Antibacterial peptide SMGAP from scophthalmus maximus, fusion type antibacterial peptide and application of antibacterial peptide SMGAP and fusion type antibacterial peptide

Abstract

The invention provides a scophthalmus maximus-derived antibacterial peptide SMGAP. The amino acid sequence of the scophthalmus maximus-derived antibacterial peptide SMGAP is as shown in SEQ ID NO: 1. The invention provides a scophthalmus maximus-derived fused antibacterial peptide. The amino acid sequence of the scophthalmus maximus-derived fused antibacterial peptide is as shown in SEQ ID NO: 2. The invention provides application of the scophthalmus maximus-derived antibacterial peptide SMGAP or the fusion type antibacterial peptide in preparation of an antibacterial product, and in the antibacterial product, the antibacterial peptide SMGAP or the fusion type antibacterial peptide is an active component. The antibacterial peptide SMGAP disclosed by the invention has an obvious effect of inhibiting the growth of Edwardsiella piscicida EIB202, Salmonella Typhimurium SL1344, and Escherichia coli O157: H7EDL933, and the antibacterial peptide SMGAP disclosed by the invention has an obvious effect of inhibiting the growth of the Edwardsiella piscicida EIB202, the Salmonella Typhimurium SL1344 and the Escherichia coli O157: H7EDL933.

Description

Technical Field

[0001] The invention belongs to the technical field of antimicrobial peptides, and particularly relates to an antimicrobial peptide SMGAP derived from turbot, a fusion antimicrobial peptide and applications thereof. Background Art

[0002] Turbot (Scophthalmus maximus), also known as turbot, is a fish of the genus Turbot of the family Cyprinidae. As an important marine farmed fish, it plays an important role in the development of fisheries in my country. However, the frequent occurrence of fish diseases in recent years has become a major pain point restricting its development. In the field of aquaculture disease prevention and control technology, antibiotics have been widely used as a drug with significant efficacy against pathogens, but the massive abuse of antibiotics has also created new difficulties for aquaculture. Given that the new vaccine development process is slow and new vaccine targets need to be continuously developed, antimicrobial peptides naturally present in fish as a bactericidal substance have become a research hotspot to replace antibiotics.

[0003] As a new type of antibacterial substance, antimicrobial peptides exist naturally in organisms and are one of the important components of the nonspecific immune defense system in animals. They play an important role in defending against the invasion of pathogens. They have the advantages of small molecular weight, broad antibacterial spectrum, no toxicity or cell damage to the host, and have good medicinal prospects. In 1972, Swedish scientist Boman and others first discovered antimicrobial peptides in fruit flies and identified their immune and bactericidal functions. Since then, at least more than a thousand antimicrobial peptides have been found in various species.

[0004] Lipids are important substances in the metabolism of organisms, and their storage place, lipid droplets, is not only an organelle for storing lipids, but also an important hub connecting cell metabolism and natural immunity, and is widely involved in host anti-infection and inflammatory responses. It actively participates in the innate immune response to support the cell's antimicrobial defense, mediates the interaction between pathogens and hosts, and also changes the host's metabolism during the infection process. Therefore, it is of great significance to study the role of lipid droplets in pathogen-host competition. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides an antimicrobial peptide SMGAP derived from turbot, a fusion antimicrobial peptide and applications thereof.

[0006] In order to solve the above technical problems, the first aspect of the present invention provides an antimicrobial peptide SMGAP from turbot, the main feature of which is that the amino acid sequence is shown in SEQ ID NO: 1. The present invention is an antimicrobial peptide SMGAP screened from the turbot proteome. The nucleotide sequence of the gene encoding the antimicrobial peptide SMGAP is shown in SEQ ID NO: 3.

[0007] The second aspect of the present invention provides a fusion antimicrobial peptide derived from turbot, the main characteristics of which are that the amino acid sequence is shown in SEQ ID NO: 2 and the nucleotide sequence is shown in SEQ ID NO: 4.

[0008] The third aspect of the present invention provides the use of the turbot-derived antimicrobial peptide SMGAP or the fusion antimicrobial peptide in the preparation of an antimicrobial product, the main feature of which is that in the antimicrobial product, the antimicrobial peptide SMGAP or the fusion antimicrobial peptide is an active ingredient.

[0009] Preferably, the antibacterial product is an antibacterial product that inhibits Edwardsiella piscicola, Salmonella typhimurium, and Escherichia coli.

[0010] Preferably, the antibacterial product is an antibacterial product for aquatic products.

[0011] Preferably, the antibacterial product is a feed additive, a veterinary drug or a preservative.

[0012] The fourth aspect of the present invention further provides a recombinant expression vector PCDH, into which a nucleic acid fragment encoding a fusion antimicrobial peptide gene is inserted.

[0013] The turbot-derived antimicrobial peptide SMGAP obtained by the present invention has obvious inhibitory effects on the growth of Edwardsiella piscicida EIB202, Salmonella Typhimurium SL1344, and enterohemorrhagic Escherichiacoli O157:H7 EDL933, and can be applied to the preparation of antibacterial products in aquaculture, such as feed additives, veterinary drugs, preservatives, pharmaceutical products, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the nucleic acid sequence and amino acid sequence of the antimicrobial peptide SMGAP.

[0015] Figure 2 Schematic diagram of PCDH-SMGAP-FLAG expression vector.

[0016] Figure 3 This is a detection diagram of the expression of the antimicrobial peptide SMGAP in RAW264.7 cells, wherein lane 1 is the Marker, lane 2 is the RAW264.7 transfected with the SMGAP-fusion peptide-FLAG plasmid, and lane 3 is the untransfected RAW264.7.

[0017] Figures 4 to 6Schematic diagrams of the detection of the effects of the antimicrobial peptide SMGAP on the growth of Edwardsiella piscicida EIB202, Salmonella Typhimurium SL1344, and enterohemorrhagic Escherichia coli O157:H7 EDL933.

[0018] Figure 7 Schematic diagram of the detection of the number of Edwardsiella piscicidaEIB202, Salmonella Typhimurium SL1344, and enterohemorrhagic Escherichia coli O157:H7 EDL933 invading cells after the antimicrobial peptide SMGAP was expressed in RAW264.7 cells.

[0019] Figure 8 Schematic diagram of the detection of the effect of antimicrobial peptide SMGAP on cell proliferation.

[0020] Fig. 9 Schematic diagram for the detection of the cytotoxic effects of the antimicrobial peptide SMGAP. DETAILED DESCRIPTION

[0021] In order to make the technical problem solved by the present invention clearer, the present invention is further described below in conjunction with embodiments and drawings. The specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0022] The antimicrobial peptide SMGAP is located on the lipid droplets, and protein sequence comparison analysis shows that the antimicrobial peptide overlaps with the N-terminus of 3-phosphoglyceraldehyde dehydrogenase in turbot fish, so it is a GAPDH-related antimicrobial peptide. The present invention first identifies the antimicrobial peptide SMGAP on the lipid droplets through proteomics technology, and uses gene function technology to recombinantly express it in cells, studies its intracellular bactericidal effect, and co-incubates it with Edwardsiella piscicida that infects turbot. Its bactericidal effect shows that it has great significance in the research and development and application of actual new antimicrobial drugs.

[0023] Example 1

[0024] Determination of the amino acid sequence of the antimicrobial peptide SMGAP

[0025] Lipid droplets were extracted from turbot (Yantai Development Zone Tianyuan Aquatic Products Co., Ltd.) 7 days after being injected with Edwardsiella piscicida (CCTCC M208068). An amino acid sequence encoding the antimicrobial peptide SMGAP was screened out through its differential peptides. Then, it was compared with the protein sequence of GAPDH by BLAST technology. The following results were obtained by BLAST technology: Figure 1 The nucleotide sequence is shown.

[0026] Example 2

[0027] Construction of expression plasmid for antimicrobial peptide SMGAP

[0028] like Figure 2 As shown, the nucleotide sequences of the antimicrobial peptide and the fusion peptide were synthesized by Qingke Biotechnology Co., Ltd. and integrated into the PCDH-FLAG plasmid, which can express the antimicrobial peptide SMGAP and the fusion peptide in cells.

[0029] The high-concentration plasmid without endotoxin was extracted using the large extraction kit of Novozymes, and then it was transfected with ZETAlife transfection reagent. 8ul of transfection reagent and 8ug of PCDH-SMGAP-fusion peptide-FLAG plasmid were added to a 6-well plate with 1 million cells / well. Its expression was detected by Western Blot technology 48h after transfection. The results are as follows Figure 3 As shown, it showed that the antimicrobial peptide SMGAP had been successfully expressed.

[0030] Example 3

[0031] Antibacterial activity detection of antimicrobial peptide SMGAP

[0032] 0.75 mg / ml of synthetic antimicrobial peptide SMGAP was added to 200 μL of LB medium, and the growth curve of bacteria was measured for 12 hours. The initial bacterial concentration was 10 8 The control group was 200 μL of LB medium without antimicrobial peptide SMGAP. The bacteria selected were Edwardsiella piscicida EIB202, Salmonella TyphimuriumSL1344, and enterohemorrhagic Escherichia coli O157:H7 EDL933. Figures 4 to 6 As shown, the antimicrobial peptide SMGAP of the present invention can significantly inhibit the growth of these three strains.

[0033] Example 4

[0034] The antimicrobial peptide SMGAP verifies its bactericidal function in cells

[0035] The plasmid was transfected with ZETA life transfection reagent. 8ul of transfection reagent and 8ug of PCDH-SMGAP-FLAG plasmid were added to 1 million RAW264.7 cells / well in a 6-well plate. Edwardsiella piscicida EIB202, Salmonella Typhimurium SL1344, enterohemorrhagic Escherichia coli O157:H7 EDL933 were added at a ratio of MOI = 10:1 48h after transfection. 7 bacteria, and add 1ml Opti-MEM medium to each well plate, and infect in an incubator containing 5% CO2 at 35°C. After 2 hours of infection, rinse the cells twice with PBS, and then add 1ml of Opti-MEM medium containing 1000ug / ml gentamicin. After 20 minutes, rinse the cells twice with PBS, and then add 1ml of Opti-MEM medium containing 10ug / ml gentamicin for 4 hours. After 4 hours, rinse the cells twice with PBS, add 500ul of PBS containing 0.5% Triton to each well of cells to lyse the cells, dilute the lysate 2-3 times, take 5ul and drop it on the LB solid culture medium, invert the plate, place it in a 30°C incubator for overnight culture, and then count the number of colonies on the plate by drop plate. The counting results are as follows Figure 7 As shown, the untransfected original RAW264.7 cells were used as the infection control group, indicating that after the cells expressed the antimicrobial peptide SMGAP, the invading cells of Edwardsiella piscicida EIB202, Salmonella Typhimurium SL1344, and enterohemorrhagic Escherichia coli O157:H7 EDL933 were significantly killed.

[0036] Example 5

[0037] Effects of antimicrobial peptide SMGAP on cell proliferation and cytotoxicity

[0038] After adding 0.75 mg / ml SMGAP to 100,000 RAW264.7 cells / well of a 96-well plate, 10 ul of CCK8 reagent was added, and the cells were incubated in a 37°C incubator for 1 hour. The effect of SMGAP on cell proliferation was detected at absorbance A450 nm. The results are shown in Figure 8 As shown, the cells without SMGAP were used as the control group, and the absorbance of the cells with SMGAP did not change significantly, indicating that the antimicrobial peptide SMGAP of the present invention did not affect the cell proliferation ability.

[0039] After adding 0.75 mg / ml SMGAP to 100,000 RAW264.7 cells / well of a 96-well plate, 20 ul of neutral red staining solution was added, and the cells were incubated in a 37°C incubator for 2 hours. The cells were rinsed twice with PBS, and then 200 ul / well of neutral red lysis solution was added. The cells were stained on a shaker for 10 minutes, and then the effect of SMGAP on cytotoxicity was detected at absorbance A690 nm. The results are shown in Figure 2. Fig. 9 As shown, the cells without SMGAP were used as the control group, and the absorbance of the cells with SMGAP did not change significantly, indicating that the antimicrobial peptide SMGAP of the present invention did not affect cytotoxicity.

[0040] The above description is a specific embodiment of the present invention and does not constitute a limitation on the protection scope of the present invention. Any modification and variation made to the technical concept of the present invention should be included in the protection scope of the present invention.

Claims

1. An antimicrobial peptide SMGAP derived from turbot, characterized in that: The amino acid sequence is shown in SEQ ID NO:

1.

2. A fusion antimicrobial peptide derived from turbot, characterized in that: The amino acid sequence is shown in SEQ ID NO:

2.

3. Use of the antimicrobial peptide SMGAP derived from turbot according to claim 1 or the fusion antimicrobial peptide according to claim 2 in the preparation of antimicrobial products, characterized in that: In the antibacterial product, the antibacterial peptide SMGAP or fusion antibacterial peptide is an active ingredient.

4. The use according to claim 3, characterized in that: The antibacterial product is an antibacterial product that inhibits Edwardsiella piscicola, Salmonella typhimurium and Escherichia coli.

5. The use according to claim 3, characterized in that: The antibacterial product is an antibacterial product for aquatic products.

6. The use according to claim 3, characterized in that: The antibacterial product is a feed additive, a veterinary drug or a preservative.

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