solublely expressed in Escherichia coli expression host, while retaining the PAMPs binding activity and Dorsal signaling pathway activation function.

[0012] The present invention provides an isolated nucleic acid molecule that encodes the above-mentioned recombinant protein LRR-IG4 of the Chinese mitten crab or its functionally active fragment; the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:5, the open reading frame of the sequence shown in SEQ ID NO:5 is 3018 bp in length, and encodes a type I transmembrane protein of 1005 amino acid residues shown in SEQ ID NO:1.

[0013] The present invention provides a recombinant expression vector containing the above-mentioned nucleic acid molecules.

[0014] The present invention provides a recombinant host cell containing the above-mentioned recombinant expression vector.

[0015] The present invention provides a kit for in vitro identification or binding of pathogenic microorganisms and their surface pathogen-related molecular patterns, the kit comprising the above-mentioned Chinese mitten crab LRR-IG4 recombinant protein or its functionally active fragment, and a solid-phase carrier or detection marker.

[0016] This invention provides the application of the above-mentioned recombinant protein LRR-IG4 from the Chinese mitten crab in the preparation of an immune enhancer for crustaceans.

[0017] The present invention provides an aquatic immune-enhancing composition comprising the above-mentioned recombinant protein LRR-IG4 of Chinese mitten crab or its functionally active fragment, and aquatic-acceptable excipients or carriers.

[0018] This invention discloses a method for detecting the expression level of the EsLRR-IG4 gene in isolated crustacean biological samples, the method comprising the following steps: Nucleic acid was extracted from isolated crustacean biological samples for testing. The transcriptional expression level of the EsLRR-IG4 gene in the nucleic acid was detected; The obtained EsLRR-IG4 gene expression level data were output as molecular marker information.

[0019] In some embodiments, the ex vivo biological sample is blood cells, hepatopancreatic tissue, gill tissue, or muscle tissue, and the crustacean is Chinese mitten crab, whiteleg shrimp, giant freshwater prawn, or swimming crab.

[0020] Compared with the prior art, the present invention has achieved the following breakthroughs and significant beneficial effects: (1) This invention, for the first time through prokaryotic recombinant expression and in vitro functional verification, demonstrates that the EsLRR-IG4 protein of the Chinese mitten crab not only possesses LPS binding activity but also PGN binding activity, exhibiting a **broad-spectrum recognition ability** of surface molecules of Gram-negative and Gram-positive bacteria. This result exceeds the expectations of those skilled in the art regarding the recognition spectrum of a single receptor, because receptors with LRR domains typically tend to recognize specific types of PAMPs, while the EsLRR-IG4 protein of this invention can simultaneously recognize two structurally different PAMPs (LPS is lipopolysaccharide, and PGN is peptidoglycan), suggesting that there may be a synergistic recognition mechanism between the LRR and IG dual domains.

[0021] (2) This invention is the first to demonstrate that knockdown of the EsLRR-IG4 protein in the hemocytes of the Chinese mitten crab significantly inhibits the expression of the downstream anti-lipopolysaccharide factor ALF in the Toll pathway and hinders the nuclear translocation of the Dorsal transcription factor, thereby constructing a **complete immune signaling axis** from "pathogen recognition (PAMP binding) - signal transduction (Dorsal activation) - effector output (ALF upregulation)". No prior art has reported any Chinese mitten crab receptor capable of simultaneously performing these three functional steps. This discovery provides a new perspective for understanding the molecular mechanisms of innate immunity in crustaceans.

[0022] (3) This invention utilizes the functionally validated recombinant protein to develop pathogen detection reagents and immune enhancers, overcoming the shortcomings of existing technologies that rely solely on sequence prediction and lack functional experimental support. Compared to unvalidated hypothetical proteins, the recombinant protein of this invention has a clearly defined minimum effective binding concentration (3.125 μg / mL for LPS and 1.5625 μg / mL for PGN), providing reliable technical parameters for practical applications.

[0023] (4) The method for detecting the expression level of the EsLRR-IG4 gene established in this invention provides an objective molecular indicator for assessing the immune status of crustaceans. Existing technologies lack experimental evidence regarding the correlation between gene expression and immune response. This invention, through in vivo injection experiments and temporal expression analysis, demonstrates for the first time that the EsLRR-IG4 gene exhibits a significant temporal upregulation pattern after pathogen infection, providing an experimental basis for the application of this gene as a molecular marker. Attached image description: To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0024] Figure 1 The expression diagram of recombinant proteins with LRR and IG domains is shown. (A) shows the Western blot analysis of the recombinant protein, and (B) shows the SDS-PAGE analysis. M: Protein molecular weight standard (kDa); Lane 1: pET-32a empty tag protein His-rTrx; Lane 2: rEsLRR1; Lane 3: rEsIG1; Lane 4: rEsLRR2; Lane 5: rEsIG2; Lane 6: rEsLRR3; Lane 7: rEsIG3; Lane 8: rEsLRR4; Lane 9: rEsIG4; Lane 10: rEsLRR5; Lane 11: rEsIG5.

[0025] Figure 2 This is a diagram showing the bacterial binding activity analysis of recombinant proteins with LRR and IG domains.

[0026] Figure 3 The binding activities of rEsLRRs and rEsIGs to PAMPs are given, where (AB) rEsLRRs and rEsIGs bind to LPS; and (CD) rEsLRRs and rEsIGs bind to PGN. Values ​​are expressed as mean ± standard deviation (n = 3).

[0027] Figure 4 A bar chart showing the changes in the expression levels of anti-lipopolysaccharide factors (ALF1, ALF2) and Crustin antimicrobial peptide genes in blood cells after knocking down EsLRR-IG4 via RNA interference.

[0028] Figure 5 Laser confocal microscopy image used to detect the effect of EsLRR-IG4 knockdown on the nuclear translocation of Dorsal transcription factor induced by Vibrio parahaemolyticus.

[0029] Figure 6 Figure 1 shows the results of TMHMM (A) and Phobius (B) software prediction and immunofluorescence experiments verifying the localization of EsLRR-IG4 protein in the cell membrane (C).

[0030] Figure 7 This is a schematic diagram of the signaling pathway mediated by EsLRR-IG4 to regulate the expression of anti-lipopolysaccharide factors. Detailed Implementation

[0031] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings and specific embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods are conventional molecular biology, cell biology, and immunology methods.

[0032] In this invention, the GenBank accession numbers of the EsLRR-IG1~5 genes are OP921207, OP921208, OP921209, OP921210 and OP921211, respectively.

[0033] In this invention, "Chinese mitten crab LRR-IG4 recombinant protein" refers to a protein obtained through recombinant expression via genetic engineering, having the amino acid sequence shown in SEQ ID NO:1, or a functionally active fragment containing the LRR domain shown in SEQ ID NO:2 or the IG domain shown in SEQ ID NO:3. The "functionally active fragment" refers to a fragment that retains pathogen-associated molecular patterns (PAMPs) binding activity, i.e., a fragment capable of specifically binding lipopolysaccharide (LPS) and / or peptidoglycan (PGN). The LRR domain consists of amino acid residues from position 45 to 378 in the amino acid sequence shown in SEQ ID NO:1; the IG domain consists of amino acid residues from position 451 to 554 in the amino acid sequence shown in SEQ ID NO:1; and the intracellular region consists of amino acid residues from position 593 to 1005 in the amino acid sequence shown in SEQ ID NO:1. The recombinant protein also includes a single transmembrane region located at amino acids 571 to 592.

[0034] In this invention, "crustaceans" include, but are not limited to, Chinese mitten crabs and other freshwater and saltwater crabs, shrimps and other economically important crustaceans, with Chinese mitten crabs being the preferred species.

[0035] In this invention, the "pathogen-associated molecular model" includes lipopolysaccharides on the surface of Gram-negative bacteria and peptidoglycans on the surface of Gram-positive bacteria.

[0036] Example 1: Cloning and sequence analysis of the EsLRR-IGs gene Total RNA was extracted from the blood cells of the Chinese mitten crab (Eriocheir sinensis) and cDNA was obtained by reverse transcription.

[0037] The full-length cDNA sequence of the EsLRR-IGs gene was obtained by cloning using RACE (rapid amplification of cDNA ends) technology.

[0038] The open reading frame (ORF) of the EsLRR-IG4 gene is 3018 bp in length (GenBank accession number: OP921210), and its nucleotide sequence is shown in SEQ ID NO:5. This ORF encodes a protein consisting of 1005 amino acid residues, the amino acid sequence of which is shown in SEQ ID NO:1, with a theoretical molecular weight of approximately 113.2 kDa.

[0039] Example 2: Recombinant expression of EsLRR-IGs and identification of bacterial / PAMP binding activity 2.1 Recombinant protein expression and purification: (1) The cDNA sequences of the extracellular LRR and IG domains of EsLRR-IGs were amplified using specific primers with restriction enzyme sites. The amplified sequence fragments were ligated into pMD19-T. The amplified LRR domain clone fragment and pET-32a vector were digested with BamHI and EcoRI, respectively. The IG domain clone fragment and pET-32a vector were digested with EcoRI and HindIII, respectively. The enzyme digestion system is shown in Table 1. The digestion temperature was 37℃ and the digestion time was 5 h. The digested target fragments were recovered and ligated into the pET-32a expression vector digested with the corresponding restriction endonuclease using T4 DNA ligase. After successful sequencing, subsequent experiments were performed. Table 1 Plasmid digestion system

[0040] (2) The plasmid was transformed into E. coli BL21(DE3)pLysS ChemicallyCompetent Cells and cultured at 37°C and 180 rpm for 3 h. (3) Spread 100 μL of bacterial culture onto a solid LB medium plate containing ampicillin resistance, incubate upright for 30 min, then invert and continue incubation for 12 h; (4) Using a sterilized toothpick, pick up one colony in the clean bench and immerse it in 500 μL of LB liquid medium containing ampicillin. Incubate in a shaker at 37°C until turbid. Then inoculate the bacterial solution into 100 mL of LB liquid medium containing ampicillin and continue to incubate under the same conditions. (5) Detect the absorbance of bacteria. When the absorbance value at 600 nm reaches 0.8, add 10 μL of IPTG inducer (concentration of 0.1 mM) and culture in a shaker at 180 rpm for 12 h to induce the bacterial culture to express recombinant protein. (6) Centrifuge the induced bacterial culture at 4℃, 12000 ×g for 10 min, collect the bacterial pellet, and resuspend the bacteria in 0.01 M PBS; (7) Bacteria obtained by ultrasonic disruption on ice, power 40%, ultrasonic disruptor paused for 2 seconds every 3 seconds, total instrument running time 40 min; (8) After sonication, centrifuge at 4°C, 10000 ×g for 30 min and collect the protein supernatant; (9) The expression of recombinant proteins in the supernatant was analyzed by 12.5% ​​sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. (10) The protein was incubated with high-affinity Ni-NTA resin (Sangon Biotech, China) according to the instructions and purified by elution with a gradient of imidazole from 20 mM to 500 mM. (11) The concentration of recombinant protein was detected using the BCA protein concentration detection kit, and the purity and size of the target protein were analyzed by 12.5% ​​SDS-PAGE and Coomassie blue staining.

[0041] 2.2 Western blot procedure: (1) Transfer the protein on the SDS gel to the PVDF membrane. The PVDF membrane was soaked in methanol for 2 min beforehand. The materials used for the transfer were laid out in the order of sponge-filter paper-protein gel-PVDF membrane-filter paper-sponge. The transfer voltage was 100 V and the transfer time was 1 h. (2) After the transfer is completed, carefully remove the PVDF membrane with tweezers, soak it in 5% skim milk powder, and slowly shake it on a shaker for 3 hours to seal it. (3) After the blocking was completed, the PVDF membrane was immersed in anti-His-tagged mouse monoclonal antibody (1:5000) and incubated on a horizontal shaker for 2 h with slow shaking. (4) Immerse the PVDF membrane in 1 × PBST cleaning solution, place it on a horizontal shaker and shake it slowly for 5 min, repeat the cleaning 6 times; (5) Immerse the PVDF membrane in horseradish peroxidase (HRP)-labeled goat anti-mouse enzyme-labeled secondary antibody (1:2000) and incubate on a horizontal shaker for 2 h; (6) Immerse the PVDF membrane in 1 × PBST cleaning solution, place it on a horizontal shaker and shake it slowly for 5 min, repeat the cleaning 6 times; (7) Use the ECL chemiluminescence kit for development. Prepare the developing solution according to the kit instructions. Add the developing solution to the PVDF membrane and react in the dark for 1 min. Then use the chemiluminescence imaging system to image and record the results.

[0042] 2.3 Coomassie Brilliant Blue Staining: (1) Carefully peel the protein glue off the slide and put it into the glue box. Add Coomassie brilliant blue staining solution to cover the protein glue. Heat in a water bath until small bubbles appear in the staining solution. Place on a shaker and shake for 15 minutes to stain. (2) Place the stained protein glue in the decolorizing solution to decolorize it. The decolorizing solution should cover the protein glue. After heating to boiling in a water bath, place it on a horizontal shaker to decolorize until the protein bands are clearly visible. During this process, the decolorizing solution can be replaced and this decolorization step can be repeated. (3) Use a protein gel imaging system to take pictures and record.

[0043] 2.4 Detection of bacterial binding activity: (1) Three Gram-positive bacteria (Staphylococcus aureus, Corynebacterium glutamicum, and Micrococcus lysate) and three Gram-negative bacteria (Vibrio parahaemolyticus, Vibrio alginolyticus, and Vibrio harveyi) were cultured separately, collected, washed, and resuspended in PBS to 5 × 10⁻⁶. 7 CFU / mL. (2) Mix 50 μL of recombinant protein rEsLRR4 or rEsIG4 (300 μg / mL) with an equal volume of bacterial suspension and incubate with gentle shaking at a suitable temperature for 2 h. Use protein containing the empty His-Trx tag as a negative control. (3) After incubation, centrifuge at 3000×g for 5 minutes, and wash the bacterial cells thoroughly with PBS to remove unbound proteins. The final bacterial pellet is resuspended in SDS-PAGE loading buffer and boiled for 10 minutes for Western blot analysis, and detected using anti-His tag antibody. The expression of recombinant proteins with LRR and IG domains is as follows: Figure 1 As shown.

[0044] like Figure 2 As shown, both rEsLRRs and rEsIGs exhibited binding activity against all tested Gram-negative and Gram-positive bacteria, while the negative control showed no band, confirming their broad-spectrum bacterial binding ability.

[0045] 2.5 PAMP binding activity assay (ELISA method): (1) Coagulate 100 μL of Escherichia coli LPS or Micrococcus luteus PGN at a concentration of 80 μg / mL onto a 96-well microplate and incubate overnight at 4°C. After washing with PBST, block with 5% BSA at 37°C for 2 h. (2) Add 100 μL of serially diluted (1.5625 μg / mL ~ 200 μg / mL) rEsLRR4 or rEsIG4 protein, with His-Trx at the same concentration as a negative control, and incubate at 37℃ for 1 h. (3) After washing, mouse anti-His tag monoclonal antibody (1:2000, 37℃, 1h) and HRP-labeled goat anti-mouse IgG (1:5000, 37℃, 1h) were added sequentially for incubation. After each incubation, the mice were washed with PBST. (4) Add TMB substrate and develop color for 10 minutes, then terminate the reaction with 2M H2SO4. Read the absorbance value at 450 nm.

[0046] Experimental results are as follows Figure 3 As shown, both rEsLRRs and rEsIGs bind to LPS and PGN, with rEsLRR4 and rEsIG4 exhibiting the strongest binding affinity, which is dose-dependent. The lowest significant binding concentration for LPS is approximately 3.125 μg / mL, and for PGN, it is approximately 1.5625 μg / mL.

[0047] Example 3: Identification of humoral immune function by regulating antimicrobial peptide expression with EsLRR-IG4 3.1 In vivo RNA interference (RNAi): (1) Based on the EsLRR-IG4 sequence, a sequence-specific siRNA-LRR-IG4 was designed, and a random siRNA-NC sequence was used as a negative control. The siRNA used in the experiment was designed and synthesized by Suzhou GeneGene Co., Ltd. (2) Following the instructions provided by Gemma Gene Co., Ltd., dilute the siRNA to 20 μM using RNase-free water; (3) Slowly add the transfection reagent SiRNA-Mate™ to the SiRNA interference reagent according to the ratio, mix gently, and incubate at room temperature for 30 min to form the SiRNA-SiRNA-Mate complex. (4) The experiment was set up as siRNA-LRR-IG4 injection group, siRNA-NC injection group, Vibrio parahaemolyticus injection group and Blank control group. The incubated siRNA reagent or an equal volume of DEPC water was injected into the Chinese mitten crab using a microsyringe. 4.51 μL of siRNA was injected per gram of body weight, that is, about 1 OD of siRNA reagent was injected for every 30 g of body weight. (5) 24 h after injection of siRNA reagent or an equal volume of DEPC water, the experimental group was injected with 50 μL of Vibrio parahaemolyticus (1 × 10⁻⁶). 7 (CFU / mL), and blood cells and liver and pancreas tissue were collected 3 hours after injection; (6) The knockdown efficiency of the EsLRR-IG4 gene was detected by qRT-PCR experiment.

[0048] Gene expression analysis (qRT-PCR): After successfully knocking down EsLRR-IG4, RNA was extracted and reverse transcribed. The knockdown efficiency of EsLRR-IG4 and the expression levels of downstream effector molecules such as antimicrobial peptides ALF1 (GenBank accession number: DQ793214), ALF2 (GenBank accession number: GU014699), ALF3 (GenBank accession number: HQ850572), Crustin1 (GenBank accession number: GQ200832) and Crustin2 (GenBank accession number: GQ200833) were detected by qRT-PCR.

[0049] The results showed that siRNA-LRR-IG4 could efficiently silence target genes, and the expression levels of ALF1 and ALF2 were significantly downregulated after knockdown compared with the siRNA-NC control group. Figure 4 While changes in genes such as Crustin were not significant, this reveals the specificity of their regulation.

[0050] 3.2 Preparation of Dorsal antibody: (1) The ORF sequence of EsDorsal (GenBank accession number: KC900086.1) was amplified using the primer sequences in Table 2, and the amplified product was sequenced. The correctly sequenced target fragment was ligated into the pET-28a vector, and the ligation product was sequenced to verify whether the recombinant plasmid was successfully constructed. The successfully constructed recombinant plasmid was transformed into E. coli BL21(DE3)pLysSChemically Competent Cells competent cells to induce the expression of the recombinant protein, and then the recombinant protein was purified. The construction of the recombinant plasmid and the induction and expression of the recombinant protein were the same as those in Example 2. Table 2 Primers used in this experiment

[0051] (2) Polyclonal antibodies were prepared using purified EsDorsal recombinant protein (rEsDorsal). 400 μgr EsDorsal and 400 μL of complete Freund's adjuvant were thoroughly mixed and injected subcutaneously into 3-week-old ICR mice. (3) After 7 days, 200 μg rEsDorsal and 200 μL of incomplete Freund's adjuvant were thoroughly mixed and injected subcutaneously into mice. The above injection steps were repeated after 7 days. (4) Seven days after the third injection, blood was collected from the mouse eyeballs, and the blood was allowed to stand in a 4°C refrigerator for 12 h to settle. Then, the blood was centrifuged at 4°C, 4,000 × g for 10 min to obtain serum. (5) The effect of the prepared EsDorsal polyclonal antibody was detected by Western blot. The serum was dispensed into 20 μL tubes and stored at -80℃ for subsequent experiments.

[0052] 3.3 Cell immunofluorescence assay (Dorsal nuclear translocation detection): (1) Based on in vivo RNA interference, 50 μL of Vibrio parahaemolyticus (1 × 10⁻⁶) was added. 7 (CFU / mL) was injected into the Chinese mitten crab. Three hours after the injection, hemolymph and blood cells were extracted. (2) Wash blood cells twice with sterile 0.01 M PBS, centrifuge at 800 ×g for 10 min at 4℃, and collect blood cells; (3) Resuspend the cells in 200 μL of 4% paraformaldehyde and incubate at 37°C in the dark for 15 min. Dilute the cell concentration to 6 × 10⁻⁶. 5 cell / mL; (4) Gently drop the cell suspension onto the adhesive slide and air dry in a fume hood for 30 min. Observe the air-drying state of the cells. Gently drop 2 mL of sterile 0.01 M PBS onto the slide and wash the cells 3 times, 5 min each time. (5) Gently drop 0.2% Triton X-100 onto a glass slide to permeate the cells, incubate in a 37°C incubator for 10 min, and wash the cells three times with sterile 0.01 M PBS for 5 min each time to remove residual 0.2% Triton X-100; (6) Use 2% BSA to block blood cells and incubate at 37°C for 30 min. (7) Dilute the anti-Dorsal antiserum in 2% BSA at a ratio of 1:200, then incubate the blood cells with antibody dilution buffer, stand at 4℃ for 12 h, and gently wash the slides 3 times with sterile 0.01 M PBS for 5 min each time. (8) Block blood cells with 2% BSA and incubate at room temperature for 10 min; (9) Incubate with FITC-labeled goat anti-mouse secondary antibody (1:1,000) at 37°C in the dark for 2 h, and wash with sterile PBST 5 times for 5 min each time; (10) Add DAPI (1 mg / mL stock solution, diluted 10,000 times with 0.01 M PBS) to stain the cell nuclei and incubate at room temperature in the dark for 10 min; (11) Wash the slide 5 times with sterile 0.01 M PBS, 5 min each time; finally, mount the slide with one drop of mounting glycerol (80%, prepared with 0.01 M PBS), and observe the imaging with a laser confocal microscope after mounting. (12) Use ZEN software to process images.

[0053] Experimental results are as follows Figure 5 As shown, observations under a laser confocal microscope revealed that in the siRNA-NC control group, stimulation by Vibrio parahaemolyticus led to the translocation of Dorsal from the cytoplasm to the nucleus; however, in the siRNA-LRR-IG4 knockdown group, even with the same stimulation, Dorsal protein remained primarily in the cytoplasm, and nuclear translocation was significantly inhibited. This directly demonstrates that EsLRR-IG4 acts upstream of Dorsal, regulating its activation and nuclear translocation, thereby initiating the expression of humoral immune effector molecules such as ALF.

[0054] Example 4: Predictive Analysis of Transmembrane Domains of EsLRR-IG4 Protein The EsLRR-IG4 protein domains were predicted using online software TMHMM and Phobius, with results as follows: Figure 6 As shown in Figures A and B, this result demonstrates that EsLRR-IG4 is a type I transmembrane protein comprising a signal peptide, an extracellular region, a single transmembrane region, and an intracellular region. Its extracellular region contains an LRR domain (amino acid positions 45-378) and an IG domain (amino acid positions 451-554), while its intracellular region comprises amino acid positions 593-1005.

[0055] Example 5: Detection of EsLRR-IG4 cell membrane localization (1) Extract blood lymph from Chinese mitten crab, centrifuge at 800 ×g for 10 min at 4℃, discard the supernatant, collect blood cells, and wash the blood cells with sterile 0.01 M PBS. Wash the blood cells 3 times, resuspend the cells in L-15 medium, and count them (5 × 105 cells / mL). (2) Place cell crawlers in a 6-well cell culture plate, and drop a small amount of 0.01 M PBS between the cell crawler and the contact surface of the 6-well plate to allow the cell crawler to adhere to the bottom of the 6-well plate and prevent the cells from adhering to the bottom of the 6-well plate. Then add 2 mL of cell suspension to each well and place the plate in a cell culture incubator at 28°C for 12 h to allow the cells to adhere to the cell crawler. (3) Discard the culture medium, wash twice with 0.01 M PBS, stain with cell membrane staining solution DiI in a 37°C incubator for 10 min in the dark, and then wash twice with sterile 0.01 M PBS for 5 min each time; (4) Fix blood cells with 4% paraformaldehyde, place them in a 37°C incubator and incubate for 15 min to form a fixed structure. Then wash the slides three times with 0.01 M PBS for 5 min each time. (5) Block blood cells with 2% BSA, and incubate at 37°C for 2 h. (6) The mouse antiserum against EsLRR-IG4 was diluted in 2% BSA at a ratio of 1:100. Blood cells were incubated with the diluted antibody and incubated at 4°C for 12 h. Then the cell smears were washed with sterile 0.01 M PBS three times for 5 min each time. (7) Continue to block blood cells with 2% BSA for 10 min at room temperature; (8) Incubate with FITC-labeled goat anti-mouse secondary antibody (1:1,000) at 37°C in the dark for 1 h, and wash the cell smears three times with sterile 0.01 M PBS for 5 min each time; (9) Stain the cell nuclei with DAPI at room temperature in the dark for 10 min. Wash the slides three times with sterile 0.01 M PBS for 5 min each time. (10) Mount the slide with glycerin, observe the image with a laser confocal microscope, and take pictures to record the image.

[0056] like Figure 6 As shown in Figure C, EsLRR-IG4 is located on the cell membrane and is a membrane receptor.

[0057] Based on the above experimental results, a schematic diagram of the signaling pathway regulating the expression of anti-lipopolysaccharide factor mediated by EsLRR-IG4 is shown below. Figure 7 As shown.

[0058] In summary, based on the experimental results of Examples 1 to 5, the present invention possesses outstanding substantive features and significant progress compared to the prior art, and its inventiveness is mainly reflected in the following aspects: First, selective screening of homologous families has yielded unexpected technical results.

[0059] This invention, through systematic prokaryotic expression and parallel gradient titration experiments among numerous homologous members of the EsLRR-IG gene family (EsLRR-IG1~5) of the Chinese mitten crab, confirmed that although both rEsLRRs and rEsIGs can bind to pathogen-associated molecular patterns, the recombinant proteins (rEsLRR4 and rEsIG4) encoded by EsLRR-IG4 exhibit a significantly enhanced binding activity against lipopolysaccharide (LPS) and peptidoglycan (PGN). Figure 3(A~D), and the binding exhibits a strictly dose-dependent relationship. The minimum significant binding concentration for LPS is as low as 3.125 μg / mL, and the minimum significant binding concentration for PGN is as low as 1.5625 μg / mL. Given the high homologous sequence similarity, those skilled in the art could not directly expect EsLRR-IG4 to possess such excellent broad-spectrum, high-affinity binding properties based on the gene sequence. This scheme of selecting the most active functional individual from the homologous gene cluster constitutes a typical selective invention, achieving unexpected technical effects.

[0060] Second, for the first time in crustaceans, the complete transmembrane receptor signaling axis of "recognition-transduction-effect" was elucidated and confirmed.

[0061] While existing technologies know that the LRR and IG domains can participate in pattern recognition, and that the Toll / Dorsal pathway is involved in crustacean immunity, no literature has ever disclosed what kind of dual-domain transmembrane receptor exists on the cell membrane of the Chinese mitten crab that can act as an upstream signaling switch to initiate this cascade reaction. This invention not only confirms that EsLRR-IG4 is located on the cell membrane (… Figure 6 Furthermore, through in vivo RNA interference experiments ( Figure 4 , Figure 5 Rigorous evidence demonstrates that specific knockdown of EsLRR-IG4 completely blocks the translocation of the Vibrio parahaemolyticus-induced transcription factor Dorsal from the cytoplasm to the nucleus, and significantly and specifically downregulates the transcriptional levels of downstream effector molecules ALF1 and ALF2, while unrelated antimicrobial peptides such as Crustin remain unaffected. This experiment eliminates nonspecific toxicity and, for the first time, provides conclusive evidence of loss-of-function in vivo, confirming that EsLRR-IG4 is an essential upstream membrane receptor mediating humoral immune responses in crustaceans. This overcomes the significant technical limitations of existing techniques, such as "known sequence but unknown function" and "only in vitro binding without in vivo functional verification."

[0062] Third, the non-obviousness of the spatial arrangement of dual domains and the broad-spectrum collaborative recognition mechanism.

[0063] The EsLRR-IG4 recombinant protein of this invention, as a type I transmembrane receptor, has an extracellular region composed of a specific amino acid segment consisting of an LRR domain (positions 45-378) and an IG domain (positions 451-554) linked together. This specific spatial configuration not only endows the receptor with the ability to recognize LPS, a characteristic molecule of Gram-negative bacteria, but also with the ability to recognize PGN, a characteristic molecule of Gram-positive bacteria. Furthermore, it exhibits highly efficient cell-binding activity against various common aquatic pathogens, including Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio harveyi, Staphylococcus aureus, Corynebacterium glutamicum, and Micrococcus lysate. Figure 2This single receptor possesses the high sensitivity characteristic of broad-spectrum recognition across multiple PAMPs, exceeding the conventional understanding of those skilled in the art based on single-pattern recognition receptors. It provides a technical entity with definite performance and reliable parameters for the development of aquatic broad-spectrum pathogen detection kits and aquatic immune enhancers.

[0064] The sequence of this application is as follows:

[0065] SEQ ID NO:2—LRR domain amino acid sequence (corresponding to positions 45-378 of SEQ ID NO:1): HCPESCWCLLPGELECLATGEKYPPRPPSDITKLVLHGYEFVPAHFLADLKRLRHLTVSGSQVTSLAFLPLLPELEVLEVRYNRLQSLGGGYLKASTPALTHLDASHNEITLLNATDLEGLKKLQNLSLHHNPISEASADALKDLTSLTHLDISKSKISKLHSNWLADA KNLQYLNVSNAKVREVPRIHGENIRVLDASHNFLANLPDGLIMATCLQSLFLHHNPFEAVNEQTLMNSKCLQELHLSHTHIVNIDEFVFADMPLLQKLHLEQNERLIRVEHGAFTGLHNLQYLNLAETPNLKEIEEVAFAGLPNLISLDLRKSGLMVLPLSLTKL (SEQ ID NO:2).

[0066] SEQ ID NO:3—IG domain amino acid sequence (corresponding to positions 451-554 of SEQ ID NO:1): GDIVMAKSRQSALLECNMTANPSHSILWLSKEHRVFRYNDTSGDHDDEWISYHIEQVAASASIDPRFEVLASGHLLIREVDHSDIGWYKCFAYNSVGNTSILVF (SEQ ID NO: 3).

[0067] SEQ ID NO:4—Intracellular amino acid sequence (corresponding to positions 593-1005 of SEQ ID NO:1): LMDRLGWECCCCRYRVSPKARQIKKLLESVESYKSQQLDRLRENYNYQVVSIKDSCYQQMERIRESYGTQGRNLRELRDYSTQGLTSMRDQYLDQVNKVRDYSVSQMNRVRENYVFQRHRIRKFSAHQLLRLRETYKYQQKTLNKILENLPDLYLQNCRTGACQRTDSIIFDDALHGIDAYYRVDFLDTQSHCSDYYTPASTLTHSFRSNKGQDPVKQHSRTSSNTSSDFVEAQAWLRRDSSALSGNSPSRTLPSYSKIHGRSLSVAGPSFMVSDPVKVHKRSLSASHPSHRIAPAPETRGIPARITLCEETSDYNNQTCPNSPISVPNTPVHKKKESSSPAPSLQALKVQSVDNLEPIHQKTEVPSTITEVPSVSSGENNASNTTSDETNSVASDVSVQPDVVDRVAFETSL (SEQ ID NO:4).

[0068] SEQ ID NO:5 —— Nucleotide sequence of the open reading frame of EsLRR-IG4 gene (3018 bp, GenBank accession No. OP921210):

[0069] SEQ ID NO:6——EsDorsal-F: AGACTGTCAGACAAAATAAAAATGGAG` SEQ ID NO:7——EsDorsal-R: TCAATGAGCTGATGACAGCTGGT` SEQ ID NO:8——EsDorsal-F-EcoRI: CCGGAATTCAGACTGTCAGACAAAATAAAAATGGAG` SEQ ID NO:9——EsDorsal-R-HindIII: CCCAAGCTTTCAATGAGCTGATGACAGCTGGT` In summary, any combination of various embodiments of the present invention without departing from the inventive concept of the present invention shall be considered as the disclosure of the present invention; any simple modifications to the technical solutions and any combination of different embodiments without departing from the inventive concept of the present invention shall be within the protection scope of the present invention.

Claims

1. A recombinant protein of LRR-IG4 from the Chinese mitten crab, characterized in that, The recombinant LRR-IG4 protein from the Chinese mitten crab is a type I transmembrane protein comprising a signal peptide, an extracellular region, a single transmembrane region, and an intracellular region. The extracellular region includes a leucine-rich repeat-sequence LRR domain (SEQ ID NO:2) and an immunoglobulin-like IG domain (SEQ ID NO:3). Furthermore, the protein exhibits broad-spectrum pathogen-associated molecular pattern (PAMP) binding activity, specifically lipopolysaccharide (LPS) and peptidoglycan (PGN). The LRR domain is located at amino acids 45 to 378 at the N-terminus of the protein, the IG domain is located at amino acids 451 to 554 at the protein, the single transmembrane region is located at amino acids 571 to 592 at the protein, and the intracellular region is located at amino acids 593 to 1005 at the protein. The protein can activate the nuclear translocation of the Dorsal transcription factor, a downstream component of the Toll signaling pathway in the Chinese mitten crab, by binding to lipopolysaccharide and peptidoglycan, and upregulate the expression of the anti-lipopolysaccharide factor ALF.

2. The recombinant LRR-IG4 protein from the Chinese mitten crab according to claim 1, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO:

1. The amino acid sequence shown in SEQ ID NO:1 is codon-optimized and can be solublely expressed in Escherichia coli expression host, while retaining the PAMPs binding activity and Dorsal signaling pathway activation function.

3. An isolated nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the recombinant protein LRR-IG4 of the Chinese mitten crab as described in claim 1 or its functionally active fragment. The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:

5. The open reading frame of the sequence shown in SEQ ID NO:5 is 3018 bp in length and encodes a type I transmembrane protein of 1005 amino acid residues as shown in SEQ ID NO:

1.

4. A recombinant expression vector, characterized in that, The recombinant expression vector contains the nucleic acid molecule as described in claim 3.

5. A recombinant host cell, characterized in that, It contains the recombinant expression vector as described in claim 4.

6. A kit for in vitro identification or binding of pathogenic microorganisms and their surface pathogen-associated molecular patterns, characterized in that, The kit contains the recombinant LRR-IG4 protein of the Chinese mitten crab as described in claim 1 or 2, or a functionally active fragment thereof, as well as a solid-phase carrier or detection marker.

7. The use of the recombinant LRR-IG4 protein of the Chinese mitten crab as described in claim 1 or 2 in the preparation of an immune enhancer for crustaceans.

8. An aquatic immune-enhancing composition, characterized in that, It comprises the recombinant LRR-IG4 protein of Chinese mitten crab as described in claim 1 or its functionally active fragment, as well as aquatic-acceptable excipients or carriers.

9. A method for detecting the expression level of the EsLRR-IG4 gene in isolated crustacean biological samples, characterized in that, The method includes the following steps: Nucleic acid was extracted from isolated crustacean biological samples for testing. The transcriptional expression level of the EsLRR-IG4 gene in the nucleic acid was detected; The obtained EsLRR-IG4 gene expression level data were output as molecular marker information.

10. The method according to claim 9, characterized in that, The ex vivo biological samples are blood cells, hepatopancreatic tissue, gill tissue, or muscle tissue, and the crustaceans are Chinese mitten crab, whiteleg shrimp, giant freshwater prawn, or swimming crab.

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