L-type lectin vip36 from takifugu obscurus and its encoding gene and application

Abstract

The present application discloses a dark striped pufferfish L-type agglutinin VIP36, the amino acid sequence of which is shown as SEQ ID NO. 1, and the coding gene of which is shown as SEQ ID NO. 2. The present application also discloses the application of the dark striped pufferfish L-type agglutinin VIP36 in the preparation of a preparation with the efficacy of inhibiting Staphylococcus aureus, Vibrio parahaemolyticus, Aeromonas hydrophila and Vibrio harveyi. The present application firstly clones the coding gene of the dark striped pufferfish L-type agglutinin VIP36, constructs a prokaryotic expression vector of the dark striped pufferfish L-type agglutinin VIP36 and develops a recombinant protein; studies the use of the VIP36 recombinant protein in the immune defense function of the dark striped pufferfish, and the immune defense function includes regulating the homeostasis and health of the body, and the present application provides a theoretical basis for preventing diseases caused by pathogenic bacteria and a theoretical guidance for the healthy breeding of the dark striped pufferfish.

Description

Technical Field

[0001] This invention relates to the L-type lectin VIP36 of the dark-spotted pufferfish, its encoding gene, and its applications, belonging to the field of lectin activity research technology. Background Technology

[0002] The dark-spotted pufferfish (Takifugu obscurus), commonly known as the river pufferfish, belongs to the class Osteichthyes and the genus Takifugu. It is mainly distributed in the coastal waters of China and the middle and lower reaches of the Yangtze River, and is a typical anadromous fish. Pufferfish meat is delicious and has high medicinal value, making it of significant economic importance. However, with the continuous expansion of aquaculture and the gradual deterioration of the aquatic environment, farmed diseases such as bacterial fish diseases occur frequently. Pufferfish are lower vertebrates with both innate and acquired immunity; their acquired immunity is relatively incomplete, and the innate immune system plays a crucial role in their defense against external pathogens. Therefore, the development and utilization of pufferfish immune-related genes are particularly important.

[0003] L-type lectins (LTLs) were first discovered in the seeds of legumes and contain a lecithin-leg-like domain (LTLD). Currently, over 100 LTLs have been isolated from plants, each with varying lecithin-binding activities but similar physicochemical properties and structures. Proteins containing LTLDs have also been found in animals; these proteins are mostly membrane-bound proteins, distinct from the soluble LTLs found in plants. In higher mammals, four L-type lectins have been identified: the endoplasmic reticulum-Golgi apparatus intermediate compartment 53 kDa protein ERGIC-53, ERGIC-53-like proteins, the 36 kDa vesicle-integrated membrane protein VIP36, and VIP36-like proteins. These lectins are L-type membrane proteins, containing an extracellular LTLD domain and a cytosol domain. L-type lectins participate in the sorting and transport of animal proteins. ERGIC-53 and VIP36 were the first animal L-type lectins discovered. In Ca 2+In the presence of VIP36, ERGIC-53 can recognize mannose-rich proteins and promote the transport of certain glycoproteins from the endoplasmic reticulum to the Golgi apparatus. VIP36 is also involved in protein transport during secretion. In recent years, many studies have found that plant L-lectin receptor kinases are involved in the immune response against pathogenic microorganisms in plants, while research on animal L-lectins is still in the exploratory stage. Some studies have reported that the L-lectins ERGIC-53, VIP36, and VIP36-like in the channel catfish (Ictalurus punctatus) participate in the immune response against the invasion of the Gram-negative bacterium Edwardsiella ictaluri; in the Japanese prawn (Marsupenaeus japonicas), L-lectins participate as opsonins in shrimp antimicrobial immunity.

[0004] Currently, there are no reports on L-lectin and its immune function in the dark-spotted pufferfish. Research on the L-lectin gene and its related immune function is of great significance for further elucidating the regulatory mechanism of the pufferfish's innate immune system and understanding the impact of the L-lectin gene on pufferfish immune metabolism. It can also provide a theoretical basis for preventing diseases caused by pathogens and offer theoretical guidance for revealing healthy pufferfish aquaculture. Summary of the Invention

[0005] In response to the aforementioned prior art, this invention cloned the open reading frame sequence (full length 1002 bp, encoding 333 amino acids) of the L-type lectin VIP36 gene of *Putra tumefaciens* using RACE technology, and investigated the role of *Putra tumefaciens* L-type lectin VIP36 in the host's innate antibacterial immune defense.

[0006] This invention is achieved through the following technical solution:

[0007] The amino acid sequence of L-type lectin VIP36 from the dark-spotted pufferfish is shown in SEQ ID NO.1.

[0008] Application of L-type lectin VIP36 from Pufferfish obscurus in the preparation of formulations with inhibitory effects against Staphylococcus aureus.

[0009] Application of L-type lectin VIP36 from pufferfish (Pueraria lobata) in the preparation of formulations with inhibitory effects against Vibrio parahaemolyticus.

[0010] Application of L-type lectin VIP36 from pufferfish (Tetraodon spp.) in the preparation of formulations with inhibitory effects on Aeromonas hydrophila.

[0011] Application of L-type lectin VIP36 from pufferfish (Pueraria lobata) in the preparation of formulations with inhibitory effects against Vibrio harzianum.

[0012] The preparation can be a bacterial inhibitor, antibacterial agent, bactericide, pharmaceutical preparation, immune enhancer, feed additive, etc.

[0013] An active ingredient of a preparation that inhibits Staphylococcus aureus, Vibrio parahaemolyticus, Aeromonas hydrophila and / or Vibrio harveyi is VIP36, an L-type lectin from Pufferfish pufferfish.

[0014] The encoding gene for the L-type lectin VIP36 of the dark-spotted pufferfish is shown in SEQ ID NO.2.

[0015] Application of the gene encoding L-type lectin VIP36 of pufferfish in the preparation of L-type lectin VIP36 of pufferfish.

[0016] A recombinant expression vector containing the encoding gene for VIP36, an L-type lectin from *Putrachea obtusifolia*. The application of this recombinant expression vector in the preparation of VIP36, an L-type lectin from *Putrachea obtusifolia*.

[0017] A recombinant engineered bacterium containing the aforementioned recombinant expression vector or the encoding gene for pufferfish L-lectin VIP36, capable of stably expressing pufferfish L-lectin VIP36. The application of this recombinant engineered bacterium in the preparation of pufferfish L-lectin VIP36.

[0018] Furthermore, the host of the recombinant engineered bacteria is Escherichia coli.

[0019] This invention is the first to clone the encoding gene of L-lectin VIP36 from *Putrachea obtusifolia*, and introduce it into *E. coli* for heterologous expression. The L-lectin VIP36 was purified for the first time. The antibacterial activity of the VIP36-LTLD recombinant protein was then investigated using bacterial agglutination, bacterial binding, direct sugar binding, bacterial inhibition, and bacterial biofilm detection experiments. The results show that the VIP36-LTLD recombinant protein exhibits antibacterial activity against calcium ions (Ca). 2+ In its presence, it can agglutinate one Gram-positive bacterium (Staphylococcus aureus) and three Gram-negative bacteria (Vibrio parahaemolyticus, Aeromonas hydrophila, and Vibrio harbinae), in Ca 2+ In the absence of these substances, it can directly bind to lipopolysaccharide, peptidoglycan, mannose, and galactose, and bind to Staphylococcus aureus, Vibrio harzianum, and Vibrio parahaemolyticus. Its binding to Aeromonas hydrophila is weaker. It can also directly inhibit the growth of Vibrio harzianum and Staphylococcus aureus and the growth of bacterial biofilms, but it has no significant inhibitory effect on the growth of Vibrio parahaemolyticus and Aeromonas hydrophila and the growth of biofilms.

[0020] This invention uses RACE technology to clone the VIP36 gene sequence; it constructs for the first time a prokaryotic expression vector of the pufferfish VIP36 gene and develops a recombinant protein, which is stably expressed in Escherichia coli; it studies the use of the VIP36 recombinant protein in the immune defense function of pufferfish, which includes regulating innate immunity and maintaining the body's homeostasis and health. This invention provides a theoretical basis for preventing diseases caused by pathogens and also provides theoretical guidance for the healthy aquaculture of pufferfish.

[0021] The various terms and phrases used in this invention have their general meanings known to those skilled in the art. Attached Figure Description

[0022] Figure 1 : Schematic diagram of SDS-PAGE gel results, where M: protein molecular weight standard; 1: Escherichia coli Transetta (DE3) strain containing pET32a-VIP36-LTLD recombinant plasmid before induction; 2: E. coli Transetta (DE3) strain containing pET32a-VIP36-LTLD recombinant plasmid after induction with 0.5 mM IPTG; 3: purified rVIP36-LTLD protein.

[0023] Figure 2 Schematic diagram of bacterial agglutination experiment results.

[0024] Figure 3 : Schematic diagram of the experimental results of direct sugar binding, where A: lipopolysaccharide; B: peptidoglycan; C: mannose; D: galactose.

[0025] Figure 4 : Schematic diagram of bacterial binding assay results, where A: Western Blot detection of the bacterial binding ability of rVIP36-LTLD protein; B: ELISA detection of the bacterial binding ability of rVIP36-LTLD protein.

[0026] Figure 5 Schematic diagram of in vitro bacterial growth inhibition experiment results, where A: Staphylococcus aureus; B: Vibrio harzianum; C: Vibrio parahaemolyticus; D: Aeromonas hydrophila.

[0027] Figure 6 : Schematic diagram of bacterial biofilm detection results, where A: Staphylococcus aureus; B: Vibrio harzianum; C: Vibrio parahaemolyticus; D: Aeromonas hydrophila. Detailed Implementation

[0028] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.

[0029] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0030] Experiment 1: Preparation of VIP36-LTLD recombinant protein

[0031] (I) VIP36 gene cloning

[0032] 1. Total RNA extraction

[0033] a) Take 30 mg of tissue sample (mixed liver and kidney tissue) from the dark-spotted pufferfish into a 1.5 mL RNase-free centrifuge tube;

[0034] b) Add 200 μL of Trizol Regent to the centrifuge tube;

[0035] c) After rapid and thorough grinding with an electric grinder, add 800 μL of Trizol Regent;

[0036] d) Use a pipette to repeatedly blow and aspirate the precipitate to resuspend it, and let it stand at room temperature for 5 minutes;

[0037] e) Add 200 μL of chloroform, seal the centrifuge tube tightly, shake vigorously to mix for 15 seconds, and let stand at room temperature for 10 minutes.

[0038] f) Centrifuge at 4℃ and 12,000 rpm for 10 min to separate the solution into upper, middle and lower layers;

[0039] g) Carefully aspirate the upper aqueous phase solution into another centrifuge tube, add 1 volume of 70% ethanol, and mix by inverting.

[0040] h) Transfer the mixed solution into the adsorption column RA (the adsorption column is fitted inside the collection tube);

[0041] i) Centrifuge at 4℃, 10,000 rpm for 45s, discard the waste liquid, and put the adsorption column back into the collection tube;

[0042] j) Add 500 μL of protein removal solution RE, centrifuge at 12,000 rpm for 1 min at 4 °C, and discard the waste liquid;

[0043] k) Add 700 μL of rinsing buffer RW, centrifuge at 12,000 rpm for 1 min at 4 °C, and discard the waste liquid;

[0044] l) Add 500 μL of rinsing buffer RW, centrifuge at 12,000 rpm for 1 min at 4 °C, and discard the waste liquid;

[0045] m) After discarding the solution, briefly centrifuge, place at room temperature for several minutes, and then air dry completely;

[0046] n) Remove the adsorption column RA and place it in a new RNase-free centrifuge tube. Add 50-80 μL of RNase-free water to the middle of the adsorption membrane and let it stand at room temperature for 2 minutes.

[0047] o) Centrifuge at 4℃, 12,000 rpm for 1 min to obtain RNA samples from each tissue;

[0048] p) The total RNA obtained was detected by 1.5% agarose gel electrophoresis, and the RNA concentration was quantified by measuring the OD value. The RNA quality was judged by the A260 / A280 ratio, and the ratio was kept between 1.8 and 2.0.

[0049] 2. cDNA Synthesis

[0050] The extracted RNA was reverse transcribed to obtain the corresponding first-strand cDNA. The reverse transcription reaction steps are as follows:

[0051] a) Mix the following reagents into an RNase-free PCR tube, centrifuge slightly, and set aside at room temperature for use in step f: 5× First-Strand Buffer, 2.0 μL;

[0052] DTT (20mM), 1.0μL;

[0053] dNTP Mix (10 mM), 1.0 μL;

[0054] Total volume, 4.0 μL;

[0055] b) Add the following reagents to two RNase-free PCR tubes respectively:

[0056] For preparation of 5'-RACE-Ready cDNA: RNA, 1.0~2.75μL; 5'-CDS PrimerA, 1.0μL;

[0057] For preparation of 3'-RACE-Ready cDNA: RNA, 1.0~3.75μL; 3'-CDS PrimerA, 1.0μL;

[0058] c) Add sterile water to the centrifuge tube from step b, and bring the 5'-RACE-Ready cDNA and 3'-RACE-Ready cDNA to 3.75 μL and 4.75 μL, respectively;

[0059] d) Mix by slight centrifugation, place the centrifuge tube in a PCR instrument for incubation at 72°C for 3 min, 42°C for 2 min, cool the sample to 4°C, and centrifuge at 14,000g for 10 s;

[0060] e) Add 1 μL of SMARTer IIA oligo to a 5'-RACE-Ready cDNA centrifuge tube;

[0061] f) Add the following reagents sequentially to the centrifuge tube from step a:

[0062] Buffer Mix from Step 1, 4.0μL;

[0063] RNase Inhibitor (40U / μL), 0.25μL;

[0064] SMARTScribe Reverse Transcriptase (100U), 1.0μL;

[0065] Total volume: 5.25 μL;

[0066] g) Take the mixture from step f and add it to step d (3'-RACE-Ready cDNA) or step e (5'-RACE-Ready cDNA), with a total volume of 10 μL;

[0067] h) Gently centrifuge to mix, and collect the sample at the bottom of the tube;

[0068] i) Place the centrifuge tubes in the PCR instrument and incubate at 42℃ for 90 min, then at 70℃ for 10 min. Cool the samples to 4℃ and centrifuge at 14,000g for 10 s.

[0069] j) Dilute the synthesized cDNA template with 100 μL of Tricine-EDTA Buffer and store the sample at -20°C.

[0070] 3. VIP36 gene amplification and purification

[0071] The partial sequence of the VIP36 gene was obtained by analyzing the transcriptome database of *Pueraria lobata*. Primers for amplifying the complete VIP36 gene ORF were then designed using Primer 5.0 software: VIP36-F: 5'-ATGGGGCATTTCGGCGCATTACTCAGC-3' (as shown in SEQ ID NO.3) and VIP36-R: 5'-TCAGTAGAACCTCTTGTTCCTCTCC-3' (as shown in SEQ ID NO.4). The VIP36 gene sequence was cloned using cDNA as a template, and the PCR system is shown in Table 1.

[0072] Table 1

[0073]

[0074]

[0075] The PCR amplification program was as follows: 94℃ for 30s, 72℃ for 2min, 5 cycles; 94℃ for 30s, 70℃ for 30s, 72℃ for 2min, 5 cycles; 94℃ for 30s, 68℃ for 30s, 72℃ for 2min, 25 cycles; 72℃ for 10min; 4℃ for 10min.

[0076] After the reaction, the PCR products were examined by electrophoresis on a 1.2% agarose gel. The target band was purified using an agarose gel extraction kit (TIANGEN, China). The specific steps are as follows:

[0077] a) Column equilibration: Add 500 μL of equilibration solution BL to the adsorption column CA2 (the adsorption column is placed in the collection tube), centrifuge at 12,000 rpm for 1 min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0078] b) Cut a single target DNA band from the agarose gel, place it in a clean centrifuge tube, and weigh it;

[0079] c) Add 3 times the volume of sol solution PN to the gel, and place in a 50°C water bath for about 10 minutes, gently turning the centrifuge tube up and down continuously during this time to ensure that the gel is fully dissolved.

[0080] d) Add the obtained solution to the CA2 adsorption column, let it stand at room temperature for 2 min, centrifuge at 12,000 rpm for 1 min, discard the waste liquid in the collection tube, and put the CA2 adsorption column into the collection tube;

[0081] e) Add 600 μL of bleaching solution PW, centrifuge at 12,000 rpm for 1 min, discard the waste liquid, and place the adsorption column CA2 into the collection tube;

[0082] f) Repeat step e;

[0083] g) Place the adsorption column back into the tube, centrifuge at 12,000 rpm for 2 min to remove as much of the washing solution as possible, and place the CA2 adsorption column at room temperature for a few minutes to dry completely.

[0084] h) Place the adsorption column in a clean centrifuge tube, add 30 μL of elution buffer EB to the center of the adsorption membrane, and incubate at room temperature for 2 min.

[0085] i) Centrifuge at 12,000 rpm for 2 min, collect the DNA solution, and store it in a -20°C freezer.

[0086] 4. TA Cloning

[0087] The purified product was subjected to TA cloning. First, the recombinant vector was constructed using the TaKaRa pMD18-T Vector ligation kit (TaKaRa, Japan). The ligation system (total volume 10 μL) consisted of 5.0 μL Solution I, 4.2 μL PCR product, and 0.8 μL TaKaRa pMD18-T Vector. After gentle mixing, the mixture was ligated overnight at 16°C. After ligation, heat shock transformation was performed. Take competent DH5α cells (TIANGEN, China), thaw them on ice for 5 min, add 10 μL of ligation product to 50 μL of DH5α; heat shock at 42℃ for 45 s, then quickly place on ice and let stand for 2 min; add the liquid (ligation product + DH5α) from the PCR tube to LB liquid medium without ampicillin (Amp-), and incubate at 37℃ with shaking at 180 rpm for 1-2 h; after the shaking incubation, centrifuge the bacterial solution at 4,000 rpm for 5 min, remove most of the supernatant, and aspirate 50 μL of the bacterial solution onto an Amp+LB solid plate and spread it evenly; incubate inverted at 37℃ overnight.

[0088] Colonies will appear after overnight incubation. Use a sterile toothpick to pick white colonies from the transformed plate and mix them in a PCR tube containing 10 μL of sterile water. This mixture will be used as a template for PCR amplification. PCR amplification is performed using the universal vector primers M13 Forward Primer: 5'-CAGGGTTTTCCCAGTCACG-3' and M13 Reverse Primer: 5'-GAGCGGATAACAATTTCACAC-3'. The 25 μL reaction mixture consists of: 12.5 μL ExTaq (TaKaRa), 1.0 μL bacterial culture, 1.0 μL M13F, 1.0 μL M13R, and 9.5 μL ddH2O. After mixing and centrifugation, the mixture is placed on a PCR instrument. The amplification conditions are: 95℃ pre-denaturation for 4 min; 94℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 2 min, repeated 35 times; and a final extension at 72℃ for 10 min. After the reaction was completed, the PCR products were detected by 1.2% agarose gel electrophoresis. The recombinants containing appropriate fragment sizes were sent to Nanjing Sipujin Biotechnology Co., Ltd. for DNA sequencing.

[0089] The present invention uses the RACE method to amplify the full-length sequence of the coding region of the VIP36 gene, which is 1002 bp and encodes a VIP36 protein composed of 333 amino acids with a molecular weight of 37.4 kDa.

[0090] Nucleotide sequence (5'-3') (as shown in SEQ ID NO.2):

[0091] ATGGGGCATTTCGGCGCATTACTCAGCACATCCCTGGTTTTACTGACGTCTCGGTTCGGTTCGGTTTCCTGCGATCTGACGGACGGAAACACG GAGCACCTAAAAAGGGAGCACTCGTTAACGAAGCCCTACCAAGGTGTGGGC AGCCAGTGGGACTTCTGGGGCAGCACCTTGGTGACCAACTCGTACGTGCGCCTCACCCCCGATGAGAGGAGCAAGC AGGGCTCCATCTGGAACACTGTGCCGTGTCACCTGAAGGACTGGGAGATGCACGTGCAGTTTAAAGTGCACGGTTC TGGGAAGAAGAATCTCCATGGCGACGGCATCGCTCTGTGGTACACAAGAGACAGACTGCAAGCAGGACCCGTGTTT GGAAACCGGGACCATTTTGTCGGCCTGACTATTTTGTTGGACACCTTCCGCAACGACCTCCACGGGATGGATCGTT CCTTCCCTTACATCTCAGCCATGGTGAACAACGGCTCCGTCAGTTACGACCACGGCAAAGACGGACGCTCCTCCGA GATGGGCGGCTGCTCCGCTGAGATCCGGAACCGGGACCACGACACGTACCTGGCCATCCGCTACTCCAAGGGCAGA CTGACGGTGATGGTGGACGTGGACGACAAGAACGAGTGGAGGGAATGCATCGACGTCGGCGGCGTGCGCCTTCCCA CCGGATACTTCTTTGGTGCTTCCGCCGCCACAGGAGATCTGTCAGATAACCACGACATCATCTCCATGAAGCTCTA CCAGCTGATGGTGGAGCACAGCCCAGAGGAGGAGAACCAGGACTGGTCCAAGATCGAGCCCAGCGTCAGCCTCCTCAAGTCCCCCAAAGACAACATCGATGACCCCACCGGGAACTTCCGGGGAACGCCGCTGACCGGCTGGAAGGTCTTCCTGCTGCTGCTCTGCTCTCTGCTGGGAATCGTGGTGTGTGGAGTGGTGGGAGCGGTGGTGTTCCAGAAGAGGCAGGAGAGGAACAAGAGGTTCTACTGA.

[0092] Amino acid sequence (as shown in SEQ ID NO.1) (the underlined portion is the LTLD domain):

[0093] MGHFGALLSTSLVLLTSRFGSVSCDLTDGNT EHLKREHSLTKPYQGVGSQWDFWGSTLVTNSYVRLTP DERSKQGSIWNTVPCHLKDWEMHVQFKVHGSGKKNLHGDGIALWYTRDRLQAGPVFGNRDHFVGLTILLDTFRNDL HGMDRSFPYISAMVNNGSVSYDHGKDGRSSEMGGCSAEIRNRDHDTYLAIRYSKGRLTVMVDVDDKNEWRECIDVG GVRLPTGYFFGASAATGDLSDNHDIISMKLYQLMV EHSPEEENQDWSKIEPSVSLLKSPKDNIDDPTGNFRGTPLTGWKVFLLLLCSLLGIVVCGVVGAVVFQKRQERNKRFY.

[0094] (II) Expression and purification of VIP36 recombinant protein

[0095] 1. PCR amplification of the VIP36 gene

[0096] VIP36-LTLD expression primers were designed based on the coding region sequence of the VIP36 gene.

[0097] VIP36-LTLD-exF: 5'-GCCATGGCTGATATCGGATCCGAGCACCTAAAAAGGGAGCACTC-3', as shown in SEQ ID NO. 5;

[0098] VIP36-LTLD-exR: 5'-ACGGAGCTCGAATTCGGATCCCACCATCAGCTGGTAGAGCTTCA-3', as shown in SEQ ID NO,6.

[0099] PCR amplification yielded the target band corresponding to the LTLD domain of VIP36 (i.e., the underlined portion of the nucleotide sequence shown in SEQ ID NO.2 above). The total amplification system was 50 μL, and the contents of each component were as follows:

[0100] Max Premix (2×), 25μL;

[0101] Upstream primer, 1 μL;

[0102] Downstream primer, 1 μL;

[0103] cDNA, 2 μL;

[0104] ddH2O, 21 μL;

[0105] The PCR amplification conditions were: 95℃ for 3 min, 95℃ for 15 s; 58℃ for 15 s; 72℃ for 1 min, 34 cycles, followed by 72℃ for 5 min and 4℃ for 2 min. The PCR products were subjected to 1.2% agarose gel electrophoresis, and the VIP36-LTLD expression fragment was recovered using the gel.

[0106] 2. Plasmid preparation

[0107] Inoculate LB broth containing the pET-32a vector into Amp+-containing medium and incubate overnight at 37°C with a shaker at 200 rpm. Extract plasmids using a plasmid miniaturization kit. Collect the liquid obtained from the final elution column using a centrifuge tube; this is the solution containing the pET-32a plasmid. Measure the OD value of the solution using a UV spectrophotometer to quantify the DNA concentration. Perform agarose gel electrophoresis to assess the plasmid extraction quality. The extracted plasmid can be purified using gel recovery. Store the product at -20°C or directly digest with plasmid enzymes.

[0108] 3. Recombination reaction of VIP36-LTLD with pET-32a vector

[0109] a) The purified DNA fragment and pET-32a vector were digested with BamHI. The system was as follows:

[0110] 10× Buffer BamH I, 5 μL;

[0111] BamH I, 1 μL;

[0112] pET-32a vector / DNA fragment, 20 μL (depending on concentration, ≤1 μg);

[0113] Sterilize ddH2O and add to a final volume of 50 μL;

[0114] Enzyme digestion reaction conditions: 37℃ water bath for 4 hours.

[0115] b) After the reaction, all products were subjected to 1.2% agarose gel electrophoresis, and the target band was rapidly excised using a UV gel cutter. DNA was then recovered and purified using a gel extraction kit.

[0116] c) Recombination reaction:

[0117] The VIP36-LTLD fragment was recombined with the linearized pET-32a vector. Using II. Similar recombination reaction system (Vazyme), reaction system (10 μL):

[0118] Linearized vector, 4 μL;

[0119] Insert fragment (VIP36-LTLD cDNA), 3 μL;

[0120] 5×CEⅡBuffer, 2μL;

[0121] Exnase II, 1 μL;

[0122] The recombinant reaction system was incubated at 37°C for 30 min in a PCR instrument. After the reaction was completed, the system was immediately removed and placed on ice to cool.

[0123] 4. Transformation of the recombinant product into the expression vector

[0124] 10 μL of the ligation product was transferred into 50 μL of DH5α competent cells, gently mixed, and incubated on ice for 30 min; then heat-shocked in a 42°C water bath for 45 s, followed immediately by an ice bath for 2 min; 500 μL of liquid LB medium was added, and the mixture was placed in a shaker at 37°C and shaken at 200 rpm for 1 h; 100 μL of the cultured bacterial solution was taken, spread on an Amp+LB plate, and incubated upright in a 37°C incubator for 1 h, then inverted and incubated overnight.

[0125] Five single-clone strains were selected from the plate as PCR templates, and a 25 μL PCR reaction system was prepared on ice. Reaction conditions: 95℃ for 15 s, 58℃ for 15 s, 72℃ for 1 min, 34 cycles, followed by 72℃ for 5 min, and 4℃ for 5 min. Electrophoresis was performed on a 1.2% agarose gel, and positive clones were sequenced. After sequencing results were returned, sequence information was compared using NCBI, and plasmids with good sequencing results were selected. These plasmids were transformed into E. coli Transetta (DE3) competent cells, cultured overnight at 37℃ on LB plates containing Amp, and single clones were picked. PCR-positive single clones were used for further experiments.

[0126] 5. Protein expression assay

[0127] The expression strain was inoculated into 3 mL of AMP+LB medium and cultured overnight at 37°C and 200 rpm. 50 μL of the bacterial culture was transferred to a fresh 5 mL AMP+LB medium at a 1:100 ratio and cultured at 37°C for approximately 3 hours, until the OD600 of the bacterial culture reached 0.6–0.8. 500 μL of the bacterial culture was then taken as a control and stored at 4°C. Isopropyl-β-D-thiosesquiglycoside (IPTG) was added to the remaining portion to a final concentration of 0.5 mM, and the culture was continued at 37°C with shaking to induce expression for 5 hours. 500 μL of the bacterial culture was then reserved for assays.

[0128] Before and after induction, the bacterial cultures were centrifuged at 6,000 rpm for 3 min, the supernatant was discarded, and the collected bacterial cells were resuspended in 100 μL of distilled water. Pre-loading treatment was then performed: 30 μL of protein loading buffer was added, mixed, and the mixture was incubated in a metal bath at 100℃ for 10 min to denature the protein. Following this, 12% SDS-PAGE electrophoresis was performed. The gel was then stained with Coomassie brilliant blue for 20 min, and destained until the bands were clear. Strains with high expression levels were selected and incubated with 1 / 10 volume of sterile glycerol at -80℃.

[0129] 6. Inclusion body detection and large-scale protein expression

[0130] The expression system was expanded to 300 mL of Amp+LB liquid medium according to the experimental expression method, and the bacterial culture before IPTG induction was used for subsequent detection. After centrifugation, the bacterial cells were resuspended in 20 mL of sterile 1×PBS + 0.2% Triton X-100. The bacterial cells were sonicated in an ice bath using an ultrasonic cell disruptor with an amplitude transformer of 2, a power of 200 W, a sonication time of 5 seconds followed by a 5-second interval, and a total working time of 50 min. After cell disruption, the supernatant and precipitate were separated by centrifugation. Inclusion bodies were present in the precipitate. An appropriate amount of supernatant and precipitate was taken for high-temperature protein denaturation.

[0131] 7. Inclusion body denaturation and renaturation

[0132] Prepare Buffer A (50.0 mM Tris-HCl, 5.0 mM EDTA; pH 8.0), Buffer B (50.0 mM Tris-HCl, 5.0 mM EDTA, 2.0 M urea, pH 8.0), and Buffer C (0.1 M Tris-HCl, 10.0 mM DTT, 8.0 M urea, pH 8.0). Wash the precipitate after cell disruption with Buffer A and Buffer B, and dissolve the inclusion bodies with Buffer C. Maintain a constant temperature of 37°C with shaking until the precipitate is completely dissolved. Then, place the solution into a pre-treated dialysis bag and dialyze to 1 L of dialysis buffer (0.1 M Tris-HCl, 5.0 mM EDTA, 5.0 mM L-Cysteins, 10% glycerol) at 4°C for at least 16 hours to refold, and repeat twice.

[0133] 8. Western Blot detection of recombinant proteins

[0134] The purified proteins were separated by 12% SDS-PAGE electrophoresis at 120V. After electrophoresis, a semi-dry transfer method was performed using a versatile protein transfer system (Bio-Rad). The instrument program was: 25V, 2.5A, transfer for 20 min. After the protein was transferred to a PVDF membrane, it was blocked with 5% skim milk powder, followed by incubation with primary antibody (Anti-His Mouse Monoclonal Antibody) and secondary antibody (Goat Anti-Mouse IgG (H+L), HRP Conjugate). After incubation, the membrane was washed with PBST, and chromogenic buffer was added. The membrane was then developed and photographed using a chemiluminescent gel imaging system. The results are shown below. Figure 1 As shown.

[0135] In this experiment, the E. coli expression vector of VIP36-LTLD was successfully constructed, and recombinant expression and protein purification were performed to obtain the VIP36-LTLD recombinant protein.

[0136] Experiment 2 Bacterial Agglutination Experiment

[0137] Staphylococcus aureus, Vibrio parahaemolyticus, Aeromonas hydrophila, and Vibrio harveyi were selected to detect the agglutination activity of the VIP36-LTLD recombinant protein. The four bacteria were cultured to the logarithmic growth phase, centrifuged, washed with sterile PBS, and resuspended until the bacterial concentration reached 2 × 10⁻⁶. 8The recombinant protein was diluted to 100 μg / mL and stored at room temperature until use. The experimental group consisted of two groups: Group 1, where 25 μL of recombinant protein was added to each well of a 96-well cell culture plate, followed by incubation with 25 μL of bacteria in each well; Group 2, where CaCl2 was added to each well to a final concentration of 10 mM, was added to Group 1. The negative control was achieved by replacing the recombinant protein in each group with thioredoxin (TRX) (100 μg / mL), and the experiment was performed simultaneously. The 96-well plates were incubated at room temperature for 1 hour, and agglutination was observed and photographed under an inverted microscope. The results are shown below. Figure 2 As shown.

[0138] Depend on Figure 2 It can be seen that in Ca 2+ In its presence, the VIP36-LTLD recombinant protein can agglutinate Staphylococcus aureus, Vibrio parahaemolyticus, Aeromonas hydrophila, and Vibrio harveyi.

[0139] Experiment 3: Direct Sugar Binding Experiment

[0140] The direct sugar-binding activity of the VIP36-LTLD recombinant protein was confirmed using enzyme-linked immunosorbent assay (ELISA). Lipopolysaccharide, peptidoglycan, mannose, and galactose were selected for the experiment, and the procedures are as follows:

[0141] a) Dilute the sugar with sterile distilled water to 80 μg / mL and sonicate (3 × 15 s).

[0142] b) Add the sonicated sugar to a 96-well plate, 50 μL per well; place the plate at 37°C overnight until the moisture has completely evaporated, then place it at 60°C for 30 min to coat the wells with sugar.

[0143] c) Add 200 μL of BSA (1 mg / mL, prepared with TBS) to each well and seal at 37°C for 2 h.

[0144] d) Remove the blocking solution and wash each well four times with 200 μL of TBS.

[0145] e) Add serially diluted protein solution (0-50 μg / mL, dissolved in TBS containing 0.1 mg / mL BSA) to each well, 50 μL per well, and incubate at room temperature for 3 h.

[0146] f) Wash four times with TBS, add 100 μL of anti-His-tag mouse monoclonal antibody (1:1000 diluted in TBS containing 0.1 mg / mL BSA) to each well, and incubate at 37°C for 1 h.

[0147] g) Wash four times with TBS, add horseradish peroxidase-labeled goat anti-mouse IgG (1:5000, prepared with TBS) to each well, and incubate at 37°C for 1 h.

[0148] h) Wash four times with TBS, add 100 μL of citrate-disodium hydrogen phosphate buffer containing 0.01% 5,5',5,5'-tetramethylbenzidine (Sigma), and develop color at room temperature. When the color development is moderate, terminate the color development reaction with 50 μL of 2M sulfuric acid.

[0149] I) Use an ELISA reader to read the absorbance at 450 nm.

[0150] The control group used TRX instead of the VIP36-LTLD recombinant protein in the same experiment. The experiment was repeated three times. The results are as follows. Figure 3 As shown.

[0151] Depend on Figure 3 It is evident that the VIP36-LTLD recombinant protein can directly bind to lipopolysaccharide, peptidoglycan, mannose, and galactose, with a slightly stronger binding ability to mannose.

[0152] Experiment 4 Bacterial Binding Experiment

[0153] *S. aureus*, *V. parahemolyticus*, *A. hydrophila*, and *V. harvestyi* were selected as experimental strains to detect the bacterial binding ability of recombinant proteins. The experimental strains cultured overnight were centrifuged, washed with sterile PBS, and resuspended to a bacterial concentration of 2 × 10⁻⁶. 8 The VIP36-LTLD recombinant protein was diluted to a concentration of 500 μg / mL with sterile PBS and incubated at room temperature until use. In the experimental group, 500 μL of bacterial resuspension and 500 μL of recombinant protein sample were mixed in a sterile centrifuge tube and incubated at room temperature for 30 min. In the control group, 500 μL of bacterial resuspension and 500 μL of TRX carrier protein were mixed in a sterile centrifuge tube and incubated at room temperature for 30 min. After incubation, the mixture was centrifuged, and the precipitate was washed four times with 1 mL of sterile PBS. The wash solution was then subjected to high-temperature denaturation of the protein before 12% SDS-PAGE electrophoresis and Western blotting. The results are as follows: Figure 4 As shown.

[0154] The bacterial binding activity of the VIP36-LTLD recombinant protein was detected using a modified ELISA method. *S. aureus*, *V. parahemolyticus*, *A. hydrophila*, and *V. harvestyi* (2 × 10⁻⁶) were used. 8Add 100 μL of cells / mL to each well of a 96-well plate and incubate overnight at 4°C. Fix bacteria with 50 μL of 0.05% glutaraldehyde, block with 100 μL of TBS solution containing 3% BSA for 2 h, then add 100 μL of VIP36-LTLD recombinant protein (100 μg / mL) to each well and incubate at room temperature for 3 h. Use 100 μL of rTRX and TBS as controls. Then add 100 μL of anti-His-tag mouse monoclonal antibody and incubate at 37°C for 1 h. After washing four times with TBS, add 100 μL of horseradish peroxidase-labeled goat anti-mouse IgG and incubate at 37°C for 1 h. After washing four times with TBS, add 100 μL of citrate-disodium hydrogen phosphate buffer containing 0.01% 5,5',5,5'-tetramethylbenzidine and develop color at room temperature. When the color development is moderate, stop the color development reaction with 50 μL of 2M sulfuric acid. The absorbance was read using a microplate reader at 450 nm. The results are as follows: Figure 4 As shown.

[0155] Depend on Figure 4 It can be seen that in Ca 2+ In the absence of these bacteria, Western blotting (WB) results showed that the VIP36-LTLD recombinant protein could bind to Staphylococcus aureus, Vibrio harveyi, Vibrio parahaemolyticus, and Aeromonas hydrophila, with slightly weaker binding to Aeromonas hydrophila. ELISA results showed that the VIP36-LTLD recombinant protein had strong binding ability to all of these bacteria.

[0156] Experiment 5 Bacterial Inhibition Experiment

[0157] The purified VIP36-LTLD recombinant protein was used for bacterial inhibition experiments. *S. aureus*, *V. parahemolyticus*, *A. hydrophila*, and *V. harvestyi* were selected as experimental strains, and a control group was set up. Fresh bacterial culture that had been cultured overnight was transferred to BHI medium at a ratio of 1:100, and then the recombinant protein was added to the medium to a final concentration of 200 μg / mL. The control group was treated with sterile PBS. Measurements were taken at 0h, 1h, 2h, 3h, 4h, 5h, and 6h. At each time point, the absorbance of the bacterial culture was measured at a wavelength of 600 nm using a microplate reader. Three independent replicate experiments were performed. Results are shown below. Figure 5 As shown.

[0158] Depend on Figure 5 It is evident that the VIP36-LTLD recombinant protein can inhibit the growth of Vibrio harveyi and Staphylococcus aureus, with a more pronounced inhibitory effect on Staphylococcus aureus, but it cannot inhibit the growth of Vibrio parahaemolyticus and Aeromonas hydrophila.

[0159] Experiment 6 Crystal Violet Staining for Detection of Biofilms

[0160] S. aureus, V. parahemolyticus, A. hydrophila, and V. harvestyi were selected as experimental strains. First, 160 μL of LB medium was added to a sterile 96-well polystyrene plate, followed by 20 μL of the bacterial culture medium (approximately 10⁻⁶ oz). 6 The culture medium was prepared by adding 20 μg of recombinant protein (CFU / ml) and gently shaking to mix. The mixture was then incubated at 37°C for 36 h. The control group received the same concentration of TRX solution. The culture medium was gently discarded, and the 96-well plate was washed with sterile PBS to remove excess unadsorbed cells. The remaining liquid at the bottom of the wells was gently aspirated with a pipette, and 200 μL of anhydrous methanol was added for fixation for 15 min. The methanol was discarded, and the 96-well plate was inverted to air dry. Then, 200 μL of 0.1% crystal violet solution (Shanghai Sangon Biotech) was added to each well for staining for 30 min. After staining, the remaining staining solution was washed off with sterile water, and the remaining liquid was aspirated with a pipette. After air drying, 150 μL of 30% glacial acetic acid was added to each well in a fume hood and allowed to stand for 30 min to fully dissolve the crystal violet stain. The absorbance at 600 nm was measured using a BioTek microplate reader. Results are as follows: Figure 6 As shown.

[0161] Depend on Figure 6 It is evident that the recombinant protein can inhibit the growth of Vibrio harzianum and Staphylococcus aureus biofilms, but has no significant inhibitory effect on the growth of Vibrio parahaemolyticus and Aeromonas hydrophila biofilms.

[0162] The above embodiments are provided to those skilled in the art to fully disclose and describe how the claimed implementations can be carried out and used, and are not intended to limit the scope of the disclosure herein. Modifications that will be obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. The application of L-type lectin VIP36 from the dark-spotted pufferfish in the preparation of an antibacterial agent for inhibiting Staphylococcus aureus, characterized in that... The amino acid sequence of the L-type lectin VIP36 of the dark-spotted pufferfish is shown in SEQ ID NO.

1.

2. The application of L-type lectin VIP36 from *Pueraria lobata* in the preparation of an antibacterial agent for inhibiting *Vibrio harzianum*, characterized in that... The amino acid sequence of the L-type lectin VIP36 of the dark-spotted pufferfish is shown in SEQ ID NO.

1.

3. The application according to claim 1 or 2, characterized in that: The antibacterial agent is a feed additive.

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