PtALF-8 gene for coding anti-lipopolysaccharide factor of portunus trituberculatus and protein and application of PtALF-8 gene

Abstract

The invention discloses a PtALF-8 gene for coding a portunus trituberculatus anti-lipopolysaccharide factor as well as a protein and application of the PtALF-8 gene, and belongs to the technical field of molecular biology. The nucleotide sequence of the PtALF-8 gene is as shown in SEQ ID No. 1, and the amino acid sequence of the portunus trituberculatus anti-lipopolysaccharide factor protein obtained by encoding the PtALF-8 gene is as shown in SEQ ID No. 2. In-vitro antibacterial experiments prove that the anti-lipopolysaccharide factor protein PtALF-8 has a remarkable inhibition effect on the growth of vibrio parahaemolyticus and staphylococcus caprae. Therefore, the invention lays an application foundation for disease control and gene-assisted breeding of portunus trituberculatus, and has potential market application value in the aspects of development of antibacterial drugs, production of feed additives and the like.

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: 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.

[0007] 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.

[0008] Further, the construction steps of the Portunus trituberculatus anti-lipopolysaccharide factor protein PtALF-8 are as follows: chemically synthesizing the gene sequence of the anti-lipopolysaccharide factor protein PtALF-8 PtALF-8 , adding a BamH I (GGATCC) and a Xho I (CTCGAG) enzyme cutting site at the 5' end and the 3' end of the sequence respectively, cutting the synthesized gene sequence, inserting the gene sequence into a plasmid pET32a to form a recombinant plasmid, and transforming the recombinant plasmid into an engineering bacterium E.Coli to form a recombinant engineering bacterium.

[0009] The application further provides a recombinant plasmid containing the gene. PtALF-8

[0010] The application further provides a recombinant engineering bacterium containing the gene. PtALF-8

[0011] The application further provides an application of the gene or the Portunus trituberculatus anti-lipopolysaccharide factor protein PtALF-8 in preparation of an antibacterial agent for inhibiting gram-negative bacteria and gram-positive bacteria. PtALF-8

[0012] Further, the gram-negative bacteria are Vibrio parahaemolyticus, and the gram-positive bacteria are Staphylococcus caprae.

[0013] Further, the use concentration of the anti-lipopolysaccharide factor protein PtALF-8 ranges from 0.05 mg / mL to 2.0 mg / mL.

[0014] The application further provides an application of the gene or the Portunus trituberculatus anti-lipopolysaccharide factor protein PtALF-8 in preparation of a feed additive for aquatic animals. PtALF-8

[0015] Further, the aquatic animals include Portunus trituberculatus and Penaeus.

[0016] Compared with the prior art, the application has the following advantages and beneficial technical effects: 1、 the anti-lipopolysaccharide factor gene sequence of the Portunus trituberculatus is chemically synthesized; PtALF-8 2、 the anti-lipopolysaccharide factor protein PtALF-8 is obtained by using a prokaryotic expression technology. PtALF-8 ​​​​The recombinant protein of the gene; and the bacteriostatic experiment in vitro proves that the bacteriostatic rates of the PtALF-8 protein on Vibrio parahaemolyticus are 97.0%, 77.1%, 75.3% and 61.6% respectively at the concentrations of 1.624 mg / mL, 0.812 mg / mL, 0.406 mg / mL and 0.0812 mg / mL. The bacteriostatic rates on Staphylococcus caprae are 77.2%, 76.7%, 66.1% and 63.2% respectively at the same concentrations, and the PtALF-8 protein has a significant inhibitory effect on the growth of Vibrio parahaemolyticus and Staphylococcus caprae.

[0017] 2. The anti-lipopolysaccharide factor of Portunus trituberculatus provided by the application PtALF-8 The gene and the coded protein can be used for producing bacteriostatic drugs, treating diseases in the breeding process of Portunus trituberculatus, producing feed additives and preservatives, providing data support for analyzing the immune mechanism of Portunus trituberculatus, and providing a theoretical reference for the disease-resistant breeding and disease prevention of Portunus trituberculatus. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Fig. 2 is a photograph of the protein PtALF-8 after purification; M: marker; 1: protein after purification by Ni column before induction; 2: protein after purification by Ni column after induction.

[0019] Figure 2 Fig. 4 is an experimental graph of the protein PtALF-8 inhibiting Staphylococcus caprae; from left to right, the culture dishes are added with 0.0812 mg / mL, 0.406 mg / mL, 0.812 mg / mL and 1.624 mg / mL of the protein and PBS respectively.

[0020] Figure 3 Fig. 4 is an experimental graph of the protein PtALF-8 inhibiting Staphylococcus caprae; from left to right, the culture dishes are added with 0.0812 mg / mL, 0.406 mg / mL, 0.812 mg / mL and 1.624 mg / mL of the protein and PBS respectively. DETAILED DESCRIPTION

[0021] The technical solutions of the application are further described in detail in combination with the following specific examples. In the following examples, the experimental methods used are conventional methods, and the materials and reagents used can be purchased from biological or chemical reagent companies.

[0022] Example 1 The target gene sequence used in this example was synthesized by a commercial company using a chemical approach. The chemically synthesized gene sequence was added with restriction enzyme recognition sequences matching the pET32a multiple cloning site at the 5' end and 3' end, respectively, to facilitate subsequent vector construction. In this example, BamH I (GGATCC) and Xho I (CTCGAG) restriction sites were added at the 5' end and 3' end of the sequence, respectively. The synthesized gene sequence was purified by PAGE and provided in the form of freeze-dried DNA. The chemically synthesized gene fragment was dissolved in nuclease-free water, and then subjected to double digestion with the same restriction enzymes as the pET32a vector. After purification of the digestion products, T4 DNA ligase system was used for ligation at 16°C overnight, to obtain the recombinant plasmid pET32a-PtALF-8.

[0023] The ligation product was transformed into chemically competent E. coli DH5a, and plated on LB solid medium containing kanamycin and incubated at 37°C for 12-16 h. Single colonies were picked for colony PCR and digestion identification to confirm that the target insert 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. The recombinant plasmid confirmed by sequencing to be free of mutations and correctly ligated was used as the subsequent expression plasmid, and was transformed into ArcticExpress(DE3)pRARE2 for protein expression.

[0024] Example 2 This example used 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 containing kanamycin (final concentration 50 μg / mL) and incubated at 37°C, 180 rpm for 8 h as a primary seed solution. Then 200 μL of the primary seed solution was transferred to a flask containing 60 mL of LB and kanamycin, and incubated at 28°C, 150 rpm for 16 h to obtain a secondary seed solution. The secondary seed solution 60 mL was inoculated into 900 mL of LB medium (containing the same resistance), and shaken at 28°C, 150 rpm until the OD600 reached 0.7-0.8. Then 1 MIPTG 0.5 mL was added to make the final concentration 0.7 mM, and the expression was continued at 28°C, 150 rpm for 4.5 h, or at 37°C, 200 rpm overnight. After induction, the bacterial cells were collected by centrifugation at 7500 rpm, 20°C for 15 min, and 1 L of culture could obtain about 3.7-4.0 g of wet bacterial cells.

[0025] The collected cells were resuspended in 80 mL of lysis buffer and lysed by high pressure homogenization at 750-850 bar for three times with 4°C. The lysate was centrifuged at 8000 rpm for 30 min at 4°C to separate the supernatant and the precipitate. The precipitate was resuspended in 40 mL of solubilization buffer at 4°C overnight and used as the inclusion body sample. The solubilized precipitate sample was then loaded onto a 5 mL Ni-NTA column equilibrated with binding buffer, and the flow-through was collected. Elution was performed using elution buffer containing 20 mM, 50 mM, 100 mM, 250 mM, 500 mM and 1000 mM imidazole, respectively, and the elution fractions were collected. The 100 mM imidazole elution peak contained the main target protein. After elution, the column was washed with column treatment solution and stored with 20% ethanol.

[0026] The 100 mM imidazole elution fraction with high concentration was combined, and L-arginine, EDTA and DTT were added to the system to achieve a final concentration of 1%, 5 mM and 5 mM, respectively. After standing at 4°C for 30 min, the urea gradient dialysis was performed. The dialysis buffer was 20 mM Tris-HCl (pH 8.0) containing 10% glycerol and 0.02% sodium azide, and the urea concentration was 8 M, 6 M, 4 M, 2 M and 0 M, respectively, for 2-4 h at each level. After dialysis, the protein was further concentrated using an ultrafiltration tube, and the protein concentration was determined. Finally, about 5.68 mg / mL of refolded protein was obtained, which could be stored at -20°C or -80°C.

[0027] To verify the expression, 200 μL of the fermentation broth was centrifuged and resuspended, and an equal volume of 2x SDS lysis buffer was added, and the sample was treated at 100°C for 5 min and then loaded onto SDS-PAGE. An obvious expression band with a molecular weight of about 26.3 kDa was observed in the gel Figure 1 , indicating that the anti-lipopolysaccharide factor protein PtALF-8 was successfully expressed and could be purified by the above method.

[0028] Example 3: In vitro antibacterial test of Portunus trituberculatus PtALF-8 protein 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.

[0029] 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. 3 CFU / 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.

[0030] 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.

[0031] 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.

[0032] The above examples are only used to illustrate the technical solutions of the present application, but not to limit the present application; although the present application has been described in detail with reference to the foregoing examples, the technical solutions recorded in the foregoing examples can still be modified by those of ordinary skill in the art, or some technical features thereof can be replaced by equivalent replacements; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions claimed by the present application.

Claims

1. A gene encoding an anti-lipopolysaccharide factor from the swimming crab *Portunus trituberculatus*. PtALF-8 Genes are characterized by: The PtALF-8 The nucleotide sequence of the gene is shown in SEQ ID No.

1.

2. A protein PtALF-8, an anti-lipopolysaccharide factor from the swimming crab *Portunus trituberculatus*, characterized in that: The anti-lipopolysaccharide protein PtALF-8 of the swimming crab described in claim 1 is... PtALF-8 The gene is encoded, and its amino acid sequence is shown in SEQ ID No.

2.

3. The anti-lipopolysaccharide factor protein PtALF-8 of the swimming crab according to claim 2, 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*.

4. A recombinant plasmid, characterized in that: The recombinant plasmid contains the contents of claim 1. PtALF-8 Gene.

5. A recombinant engineered bacterium, characterized in that: The recombinant engineered bacteria contain the contents described in claim 1. PtALF-8 Gene.

6. The claim 1 PtALF-8 The use of the gene or the anti-lipopolysaccharide factor protein PtALF-8 of the swimming crab as described in claim 2 in the preparation of an antibacterial agent that inhibits Gram-negative and Gram-positive bacteria.

7. The application according to claim 6, characterized in that: The Gram-negative bacteria are Vibrio parahaemolyticus, and the Gram-positive bacteria are Staphylococcus aureus.

8. The application according to claim 6, characterized in that: The concentration range for the anti-lipopolysaccharide factor protein PtALF-8 is 0.05 mg / mL to 2.0 mg / mL.

9. The claim 1 PtALF-8 The application of the gene or the anti-lipopolysaccharide factor protein PtALF-8 of the swimming crab as described in claim 2 in the preparation of aquatic animal feed additives.

10. The application according to claim 9, characterized in that: The aquatic animals include swimming crabs and prawns.

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