Pinctada martensii C-type lectin PmPLP and its application

Abstract

The present invention discloses a Pinctada martensii C-type lectin PmPLP, as well as its application in the preparation of drugs for inhibiting microorganisms and in promoting the release of pro-inflammatory factors induced by nucleation in Pinctada martensii. The present invention conducts preliminary research on the identification and immune function of Pinctada martensii C-type lectin PmPLP to determine the immune response of PmPLP molecules in the immune response of Pinctada martensii against the invasion of external pathogens, and to understand the antibacterial activity of PmPLP after in vitro expression, providing data support and research basis for the study of the immune defense mechanism of Pinctada martensii and other marine mollusks C-type lectins. The present invention conducts in-depth research on the immune response mechanism of Pinctada martensii, which helps to reveal its molecular regulatory mechanism in response to pathogen invasion, and is of great significance for improving aquaculture efficiency and disease prevention and control.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and in particular relates to a Pinctada martensii C-type lectin PmPLP and an application thereof. Background Art

[0002] The Pinctada martensii (Pinctada martensii) is one of the primary pearl oysters cultivated in artificial seawater in my country. In recent years, the deteriorating water quality of coastal areas has led to the frequent occurrence of various diseases, resulting in a large number of deaths among the oysters, causing significant economic losses to my country's pearl industry. As a typical filter-feeding organism, the Pinctada martensii is susceptible to infection by a variety of pathogens, including Vibrio (such as Vibrio alginolyticus and Vibrio parahaemolyticus), Rickettsia, and herpes viruses. Similar to other invertebrates, the Pinctada martensii possesses only an innate immune system, comprised of a coordinated defense mechanism comprised of cellular and humoral immunity. Therefore, in-depth research on the immune response mechanisms of the Pinctada martensii oyster will help reveal the molecular regulatory mechanisms underlying its response to pathogen invasion, which is of great significance for improving aquaculture efficiency and disease prevention and control.

[0003] The pearl formation process is regulated by multiple environmental and biological factors, and nucleus implantation influences the expression profiles of key genes. Artificial nucleus implantation has garnered widespread attention worldwide. Pinctada martensii is a commercially cultivated species, producing over 90% of saltwater pearls in China. Nucleus implantation involves transplanting a small fragment of donor oyster's mantle and an artificial nucleus into a recipient oyster. After implantation, the mantle epidermal cells secrete nacre, which undergoes biomineralization and ultimately forms a pearl. During the implantation procedure, the tissue fragment, nucleus, and pathogenic microorganisms introduced into the recipient oyster can trigger a cascade of immune responses in the host pearl oyster. This intense immune response can lead to nucleus expulsion, difficulty forming a pearl sac, or even death. Therefore, researchers and aquaculture practitioners have been studying the immune response mechanisms of Pinctada martensii after nucleus implantation, actively implementing various measures to reduce immune rejection during nucleus implantation and enhance the species' regulatory capacity, thereby increasing survival and yield.

[0004] C-type lectins regulate the antibacterial immune response in invertebrates through multiple mechanisms and are a crucial component of the invertebrate innate immune system. C-type lectins are a large class of metazoan extracellular proteins that function as important pattern recognition receptors (PRRs), specifically recognizing pathogen-associated molecular patterns on bacterial surfaces, such as lipopolysaccharide (LPS) and peptidoglycan (PGN), as well as β-glucan, a major component of fungal cell walls. Their carbohydrate recognition domains bind to carbohydrate molecules on the surface of microorganisms, thereby initiating an immune response. Once pathogens are recognized, C-type lectins directly participate in the agglutination of microorganisms, causing them to aggregate and restrict their spread.

[0005] However, the role of lectins in the immune system of the Pinctada martensii oyster remains largely unexplored. Studying Pinctada martensii lectins is not only crucial for understanding the mechanisms of immune control against pathogens and its healthy aquaculture, but also indirectly impacts human life. Therefore, the discovery and utilization of pattern recognition receptor C-type lectins holds significant theoretical and practical implications for understanding the immune defense mechanisms of Pinctada martensii and for disease prevention and control. Summary of the Invention

[0006] To address the threat posed by diseases, such as bacterial infections, to Pinctada martensii aquaculture, the present invention conducted preliminary research on the identification and immune function of the Pinctada martensii C-type lectin, PmPLP. Based on this research, the present invention provides the following technical solutions.

[0007] In a first aspect, the present invention provides a Pinctada martensii C-type lectin PmPLP, the amino acid sequence of the C-type lectin being shown in SEQ ID NO.1.

[0008] In a second aspect, the present invention provides a gene encoding the C-type lectin PmPLP, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0009] In a third aspect, the present invention provides an expression vector comprising the gene encoding the C-type lectin PmPLP. Preferably, the vector is a PET series vector.

[0010] In a fourth aspect, the present invention provides a host cell comprising the expression vector. Preferably, the host cell is selected from Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, Yarrowia lipolytica or Bacillus subtilis.

[0011] In a fifth aspect, the present invention provides a use of the Pinctada martensii C-type lectin PmPLP in the preparation of a drug for inhibiting microorganisms; the microorganism is at least one of Gram-positive bacteria, Gram-negative bacteria and fungi.

[0012] In one or more embodiments, the Gram-positive bacteria are selected from Micrococcus luteus, Staphylococcus aureus, Bacillus subtilis and Streptococcus agalactiae; the Gram-negative bacteria are selected from Escherichia coli, Pseudomonas aeruginosa, Aeromonas hydrophila and Vibrio harveyi; and the fungi are selected from Aspergillus niger and Rhizopus.

[0013] In a sixth aspect, the present invention provides a pharmaceutical composition comprising an effective amount of the Pinctada martensii C-type lectin PmPLP as described above, and a pharmaceutically acceptable carrier.

[0014] In one or more embodiments, the concentration of the Pinctada martensii C-type lectin PmPLP in the composition is less than 1 mg / mL.

[0015] In a seventh aspect, the present invention provides the use of the Pinctada martensii C-type lectin PmPLP in promoting the release of pro-inflammatory factors induced by Pinctada martensii nucleus implantation.

[0016] In one or more embodiments, the pro-inflammatory factor is at least one of Caspase-2, IL-17, IRAK1, IκK, NF-κB, TRAF3 and TNF-α.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] This paper conducts preliminary research on the identification and immune function of the C-type lectin PmPLP of the Pinctada martensii to determine the immune response of the PmPLP molecule in the immune response of the Pinctada martensii against the invasion of external pathogens, and to understand the antibacterial activity of PmPLP after in vitro expression, providing data support and research basis for the study of the immune defense mechanism of C-type lectins in Pinctada martensii and other marine mollusks. This paper deeply studies the immune response mechanism of the Pinctada martensii, which helps to reveal its molecular regulatory mechanism in response to pathogen invasion, and is of great significance for improving aquaculture efficiency and disease prevention and control. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 These are the structural features of the PmPLP gene in Example 1.

[0020] Figure 2 The transmembrane region and signal peptide of PmPLP in Example 1 were predicted.

[0021] Figure 3 This is the protein hydrophilicity / hydrophobicity analysis of PmPLP in Example 1.

[0022] Figure 4 This is a three-dimensional structural model diagram of PmPLP in Example 1.

[0023] Figure 5 This is the phylogenetic tree analysis of PmPLP in Example 1.

[0024] Figure 6 is the expression level of PmPLP in different tissues in Example 2.

[0025] Figure 7 This is a diagram showing the expression pattern of PmPLP after nuclear transplantation in Example 2.

[0026] Figure 8 This is a diagram of the prokaryotic expression and purification of PmPLP in Example 3.

[0027] Figure 9 This is a graph showing the potency of the PmPLP polyclonal antibody in Example 3.

[0028] Figure 10 This is the Western blot analysis of the PmPLP polyclonal antibody in Example 3.

[0029] Figure 11 This is the binding activity of rPmPLP with microorganisms in Example 4.

[0030] Figure 12 This is the bacterial agglutination activity analysis of rPmPLP in Example 5.

[0031] Figure 13 This is the analysis of the antibacterial effect of rPmPLP in Example 6.

[0032] Figure 14 This is the PmPLP antibacterial activity test result 1 in Example 7.

[0033] Figure 15 This is the PmPLP antibacterial activity test result 2 in Example 7.

[0034] Figure 16 This is the scanning electron microscopy observation of the effect of rPmPLP on bacteria in Example 8.

[0035] Figure 17 This is the inflammatory factor expression result 1 after injection of rPmPLP and nuclear transplantation in Example 9.

[0036] Figure 18 This is the inflammatory factor expression result 2 after injection of rPmPLP and nuclear transplantation in Example 9. DETAILED DESCRIPTION

[0037] The economic value of Pinctada martensii is mainly reflected in pearl production, but disease problems such as bacterial infection pose a serious threat to Pinctada martensii aquaculture. Therefore, in-depth study of the immune response mechanism of Pinctada martensii will help to reveal its molecular regulatory mechanism for responding to pathogen invasion, which is of great significance for improving aquaculture efficiency and disease prevention and control. The present invention conducts preliminary research on the identification and immune function of Pinctada martensii C-type lectin PmPLP to determine the immune response of PmPLP molecules in the immune response of Pinctada martensii against invasion of external pathogens, as well as to understand the antibacterial activity of PmPLP after in vitro expression, so as to provide data support and research basis for the study of the immune defense mechanism of Pinctada martensii and other marine mollusks C-type lectin.

[0038] Specifically, the present invention includes but is not limited to the following research contents:

[0039] The present invention has identified a new type of C-type lectin PmPLP from Pinctada martensii. Figure 1 As shown, its ORF is 486 bp and encodes a 161-amino acid polypeptide with an isoelectric point of 7.58 and a theoretical molecular weight of 18.81 kDa. PmPLP contains a single CTLD domain and a galactose-binding motif, "-QPD-," and a mannose-binding motif, "-WND-." PmPLP is expressed at its highest level in normal gills.

[0040] The present invention prepared rPmPLP and its polyclonal antibody anti-rPmPLP through in vitro prokaryotic expression. The recombinant protein rPmPLP has binding activity with Aeromonas hydrophila, Pseudomonas aeruginosa, Streptococcus agalactiae, and Micrococcus luteus; rPmPLP also has agglutination effect with Escherichia coli, Aeromonas hydrophila, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis, and Micrococcus luteus.

[0041] The present invention significantly upregulates the expression of PmPLP and inflammatory factors Caspase-2, IL-17, IRAK1, IκK, NF-κB, TRAF3, and TNF-α after rPmPLP is injected into the nucleus-implanted shellfish.

[0042] The rPmPLP described in the present invention has direct antibacterial activity and can cause different morphological damage to different bacteria. rPmPLP has broad-spectrum antibacterial properties, inhibiting the growth of four Gram-positive bacteria, four Gram-negative bacteria, and two fungi. Example

[0043] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0044] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific embodiments rather than for limiting the scope of protection of the present invention.

[0045] When numerical ranges are given in the examples, it should be understood that, unless otherwise specified herein, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0046] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0047] The examples involve the addition amounts, contents and concentrations of various substances, wherein the percentages mentioned are by mass unless otherwise specified.

[0048] The Pinctada martensii oysters used in the following examples were obtained from the Dajing breeding base in Xilian Town, Xuwen County, Zhanjiang City, Guangdong Province. The selected individuals were all cultured for approximately 24 months. The present invention selected 10 experimental oysters with good vitality for dissection, and tissue samples were collected in sequence, including hemolymph, gills, mantle, adductor muscle, and hepatopancreas. The collected tissues were placed in pre-labeled cryovials and quickly frozen in liquid nitrogen for use in subsequent experiments.

[0049] The reagents and sources used in the following examples are as follows:

[0050] Table 1 Reagents and sources

[0051]

[0052] The instruments and equipment used in the following examples are as follows:

[0053] Table 2 Instruments and equipment

[0054]

[0055] The reagents used in the following examples were prepared as follows:

[0056] (1) LB liquid medium: 10 g of NaCl and tryptone and 5 g of yeast extract, add 800 mL of distilled water, adjust the pH to 7.0 with NaOH solution, make up to 1000 mL, and aliquot. Sterilize by autoclaving at 121 °C for 15 min and store at 4 °C.

[0057] (2) LB solid medium: Add agar powder to LB liquid medium at a ratio of 1.5:100, sterilize with high-pressure steam at 121°C for 15 min, and add the corresponding antibiotics to their working concentration in an ultra-clean workbench when the temperature cools to 50°C - 60°C. Pour the mixture into a bacterial culture dish, wait for the culture medium to solidify, seal it with sealing film, and store it upside down at 4°C.

[0058] (3) BHI liquid medium: Weigh 38.5 g of brain heart infusion broth, heat and stir in 1000 mL of distilled water, autoclave at 121 °C for 15 min, and store at room temperature.

[0059] (4) TSB liquid medium: 20 g NaCl; 15 g tryptone; 5 g soy peptone; add distilled water to 800 mL, then adjust the pH to 7.2 with NaOH, make up to 1000 mL, aliquot, autoclave at 121°C for 15 min, and store at 4°C.

[0060] (5) Ampicillin solution: 1 g of ampicillin sodium; add distilled water to 10 mL, mix well, filter through a 0.22 μm filter membrane to sterilize, and store at -20°C in the dark.

[0061] (6) TBE buffer: 27.5 g boric acid, 54 g Tris, 20 mL 0.5 mol / L EDTA (pH 7.9), and dilute to 1000 mL with water.

[0062] (7) DEPC solution: Prepare 0.1% DEPC stock solution per 100 ml of distilled water, mix well using a shaker, ventilate overnight, sterilize at high temperature for 30 min, and store at 4°C.

[0063] (8) Lysis buffer: 20 mM phosphate buffer, 150 mM NaCl, pH 8.0.

[0064] (9) Inhibitors: 2.08 g glucose, 0.8 g sodium citrate, 0.336 g EDTA, 2.25 g chlorine. Stir until dissolved, dilute to 100 mL, adjust pH to 7.5, filter sterilize, and store in a refrigerator.

[0065] The primers involved in the following examples were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The specific primer information is as follows:

[0066] Table 3 Main primer information

[0067]

[0068] Example 1: Gene cloning and physicochemical property analysis of PmPLP

[0069] 1.1 Synthesis of first-strand cDNA

[0070] The steps for preparing cDNA template are as follows:

[0071] (1) Add the following reagents to an enzyme-free EP tube in an ice bath: Table 4

[0072]

[0073] (2) The reverse transcription program of the PCR instrument is as follows: Table 5

[0074]

[0075] After the procedure, place on ice or store in a -20 ℃ refrigerator for future use.

[0076] 1.2 Intermediate fragment cloning

[0077] (1) Intermediate fragment PCR reaction system: Table 6

[0078]

[0079] (2) Intermediate fragment PCR reaction conditions: Table 7

[0080]

[0081] 3) After PCR amplification of the intermediate fragment, perform electrophoresis analysis on the reaction product to confirm the target band. Expand the reaction system for secondary PCR amplification to obtain a DNA product with a higher concentration. After that, ligate the product to the pMD-19T Vector overnight.

[0082] (4) Gently mix the Fast-T1 chemically competent cells and the ligation product, place on ice for 30 minutes, heat shock in a 42°C water bath for 30 seconds, and then immediately transfer to ice to cool for 3 minutes.

[0083] (5) Add LB liquid culture medium (make sure it is sterile) to the mixture tube and then place it in a 37°C constant temperature shaking incubator for 1 hour. Spread the cultured bacterial liquid onto LB solid culture medium (containing ampicillin) and place it upright in a 37°C constant temperature incubator for 15 to 30 minutes to allow the bacterial liquid to absorb. Then, place it inverted and incubate for 12 hours.

[0084] (6) Select a single colony on the culture plate and inoculate it into LB medium (containing ampicillin) (make sure it is sterile) and culture at 37°C for 5 to 6 hours. Use the laboratory universal primers to construct a colony PCR system for amplification. After agarose gel electrophoresis, take a bacterial sample with the correct target band and send it to a sequencing company for sequencing.

[0085] Colony PCR reaction system: Table 8

[0086]

[0087] Reaction conditions: Table 9

[0088]

[0089] 1.3 RACE amplification

[0090] Refer to the method in "Wu Y, He J, Yao G, et al. Molecular cloning, characterization, and expression of two TNFRs from the pearl oyster Pinctada fucata martensii[J]. Fish & Shellfish Immunology, 2020, 98: 147-159", prepare 5' and 3' RACE templates according to the instructions of the RACE amplification kit, add the corresponding reagents and universal primers (NUP and UPM) according to the preparation system in the literature, perform secondary amplification by nested PCR, and send the products for testing according to the results.

[0091] 1.4 Bioinformatics Analysis

[0092] This study obtained sequence information for each species from the NCBI online website and performed bioinformatics analysis of PmPLP using online analysis websites and related software. The websites and software used are listed in the table below.

[0093] Table 10. Software used for bioinformatics analysis

[0094]

[0095] 1.4 Results and Analysis

[0096] The full-length of the Pinctada martensii C-type lectin PmPLP gene cloned was 646 bp, and its open reading frame (ORF) was 486 bp, encoding a polypeptide consisting of 161 amino acids (aa) ( Figure 1 -A). SMART online software analysis showed that PmPLP contains a CRD domain ( Figure 1 -B). The theoretical isoelectric point (pI) predicted by Expasy protein analysis software is 7.58, and the calculated molecular weight is 18.81 kDa. According to the prediction analysis of protein transmembrane regions and signal peptides on the DeepTMHMM website, PmPLP does not contain a transmembrane domain, and its N-terminus is located outside the cell. The predicted signal peptide is located in the first 23 amino acids ( Figure 2 ). According to ProtScale hydrophobicity, PmPLP protein is predicted to be a hydrophilic protein with a GRAVY coefficient of -0.490 ( Figure 3 ). The protein structure was predicted using SWISS-MODEL homology modeling, with a GMQE value of 0.87 and a sequence homology of 96.89%. The prediction model ( Figure 4 ) is highly credible.

[0097] 1.5 Homology comparison and phylogenetic tree construction

[0098] The amino acid sequence of PmPLP was compared with the blast sequence in the NCBI database using DNAMAN6.0. The results showed that PmPLP of Pinctada martensii had a high similarity with the molluscs Magallana gigas and Mediterranean mussels (Mytilus galloprovincialis), with similarities of 32.14% and 29.26% respectively. The highest similarity with vertebrates was Oreochromis aureus, with similarity of 32.52%. The phylogenetic tree was constructed using MEGA 6.0, as shown in Figure 5. Figure 5 As shown in the figure, the analysis showed that the amino acid sequences of the Pinctada martensii PmPLP and the C-type lectin domain protein of the long oyster clustered into one branch.

[0099] Example 2: Expression pattern of PmPLP

[0100] 2.1 PmPLP expression pattern in normal tissues

[0101] qRT-PCR was used to detect the expression profile of PmPLP in the main tissues of Pinctada martensii, and five tissues including mantle, gill, hepatopancreas, blood cells and adductor muscle were selected for detection.

[0102] (1) Prepare fluorescent quantitative cDNA template according to the instructions of All-in-One RT MasterMix. The PCR reaction procedure is as follows: incubate at 37°C for 2 min, incubate at 55°C for 15 min, and incubate at 85°C for 5 min. Quickly remove the cDNA template and place it on ice for use, or store it in a -20°C refrigerator.

[0103] The reaction system is as follows: Table 11

[0104]

[0105] (2) Fluorescence quantitative PCR procedure

[0106] GAPDH was selected as the internal reference gene, and the qRT-PCR reaction system and reaction procedure were as follows:

[0107] Reaction system: Table 12

[0108]

[0109] Reaction conditions: Table 13

[0110]

[0111] 3) Data statistics and analysis

[0112] After the real-time fluorescence quantitative PCR reaction was completed, the data that met the standards were screened and 2 -ΔΔCT The relative gene expression levels were calculated using SPSS 27.0 software, and the experimental data were statistically analyzed.

[0113] (4) The results are as follows Figure 6 As shown in the data, the mRNA expression level of the PmPLP gene in the gill tissue of Pinctada martensii was much higher than that in other tissues, followed by the mantle and blood cells, and was almost not expressed in the hepatopancreas and adductor muscle.

[0114] 2.2 PmPLP expression pattern after nuclear transplantation

[0115] Specialized technicians performed nucleation procedures on 70 pearl oysters. They implanted a spherical nucleus and a fragment of mantle tissue into the host pearl oysters. Gill tissues were collected from eight oysters at 0, 6, 12, 24, 48, 72, and 96 hours after nucleation. The expression profile of PmPLP in the gill tissue of Pinctada martensii after nucleation was determined using qRT-PCR, as described above.

[0116] The results are as follows Figure 7 As shown in the figure, the expression of PmPLP was significantly downregulated at 24 h and 48 h after nuclear implantation (P < 0.05), and then basically returned to the level before nuclear implantation at 72 h and 96 h.

[0117] Example 3: Prokaryotic expression, purification and antibody preparation of PmPLP

[0118] 3.1 Construction of expression vector

[0119] Based on the known full-length cDNA sequence of PmPLP, its open reading frame (ORF) was predicted and gene synthesis was performed using Escherichia coli BL21 as the host strain. The PmPLP gene fragment was amplified by PCR and the PCR product was purified and recovered. The PmPLP gene fragment and the pET-28a(+) expression vector were digested with restriction endonucleases NdeI and BamHI, respectively. A fusion expression vector was then constructed by ligation at 37°C. In the ligation product, the PmPLP gene was expressed as a fusion with the His tag on the vector, forming the recombinant expression vector pET-28a-PmPLP.

[0120] 3.2 Transformation of expression strains

[0121] Take 100 μL of BL21 (Rosetta) competent cells stored at -80°C and place on ice to slowly thaw. In a clean bench, divide the competent cells into two 50 μL aliquots in a sterile 1.5 mL centrifuge tube. Next, add 20 μL of the recombinant plasmid pET-28a-PmPLP to the competent cells, mix gently, and incubate in an ice bath for 30 minutes. Then, heat shock the mixture at 42°C for 90 seconds and quickly cool it in an ice bath for 5 minutes. Finally, spread 30 μL of the transformation mixture evenly on a TB solid medium plate containing 50 μg / mL kanamycin. Incubate the plate in a 37°C incubator for 14 hours (overnight) to obtain single clonal colonies. Select single clonal colonies for subsequent activation culture.

[0122] 3.3 Expression identification

[0123] Selected single clones were inoculated into 2 mL of TB liquid medium containing kanamycin (50 μg / mL) and cultured at 37°C with shaking until the OD600 reached 0.6-0.8. The culture was then transferred to 5 mL of expansion medium (containing the same antibiotics) at a 1:10 ratio and cultured with shaking until the logarithmic growth phase. IPTG was then added to a final concentration of 1 mM and expression was induced at 37°C for 4 hours. Cell samples were collected for subsequent protein expression analysis.

[0124] 3.4 Scale-up expression and purification

[0125] Select a BL21 strain expressing the target protein and inoculate it into 1 L of TB medium containing 50 μg / mL kanamycin. Incubate the culture at 37°C and 220 rpm until the OD600 reaches 0.6-0.8. Add IPTG to a final concentration of 1 mM and induce the culture at 30°C for 12 hours. After induction, harvest the cells by centrifugation at 4000 rpm for 10 minutes.

[0126] 3.5 Bacterial lysis and protein extraction

[0127] Resuspend the collected bacterial slurry in 40 mL of lysis buffer, place it in an ice bath, and process it using a 420 W ultrasonic disruptor. Ultrasonication conditions: 3 seconds per pulse, 5 seconds per interval, for 15 minutes to ensure thorough bacterial disruption. Centrifuge the disrupted bacterial slurry at 4°C and 12,000 rpm for 20 minutes. Collect the supernatant, which is the crude extract containing the target protein. Filter through a 0.22 μm filter membrane to remove unbroken cell debris and other impurities to ensure smooth subsequent purification.

[0128] 3.6 Purification and analysis of target protein

[0129] The filtered supernatant was loaded onto a Ni-NTA affinity chromatography column pre-equilibrated in lysis buffer. The target protein was enriched by the specific binding of the His tag to the Ni column. Nonspecifically bound impurities were removed by flow-through elution with lysis buffer. The target protein was eluted using a gradient elution method (imidazole concentrations of 20 mM, 50 mM, 100 mM, and 250 mM), and the purified sample was collected based on the elution peak. The purified sample was added to an appropriate amount of loading buffer, boiled at 100°C for 5 minutes, and analyzed by electrophoresis on a 12% SDS-PAGE gel.

[0130] The results are as follows Figure 8 Figure 1 shows the SDS-PAGE results of protein induced expression, with lane 1 representing the uninduced strain and lanes 2-5 representing purifications from different induced strains. Lane B shows the SDS-PAGE results of protein purification, with lane 1 representing the flow-through and lanes 2-7 representing purified samples. Lane C shows the SDS-PAGE results of the final product of protein amplification and purification. SDS-PAGE electrophoresis analysis revealed that the recombinant protein rPmPLP was expressed at approximately 19 kDa, with a molecular weight nearly consistent with the predicted size, indicating the correct target band. The bright band indicates successful induced expression of the target protein. The purified protein exhibits a single band, suitable for subsequent validation experiments.

[0131] 3.7 Preparation of polyclonal antibodies

[0132] In this study, healthy, glossy-coated, and freely moving female Balb / c mice, 6-8 weeks old, were immunized using multiple subcutaneous injections. The first immunization consisted of a 100 μL emulsified mixture of 1 mg / mL antigen and Freund's complete adjuvant (FCA) at a 1:1 ratio. Fourteen days later, the second immunization involved a 100 μL emulsified mixture of 500 μg / mL antigen and Freund's incomplete adjuvant (FIA) at a 1:1 ratio. One week after the second immunization, the third immunization involved a 100 μL emulsified mixture of 250 μg / mL antigen and FIA at a 1:1 ratio. One week later, the fourth immunization was repeated, with the three-dose regimen repeated. Five days after the fourth immunization, eye bleeding was performed, and the supernatant, which constituted the polyclonal antibodies, was collected.

[0133] 3.8 ELISA detection of polyclonal antibody titer

[0134] (1) Dilute the antigen to 10 μg / mL with coating solution, add 50 μL to each well, and place on a shaker at room temperature for 1 h;

[0135] (2) Discard the antigen, add 200 μL of blocking solution to each well, and place on a shaker at room temperature for 1 h;

[0136] (3) Wash the plate three times with PBST, add 200 μL of the test serum to each well (initial dilution ratio is 1:50), and place 50 μL per well on a shaker at room temperature for 1 h. Each sample has two parallel controls.

[0137] (4) Wash the plate three times with PBST, add 50 μL of HRP-labeled goat anti-mouse IgG secondary antibody (1:250 dilution) to each well, and place on a shaker at room temperature for 1 h;

[0138] (5) Wash the plate three times with TTBS, pat dry, and add 100 μL of TMB colorimetric solution to each well. When a blue color is observed, add sulfuric acid to terminate the reaction. When the liquid turns yellow, immediately place the plate in a microplate reader for reading.

[0139] (6) Rat negative serum was used as a negative control, and the PBS buffer group was used as a blank when reading. When analyzing the data, wells with a ratio of experimental group (P) / negative control group (N) > 2.1 were considered positive, and the positive result with the largest dilution factor was considered the antibody titer.

[0140] 3.9 Western Blot Detection Method

[0141] The specific operation of Western Blot technology used in the specific detection of polyclonal antibodies and target protein detection experiments is as follows:

[0142] (1) After mixing the protein sample with 5× loading buffer, boil it at 100°C for 10 min to completely denature the protein, and then perform 12% SDS-PAGE;

[0143] (2) After electrophoresis, the PAGE gel containing the protein sample is cut according to the molecular weight of the target protein and transferred to a polyvinylidene fluoride membrane (PVDF membrane) using the "transfer sandwich" wet transfer method;

[0144] (3) After transfer, wash the PVDF membrane three times with TBST buffer for 5 minutes each time to remove unbound proteins and impurities;

[0145] (4) Use the no-wash rapid blocking solution and place the PVDF membrane on a horizontal shaker to block for 10 minutes;

[0146] (5) Incubate the blocked PVDF membrane with the diluted primary antibody (polyclonal antibody) in a 4°C refrigerator overnight;

[0147] (6) Wash thoroughly with TBST buffer three times, 10 min each time, shaking rapidly on a shaker to remove unbound primary antibody;

[0148] (7) Add diluted horseradish peroxidase-labeled goat anti-mouse IgG secondary antibody and incubate at room temperature for 1 h;

[0149] (8) Repeat step (6);

[0150] (9) Add ECL chemiluminescent solution onto the PVDF membrane, place the membrane in a chemiluminescent gel imaging system for exposure and imaging, and save the image results.

[0151] In this example, mice were immunized with the recombinant protein rPmPLP to prepare polyclonal antibodies against rPmPLP in mouse serum. The titer of the polyclonal antibody against PmPLP was determined by ELISA. The results are shown in Figure 9. The titer was 1:4050, and the dilution ratio was 1:1000. The specificity of the polyclonal antibody was determined by Western blotting. Figure 10 As shown, a clear, single reactive band was observed near the molecular weight of PmPLP (19.2 kDa), with no nonspecific reactive bands detected. This indicates that the antibody specifically binds only to the recombinant protein rPmPLP expressed in the bacteria and can be used in subsequent immunoassays.

[0152] Example 4: Microbial binding experiment of rPmPLP

[0153] The microbial binding activity of rPmPLP was tested using Streptococcus agalactiae, Micrococcus luteus, Aeromonas hydrophila, and Pseudomonas aeruginosa. Bacteria grown to the logarithmic phase were collected by centrifugation (3000 × g, 10 min), washed with PBS buffer, and resuspended to adjust the concentration to 2 × 10 8 CFU / mL; 20 μL of various bacterial suspensions were taken and incubated with 20 μL of rPmPLP at 4 °C overnight; the incubated bacterial suspension was thoroughly washed three times with PBS, and the bacterial suspension was mixed with loading buffer at a ratio of 4:1, boiled at 99 °C for 10 min, and after 12% SDS-PAGE, the binding activity of rPmPLP with microorganisms was detected by Western blot.

[0154] The results are as follows Figure 11 As shown, after incubation with Streptococcus agalactiae, Micrococcus luteus, Aeromonas hydrophila, and Pseudomonas aeruginosa, rPmPLP all displayed bands with similar molecular weights to rPmPLP, indicating that rPmPLP has binding activity against both Gram-negative and Gram-positive bacteria. The thickness of the bands indicates the strength of rPmPLP's binding to microorganisms. The results showed that the band bound to Micrococcus luteus was the thickest, indicating that rPmPLP had the strongest binding to M. luteus, while its binding to P. aeruginosa was relatively weak. No bands were evident in the control group.

[0155] Example 5: Bacterial Agglutination Experiment of rPmPLP

[0156] Bacillus subtilis, Staphylococcus aureus, Micrococcus luteus, Aeromonas hydrophila, Pseudomonas aeruginosa, and Escherichia coli were cultured to the logarithmic growth phase, the bacterial suspension was collected, and fixed with formaldehyde solution for 10 min; the bacterial suspension was washed three times with TBS, then stained with fluorescein isothiocyanate (FITC) in the dark for 2 h, and then washed three times with TBS buffer; the above bacterial suspension was adjusted to 1×10 8 CFU / mL concentration, stored at 4 ℃ for later use; 20 μL of bacterial suspension was mixed with 20 μL of recombinant protein in a 96-well plate, 10 mM CaCl2 solution was added to the control group, TBS was used as a negative control, and the 96-well plate was incubated at room temperature for 2 hours; the 96-well plate was placed under an inverted fluorescence microscope for observation and photography.

[0157] FITC fluorescence labeling technology was used to label microorganisms and detect the agglutination effect of rPmPLP on microorganisms. Figure 12 As shown in the figure, Bacillus subtilis, Staphylococcus aureus, Aeromonas hydrophila, Pseudomonas aeruginosa, Micrococcus luteus and Escherichia coli showed different degrees of agglutination reaction under the action of rPmPLP. 2＋Under the conditions of , it was observed that rPmPLP had more obvious agglutination phenomenon against Bacillus subtilis, Staphylococcus aureus, Aeromonas hydrophila, Pseudomonas aeruginosa, and Micrococcus luteus, but had no obvious effect on Escherichia coli.

[0158] Example 6: Oxford cup antibacterial test

[0159] This example refers to the method in “Tang Xin. Study on the immune function of large yellow croaker lectin Galectin-1, Ladderlectin and Perforin-1 [D]. Xiamen: Jimei University, 2023” and uses the Oxford cup agarose diffusion method to detect antibacterial activity. When the experimental bacteria are cultured to the logarithmic growth phase, the bacterial concentration is adjusted to 2×10 6 CFU / mL. Prepare LB solid medium containing the bacterial suspension. Once the medium is completely solidified, place it in an Oxford cup. Add 100 μg of recombinant protein to the Oxford cup, with and without calcium ions. Also include ampicillin as a positive control and PBS as a blank control. Place the culture dish in a 37°C biological incubator and incubate upright for 12 hours. Observe the inhibition zone in the Oxford cup and measure its diameter.

[0160] According to the Oxford cup agar diffusion experiment analysis, the results are as follows Figure 13 As shown in Table 14, the recombinant protein rPmPLP has a direct antibacterial effect, while calcium ions do not enhance the antibacterial effect of rPmPLP.

[0161] Table 14 Analysis of inhibition zone experiment

[0162]

[0163] Example 7: Inhibition curve of rPmPLP

[0164] This example measured the effect of the recombinant protein rPmPLP on the growth curves of bacteria and fungi (including Micrococcus luteus, Staphylococcus aureus, Bacillus subtilis, Streptococcus agalactiae; Escherichia coli, Pseudomonas aeruginosa, Aeromonas hydrophila, Vibrio harveyi; Aspergillus niger, and Rhizopus). Glycerol-preserved bacteria and fungi were activated in culture medium and inoculated into liquid culture overnight to the logarithmic growth phase. The culture was resuspended, washed three times with PBS, and diluted to a concentration of 5 x 10 5 CFU / mL of bacterial suspension. In a 96-well plate, mix 50 μL of recombinant protein (1 mg / mL) with 10 μL of each bacterial suspension. Incubate in a 37°C (30°C for fungi) incubator for 2 h. Then, add 140 μL of liquid culture medium, mix thoroughly, and measure the OD600 value using a microplate reader. PBS was used as a negative control. Each sample was repeated three times.

[0165] The results are as follows Figure 14 、 15 As shown, the recombinant protein rPmPLP exhibited significant inhibitory effects against four Gram-positive bacteria (Micrococcus luteus, Staphylococcus aureus, Bacillus subtilis, and Streptococcus agalactiae), four Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Aeromonas hydrophila, and Vibrio harveyi), and two fungi (Aspergillus niger and Rhizopus harveyi) (P < 0.01). The growth of Gram-positive bacteria was inhibited to varying degrees by rPmPLP. The growth of Micrococcus luteus was most rapidly inhibited, beginning after just 2 hours. The growth of Staphylococcus aureus and Bacillus subtilis began to be inhibited after 3 hours, while the growth of Streptococcus agalactiae was inhibited slightly later, at 4 hours. Among Gram-negative bacteria, Aeromonas hydrophila was the most rapidly inhibited, also beginning after 2 hours. Vibrio harveyi's growth was inhibited after 3 hours, Escherichia coli after 4 hours, and Pseudomonas aeruginosa's growth was the last to be inhibited, at 6 hours. After 12 hours of rPmPLP treatment, the growth of the remaining eight bacterial species, with the exception of E. coli, almost completely ceased, demonstrating a significant inhibitory effect that contrasted sharply with the rapid growth of the control group. While E. coli's growth rate was inhibited, it remained slow. The growth of the two fungi gradually declined after 12 hours and reached significant levels after 24 hours. The inhibitory effect on Aspergillus niger weakened after 48 hours, while Rhizopus was continuously inhibited throughout the entire 48 hours.

[0166] In summary, rPmPLP has broad-spectrum antibacterial activity, including Gram-positive and Gram-negative bacteria and fungi. The Gram-positive bacteria are selected from Micrococcus luteus, Staphylococcus aureus, Bacillus subtilis, and Streptococcus agalactiae; the Gram-negative bacteria are selected from Escherichia coli, Pseudomonas aeruginosa, Aeromonas hydrophila, and Vibrio harveyi; and the fungi are selected from Aspergillus niger and Rhizopus.

[0167] Example 8: Scanning electron microscopy experiment

[0168] Prepare bacterial suspension according to the method of Example 7 and dilute with PBS to 1×10 7CFU / mL. Mix 100 μL of bacterial suspension with 100 μL of protein solution, incubate in a 37°C constant temperature incubator for 2 h, and collect the bacteria by centrifugation. Add 1 mL of 2.5% glutaraldehyde to resuspend the bacteria and place at 4°C for 12 h. After another centrifugation, collect the bacteria and wash three times with PBS. Dehydrate once with ethanol concentrations of 30%, 50%, 70%, 80%, and 90% for 10 min each, and finally dehydrate thoroughly with 100% ethanol for 15 min. Add 10 μL of the sample to the silicon wafer. If the bacterial concentration is low (little precipitation after centrifugation), continue adding the sample 3-5 times after the ethanol evaporates. Place the sample silicon wafer in a 70°C oven to dry overnight. After gold spraying, observe using a scanning electron microscope.

[0169] rPmPLP was co-incubated with four Gram-positive bacteria (Micrococcus luteus, Staphylococcus aureus, Bacillus subtilis, Streptococcus agalactiae) and four Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Aeromonas hydrophila, Vibrio harveyi), and the bacterial morphology was observed using SEM. Figure 16 As shown in the figure, the bacterial surface of the blank control group was smooth and plump, and the bacteria were intact. The surfaces of Bacillus subtilis and Pseudomonas aeruginosa in the experimental treatment group had holes; the spherical structure of Streptococcus agalactiae and Micrococcus luteus was incomplete, with roughened surfaces, wrinkles, and fractures; the cell wall of Escherichia coli peeled off, and the cell membrane showed obvious dissolution; the cell membrane of Vibrio harveyi also showed slight dissolution, and the cell membranes of the bacteria adhered together; the surface of Pseudomonas aeruginosa was concave, and the bacteria were not obviously broken; Staphylococcus aureus in the experimental group did not change significantly compared with the control group, but the bacterial surface of the experimental group was rough and slightly wrinkled and deformed.

[0170] Example 9: rPmPLP injection, nuclear implantation experiment and inflammatory factor gene expression detection

[0171] Ninety Pinctada martensii oysters were randomly divided into three groups, 30 in each: a blank group (nucleus implant only), a control group (PBS + nucleus implant), and an experimental group (rPmPLP + nucleus implant). The recombinant protein rPmPLP was diluted to 100 ng / mL. Each Pinctada martensii oyster in the experimental group was injected with 100 μL of rPmPLP solution into the adductor muscle, while the control group was injected with an equal volume of PBS.

[0172] To investigate the inflammatory response of Pinctada martensii to rPmPLP immunization and nuclear implantation, this study analyzed the expression patterns of PmPLP genes and various inflammatory factors in rPmPLP-injected cells after nuclear implantation (qRT-PCR detection). Figure 17 、 18As shown, AH represent the mRNA expression patterns of PmPLP (A) and inflammatory factors Caspase-2 (B), IL-17 (C), IRAK1 (D), IκK (E), NF-κB (F), TRAF3 (G), and TNF-α (H), respectively.

[0173] After immune stimulation, PmPLP increased significantly at 12 hours. The inflammatory factor TNF-α increased rapidly and significantly at 6 hours and remained upregulated until it returned to the same level as the PBS group at 72 hours. The expression of inflammatory factors Caspase-2 and IRAK1 in the stimulation group was significantly higher than that in the PBS group at 6 hours. IL-17 expression was significantly higher than that in the PBS group at 12, 24, 48, and 72 hours. The inflammatory factors TRAF3 and NF-κB were significantly upregulated at 24 hours after stimulation, almost returned to the same level as the PBS group at 48 hours, and then significantly upregulated again at 72 hours. IκK was significantly upregulated at 12 and 24 hours and returned to the level of the PBS group at 72 hours.

[0174] In summary, after co-treatment with rPmPLP and nuclear implantation, the expression of PmPLP and Caspase-2, IL-17, IRAK1, IκK, NF-κB, TRAF3, and TNF-α were significantly upregulated, indicating that PmPLP can promote the release of proinflammatory factors induced by nuclear implantation in Pinctada martensii.

[0175] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by any technician familiar with this technical field within the technical scope disclosed by the present invention should be covered by the scope of protection of the present invention.

Claims

1. A Pinctada martensii C-type lectin PmPLP, characterized in that The amino acid sequence of the C-type lectin is shown in SEQ ID NO.

1.

2. The gene encoding the C-type lectin PmPLP according to claim 1, characterized in that The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

3. An expression vector comprising the gene according to claim 2, wherein the vector is a PET series vector.

4. A host cell comprising the expression vector of claim 3, wherein the host cell is selected from Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, Yarrowia lipolytica or Bacillus subtilis.

5. Use of the Pinctada martensii C-type lectin PmPLP according to claim 1 in the preparation of a drug for inhibiting microorganisms; the microorganism is at least one of Gram-positive bacteria, Gram-negative bacteria and fungi; the Gram-positive bacteria are selected from Micrococcus luteus, Staphylococcus aureus, Bacillus subtilis and Streptococcus agalactiae; the Gram-negative bacteria are selected from Escherichia coli, Pseudomonas aeruginosa, Aeromonas hydrophila and Vibrio harveyi; the fungi are selected from Aspergillus niger and Rhizopus.

6. A pharmaceutical composition, characterized in that The composition contains an effective amount of the Pinctada martensii C-type lectin PmPLP according to claim 1, and a pharmaceutically acceptable carrier.

7. The composition according to claim 6, wherein The concentration of the Pinctada martensii C-type lectin PmPLP in the composition is less than 1 mg / mL.

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