Expression system of tilapia antifungal protein mptx and application

Abstract

The application provides an expression system and application of tilapia antibacterial protein Mptx, and belongs to the technical field of recombinant proteins.The tilapia Mptx eukaryotic protein is obtained by the method of gene cloning and eukaryotic expression vector construction, the binding and agglutination effect of Mptx on related pathogenic bacteria of tilapia are detected by ELISA and agglutination experiment, and it is proved that the obtained Mptx has the effects of inhibiting the proliferation of pathogenic bacteria and protecting the organism by the methods of bacteriostatic experiment and survival rate detection.The application proves that the Mptx eukaryotic protein can significantly reduce the death of tilapia caused by streptococcus agalactiae infection, and has important significance in the aquaculture industry.

Description

Technical Field

[0001] This invention relates to the field of recombinant protein technology, and in particular to the expression system and application of the tilapia antibacterial protein Mptx. Background Technology

[0002] Tilapia, also known as African carp, Vietnamese fish, and South Sea carp, is a key freshwater aquaculture fish species cultivated through research in the global aquaculture industry and is hailed as one of the major sources of animal protein in the future. It typically lives in freshwater but can also inhabit brackish water with varying salinity, and can survive in the shallow waters of lakes, rivers, and ponds. It has a strong adaptability, capable of reproducing even in small bodies of water, and can even grow in rice paddies. It also exhibits remarkable adaptability to water with low dissolved oxygen levels. Most tilapia are omnivorous, feeding primarily on aquatic plants and detritus.

[0003] Tilapia are highly resistant to disease, but during winter, due to factors such as water temperature and quality, coupled with intensive farming, their physical condition deteriorates, leading to a higher incidence of disease. This can cause mass mortality in a short period, requiring close monitoring and prevention. *Streptococcus agalactiae*, the main pathogen causing streptococcal disease in tilapia, has caused enormous economic losses and has already had a significant impact on the aquaculture industry. Currently, there is a lack of safe and effective fish disease prevention and control measures. Although the use of antibiotics can curb the occurrence and development of diseases to some extent, the overuse of antibiotics poses a significant threat and raises serious questions about the quality and safety of aquatic products. Therefore, exploring the defense mechanisms of the fish immune system and tapping into the potential of important molecules in the innate immune system to fundamentally prevent and control fish diseases, in order to adapt to long-term, safe, and sustainable development, is an urgent need for the green aquaculture industry.

[0004] Streptococcus agalactiae, the main pathogen causing streptococcal disease in tilapia, has had a significant impact on the aquaculture industry, causing substantial economic losses. Furthermore, the current lack of safe and effective fish disease prevention and control measures, along with the widespread overuse of antibiotics, has greatly threatened and cast doubt on the quality and safety of aquatic products. Therefore, exploring the defense mechanisms of the fish immune system and tapping into the potential of key molecules in the innate immune system to fundamentally prevent and control fish diseases, in order to achieve long-term, safe, and sustainable development, is an urgent need for the green aquaculture industry. Summary of the Invention

[0005] The purpose of this invention is to provide an expression system and application of the tilapia antibacterial protein Mptx, which demonstrates its important role in resisting pathogen infection and improving the survival rate of organisms.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] The present invention provides a recombinant expression vector containing the encoding gene of Nile tilapia OnMptx protein, which is obtained by amplifying Nile tilapia cDNA using primer pairs with nucleotide sequences as shown in SEQ ID NO:1 and SEQ ID NO:2.

[0008] The present invention also provides a host cell containing the above-mentioned recombinant expression vector.

[0009] Preferably, the host cell is a HEK293 cell.

[0010] The present invention also provides the use of the above-mentioned recombinant expression vector or the above-mentioned host cell in the preparation of drugs for treating aquatic pathogens.

[0011] This invention also provides a method for preparing recombinant OnMptx protein, comprising the following steps:

[0012] The host cells were transfected using the recombinant expression vector described above, and the cell culture supernatant was collected and purified to obtain the recombinant OnMptx protein.

[0013] The present invention also provides a recombinant OnMptx protein prepared by the above preparation method.

[0014] The present invention also provides a method for enhancing the resistance of aquatic organisms to pathogens by applying the above-mentioned recombinant OnMptx protein during aquaculture.

[0015] The present invention also provides the application of the above-mentioned recombinant expression vector, host cell or recombinant OnMptx protein in the preparation of reagents for agglutinating pathogens, wherein the pathogens are Streptococcus agalactiae or Aeromonas hydrophila.

[0016] The present invention also provides the application of the above-mentioned recombinant expression vector, host cell or recombinant OnMptx protein in the production of reagents that inhibit the proliferation of aquatic pathogens, wherein the pathogens are Streptococcus agalactiae or Aeromonas hydrophila.

[0017] The present invention also provides an inhibitory drug for aquatic animal pathogens, wherein the drug contains the above-mentioned recombinant OnMptx protein and other pharmaceutically acceptable carriers or excipients.

[0018] The beneficial effects of this invention are:

[0019] This invention obtains the Mptx eukaryotic protein from tilapia through gene cloning and eukaryotic expression vector construction. The binding and agglutination effects of Mptx on tilapia-related pathogens were detected using ELISA and agglutination assays. Antibacterial experiments and survival rate detection demonstrated that the obtained Mptx inhibits pathogen proliferation and protects the organism. This invention proves that the Mptx eukaryotic protein can significantly reduce mortality in tilapia caused by Streptococcus agalactiae infection, which is of great significance in the green aquaculture industry. Attached Figure Description

[0020] Figure 1 Results of cloning and identification of the Mptx gene in Nile tilapia (A: 1% agarose gel electrophoresis results, M is the marker, 1 and 2 are OnMptx PCR amplification products; B: gene sequencing results);

[0021] Figure 2 Figure 1 shows the purification and identification results of OnMptx eukaryotic protein (A: SDS-PAGE results of OnMptx protein; B: Mass spectrometry identification results).

[0022] Figure 3 Figure 1 shows the results of OnMptx protein binding and agglutination of pathogens (A: ELISA results of bacterial binding to protein; B: Fluorescence microscopy results of agglutination, objective lens 10×).

[0023] Figure 4 Figure 1: Results of OnMptx enhancing the immune protection of tilapia against Streptococcus agalactiae (A: Detection results of OnMptx protein inhibiting pathogen proliferation; B: Statistics on tilapia survival rate).

[0024] Figure 5 This is a technical roadmap for an embodiment of the present invention. Detailed Implementation

[0025] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0026] Example

[0027] Gene cloning and identification of Nile tilapia OnMptx

[0028] (1) Nile tilapia rearing: The Nile tilapia used in this experiment were purchased from the Tilapia Breeding Farm in Guangdong Province. The Nile tilapia were reared in a semi-automatic biological recirculating aquaculture system, with good water quality and temperature, and were fed floating feed at 10% of their body weight every day.

[0029] (2) Total RNA extraction from Nile tilapia liver: Liver tissue stored at -80℃ was transferred to an ice box, and 200 μL of Trizol-Regent was added to a centrifuge tube to submerge the tissue sample. After thorough grinding, 800 μL of Trizol lysis buffer was added to a final volume of 1 mL. The tissue was vortexed to ensure complete lysis, and the mixture was allowed to stand on ice for 10 min, then centrifuged at 12,000 rpm for 10 min at 4℃. 900 μL of the supernatant was transferred to an RNase-free centrifuge tube containing 300 μL of chloroform (avoiding any precipitate), thoroughly mixed, allowed to stand for 10 min, and then centrifuged at 12,000 rpm for 15 min at 4℃. Simultaneously, 500 μL of isopropanol was added to a clean 1.5 mL RNase-free centrifuge tube for later use. After centrifugation, remove the sample and carefully aspirate the upper, colorless, transparent solution (avoiding the white, flocculent middle layer). Transfer this solution to an RNase-free centrifuge tube containing 500 μL of isopropanol. Gently invert the tube 6-8 times to mix thoroughly. Centrifuge at 4°C, 12,000 rpm for 15 minutes. A white precipitate at the bottom of the tube after centrifugation is the desired RNA sample. Discard the supernatant and wash the RNA sample with 1 mL of 75% ethanol solution. Centrifuge at 4°C, 12,000 rpm for 10 minutes, then discard the supernatant (repeat this step twice). After the final centrifugation, discard the liquid in the tube and allow it to air dry at room temperature with the cap open. Then, add an appropriate amount of DEPC water to fully dissolve the RNA precipitate. Analyze the concentration and quality of the RNA sample using a NanoDrop 2000 high-precision micro-spectrophotometer. Store at -80°C for later use.

[0030] (3) cDNA synthesis: The preparation of tilapia liver cDNA template was performed according to the procedures and reaction steps provided by TaKaRa's PrimerScript™ RTreagent Kit with gDNA Eraser (Perfect RealTime). This included two parts: genomic removal and reverse transcription. The final synthesized cDNA product was stored at -20℃ for later use.

[0031] (4) Primer design: Primers were designed using Prime Premier 5 software based on the predicted nucleic acid sequence of Nile tilapia OnMptx from the NCBI database (GenBank accession number: LOC100701841), and the designed primer sequences were sent to BGI Genomics for synthesis. The upstream and downstream primers are as follows: Mptx-F, TGGTAAAATGTTCACTTTCCCACTG, as shown in SEQ ID NO.1; Mptx-R, TTCCCTGGCGTGAAGTTTAGTT, as shown in SEQ ID NO.2.

[0032] (5) OnMptx gene amplification: PCR amplification of the open reading frame (ORF) of OnMptx was performed using liver cDNA as a template and the primers designed above. The total reaction volume was 25 μL, specifically: 15 μL of 2×ExTaqMix, 2 μL of liver cDNA, 0.5 μL of Mptx-F, 0.5 μL of Mptx-R, and 7 μL of ddH2O. The reaction program was: pre-denaturation at 95℃ for 5 min, denaturation at 95℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 1 min (denaturation-extension, 30 cycles), and final extension at 72℃ for 5 min.

[0033] (6) After the PCR reaction was completed, the results were detected by 1% agarose gel electrophoresis. The voltage was set to 110V and the electrophoresis time was 30min. The agarose gel electrophoresis marker was DS2000 produced by Dongsheng Biotechnology Co., Ltd.

[0034] (7) Positive amplified bands were extracted using an agarose gel DNA recovery kit (Tiangen Biotech Co., Ltd.). The recovered product (2.5 μL) was then ligated with pMD-18T (0.5 μL) vector and incubated overnight at 4°C. The ligation product was then transformed (Top 10 competent cells), plated, and single-clone strains were picked and subjected to culture PCR.

[0035] (8) Send bacterial culture samples containing a single positive target band to BGI Genomics for sequencing.

[0036] Purification and identification of OnMptx eukaryotic protein

[0037] (1) The eukaryotic expression vector used in this experiment was pcDNA3.1. Primers were redesigned based on the restriction sites of this vector to amplify the OnMptx gene sequence containing the restriction sites BamHI (GAATCC) and HindⅢ (AAGCTT). The upstream and downstream primers are as follows: EMptx-F, CGCGGATCCGGTAAAATGTTCACTTTCCCACTG, as shown in SEQ ID NO.3; EMptx-R, CGCAAGCTTCCCTGGCGTGAAGTTTAGTT, as shown in SEQ ID NO.4.

[0038] (2) The OnMptx gene sequence containing the new restriction site was amplified by PCR (with a His tag added to the N-terminus), and it was ligated into the pMD-18T vector. Positive monoclonal strains were selected by sequencing results identified by colony PCR.

[0039] (3) The plasmid of pcDNA3.1 vector and the plasmid of OnMptx positive clone strain ligated into pMD-18T vector were extracted and digested with restriction endonucleases. Then, the pcDNA3.1-Mptx positive clone strain was obtained by gel recovery, ligation, plating and single cloning.

[0040] (4) Plasmid extraction was performed on the recombinant positive strains obtained above. The mammalian expression system selected in this study was HEK293 cells, purchased from Wuhan Yunclone Biotechnology Co., Ltd., and frozen in a liquid nitrogen tank in the laboratory. Positive cell lines containing pcDNA3.1-Mptx were obtained by cell transfection (transient transfection), and then expanded culture and expression were performed.

[0041] (5) Positive clone cell lines were transferred to cell culture flasks and passaged. 50 mL of cell culture supernatant was collected, centrifuged at 1,000 × g, and the supernatant was collected and filtered through a 0.45 μM disposable sterile filter membrane. The target protein in the cell supernatant was purified by adding an appropriate amount of Millipore His-Tag nickel column packing material to a Bio-Rad gravity chromatography column (all reagents were urea-free).

[0042] (6) Finally, the purified protein samples were detected by 12% SDS-PAGE gel electrophoresis and identified by mass spectrometry.

[0043] OnMptx binds to and agglutinates pathogens.

[0044] For the bacterial binding assay, Streptococcus agalactiae and Aeromonas hydrophila were used as representatives to test the bacterial binding ability of OnMptx.

[0045] (1) Preparation of bacterial suspension: Preserved *Streptococcus agalactiae* and *Aeromonas hydrophila* were inoculated at a ratio of 1:1000 into Brain Heart Infusion Medium (BHI) and LB broth, respectively, and incubated at 30℃ on a shaker (8-12 h) until the bacterial suspension reached the logarithmic growth phase (the suspension was shaken to a mist-like state). The suspension was then centrifuged at 4800 rpm for 5 min, and the supernatant was discarded. The suspension was resuspended in sterile TBS (10 mM Tris-HCl, 150 mM NaCl, pH 7.4), and this process was repeated 4 times to remove all culture medium. The bacterial concentration was then adjusted to 1 × 10⁻⁶. 7 CFU / mL.

[0046] (2) Coating: Take 2 mL of agalactia-free streptococci or aeromonas hydrophila, centrifuge, and resuspend in an equal volume of coating buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6). Add 100 μL to each well of a 96-well microplate and incubate overnight at 4°C.

[0047] (3) Blocking: 200 μL of blocking solution (TBS, 2 mM CaCl2, 0.5% BSA, 0.01% TWEEN 20) per well, gently shaken at 37℃ for 1 h. After blocking, wash the plate 1×TTBS, 200 μL per well, for a total of 3 times.

[0048] (4) Add the prepared OnMptx (20 μg / mL) and carrier protein Trx (20 μg / mL) to the microplate (2-fold serial dilution), TBS as blank control, 3 replicates for each sample, and incubate at 37°C with gentle shaking for 1 h.

[0049] (5) Primary antibody incubation: After washing the plate three times, add 100 μL of diluted commercial His-tagged mouse monoclonal antibody (1:1500) to each well and incubate at 37°C for 1 h. Then wash the plate 200 μL to each well for a total of 3 times.

[0050] (6) Secondary antibody incubation: Goat anti-mouse HRP-IgG (1:2000) was used as the secondary antibody, 100 μL per well, and incubated at 37℃ for 1 h. Then the plate was washed, 200 μL per well, for a total of 3 times. After patting dry, it was confirmed that there were no air bubbles in each well.

[0051] (7) Color development: Add 100 μL TMB color development solution to each well. The color development solution formula is: 9.9 mL sodium citrate buffer (pH 5.5) + 100 μL TMB + 5 μL 30% H2O2. Incubate at room temperature in the dark for 10 min. Stop the color development with 2M sulfuric acid solution. Add 50 μL / well. Immediately after adding, read the absorbance with an ELISA reader. The absorbance is 450 nm. Read three times and save the OD value.

[0052] For the agglutination test, Streptococcus agalactiae and Aeromonas hydrophila were used as representatives to detect the agglutinating bacteria function of OnMptx.

[0053] (1) Bacterial FITC labeling: Take 2 mL of prepared Streptococcus agalactiae or Aeromonas hydrophila solution (1×10⁻⁶). 8 Add 200 μL of FITC solution (CFU / mL) to the solution (dissolve FITC powder in DMSO at 1 mg / mL in the dark) and mix thoroughly. Incubate at room temperature in the dark for 30 min. Then wash the cells four times with sterile TBS. Finally, resuspend the cells in 2 mL of sterile TBS buffer and store at -20°C for later use.

[0054] (2) Take 10 μL of FITC-labeled agalactia streptococci or aeromonas hydrophila and mix with 25 μL of 10 nMptx (final concentration 5 μg), and incubate at 25℃ and 100 rpm for 2 h with shaking.

[0055] (3) Observe the aggregation of OnMptx with bacteria under a fluorescence microscope.

[0056] OnMptx protein inhibits the proliferation of pathogens and improves the survival rate of tilapia.

[0057] Antibacterial test (in vitro test)

[0058] (1) Incubate *Streptococcus agalactiae* or *Aeromonas hydrophila* in brain heart quench broth (BHI) or LB broth until the logarithmic growth phase. After centrifugation at 5000 rpm for 5 min, collect the bacterial cells and wash three times with TBS buffer. Then resuspend the cells in TBS-Ca buffer to a final volume of 1×10⁻⁶. 5 CFU / mL.

[0059] (2) Add 100 μL of the prepared bacterial resuspension to each well of a 96-well microplate. Then add 100 μL of TBS, Trx (50 μg / mL), or OnMptx (50 μg / mL), respectively, with ampicillin (Amp) and levofloxacin (Lo) serving as positive controls. After mixing, incubate with gentle shaking at 30°C.

[0060] (3) The bacterial density was detected by an OD600 microplate reader at 0h, 2h, 4h, 6h, 8h, 10h and 12h of incubation.

[0061] Survival rate testing (in vivo experiments)

[0062] (1) Healthy tilapia (approximately 30g) were randomly harvested from the aquaculture system and randomly divided into 4 groups. The groups were acclimatized for several days to allow the tilapia to fully adapt to the culture tank environment. Each group consisted of 25 fish. The groups were designated as PBS (blank control), *S. agalactiae*, Trx (empty protein) + *S. agalactiae*, and OnMptx + *S. agalactiae*. On the day of immunization, the tilapia were anesthetized with a certain concentration of MS-2,2,2. A pre-prepared agalactococcal solution (1×10⁻⁶) was then administered. 8 S. agalactiae were injected intraperitoneally with a bacterial solution containing 100 μL of S. agalactiae (CFU / mL) per fish. The experimental group was injected intraperitoneally with a bacterial solution co-incubated with OnMptx (500 μg / mL) or Trx (500 μg / mL).

[0063] (2) Starting from the day of immunization, the number of tilapia deaths in each group is counted every day for 10 days.

[0064] Figure 1 The results of the cloning and identification of the Mptx gene in Nile tilapia (A: 1% agarose gel electrophoresis results, M is the marker, 1 and 2 are OnMptx PCR amplification products; B: gene sequencing results) are presented by [Author Name - Required Documentation]. Figure 1It can be seen that the nucleotide sequence of the OnMptx gene is 675 bp, encoding 224 amino acids. The specific nucleotide sequence is as follows:

[0065] ATGAAGCTGTTCCCTGTACTGGTGATGCTGACAGCATGTGCTGCAAGACCTCAAGATTTGTCTGGTAAAATGTTCACTTTCCCACTGGAATCCAACACCGCTCATGTGAGGCTGAATGCGTCAAGATTGGATTTCAGTTCTTTAACACTCTGTCACAGATCATTTACAGACCTTAAAAGAGACCACGTTTTGTTCTCTCTGGCTACACCTGTTCATTCCAATGACTTCCTGATCTTCTGGGATTATAAAAATAAAGAGATTGAGTCCGATATCAAGGACAGGGAAGCTGTATTTGGAGGACGAGACTACAAGCCAAACATGTGGCACTCTATCTGCACCACGTGGGACTCTGAGTCTGGACTGGTGCAGATTTGGTTTAATGGGCTACCTTCGATTAGGAAATTTGTCAGCACTGGAACAAACATCGTAGGACCTGTTATAATAATTTTAGGACAGGAGCAAGATTCCCACGGTGGCGGATTTGACCTTAAGCAGTCTTTCGTTGGCATGATGTCTGACGTCCACTTGTGGGATCACATCCTTTCCTCCTGTGAAATTCAAAACTATGTGGATGAACTAAACTTCACGCCAGGGAACGTGCTGAACTGGAGCGCACTGGAGTTTGAGATCATAGATAGAGTACTGATAGAAAATAAACTCATGACGTGTCACTAA, as shown in SEQ ID NO.5;

[0066] The encoded amino acid sequence is:

[0067] MKLFPVLVMLTACAARPQDLSGKMFTFPLESNTAHVRLNASRLDFSSLTLCHRSFTDLKRDHVLFSLATPVHSNDFLIFWDYKNKEIESDIKDREAVFGGRDYKPNMWHSICTT WDSESGLVQIWFNGLPSIRKFVSTGTNIVGPVIIILGQEQDSHGGGFDLKQSFVGMMSDVHLWDHILSSCEIQNYVDELNFTPGNVLNWSALEFEIIDRVLIENKLMTCH, such as SEQ Shown as ID NO.6;

[0068] Figure 2 The images show the purification and identification results of OnMptx eukaryotic protein (A: SDS-PAGE results of OnMptx protein; B: Mass spectrometry identification results). Figure 2 It can be seen that the molecular weight of the OnMptx protein is approximately 26 kDa, and mass spectrometry analysis shows that its amino acid coverage reaches 80%.

[0069] Figure 3 The images show the results of OnMptx protein binding and agglutination of pathogens (A: ELISA results of bacterial-protein binding; B: Fluorescence microscopy results of agglutination, objective lens 10×). Figure 3 The ELISA results showed that the OnMptx protein could bind significantly to *Streptococcus agalactiae* and *Aeromonas hydrophila* in a concentration-dependent manner. Fluorescence microscopy showed that the OnMptx protein could agglutinate with *S. agalactiae* and *A. hydrophila*, while the carrier protein Trx could not bind to or agglutinate with *S. agalactiae* and *A. hydrophila*.

[0070] Figure 4 The graph shows the results of OnMptx enhancing the immunoprotective effect against Streptococcus agalactiae in tilapia (A: Detection results of OnMptx protein inhibiting pathogen proliferation; B: Statistics on tilapia survival rate). Figure 4It was found that OnMptx significantly inhibited the proliferation of S. agalactiae and A. hydrophila in a time-dependent manner, while the carrier protein Trx could not inhibit the proliferation of these two bacteria. Meanwhile, the positive control group ampicillin (Amp) or levofloxacin (Lo) could inhibit the survival of S. agalactiae or A. hydrophila. The survival rate results showed that OnMptx protein significantly improved the survival rate of tilapia (88%) after S. agalactiae infection, while Trx protein had no significant effect on the survival rate of infected tilapia (48%).

[0071] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of a recombinant OnMptx protein in the preparation of a reagent for agglutinating pathogens, characterized in that, The pathogen is *Streptococcus agalactiae* (…). Streptococcus agalactia ) or Aeromonas hydrophila ( Aeromonas hydrophila ); The amino acid sequence of the recombinant OnMptx protein is shown in SEQ ID NO.

6.

2. The application of a recombinant OnMptx protein in the production of a reagent for inhibiting the proliferation of aquatic pathogens, characterized in that, The pathogen is either Streptococcus agalactiae or Aeromonas hydrophila; The amino acid sequence of the recombinant OnMptx protein is shown in SEQ ID NO.6.

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