Pinctada martensii C-type lectin PmPLP and application thereof

Abstract

The invention discloses a Pinctada martensii C-type lectin PmPLP, an application of the Pinctada martensii C-type lectin PmPLP in preparation of drugs for inhibiting microorganisms and an application of the Pinctada martensii C-type lectin PmPLP in promotion of release of pro-inflammatory factors induced by nucleus implantation of Pinctada martensii. According to the invention, preliminary research is carried out on identification and immune functions of Pinctada martensii C-type lectin PmPLP, so that immune response of PmPLP molecules in immune response of Pinctada martensii for resisting invasion of external pathogenic bacteria is determined, and antibacterial activity of PmPLP after in-vitro expression is known; and data support and a research basis are provided for researching C-type lectin immune defense mechanisms of pinctada martensii and other marine mollusks. The immune response mechanism of the pinctada martensii is deeply studied, the molecular regulation mechanism of the pinctada martensii for coping with pathogen invasion is disclosed, and the method has important significance for improving the breeding efficiency and preventing and controlling diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and specifically relates to the C-type lectin PmPLP of Pinctada martensii and its application. Background Art

[0002] Pinctada martensii is one of the main pearl oysters for artificial seawater pearl cultivation in China. In recent years, due to the increasingly deteriorating coastal water quality, various diseases have occurred frequently, resulting in a large number of deaths of Pinctada martensii due to diseases, causing huge economic losses to China's pearl industry. As a typical filter-feeding organism, Pinctada martensii is easily infected by multiple pathogens such as Vibrio (e.g., Vibrio alginolyticus, Vibrio parahaemolyticus), Rickettsia, and herpes virus. Similar to other invertebrates, Pinctada martensii only has an innate immune system, which is composed of cell immunity and humoral immunity to jointly form its defense mechanism. Therefore, in-depth study of the immune response mechanism of Pinctada martensii helps to reveal its molecular regulation mechanism against pathogen invasion, and 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 the implant nucleus has an impact on the expression profile of key genes. The artificial nucleation technology has received extensive attention worldwide. Pinctada martensii is a commercially cultured variety, and more than 90% of seawater pearls in China are produced from Pinctada martensii. The nucleation operation is to transplant the mantle piece of the donor oyster and the artificial pearl nucleus into the recipient oyster together. After nucleation, the epidermal cells of the mantle secrete nacre, and through the process of biomineralization, pearls are finally formed. During the artificial nucleus insertion operation, the tissue pieces, pearl nuclei, and pathogenic microorganisms entering the recipient oyster body can cause a cascade reaction of the host pearl oyster's immune system. A strong immune response can lead to the expulsion of the nucleus by the oyster, making it difficult to form a pearl sac and even causing the death of the host oyster. Therefore, scientific researchers and aquaculture farmers have been studying the immune response mechanism of Pinctada martensii after nucleation, and actively taking various measures to try to reduce the immune rejection reaction in nucleation pearl cultivation, improve the regulatory ability of this species, so as to improve the survival rate of organisms and increase their yield.

[0004] C-type lectins play a regulatory role in the antibacterial immune response of invertebrates through multiple mechanisms and are an important part of the invertebrate innate immune system. C-type lectins are a large class of metazoan extracellular proteins. As important pattern recognition receptors (PRRs), they can specifically recognize pathogen-associated molecular patterns on the surface of bacteria, such as lipopolysaccharide (LPS), peptidoglycan (PGN), etc., and can also recognize the main component of the fungal cell wall, β-glucan, etc. Its carbohydrate recognition domain can bind to carbohydrate molecules on the surface of microorganisms, thereby initiating an immune response. Once a pathogen is recognized, C-type lectins can directly participate in the agglutination of microorganisms, causing the pathogens to aggregate together and restricting their spread.

[0005] However, there is no in-depth report on the role of lectins in the immune system of Pinctada fucata martensii at present. Studying the lectins of Pinctada fucata martensii not only has important significance for deeply understanding the mechanism of immune prevention and control of pathogenic microorganisms in Pinctada fucata martensii and its healthy aquaculture, but also can indirectly have important significance for human life. Therefore, the discovery and utilization of the pattern recognition receptor C-type lectin are of important theoretical and practical significance for understanding the immune defense mechanism of Pinctada fucata martensii and carrying out disease control. Summary of the Invention

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

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

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

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

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

[0011] In the fifth aspect, the present invention provides the application of the C-type lectin PmPLP of Pinctada fucata martensii in the preparation of a drug for inhibiting microorganisms; the microorganisms are 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 stolonifer.

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

[0014] In one or more embodiments, the concentration of 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 nucleation in Pinctada martensii.

[0016] In one or more embodiments, the pro-inflammatory factors are 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: The present invention conducts preliminary research on the identification and immune function of the Pinctada martensii C-type lectin PmPLP to determine the immune response of the PmPLP molecule in the immune response of Pinctada martensii against the invasion of external pathogenic bacteria, and to understand the antibacterial activity after in vitro expression of PmPLP, 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 in-depth study of the immune response mechanism of Pinctada martensii by the present invention helps to reveal its molecular regulation mechanism in response to pathogen invasion, which is of great significance for improving aquaculture efficiency and disease prevention and control. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 shows the structural characteristics of the PmPLP gene in Example 1.

[0019] Figure 2 shows the prediction of the transmembrane region and signal peptide of PmPLP in Example 1.

[0020] Figure 3 shows the hydrophilicity / hydrophobicity analysis of the PmPLP protein in Example 1.

[0021] Figure 4 shows the three-dimensional structure model diagram of PmPLP in Example 1.

[0022] Figure 5It is the phylogenetic tree analysis of PmPLP in Example 1.

[0023] Figure 6 It is the expression level of PmPLP in different tissues in Example 2.

[0024] Figure 7 It is the expression pattern diagram of PmPLP after nuclear implantation in Example 2.

[0025] Figure 8 It is the prokaryotic expression and purification diagram of PmPLP in Example 3.

[0026] Figure 9 It is the titer diagram of the polyclonal antibody of PmPLP in Example 3.

[0027] Figure 10 It is the Western blot analysis of the polyclonal antibody of PmPLP in Example 3.

[0028] Figure 11 It is the binding activity of rPmPLP to microorganisms in Example 4.

[0029] Figure 12 It is the analysis of the bacterial agglutination activity of rPmPLP in Example 5.

[0030] Figure 13 It is the analysis of the antibacterial effect of rPmPLP in Example 6.

[0031] Figure 14 It is the detection result 1 of the bacteriostatic activity of PmPLP in Example 7.

[0032] Figure 15 It is the detection result 2 of the bacteriostatic activity of PmPLP in Example 7.

[0033] Figure 16 It is the observation of the effect of rPmPLP on bacteria by scanning electron microscopy in Example 8.

[0034] Figure 17 It is the expression result 1 of inflammatory factors after injecting rPmPLP and nuclear implantation in Example 9.

[0035] Figure 18 It is the expression result 2 of inflammatory factors after injecting rPmPLP and nuclear implantation in Example 9. Detailed implementation manners

[0036] The economic value of Pinctada fucata martensii is mainly reflected in pearl production. However, disease problems such as bacterial infections pose a serious threat to the cultivation of Pinctada fucata martensii. Therefore, in-depth research on the immune response mechanism of Pinctada fucata martensii helps to reveal its molecular regulatory mechanism in response to pathogen invasion, which is of great significance for improving the cultivation efficiency and disease prevention and control. The present invention conducts preliminary research on the identification and immune function of C-type lectin PmPLP in Pinctada fucata martensii to determine the immune response of PmPLP molecule in the immune reaction of Pinctada fucata martensii against the invasion of external pathogenic bacteria, and to understand the antibacterial activity after in vitro expression of PmPLP, providing data support and research basis for the study of the immune defense mechanism of C-type lectin in Pinctada fucata martensii and other marine mollusks.

[0037] Specifically, the present invention includes but is not limited to the following research contents: The present invention identified a novel C-type lectin PmPLP from Pinctada fucata martensii. As Figure 1 shown, its ORF is 486 bp, encoding a polypeptide composed of 161 amino acids, with an isoelectric point of 7.58 and a theoretical molecular weight of 18.81 kDa. PmPLP contains a single CTLD domain, and it was found that PmPLP contains a galactose-binding motif "-QPD-" and a mannose-binding motif "-WND-". The expression level of PmPLP is the highest in the gill of normal tissue.

[0038] The present invention prepared rPmPLP and its polyclonal antibody anti-rPmPLP by in vitro prokaryotic expression. The recombinant protein rPmPLP has binding activity with Aeromonas hydrophila, Pseudomonas aeruginosa, Streptococcus agalactiae and Micrococcus luteus; rPmPLP has agglutination effects on Escherichia coli, Aeromonas hydrophila, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis and Micrococcus luteus.

[0039] After injecting rPmPLP into the nucleated shellfish in the present invention, the contents of PmPLP and inflammatory factors Caspase-2, IL-17, IRAK1, IκK, NF-κB, TRAF3, TNF-α were all significantly up-regulated.

[0040] The rPmPLP described in the present invention has direct antibacterial activity and can cause different morphological damages to different bacteria. rPmPLP has broad-spectrum antibacterial characteristics and can inhibit the growth of 4 Gram-positive bacteria, 4 Gram-negative bacteria and 2 fungi. Example

[0041] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand the other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0042] Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are for describing specific embodiments, rather than limiting the protection scope of the present invention.

[0043] When the embodiments give a numerical range, it should be understood that unless otherwise specified in the present invention, any value between the two endpoints of each numerical range and either endpoint can 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 technical field to which the present invention belongs.

[0044] For those embodiments in which specific technologies or conditions are not indicated, they are carried out according to the technologies or conditions described in the literature in this field or according to the product specifications. For reagents or instruments whose manufacturers are not indicated, they are all conventional products that can be obtained through regular channels.

[0045] In the embodiments, the addition amounts, contents, and concentrations of various substances are involved. Among them, unless otherwise specified, the percentage content refers to the mass percentage content.

[0046] The Pinctada martensii used in the following examples was from the Dajing Aquaculture Base in Xilian Town, Xuwen County, Zhanjiang City, Guangdong Province. The selected individuals all had a breeding cycle of about 24 months. Ten experimental shells with good vitality were selected for dissection in the present invention, and tissue samples were collected in sequence, including hemolymph, gills, mantle, adductor muscle, and hepatopancreas. The collected tissues were respectively placed into pre-labeled cryotubes and quickly put into liquid nitrogen for quick freezing treatment for subsequent experiments.

[0047] The reagents and their sources used in the following examples are as follows: Table 1 Reagents and Sources

[0048] The instruments and equipment used in the following examples are as follows: Table 2 Instruments and Equipment

[0049] The reagent preparations used in the following examples are as follows: (1) LB liquid medium: 10 g of NaCl and tryptone, and 5 g of yeast extract. After adding 800 mL of distilled water, adjust the pH value to 7.0 with NaOH solution, make up the volume to 1000 mL, dispense, sterilize at 121 °C under high pressure for 15 min, and store at 4 °C.

[0050] (2) LB solid medium: Add agar powder to LB liquid medium at a ratio of 1.5:100, sterilize by high-pressure steam at 121 °C for 15 min. When the temperature cools to 50 °C - 60 °C, add the corresponding antibiotic to its working concentration in a laminar flow hood, mix well and pour into a bacterial culture dish. After the medium solidifies, seal it with a sealing film and store it upside down at 4 °C.

[0051] (3) BHI liquid medium: Weigh 38.5 g of brain heart infusion broth, heat and stir in 1000 mL of distilled water, sterilize at 121 °C under high pressure for 15 min, and store at room temperature.

[0052] (4) TSB liquid medium: 20 g of NaCl; 15 g of tryptone; 5 g of soy peptone; After adding distilled water to 800 mL, adjust the pH value to 7.2 with NaOH, make up the volume to 1000 mL, dispense, sterilize at 121 °C under high pressure for 15 min, and store at 4 °C.

[0053] (5) Ampicillin solution: 1 g of sodium ampicillin; Add distilled water to 10 mL, mix well, filter and sterilize with a 0.22 μm filter membrane, and store in the dark at -20 °C.

[0054] (6) TBE buffer: 27.5 g of boric acid, 54 g of Tris, 20 mL of 0.5 mol / L EDTA (pH 7.9), make up the volume to 1000 mL with water.

[0055] (7) DEPC solution: Prepare according to the ratio of 0.1% DEPC stock solution per 100 ml of distilled water, mix well with a shaker, ventilate overnight, sterilize at high temperature for 30 min, and store at 4 °C.

[0056] (8) Lysis buffer: 20 mM phosphate buffer, 150 mM NaCl, pH 8.0.

[0057] (9) Inhibitor: 2.08 g of glucose, 0.8 g of sodium citrate, 0.336 g of EDTA, 2.25 g of chlorine. After stirring until dissolved, make up the volume to 100 mL, adjust the pH value to 7.5, filter and sterilize, and store refrigerated.

[0058] The primers involved in the following examples were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The specific primer information is as follows: Table 3 Main primer information

[0059] Example 1: Gene cloning and physicochemical property analysis of PmPLP 1.1 Synthesis of the first strand of cDNA The steps for preparing the cDNA template are as follows: (1) In an enzyme-free EP tube on ice bath, add the following reagents: Table 4

[0060] (2) The reverse transcription program of the PCR instrument is as follows: Table 5

[0061] After the program ends, place it on ice for disposal, or store it in a -20 °C refrigerator for later use.

[0062] 1.2 Cloning of the middle fragment (1) Middle fragment PCR reaction system: Table 6

[0063] (2) Middle fragment PCR reaction conditions: Table 7

[0064] (3) After PCR amplification of the middle fragment, take the reaction product for electrophoresis analysis to confirm the target band. Expand the reaction system for secondary PCR amplification. After obtaining a DNA product with a higher concentration, ligate the product with the pMD-19T Vector overnight.

[0065] (4) Gently mix the Fast-T1 chemically competent cells with the ligation product, incubate on ice for 30 min, then heat shock in a 42 °C water bath for 30 s, and then immediately transfer it to ice for cooling for 3 min.

[0066] (5) Add LB liquid medium (note aseptic) to the mixture tube, and then place it in a 37 °C constant temperature shaking incubator for 1 h. Spread the cultured bacterial solution on the LB (containing ampicillin) solid medium, place it upright in a 37 °C constant temperature incubator for 15 to 30 min until the bacterial solution is absorbed, and then incubate it upside down for 12 h.

[0067] (6) Select single colonies on the culture plate, inoculate them into LB (containing ampicillin) medium (pay attention to sterility), and culture at 37 °C for 5 to 6 h. Use common primers in the laboratory to construct a colony PCR system for amplification. After agarose gel electrophoresis of the products, take the bacterial liquid samples with correct target bands and send them to a sequencing company for sequencing.

[0068] Colony PCR reaction system: Table 8

[0069] Reaction conditions: Table 9

[0070] 1.3 RACE amplification 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 the templates for 5'-end and 3'-end RACE according to the instructions of the RACE amplification kit. Add the corresponding reagents and common primers (NUP and UPM) according to the formulation system in this literature, and perform nested PCR for secondary amplification. Send for testing according to the product situation.

[0071] 1.4 Bioinformatics analysis In this study, sequence information of each species was obtained through the NCBI online website, and bioinformatics analysis of PmPLP was performed using online analysis websites and related software. The websites and software used are shown in the following table.

[0072] Table 10 Software used for bioinformatics analysis

[0073] 1.4 Results and analysis The full length of the C-type lectin PmPLP gene cloned from Pinctada fucata martensii is 646 bp, and its open reading frame (ORF) is 486 bp, encoding a polypeptide composed of 161 amino acids (aa) ( Figure 1 -A). Analysis by SMART online software showed that PmPLP contains a CRD domain ( Figure 1-B). The predicted theoretical isoelectric point (pI) using Expasy protein analysis software is 7.58, and the calculated molecular weight is 18.81 kDa. According to the prediction and analysis of protein transmembrane regions and signal peptides on the DeepTMHMM website, PmPLP does not contain a transmembrane domain, and the N-terminus is located outside the cell. The predicted signal peptide is located within the first 23 amino acids ( Figure 2 ). According to the hydrophobicity prediction by ProtScale, the PmPLP protein belongs to a hydrophilic protein with a GRAVY coefficient of -0.490 ( Figure 3 ). The SWISS-MODEL homology modeling was used to predict the protein structure. The GMQE value was 0.87, and the sequence homology was 96.89%, indicating that the predicted model ( Figure 4 ) has a very high confidence level.

[0074] 1.5 Homology alignment and construction of phylogenetic tree The amino acid sequence of PmPLP was aligned with the sequences obtained from blast in the NCBI database using DNAMAN 6.0. The results showed that PmPLP from Pinctada fucata martensii had relatively high similarities with C-type lectin domain proteins from the mollusks Magallana gigas and Mytilus galloprovincialis, which were 32.14% and 29.26% respectively; the highest similarity with vertebrates was with Oreochromis aureus, which was 32.52%. The phylogenetic tree was constructed using MEGA 6.0. As Figure 5 shown, the analysis indicated that the amino acid sequence of PmPLP from Pinctada fucata martensii clustered together with that of the C-type lectin domain protein from Magallana gigas.

[0075] Example 2: Expression pattern of PmPLP 2.1 Expression pattern of PmPLP in normal tissues The expression profiles of PmPLP in the main tissues of Pinctada fucata martensii were detected by qRT-PCR. Five tissues, namely the mantle, gill, hepatopancreas, hemocyte, and adductor muscle, were selected for detection.

[0076] (1) Prepare the fluorescence quantitative cDNA template with reference to the All-in-One RT MasterMix instruction manual. The PCR reaction program: incubate at 37°C for 2 min, 55°C for 15 min, 85°C for 5 min, quickly take out the cDNA template and place it on ice for use, or store it in a -20°C refrigerator.

[0077] The reaction system is as follows: Table 11

[0078] (2) Fluorescence quantitative PCR program GAPDH was selected as the internal reference gene, and the qRT-PCR reaction system and reaction procedure were as follows: Reaction system: Table 12

[0079] Reaction conditions: Table 13

[0080] 3) Data statistics and analysis 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 the SPSS 27.0 software, and the experimental data were statistically analyzed.

[0081] (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.

[0082] 2.2 PmPLP expression pattern after nuclear transplantation 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.

[0083] 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.

[0084] Example 3: Prokaryotic expression, purification and antibody preparation of PmPLP 3.1 Construction of expression vector 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.

[0085] 3.2 Expression Strain Transformation Take 100 μL of BL21 (Rosetta) competent cells stored at -80 °C and place them on ice to thaw slowly. In a laminar flow hood, take a 1.5 mL sterile centrifuge tube and divide the competent cells into two equal parts, 50 μL each. Subsequently, take 20 μL of the recombinant plasmid pET-28a-PmPLP and add it to the competent cells. Gently mix and then incubate in an ice bath for 30 min. After that, heat shock the mixture at 42 °C for 90 seconds and quickly transfer it to an ice bath to cool for 5 min. Finally, spread 30 μL of the transformation mixture evenly on a TB solid medium plate containing 50 μg / mL kanamycin. Place the plate in an incubator at 37 °C and incubate for 14 h (overnight) to obtain monoclonal colonies. Select monoclonal colonies for subsequent activation culture.

[0086] 3.3 Expression Identification Inoculate the selected monoclonal into 2 mL of TB liquid medium containing kanamycin (50 μg / mL) and culture it with shaking at 37 °C until the OD600 reaches 0.6 - 0.8. Then transfer the bacterial solution to 5 mL of enlarged medium (containing the same antibiotic) at a ratio of 1:10 and continue to culture with shaking until the logarithmic growth phase. Next, add IPTG with a final concentration of 1 mM and induce expression at 37 °C for 4 h. Collect the bacterial cell samples for subsequent protein expression identification.

[0087] 3.4 Scale-up Expression and Purification Select the BL21 strain capable of expressing the target protein and inoculate it into 1 L of TB medium containing 50 μg / mL kanamycin. Scale up the culture at 37 °C and 220 rpm until the OD600 value of the bacterial solution reaches 0.6 - 0.8. Add IPTG with a final concentration of 1 mM to the medium and induce culture at 30 °C for 12 h. After induction, centrifuge at 4000 rpm for 10 min to collect the bacterial cells.

[0088] 3.5 Bacterial Cell Lysis and Protein Extraction Resuspend the collected bacterial cell pellet in 40 mL of lysis buffer and place it in an ice bath. Use a 420 W ultrasonic disruptor to process it. The ultrasonic conditions are: ultrasonic for 3 seconds, interval for 5 seconds, and continue for 15 min to ensure sufficient lysis of the bacterial cells. Centrifuge the ultrasonicated bacterial solution at 4 °C and 12000 rpm for 20 min and 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 the smooth progress of subsequent purification.

[0089] 3.6 Purification and Analysis of the Target Protein The filtered supernatant was loaded onto a Ni-NTA affinity chromatography column pre-equilibrated with lysis buffer, and the target protein was enriched by the specific binding of the His tag to the Ni column. Flow-through elution was performed with lysis buffer to remove impurity proteins with non-specific binding. The target protein was eluted by gradient elution method (imidazole concentrations were 20 mM, 50 mM, 100 mM, and 250 mM imidazole in sequence), and the purified samples were collected according to the elution peaks. An appropriate amount of loading buffer was added to the purified samples, and after boiling at 100 °C for 5 min, electrophoresis analysis was performed using a 12% SDS-PAGE gel.

[0090] The results are as Figure 8 shown. Among them, A represents the SDS-PAGE results of protein induced expression. Lane 1 is the uninduced strain, and lanes 2-5 are purifications of different induced strains; among them, B represents the SDS-PAGE results of protein purification. Lane 1 is the flow-through, and lanes 2-7 are the purified samples; among them, C represents the SDS-PAGE results of the final product of protein amplified purification. The lane is the final product of protein purification amplification. After SDS-PADE electrophoresis analysis, the recombinant protein rPmPLP was expressed at approximately 19 kDa in size, and the molecular weight was almost the same as the predicted size, indicating that the target band was correct, and the band was bright, indicating that the target protein was successfully induced and expressed. The protein purification band was single and could be used for subsequent verification experiments.

[0091] 3.7 Preparation of polyclonal antibody In this study, healthy Balb / c mice at 6-8 weeks old, female, with shiny hair and free movement were selected as immunized animals, and the subcutaneous multi-point injection method was used. For the first immunization, the antigen at a concentration of 1 mg / mL was mixed with Freund's complete adjuvant (FCA) in an equal volume ratio of 1:1, and after sufficient emulsification, 100 μL was injected into each mouse; for the second immunization, 14 days later, the antigen at 500 μg / mL was mixed with Freund's incomplete adjuvant (FIA) in an equal volume ratio of 1:1, and after sufficient emulsification, 100 μL was injected into each mouse; for the third immunization, one week after the second immunization, the antigen at 250 μg / mL was mixed with FIA in an equal volume ratio of 1:1, and after sufficient emulsification, 100 μL was injected into each mouse; the fourth immunization was carried out one week later, and the immunization protocol of the third immunization was repeated; on the 5th day after the fourth immunization, blood was collected from the mouse eyeballs, and the supernatant was collected, which was the polyclonal antibody.

[0092] 3.8 ELISA detection of polyclonal antibody titer (1) The antigen was diluted to 10 μg / mL with coating buffer, and 50 μL was added to each well and placed on a shaker at room temperature for 1 h; (2) The antigen was discarded, 200 μL of blocking solution was added to each well for blocking, and it was placed on a shaker at room temperature for 1 h; (3) Wash the plates three times with PBST. Add 200 μL of the serum to be tested (starting dilution ratio is 1:50) to each well, and incubate at room temperature on a shaker for 1 h with 50 μL per well. Set up two parallel controls for each sample. (4) Wash the plates three times with PBST. Add 50 μL of the goat anti-mouse IgG secondary antibody labeled with HRP (diluted 1:250) to each well, and incubate at room temperature on a shaker for 1 h. (5) Wash the plates three times with TTBS. After patting dry, add 100 μL of TMB chromogenic solution to each well. When blue color is observed, add sulfuric acid to terminate the reaction. When the liquid turns yellow, immediately read the values using an ELISA reader. (6) Use the rat negative serum as the negative control, and use the PBS buffer group as the blank when reading the values. During data analysis, wells with an experimental group (P) / negative control group (N) > 2.1 are considered positive, and the positive result with the highest dilution factor is the antibody titer.

[0093] 3.9 Western Blot Detection Method The specific operations of the Western Blot technique used in the specific detection of polyclonal antibodies and the detection of target proteins are as follows: (1) Mix the protein sample with 5×loading buffer, and boil at 100 °C for 10 min to completely denature the protein, and then perform 12% SDS-PAGE. (2) After electrophoresis, cut the PAGE gel containing the protein sample according to the molecular weight of the target protein, and transfer it to a polyvinylidene difluoride membrane (PVDF membrane) using the "wet transfer sandwich" method. (3) After membrane transfer, wash the PVDF membrane 3 times with TBST buffer for 5 min each time to remove unbound proteins and impurities. (4) Use a wash-free rapid blocking solution and incubate the PVDF membrane on a horizontal shaker for 10 min. (5) Incubate the blocked PVDF membrane with the diluted primary antibody (polyclonal antibody) in a 4 °C refrigerator overnight. (6) Wash thoroughly 3 times with TBST buffer for 10 min each time, and shake rapidly on a shaker to remove unbound primary antibody. (7) Add the diluted horseradish peroxidase-labeled goat anti-mouse IgG secondary antibody and incubate at room temperature for 1 h. (8) Repeat step (6). (9) Drop ECL chemiluminescent solution onto the PVDF membrane, place the membrane in a chemiluminescent gel imaging system for exposure imaging, and save the image results.

[0094] In this example, mice were immunized with recombinant protein rPmPLP to prepare mouse serum, which is a polyclonal antibody against rPmPLP. The titer of the polyclonal antibody against PmPLP was detected by ELISA. The results are shown in Figure 9. The titer was 1:4050, and the dilution factor for use was 1:1000. The specificity of the polyclonal antibody was detected by Western-blot technology. The results are as Figure 10 shown. A clear and single reaction band appeared near the molecular weight of PmPLP at 19.2 kDa, and no non-specific reaction band was detected. This indicates that the antibody specifically binds only to the recombinant protein rPmPLP in the expression bacteria and can be used for subsequent immunization experiments.

[0095] Example 4: Microbial binding experiment of rPmPLP Streptococcus agalactiae, Micrococcus luteus, Aeromonas hydrophila, and Pseudomonas aeruginosa were used to detect the microbial binding activity of rPmPLP. Bacteria grown to the logarithmic phase were collected by centrifugation (3000×g, 10 min), washed with PBS buffer and resuspended, and the concentration was adjusted to 2×10 8 CFU / mL; 20 μL of various bacterial suspensions were taken and incubated with 20 μL of rPmPLP overnight at 4 °C; the incubated bacterial suspensions were thoroughly washed 3 times with PBS, and the bacterial liquid was mixed with loading buffer at a ratio of 4:1 and boiled at 99 °C for 10 min. After 12% SDS-PAGE, Western blot was used to detect the binding activity of rPmPLP to microorganisms.

[0096] The results are as Figure 11 shown. After incubating rPmPLP with Streptococcus agalactiae, Micrococcus luteus, Aeromonas hydrophila, and Pseudomonas aeruginosa, bands similar in molecular weight to rPmPLP were shown, indicating that rPmPLP has binding activity with both Gram-negative and Gram-positive bacteria. The thickness of the bands indicates the strength of the binding ability of rPmPLP to microorganisms. The results show that the binding band of rPmPLP with Micrococcus luteus is the thickest, indicating that rPmPLP has the strongest binding ability with Micrococcus luteus and relatively weak binding ability with Pseudomonas aeruginosa. There was no obvious band in the control group.

[0097] Example 5: Bacterial agglutination experiment of rPmPLP Bacillus subtilis, Staphylococcus aureus, Micrococcus luteus, Aeromonas hydrophila, Pseudomonas aeruginosa, and Escherichia coli were cultured to the logarithmic growth phase, the bacterial liquid was collected, and fixed with formaldehyde solution for 10 min; the bacterial liquid was washed 3 times with TBS, then fluorescein isothiocyanate (FITC) was added and stained in the dark for 2 h, and then washed 3 times with TBS buffer; the above bacterial liquid was adjusted to 1x10 8At a concentration of CFU / mL, store it at 4 °C for later use; take 20 μL of the bacterial suspension and mix it with 20 μL of the recombinant protein in a 96-well plate. Add a 10 mM CaCl2 solution to the control group, and use TBS as a negative control. Incubate the 96-well plate at room temperature for 2 h; place the 96-well plate under an inverted fluorescence microscope for observation and photography.

[0098] Use the FITC fluorescence labeling technique to label microorganisms and detect the agglutination effect of rPmPLP on microorganisms. The experimental results are as Figure 12 shown. Different degrees of agglutination reactions occurred in Bacillus subtilis, Staphylococcus aureus, Aeromonas hydrophila, Pseudomonas aeruginosa, Micrococcus luteus, and Escherichia coli under the action of rPmPLP. In the presence of Ca 2＋ , more obvious agglutination phenomena of rPmPLP on Bacillus subtilis, Staphylococcus aureus, Aeromonas hydrophila, Pseudomonas aeruginosa, and Micrococcus luteus were observed, and the effect on Escherichia coli was not obvious.

[0099] Example 6: Oxford cup antibacterial experiment In this example, referring to the method in "Tang Xin. Research on the Immune Functions of Lectin Galectin-1, Ladderlectin, and Perforin-1 in Large Yellow Croaker [D]. Xiamen: Jimei University, 2023", the Oxford cup agar diffusion method was used to detect antibacterial activity. When the experimental bacteria were cultured to the logarithmic growth phase, the bacterial concentration was adjusted to 2×10 6 CFU / mL. Prepare an LB solid medium containing the bacterial suspension. Wait for the medium to solidify completely and place the Oxford cup. Add 100 μg of the recombinant protein to the Oxford cup respectively, set two groups with and without calcium ions, and set ampicillin as a positive control and PBS as a blank control. Place the culture dish in a 37 °C biological incubator and incubate it upright for 12 h. Observe the antibacterial circle of the Oxford cup and measure the diameter of the antibacterial circle.

[0100] According to the analysis of the Oxford cup agar diffusion experiment, the results are as Figure 13 and Table 14 show that the recombinant protein rPmPLP has a direct antibacterial effect, and calcium ions do not enhance the antibacterial effect of rPmPLP.

[0101] Table 14 Analysis of antibacterial circle experiment

[0102] Example 7: Bacteriostatic curve of rPmPLP This example measures 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 stolonifer). After activating each glycerol-preserved bacterium and fungus in the culture medium, they were inoculated into the liquid medium and cultured overnight until the logarithmic growth phase. After resuspending the bacterial solution, it was washed 3 times with PBS and then further diluted into a bacterial suspension with a concentration of 5x10 5 CFU / mL. In a 96-well plate, 50 μL of the recombinant protein (1 mg / mL) was mixed with 10 μL of various bacterial suspensions respectively. After incubating in an incubator at 37 °C (30 °C for fungi) for 2 h, 140 μL of the liquid medium was added. After thorough mixing, the OD 600 value was measured using an enzyme-linked immunosorbent assay (ELISA) reader. PBS was set as the negative control, and each sample was replicated three times.

[0103] The results are as Figure 14 , 15 shown. The recombinant protein rPmPLP has a significant inhibitory effect (P < 0.01) on 4 Gram-positive bacteria (Micrococcus luteus, Staphylococcus aureus, Bacillus subtilis, Streptococcus agalactiae), 4 Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Aeromonas hydrophila, Vibrio harveyi), and 2 fungi (Aspergillus niger and Rhizopus stolonifer). Under the action of rPmPLP, the growth of Gram-positive bacteria was inhibited to varying degrees. Among them, the growth of Micrococcus luteus was inhibited the fastest, starting to show only after 2 h; the growth of Staphylococcus aureus and Bacillus subtilis began to be inhibited after 3 h; the growth of Streptococcus agalactiae was inhibited slightly later, at 4 h. Among the Gram-negative bacteria, Aeromonas hydrophila was the fastest to be inhibited, also starting to be inhibited after 2 h; the growth of Vibrio harveyi was inhibited after 3 h; the growth of Escherichia coli was inhibited after 4 h; the growth of Pseudomonas aeruginosa was inhibited the latest, at 6 h. After 12 h of the action of rPmPLP, except for Escherichia coli, the growth of the other eight bacteria almost completely stopped, and the inhibitory effect was significant, forming a sharp contrast with the rapid growth state of bacteria in the control group. Although the growth rate of Escherichia coli was inhibited, it was still in a slow growth state. The growth of the two fungi was gradually inhibited after 12 h and was significantly inhibited after 24 h. The inhibitory effect on Aspergillus niger weakened after 48 h, while Rhizopus stolonifer was continuously inhibited throughout the 48 h.

[0104] In summary, rPmPLP has broad-spectrum antibacterial properties against Gram-positive bacteria, Gram-negative bacteria, and fungi. Among them, 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 stolonifer.

[0105] Example 8: Scanning electron microscopy experiment Prepare a bacterial suspension according to the method of Example 7 and dilute it to 1×10 7 CFU / mL with PBS. Take 100 μL of the bacterial suspension and mix it with 100 μL of the protein solution, and incubate it in a constant temperature incubator at 37 °C for 2 h. Centrifuge to collect the bacterial cells. Add 1 mL of 2.5% glutaraldehyde to resuspend the bacterial cells and place them at 4 °C for fixation for 12 h. Centrifuge again to collect the bacterial cells and wash them 3 times with PBS. Dehydrate the cells once with ethanol concentrations of 30%, 50%, 70%, 80%, and 90% for 10 min each time, and finally dehydrate them thoroughly with 100% ethanol for 15 min. Drop 10 μL of the sample onto a silicon wafer. If the bacterial concentration is low (less precipitate after centrifugation), continue to add the sample 3-5 times after the ethanol has evaporated. Place the silicon wafer with the sample in an oven at 70 °C overnight for drying. After sputtering with gold, observe using a scanning electron microscope.

[0106] rPmPLP was co-incubated with 4 Gram-positive bacteria (Micrococcus luteus, Staphylococcus aureus, Bacillus subtilis, Streptococcus agalactiae) and 4 Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Aeromonas hydrophila, Vibrio harveyi), and the morphology of the bacteria was observed using SEM. The results are as Figure 16 shown. The surface of the bacteria in the blank control group was flat and smooth, and the bacterial cells were plump and intact. Pores formed on the surfaces of Bacillus subtilis and Pseudomonas aeruginosa in the experimental treatment group; the spherical structures of Streptococcus agalactiae and Micrococcus luteus were incomplete, the surfaces were roughened, and shrinkage and fractures appeared; the cell wall of Escherichia coli peeled off, and obvious dissolution of the cell membrane occurred; there was also slight dissolution of the cell membrane of Vibrio harveyi, and the cell membranes between the bacterial cells adhered together; there were depressions on the surface of Pseudomonas aeruginosa, and the bacterial cells were not significantly ruptured; there were no obvious changes in Staphylococcus aureus in the experimental group compared with the control group, but the surface of the bacterial cells in the experimental group was rougher, with some shrinkage and deformation.

[0107] Example 9: rPmPLP injection, nucleation experiment, and detection of the expression of inflammatory factor genes Randomly divide 90 pearl oysters (Pinctada fucata martensii) into 3 groups, with 30 in each group, namely the blank group (only nucleation), the control group (PBS + nucleation), and the experimental group (rPmPLP + nucleation). Dilute the recombinant protein rPmPLP to 100 ng / mL. Inject 100 μL of the rPmPLP solution into the adductor muscle of each pearl oyster in the experimental group, while inject an equal volume of PBS into the control group.

[0108] To explore the inflammatory responses of Pinctada fucata martensii after rPmPLP immunization and nucleation stimulation, this study analyzed the expression patterns of the PmPLP gene and various inflammatory factors (detected by qRT-PCR) in P. fucata martensii injected with rPmPLP after nucleation. The results are as Figure 17 、 18 shown, where A - H 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), TNF-α (H), respectively.

[0109] After immune stimulation, PmPLP increased significantly at 12 h; the inflammatory factor TNF-α increased rapidly and significantly at 6 h and remained continuously up-regulated until it returned to the level of the PBS group at 72 h; the expression levels of the inflammatory factors Caspase-2 and IRAK1 in the stimulation group were significantly higher than those in the PBS group at 6 h. The expression level of IL-17 was significantly higher than that in the PBS group at 12, 24, 48, and 72 h; the inflammatory factors TRAF3 and NF-κB were significantly up-regulated at 24 h after stimulation, almost returned to the same level as the PBS group at 48 h, and were extremely significantly up-regulated again at 72 h; IκK was significantly up-regulated at 12 h and 24 h and returned to the level of the PBS group at 72 h.

[0110] In summary, after co-treatment with rPmPLP and nucleation, the contents of PmPLP, Caspase-2, IL-17, IRAK1, IκK, NF-κB, TRAF3, and TNF-α were all significantly up-regulated, indicating that PmPLP can promote the release of pro-inflammatory factors induced by nucleation in P. fucata martensii.

[0111] The above are only the preferred specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed by the present invention should be covered by the protection scope 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. A 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 containing the gene according to claim 2, and the vector is a PET series vector.

4. A host cell containing the expression vector according to claim 3, and 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 microorganisms are at least one of Gram-positive bacteria, Gram-negative bacteria and fungi.

6. The application according to claim 5, characterized in that, 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.

7. 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.

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

9. Use of the Pinctada martensii C-type lectin PmPLP according to claim 1 in promoting the release of pro-inflammatory factors induced by nucleus implantation in Pinctada martensii.

10. The application according to claim 9, characterized in that, The pro-inflammatory factors are at least one of Caspase-2, IL-17, IRAK1, IκK, NF-κB, TRAF3 and TNF-α.

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