A ptalf-8 gene encoding an anti-lipopolysaccharide factor of portunus trituberculatus and a protein and application thereof
The recombinant protein of the PtALF-8 gene was obtained through chemical synthesis and prokaryotic expression technology, which solved the disease problem caused by Vibrio parahaemolyticus in the farming of swimming crabs and achieved significant antibacterial effect and farming stability.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
- Filing Date
- 2025-12-23
- Publication Date
- 2026-05-12
AI Technical Summary
Acute hepatopancreatic necrosis caused by Vibrio parahaemolyticus is frequent in the farming of swimming crabs, resulting in low and unstable yields. Existing technologies lack effective immune defense measures.
The PtALF-8 gene was chemically synthesized and recombinant protein PtALF-8 was obtained through prokaryotic expression technology. It was then used to prepare antibacterial agents and feed additives to inhibit the growth of Vibrio parahaemolyticus and Staphylococcus aureus.
PtALF-8 protein showed significant antibacterial effects against Vibrio parahaemolyticus and Staphylococcus capus at different concentrations, providing theoretical support for disease prevention and breeding in swimming crab farming.
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Figure CN121380085B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of molecular biology technology, and specifically relates to a factor encoding an anti-lipopolysaccharide factor in the swimming crab *Portunus trituberculatus*. PtALF-8 Genes, their proteins, and their applications. Background Technology
[0002] Three-spotted swimming crab ( Portunus trituberculatus Swimming crabs (Portunus trituberculatus) are an important marine economic animal and have become one of the leading species in my country's marine aquaculture. However, due to the deterioration of pond aquaculture environments and extensive farming methods, diseases frequently occur in swimming crabs, leading to low yields and unstable farming, becoming a major bottleneck restricting the industry's development. Vibrio parahaemolyticus is one of the main pathogens causing acute hepatopancreatic necrosis in marine crustaceans, and 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 factor encoding the anti-lipopolysaccharide factor of the swimming crab *Portunus trituberculatus*. PtALF-8 Genes and their proteins and their applications. The present invention provides... PtALF-8 The protein encoded by the gene has a significant inhibitory effect on the growth of Vibrio parahaemolyticus and Staphylococcus aureus, and has good application prospects.
[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-8 The gene, whose nucleotide sequence is shown in SEQ ID No. 1.
[0008] This invention also provides a *Portunus trituberculatus* anti-lipopolysaccharide factor protein PtALF-8, which is composed of the aforementioned... PtALF-8 The gene is encoded, and its amino acid sequence is shown in SEQ ID No. 2.
[0009] Furthermore, the construction steps of the *Portunus trituberculatus* anti-lipopolysaccharide factor protein PtALF-8 are as follows: chemical synthesis of the aforementioned... PtALF-8 The gene sequence was modified by adding BamHI (GGATCC) and XhoI (CTCGAG) restriction sites at the 5' and 3' ends, respectively. The synthesized gene sequence was digested and inserted into plasmid pET32a to form a recombinant plasmid. The recombinant plasmid was then transformed into the engineered bacteria E. coli to form a recombinant engineered bacteria. After culturing the recombinant engineered bacteria, the bacteria were broken up, separated, washed, and purified to obtain the anti-lipopolysaccharide factor protein.
[0010] The present invention also provides a recombinant plasmid containing the aforementioned... PtALF-8 Gene.
[0011] The present invention also provides a recombinant engineered bacterium containing the aforementioned... PtALF-8 Gene.
[0012] The present invention also provides the aforementioned PtALF-8 The application of the gene or the aforementioned three-spined swimming crab anti-lipopolysaccharide factor protein PtALF-8 in the preparation of antibacterial agents that inhibit Gram-negative and Gram-positive bacteria.
[0013] Furthermore, the Gram-negative bacteria are Vibrio parahaemolyticus, and the Gram-positive bacteria are Staphylococcus capsulatum.
[0014] Furthermore, the concentration range of the anti-lipopolysaccharide factor protein PtALF-8 is 0.05 mg / mL to 2.0 mg / mL.
[0015] The present invention also provides PtALF-8 Application of the gene or the aforementioned anti-lipopolysaccharide factor protein PtALF-8 from the swimming crab in the preparation of aquatic animal feed additives.
[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. The present invention chemically synthesizes the anti-lipopolysaccharide factor of the swimming crab *Portunus trituberculatus*. PtALF-8 Gene sequence; using prokaryotic expression technology, the anti-lipopolysaccharide factor was obtained. PtALF-8The recombinant protein of the gene was determined, and in vitro antibacterial experiments confirmed that the PtALF-8 protein exhibited inhibition rates of 97.0%, 77.1%, 75.3%, and 61.6% against Vibrio parahaemolyticus at concentrations of 1.624 mg / mL, 0.812 mg / mL, 0.406 mg / mL, and 0.0812 mg / mL, respectively. At the same concentrations, the inhibition rates against Staphylococcus aureus were 77.2%, 76.7%, 66.1%, and 63.2%, respectively, demonstrating a 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-8 The gene and its encoded protein can be used to produce antibacterial drugs for the treatment of related diseases in the farming of swimming crabs, or for 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 PtALF-8 is the purified protein; M: marker; 1: protein purified by Ni column before induction; 2: protein purified by Ni column after induction.
[0021] Figure 2 This is an experimental diagram showing the inhibition of Vibrio parahaemolyticus by protein PtALF-8. From left to right, the culture dishes contain 1.624 mg / mL, 0.812 mg / mL, 0.406 mg / mL, and 0.0812 mg / mL of protein and PBS, respectively.
[0022] Figure 3 This is an experimental diagram of the inhibition of Staphylococcus aureus by protein PtALF-8; from left to right, 0.0812 mg / mL of protein and PBS were added to the culture dishes, 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 chemically by a commercial company. The chemically synthesized gene sequence had restriction endonuclease recognition sequences matching the pET32a multiple cloning site added to its 5' and 3' ends, respectively, to facilitate subsequent vector construction. In this embodiment, BamHI (GGATCC) and XhoI (CTCGAG) restriction sites were added to the 5' and 3' ends of the sequence, respectively. The synthesized gene sequence was purified by PAGE and provided in lyophilized DNA form. The chemically synthesized gene fragment was dissolved in nuclease-free water and then double-digested with the pET32a vector using the same restriction endonuclease. After purification, the digested products were ligated overnight at 16°C in a T4 DNA ligase system to obtain the recombinant plasmid pET32a-PtALF-8.
[0026] The ligation product was transformed with 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 were further subjected to 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 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 as an example 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 to obtain the primary seed culture. Subsequently, 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 the secondary seed culture was inoculated into 900 mL of LB broth (containing the same antibiotic), and the culture was shaken at 28°C and 150 rpm until the OD600 reached 0.7–0.8. Then, 0.5 mL of 1 MIPTG was added to bring the final concentration to 0.7 mM. Induction was then continued at 28°C and 150 rpm for 4.5 h, or overnight at 37°C and 200 rpm. After induction, the cells were collected by centrifugation at 7500 rpm and 20°C for 15 min. Approximately 3.7–4.0 g of wet cells were 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 anti-lipopolysaccharide factor protein PtALF-8 was successfully expressed and can be purified using the above method.
[0032] Example 3: In vitro antibacterial test of PtALF-8 protein in 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 (commercially available) frozen at -80 °C and add it to the 2216E culture 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] The antibacterial activity of PtALF-8 protein was detected using plate counting. For the antibacterial experiment against Vibrio parahaemolyticus, the experiment was divided into a control group and an experimental group. PtALF-8 protein was diluted to final concentrations of 1.624 mg / mL, 0.812 mg / mL, 0.406 mg / mL, and 0.0812 mg / mL, and 50 μL of each solution was collected. 3CFU / mL Vibrio parahaemolyticus suspension was mixed thoroughly with an equal volume of recombinant 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-8 protein was calculated. For Staphylococcus aureus, the inhibition experiment was conducted using the same procedure as for Vibrio parahaemolyticus.
[0035] The results are as follows Figure 2 As shown, at concentrations of 1.624 mg / mL, 0.812 mg / mL, 0.406 mg / mL, and 0.0812 mg / mL, the inhibition rates of the protein against Vibrio parahaemolyticus were 97.0%, 77.1%, 75.3%, and 61.6%, respectively.
[0036] like Figure 3 As shown, the inhibition rates against Staphylococcus aureus at concentrations of 1.624 mg / mL, 0.812 mg / mL, 0.406 mg / mL, and 0.0812 mg / mL were 77.2%, 76.7%, 66.1%, and 63.2%, respectively, indicating that the anti-lipopolysaccharide factor protein PtALF-8 of the 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. Encoding the anti-lipopolysaccharide factor of the swimming crab *Portunus trituberculatus* PtALF-8 The application of the gene or the anti-lipopolysaccharide factor protein PtALF-8 from the swimming crab (Portunus trituberculatus) in the preparation of antibacterial agents that inhibit Gram-negative and Gram-positive bacteria, characterized in that: The PtALF-8 The nucleotide sequence of the gene is shown in SEQ ID No. 1; the amino acid sequence of the anti-lipopolysaccharide factor protein PtALF-8 of the 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 construction steps of the anti-lipopolysaccharide factor protein PtALF-8 from the swimming crab are as follows: chemical synthesis of the aforementioned protein... PtALF-8 The gene sequence was modified by adding restriction enzyme sites at the 5' and 3' ends of the sequence. The synthesized gene sequence was digested with enzymes and inserted into a plasmid to construct a recombinant plasmid. The recombinant plasmid was then transformed into an engineered bacterium to construct a recombinant engineered bacterium. The recombinant engineered bacterium was cultured, broken, and then separated, washed, and purified to obtain the anti-lipopolysaccharide factor protein of *Portunus trituberculatus*.
3. The application according to claim 1, characterized in that: The concentration range for the anti-lipopolysaccharide factor protein PtALF-8 is 0.05 mg / mL to 2.0 mg / mL.
4. Encoding the anti-lipopolysaccharide factor of the swimming crab *Portunus trituberculatus* PtALF-8 The application of the gene or the anti-lipopolysaccharide factor protein PtALF-8 from the swimming crab (Portunus trituberculatus) in the preparation of aquatic animal feed additives, characterized by: The PtALF-8 The nucleotide sequence of the gene is shown in SEQ ID No. 1; the amino acid sequence of the anti-lipopolysaccharide factor protein PtALF-8 of the swimming crab is shown in SEQ ID No. 2; the aquatic animals include swimming crab and shrimp.