A portunus trituberculatus anti-lipopolysaccharide factor encoding gene ptalf-7, and a protein and application thereof

Abstract

The application discloses a portunus trituberculatus anti-lipopolysaccharide factor coding gene PtALF-7 and a protein and application thereof, and belongs to the technical field of molecular biology. PtALF-7 The nucleotide sequence of the coding gene is shown as SEQ ID No. 1, and the amino acid sequence of the obtained portunus trituberculatus anti-lipopolysaccharide factor is shown as SEQ ID No. 2. PtALF-7 The in-vitro bacteriostatic experiment proves that the anti-lipopolysaccharide factor coded by the gene has a significant inhibiting effect on the growth of Vibrio parahaemolyticus and Staphylococcus caprae. Therefore, the application lays an application foundation for disease prevention of portunus trituberculatus, gene-assisted breeding and the like, and has good market application value in the development of antibacterial drugs, feed additive production and the like.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, and specifically relates to a gene encoding an anti-lipopolysaccharide factor in the swimming crab *Portunus trituberculatus*. PtALF-7 Its proteins and applications. Background Technology

[0002] Three-spotted swimming crab ( Portunus trituberculatus The swimming crab (Portunus trituberculatus) is an important marine economic animal. Vibrio parahaemolyticus is one of the main pathogens causing acute hepatopancreatic necrosis in marine crustaceans, and it is also a major pathogen causing large-scale mortality in swimming crabs, seriously hindering the healthy development of the industry.

[0003] Antilipopolysaccharide factor (ALF) is an important class of antimicrobial peptides found in crustaceans. These peptides recognize and bind to Gram-negative bacterial lipopolysaccharides (LPS), exerting their immune defense function by inhibiting bacterial growth, disrupting bacterial membrane structure, and regulating host immune responses. The ALF family is widely present in marine crustaceans such as shrimp and crabs, and numerous studies have confirmed its central role in innate immunity, making it a crucial molecule for maintaining the host's resistance to infection.

[0004] Research on anti-lipopolysaccharide factor genes and their encoded proteins is of great theoretical and practical significance for understanding the immune defense mechanism of swimming crabs, disease prevention and control, and the breeding of disease-resistant varieties. Summary of the Invention

[0005] The purpose of this invention is to provide a gene encoding an anti-lipopolysaccharide factor in the swimming crab *Portunus trituberculatus*. PtALF-7 Its proteins and applications. The encoding gene... PtALF-7 The encoded anti-lipopolysaccharide factor protein has a significant inhibitory effect on the growth of Vibrio parahaemolyticus and Staphylococcus aureus.

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

[0007] This invention provides a gene encoding an anti-lipopolysaccharide factor in the swimming crab *Portunus trituberculatus*. PtALF-7 Its nucleotide sequence is shown in SEQ ID No. 1.

[0008] This invention also provides a lipopolysaccharide-resistant protein from the swimming crab *Portunus trituberculatus*, which is encoded by the aforementioned gene. PtALF- 7 The encoded amino acid sequence is shown in SEQ ID No. 2.

[0009] Furthermore, the *Portunus trituberculatus* anti-lipopolysaccharide factor protein is obtained by the following steps: synthesizing the encoding gene. PtALF-7BamHI (GGATCC) and XhoI (CTCGAG) restriction sites were added to the 5' and 3' ends of the sequence, respectively. The synthesized gene sequence was digested with enzymes and inserted into plasmid pET32a to construct a recombinant plasmid. The recombinant plasmid was transformed into the engineered bacteria E. coli to form a recombinant engineered bacteria. After culturing the recombinant engineered bacteria, the bacteria were broken, separated, washed, dialyzed, and purified to obtain the anti-lipopolysaccharide factor protein PtALF-7 of the swimming crab.

[0010] The present invention also provides a recombinant plasmid containing the said coding gene. PtALF-7 .

[0011] The present invention also provides a recombinant engineered bacterium containing the aforementioned coding gene. PtALF-7 .

[0012] The present invention also provides the coding gene. PtALF-7 The application of *Portunus trituberculatus* anti-lipopolysaccharide factor protein in the preparation of antibacterial agents that inhibit Gram-negative and Gram-positive bacteria, wherein... PtALF-7 The nucleotide sequence of the gene is shown in SEQ ID No. 1; the amino acid sequence of the anti-lipopolysaccharide factor protein of the swimming crab is shown in SEQ ID No. 2.

[0013] Furthermore, the Gram-negative bacteria are Vibrio parahaemolyticus, and the Gram-positive bacteria are Staphylococcus capsulatum.

[0014] Furthermore, the concentration of the anti-lipopolysaccharide factor protein from the swimming crab *Portunus trituberculatus* is 0.05 mg / mL to 1.5 mg / mL.

[0015] The present invention also provides the coding gene. PtALF-7 Or the application of *Portunus trituberculatus* anti-lipopolysaccharide factor protein in the preparation of aquatic animal feed additives, the aforementioned PtALF-7 The nucleotide sequence of the gene is shown in SEQ ID No. 1; the amino acid sequence of the anti-lipopolysaccharide factor protein of the swimming crab is shown in SEQ ID No. 2.

[0016] Furthermore, the aquatic animals include swimming crabs and prawns.

[0017] Compared with existing technologies, the advantages and beneficial technical effects of the present invention are:

[0018] 1. This invention chemically synthesizes anti-lipopolysaccharide factors from the swimming crab *Portunus trituberculatus*. PtALF-7 Gene sequence; using prokaryotic expression technology, the anti-lipopolysaccharide factor was obtained. PtALF-7The recombinant protein encoded by the gene; further, this invention demonstrates through in vitro antibacterial experiments that the anti-lipopolysaccharide factor protein PtALF-7 exhibits inhibition rates of 91.5%, 74.8%, 60.4%, and 17.2% against Vibrio parahaemolyticus at concentrations of 1.136 mg / mL, 0.568 mg / mL, 0.284 mg / mL, and 0.0568 mg / mL, respectively. At concentrations of 1.136 mg / mL, 0.568 mg / mL, 0.284 mg / mL, and 0.0568 mg / mL, it shows inhibition rates of 53.6%, 33.2%, 25.4%, and 29.3% against Staphylococcus aureus, respectively, demonstrating its significant inhibitory effect on the growth of both Vibrio parahaemolyticus and Staphylococcus aureus.

[0019] 2. The anti-lipopolysaccharide factor of the swimming crab *Portunus trituberculatus* provided by this invention PtALF-7 The gene and its encoded protein can be used to produce antibacterial drugs, which can be applied to the treatment of related diseases in the farming of swimming crabs, or to the production of feed additives and preservatives. In addition, it can provide data support for the analysis of the immune mechanism of swimming crabs and provide theoretical reference for disease-resistant breeding and disease prevention and control of swimming crabs. Attached Figure Description

[0020] Figure 1 This is the purified protein PtALF-7. M: marker; 1: protein purified by Ni column before induction; 2: protein purified by Ni column after induction.

[0021] Figure 2 This is a diagram of the experiment showing the inhibition of Vibrio parahaemolyticus by protein PtALF-7. From left to right, the culture dishes contain 1.136 mg / mL of protein, 0.568 mg / mL of PBS, 0.284 mg / mL of protein, and 0.0568 mg / mL of PBS, respectively.

[0022] Figure 3 This is a diagram of an experiment showing the inhibition of Staphylococcus aureus by protein PtALF-7. From left to right, the culture dishes contain 0.0568 mg / mL of protein, 0.284 mg / mL of protein, 0.568 mg / mL of protein, and 1.136 mg / mL of PBS, respectively. Detailed Implementation

[0023] The technical solution of the present invention will be further described in detail below with reference to specific examples. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials and reagents used can be purchased from biological or chemical reagent companies.

[0024] Example 1

[0025] The target gene sequence used in this embodiment was synthesized by a commercial company using a chemical approach. The chemically synthesized gene sequence (as shown in SEQ ID No. 1) had BamHI (GGATCC) and XhoI (CTCGAG) restriction sites added to its 5' and 3' ends, respectively, to facilitate the subsequent construction of the pET32a vector. The synthesized gene sequence was purified by PAGE and provided as lyophilized DNA. This chemically synthesized gene fragment was dissolved in nuclease-free water and then double-digested with the pET32a vector using the same restriction endonuclease. The digested products were purified and ligated overnight at 16°C in a T4 DNA ligase system to obtain the recombinant plasmid pET32a-PtALF-7.

[0026] The ligation product was transformed into chemically competent *E. coli* DH5α, plated on LB agar containing kanamycin, and incubated at 37°C for 12–16 h. Single colonies were picked for colony PCR and restriction enzyme digestion to confirm that the target insert fragment was correctly cloned into the vector. Positive clones underwent plasmid extraction, and Sanger sequencing was used to verify the sequence accuracy and reading frame consistency of the inserted gene. Recombinant plasmids confirmed by sequencing to be mutation-free and correctly ligated were used as plasmids for subsequent expression and transformed into the host strain *ArcticExpress(DE3)pRARE2* for protein expression.

[0027] Example 2

[0028] This example uses the recombinant plasmid and host strain ArcticExpress(DE3)pRARE2 obtained in Example 1 to express, purify, and renature the target protein. A single colony containing the recombinant plasmid was inoculated into 5 mL of LB medium with kanamycin (final concentration 50 μg / mL) and cultured at 37°C and 180 rpm for 8 h as the primary seed culture. Then, 200 μL of the primary seed culture was transferred to an Erlenmeyer flask containing 60 mL of LB medium and kanamycin and cultured at 28°C and 150 rpm for 16 h to obtain the secondary seed culture. 60 mL of this secondary seed culture was inoculated into 900 mL of LB medium (containing the same antibiotic) and shaken at 28°C and 150 rpm until OD (dose retardation) was reached. 600 When the concentration reaches 0.7–0.8, add 0.5 mL of 1 MIPTG to make a final concentration of 0.7 mM. Then continue to induce expression at 28℃ and 150 rpm for 4.5 h, or induce overnight at 37℃ and 200 rpm. After induction, collect the cells by centrifugation at 7500 rpm and 20℃ for 15 min. Approximately 3.7–4.0 g of wet cells can be obtained from 1 L of culture.

[0029] The collected bacterial cells were resuspended in 80 mL of disruption buffer and homogenized three times consecutively at 750–850 bar using high-pressure homogenization, maintaining a temperature of 4°C throughout the disruption process. The disruption buffer was centrifuged at 8000 rpm for 30 min at 4°C to separate the supernatant and precipitate. The precipitate was added to 40 mL of solubilization buffer and incubated overnight at 4°C for solubilization, serving as the inclusion body protein sample. The solubilized precipitate was then loaded onto a 5 mL Ni-NTA column equilibrated with binding buffer, maintaining a stable flow rate and collecting flow-through. Elution was performed sequentially using elution buffers containing 20 mM, 50 mM, 100 mM, 250 mM, 500 mM, and 1000 mM imidazole, collecting the corresponding elution fractions. The 100 mM imidazole elution peak contained the major target protein. After elution, the column was washed with column treatment buffer and stored in 20% ethanol.

[0030] The elution fractions with higher concentrations of 100 mM imidazole were combined, and L-arginine, EDTA, and DTT were added to the system to achieve final concentrations of 1%, 5 mM, and 5 mM, respectively. After standing at 4°C for 30 min, urea gradient dialysis was performed. During dialysis, 8 M, 6 M, 4 M, 2 M, and 0 M urea systems were used sequentially, with each stage lasting 2–4 h. The dialysis buffer was 20 mM Tris-HCl (pH 8.0) containing 10% glycerol and 0.02% sodium azide. After dialysis, the protein was further concentrated using ultrafiltration tubes, and the protein concentration was measured. The final refolded protein concentration was approximately 5.68 mg / mL, which can be stored at -20°C or -80°C.

[0031] To verify the expression, 200 μL of fermentation broth was centrifuged, resuspended, and an equal volume of 2× SDS lysis buffer was added. The mixture was then incubated at 100℃ for 5 min and loaded onto an SDS-PAGE gel. A distinct expression band with a molecular weight of approximately 26.3 kDa was observed in the gel. Figure 1 This indicates that the recombinant protein PtALF-7 was successfully expressed and can be purified using the above method.

[0032] Example 3: In vitro antibacterial test of PtALF-7, an anti-lipopolysaccharide factor protein from the swimming crab *Portunus trituberculatus*.

[0033] Culture and preparation of Vibrio parahaemolyticus: First, prepare 2216E liquid and solid culture media. Weigh the media and dissolve them in ultrapure water. Sterilize at 121 °C for 15 min in a pressure steam sterilizer. After sterilization, store the liquid culture media at 4 °C for later use, and pour the solid culture media into plates for later use. Take a small amount of Vibrio parahaemolyticus strain frozen at -80 °C and add it to 2216E medium. Incubate at 28 °C, 200 rpm for 4-5 h to obtain activated strains. Streak an appropriate amount of the bacterial suspension onto 2216E solid culture medium and incubate overnight at 28 °C, 200 rpm. Pick a single colony and inoculate it onto 2216E liquid culture medium. Incubate at 28 °C, 200 rpm for 8 h, then at 5000 rpm for 10 min, and collect the bacterial cells. Use the above methods and TSB medium to culture Staphylococcus aureus.

[0034] Detection using plate counting method PtALF-7 Antibacterial activity of the protein. For the antibacterial experiment against Vibrio parahaemolyticus, the experiment was divided into a control group and an experimental group. PtALF-7 protein was diluted to final concentrations of 1.136 mg / mL, 0.568 mg / mL, 0.284 mg / mL, and 0.0568 mg / mL. 50 μL of each diluted protein was used. 3 CFU / mL Vibrio parahaemolyticus suspension was mixed thoroughly with an equal volume of protein solution. The control group was treated with the same volume of PBS. The mixture was shaken well and incubated at 37 ℃ for 2 h. The mixture was then evenly spread onto solid culture medium in a clean bench and incubated upside down at 37 ℃ overnight. The number of colonies on the plates was recorded the next day, and the inhibition rate of PtALF-7 protein was calculated. For Staphylococcus aureus, the inhibition experiment was conducted using the same procedure as for Vibrio parahaemolyticus.

[0035] Experimental results are as follows Figure 2 As shown, at concentrations of 1.136 mg / mL, 0.568 mg / mL, 0.284 mg / mL, and 0.0568 mg / mL, the inhibition rates of PtALF-7 protein against Vibrio parahaemolyticus were 91.5%, 74.8%, 60.4%, and 17.2%, respectively.

[0036] like Figure 3 As shown, the inhibition rates against Staphylococcus aureus at concentrations of 1.136 mg / mL, 0.568 mg / mL, 0.284 mg / mL, and 0.0568 mg / mL were 53.6%, 33.2%, 25.4%, and 29.3%, respectively, indicating that the anti-lipopolysaccharide factor protein of Swimming crab (as shown in SEQ ID No. 2) has a significant inhibitory effect on the growth of Vibrio parahaemolyticus and Staphylococcus aureus.

[0037] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims

1. The gene encoding the anti-lipopolysaccharide factor in the swimming crab *Portunus trituberculatus* PtALF-7 The application of *Portunus trituberculatus* anti-lipopolysaccharide factor protein in the preparation of antibacterial agents that inhibit Gram-negative and Gram-positive bacteria, characterized in that: The gene encoding the anti-lipopolysaccharide factor of the swimming crab *Portunus trituberculatus* PtALF-7 The nucleotide sequence is shown in SEQ ID No. 1; the amino acid sequence of the anti-lipopolysaccharide factor protein of the three-spined swimming crab is shown in SEQ ID No. 2; the Gram-negative bacterium is Vibrio parahaemolyticus, and the Gram-positive bacterium is Staphylococcus capsulatum.

2. The application according to claim 1, characterized in that: The concentration of the anti-lipopolysaccharide factor protein from the swimming crab *Portunus trituberculatus* is 0.05 mg / mL to 1.5 mg / mL.

3. The application according to claim 1, characterized in that: The anti-lipopolysaccharide factor protein of the swimming crab *Portunus trituberculatus* is prepared by the following steps: synthesizing the encoding gene. PtALF-7 The nucleotide sequence was obtained by adding restriction enzyme sites at the 5' and 3' ends of the sequence, respectively; the synthesized gene sequence was digested with enzymes and inserted into a plasmid to construct a recombinant plasmid; the recombinant plasmid was transformed into engineered bacteria to form a recombinant engineered bacteria; the recombinant engineered bacteria were cultured and then broken, and after separation, washing and purification, the anti-lipopolysaccharide factor protein of swimming crab was obtained.

4. The gene encoding the anti-lipopolysaccharide factor in the swimming crab *Portunus trituberculatus* PtALF-7 The application of *Portunus trituberculatus* anti-lipopolysaccharide factor protein in the preparation of aquatic animal feed additives is characterized by: The gene encoding the anti-lipopolysaccharide factor of the swimming crab *Portunus trituberculatus* PtALF-7 The nucleotide sequence is shown in SEQ ID No. 1; the amino acid sequence of the anti-lipopolysaccharide factor protein of the three-spined swimming crab is shown in SEQ ID No. 2; the aquatic animals include three-spined swimming crab and shrimp.

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