Portunus trituberculatus anti-lipopolysaccharide factor coding gene PtALF-7 as well as protein and application thereof

Abstract

The invention discloses a portunus trituberculatus anti-lipopolysaccharide factor coding gene PtALF-7 as well as a protein and application thereof, and belongs to the technical field of molecular biology. The nucleotide sequence of the coding gene PtALF-7 is as shown in SEQ ID No. 1, and the amino acid sequence of the portunus trituberculatus anti-lipopolysaccharide factor obtained by coding of the coding gene PtALF-7 is as shown in SEQ ID No. 2. In-vitro antibacterial experiments prove that the anti-lipopolysaccharide factor generated by encoding the gene PtALF-7 has a remarkable inhibition effect on the growth of vibrio parahaemolyticus and staphylococcus caprae. Therefore, the invention lays an application foundation for disease control, gene-assisted breeding and the like of the portunus trituberculatus, and has good market application value in the aspects of development of antibacterial drugs, production of feed additives and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of molecular biology, and particularly relates to a Portunus trituberculatus antilipopolysaccharide factor coding gene PtALF-7 and a protein and application thereof. BACKGROUND

[0002] Portunus trituberculatus Portunus trituberculatus is an important marine economic animal. Vibrio parahaemolyticus is one of the main pathogens causing acute hepatopancreas necrosis disease of marine crustaceans, and is also the main pathogen causing large-scale death of Portunus trituberculatus, which seriously hinders the healthy development of the industry.

[0003] Antilipopolysaccharide factor (ALF) is an important antibacterial peptide existing in crustaceans, which can recognize and bind to lipopolysaccharide (LPS) of gram-negative bacteria, and play an immune defense function by inhibiting bacterial growth, destroying bacterial membrane structure and regulating host immune response. The ALF family is widely present in marine crustaceans such as shrimps and crabs, and many studies have confirmed that it plays a core role in innate immunity and is an important molecule for maintaining the host's ability to resist infection.

[0004] Research on the antilipopolysaccharide factor gene and the encoded protein has important theoretical and practical significance for understanding the immune defense mechanism of Portunus trituberculatus, disease prevention and control, and disease-resistant variety breeding. SUMMARY

[0005] The present application provides a Portunus trituberculatus antilipopolysaccharide factor coding gene PtALF-7 and a protein and application thereof. The coding gene PtALF-7 produces an antilipopolysaccharide factor protein which has a significant inhibitory effect on the growth of Vibrio parahaemolyticus and Staphylococcus caprae.

[0006] To achieve the above-mentioned application purposes, the present application adopts the following technical solutions: The present application provides a Portunus trituberculatus antilipopolysaccharide factor coding gene PtALF-7 , and the nucleotide sequence thereof is shown as SEQ ID No. 1.

[0007] The present application further provides a Portunus trituberculatus antilipopolysaccharide factor protein, which is encoded by the coding gene PtALF- 7 , and the amino acid sequence thereof is shown as SEQ ID No. 2.

[0008] Further, the Portunus trituberculatus antilipopolysaccharide factor protein is obtained by the following steps: synthesizing the coding 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.

[0009] The present invention also provides a recombinant plasmid containing the said coding gene. PtALF-7 .

[0010] The present invention also provides a recombinant engineered bacterium containing the aforementioned coding gene. PtALF-7 .

[0011] 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.

[0012] Furthermore, the Gram-negative bacteria are Vibrio parahaemolyticus, and the Gram-positive bacteria are Staphylococcus capsulatum.

[0013] 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.

[0014] 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.

[0015] Furthermore, the aquatic animals include swimming crabs and prawns.

[0016] Compared with existing technologies, the advantages and beneficial technical effects of the present invention are: 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; the application further proves that the antibacterial rate of the anti-lipopolysaccharide factor protein PtALF-7 on Vibrio parahaemolyticus is 91.5%, 74.8%, 60.4% and 17.2% respectively at the concentrations of 1.136 mg / mL, 0.568 mg / mL, 0.284 mg / mL and 0.0568 mg / mL through in-vitro antibacterial experiments. The antibacterial rate of the anti-lipopolysaccharide factor protein PtALF-7 on Staphylococcus caprae is 53.6%, 33.2%, 25.4% and 29.3% respectively at the concentrations of 1.136 mg / mL, 0.568 mg / mL, 0.284 mg / mL and 0.0568 mg / mL, which proves that the anti-lipopolysaccharide factor protein PtALF-7 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-7 The gene and the encoded protein can be used to produce antibacterial drugs, which can be applied to the treatment of related diseases in the breeding process of Portunus trituberculatus, or used for the production of feed additives and preservatives, etc. In addition, the gene and the encoded protein can provide data support for analyzing the immune mechanism of Portunus trituberculatus and provide a theoretical reference for the disease-resistant breeding and disease prevention of Portunus trituberculatus. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Figure 4 is a photograph of the protein PtALF-7 after purification. M: marker; 1: protein after purification through a Ni column before induction; 2: protein after purification through a Ni column after induction.

[0019] Figure 2 Figure 5 is an experimental diagram of the protein PtALF-7 inhibiting Vibrio parahaemolyticus. From left to right, the culture dishes are added with 1.136 mg / mL, 0.568 mg / mL, 0.284 mg / mL and 0.0568 mg / mL of the protein and PBS respectively.

[0020] Figure 3 Figure 6 is an experimental diagram of the protein PtALF-7 inhibiting Staphylococcus caprae. From left to right, the culture dishes are added with 0.0568 mg / mL, 0.284 mg / mL, 0.568 mg / mL and 1.136 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, unless otherwise specified.

[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 (as shown in SEQ ID No. 1) was added with BamH I (GGATCC) and Xho I (CTCGAG) enzyme cutting sites at the 5' end and 3' end respectively to facilitate subsequent vector pET32a construction. After the synthesis was completed, the 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 double-digested with the same restriction enzymes as the pET32a vector, and the digested products were purified and then connected in a T4 DNA ligase system at 16°C overnight to obtain the recombinant plasmid pET32a-PtALF-7.

[0023] The ligation product was transformed into chemically competent E. coli DH5a, spread on LB solid medium containing kanamycin, and incubated at 37°C for 12-16 h. Single colonies were picked for colony PCR and enzyme 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 have no mutation and correct ligation was used as the subsequent expression plasmid and transformed into the host strain ArcticExpress(DE3)pRARE2 for protein expression.

[0024] Example 2 This example used the recombinant plasmid obtained in Example 1 and the host strain ArcticExpress(DE3)pRARE2 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 liquid. Then 200 μL of the primary seed liquid 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 liquid. 60 mL of the secondary seed liquid was inoculated into 900 mL of LB culture medium (containing the same resistance), and shaken at 28°C, 150 rpm until the OD 600 0.7-0.8, 1 MIPTG 0.5 mL was added to make the final concentration 0.7 mM, and then the induction was continued at 28°C, 150 rpm for 4.5 h or at 37°C, 200 rpm overnight. After the induction was completed, the bacterial cells were collected by centrifugation at 7500 rpm, 20°C for 15 min, and about 3.7-4.0 g of wet bacterial cells were obtained from 1 L of culture.

[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, and the supernatant and precipitate were separated. The precipitate was resuspended in 40 mL of solubilization buffer at 4 °C overnight and used as the inclusion body protein 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 corresponding 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 a 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, urea gradient dialysis was performed. During dialysis, 8 M, 6 M, 4 M, 2 M and 0 M urea systems were used in sequence, and each dialysis lasted for 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 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. The sample was treated at 100 °C for 5 min and 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 recombinant protein PtALF-7 was successfully expressed and could be purified by the above method.

[0028] Example 3: In vitro antibacterial test of Portunus trituberculatus anti-lipopolysaccharide factor protein PtALF-7 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.

[0029] 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.

[0030] 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.

[0031] 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.

[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 anti-lipopolysaccharide factor of Portunus trituberculatus PtALF-7 characterized in that: The nucleotide sequence of the coding gene PtALF-7 is shown in SEQ ID No.

1.

2. A Portunus trituberculatus anti-lipopolysaccharide factor protein, characterized in that: The anti-lipopolysaccharide factor protein of Portunus trituberculatus is encoded by the gene in claim 1 PtALF-7 The amino acid sequence of the encoded protein is shown as SEQ ID No.

2.

3. The Portunus trituberculatus anti-lipopolysaccharide factor protein of claim 2, characterized in that: The anti-lipopolysaccharide factor protein of Portunus trituberculatus is prepared by the following steps: synthesizing a nucleotide sequence of the coding gene PtALF-7 , adding enzyme cutting sites at the 5' end and 3' end respectively; cutting the synthesized gene sequence, inserting into a plasmid to construct a recombinant plasmid; transforming the recombinant plasmid into an engineering bacterium to construct a recombinant engineering bacterium; culturing the recombinant engineering bacterium, crushing, and then separating, washing and purifying to obtain the anti-lipopolysaccharide factor protein of Portunus trituberculatus.

4. A recombinant plasmid, characterized by: The recombinant plasmid contains the coding gene of claim 1 PtALF- 7 .

5. A recombinant engineered bacterium, characterized in that: The recombinant engineering bacteria contain the coding gene of claim 1 PtALF-7 .

6. The coding gene of claim 1 PtALF-7 or use of the Portunus trituberculatus anti-lipopolysaccharide factor protein of claim 2 in the preparation of an antibacterial agent for inhibiting gram-negative bacteria and gram-positive bacteria.

7. Use according to claim 6, characterized in that: The gram-negative bacteria is Vibrio parahaemolyticus, and the gram-positive bacteria is Staphylococcus caprae.

8. Use according to claim 6, characterized in that: The concentration of the anti-lipopolysaccharide factor protein of Mithrax spinosus is 0.05 mg / mL to 1.5 mg / mL.

9. The coding gene of claim 1 PtALF-7 or the use of the recombinant protein of the anti-lipopolysaccharide factor of Portunus trituberculatus of claim 2 in the preparation of feed additives for aquatic animals.

10. Use according to claim 9, characterized in that: The aquatic animals include Mithrax spinosus and Penaeus.

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