Antibacterial peptide smgap derived from scophthalmus maximus, fused antibacterial peptide and application thereof

Abstract

The application provides an antibacterial peptide SMGAP derived from Scophthalmus maximus, and the amino acid sequence is shown as SEQ ID NO:1. The application provides a fusion antibacterial peptide derived from Scophthalmus maximus, and the amino acid sequence is shown as SEQ ID NO:2. The application provides application of the antibacterial peptide SMGAP derived from Scophthalmus maximus or the fusion antibacterial peptide in preparation of an antibacterial product, wherein the antibacterial peptide SMGAP or the fusion antibacterial peptide is an active ingredient. The antibacterial peptide SMGAP has obvious effects of inhibiting growth of Edwardsiella piscicida EIB202, Salmonella Typhimurium SL1344 and enterohemorrhagic Escherichia coli O157:H7 EDL933.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of antibacterial peptides, and particularly relates to an antibacterial peptide SMGAP from Scophthalmus maximus, a fusion type antibacterial peptide and application thereof. BACKGROUND

[0002] Scophthalmus maximus, also known as turbot, is a kind of fish of Scophthalmidae, and plays an important role in the development of fishery in China as an important marine aquaculture fish. However, frequent fish diseases in recent years have become a major obstacle to its development. In the field of disease prevention and treatment technology of aquaculture, antibiotics have been widely used as a drug with significant effect on pathogenic bacteria. However, the large-scale abuse of antibiotics has also caused new difficulties for aquaculture. In view of the slow development process of new vaccines and the need to continuously research new vaccine targets, the antibacterial peptide naturally existing in fish body has become a research hotspot as a bactericidal substance instead of antibiotics.

[0003] As a new antibacterial substance, the antibacterial peptide naturally exists in organisms, and is one of the important components of the non-specific immune defense system in the animal body, and plays an important role in defending against pathogenic bacteria. It has the advantages of small molecular weight, wide antibacterial spectrum, no toxicity or cell damage to the host, and has good prospects for medical use. In 1972, Swedish scientist Boman et al. first discovered antibacterial peptides in fruit flies, and identified their immune bactericidal function. Since then, more than a thousand antibacterial peptides have been discovered in various species.

[0004] Lipids are important substances for metabolism of organisms, and the lipid droplet, which is the storage site of lipids, is not only a lipid storage organelle, but also an important hub connecting cell metabolism and innate immunity, and widely participates in host anti-infection and inflammatory response. It actively participates in the innate immune response to support the antibacterial defense of cells, mediates the interaction between pathogens and hosts, and also changes the metabolism of the host during infection. Therefore, it is of great significance to study the role of lipid droplets in pathogen-host competition. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application provides an antibacterial peptide SMGAP from Scophthalmus maximus, a fusion type antibacterial peptide and application thereof.

[0006] To solve the above technical problems, the first aspect of the present application provides an antibacterial peptide SMGAP from Scophthalmus maximus, and the main feature is that the amino acid sequence is shown as SEQ ID NO: 1. The present application is a Scophthalmus antibacterial peptide SMGAP screened from the proteome of Scophthalmus maximus. The nucleotide sequence of the gene encoding the antibacterial peptide SMGAP is shown as SEQ ID NO: 3.

[0007] The second aspect of the present application provides a scophthalmus maximus-derived fusion antibacterial peptide, which is mainly characterized by an amino acid sequence as shown in SEQ ID NO: 2. Its nucleotide sequence is as shown in SEQ ID NO: 4.

[0008] The third aspect of the present application provides an application of the scophthalmus maximus-derived antibacterial peptide SMGAP or the fusion antibacterial peptide in preparing an antibacterial product, which is mainly characterized in that the antibacterial peptide SMGAP or the fusion antibacterial peptide is an active ingredient in the antibacterial product.

[0009] Preferably, the antibacterial product is an antibacterial product for inhibiting Edwardsiella tarda, 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 application further provides a recombinant expression vector PCDH, wherein a nucleic acid fragment encoding a fusion antibacterial peptide gene is inserted into the recombinant expression vector.

[0013] The scophthalmus maximus-derived antibacterial peptide SMGAP obtained by the present application has a significant effect of inhibiting the growth of Edwardsiella tarda EIB202, Salmonella typhimurium SL1344 and enterohemorrhagic Escherichia coli O157:H7 EDL933, and can be applied to the aquaculture industry to prepare antibacterial products, such as feed additives, veterinary drugs, preservatives, medical products, etc. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 It is an antibacterial peptide SMGAP nucleic acid sequence and amino acid sequence diagram.

[0015] Figure 2 It is a PCDH-SMGAP-FLAG expression vector schematic diagram.

[0016] Figure 3 It is an antibacterial peptide SMGAP expression detection diagram in RAW264.7 cells, wherein lane 1 is Marker, lane 2 is RAW264.7 transfected with SMGAP-fusion peptide-FLAG plasmid, and lane 3 is untransfected RAW264.7.

[0017] Figures 4 to 6The schematic diagram for detecting the influence of antibacterial peptide SMGAP on the growth of Edwardsiella piscicida EIB202, Salmonella Typhimurium SL1344 and enterohemorrhagic Escherichia coli O157:H7 EDL933, respectively.

[0018] Figure 7 The schematic diagram for detecting the influence of antibacterial peptide SMGAP expressed in RAW264.7 cells on the invasion number of Edwardsiella piscicida EIB202, Salmonella Typhimurium SL1344 and enterohemorrhagic Escherichia coli O157:H7 EDL933 in cells.

[0019] Figure 8 The schematic diagram for detecting the influence of antibacterial peptide SMGAP on cell proliferation.

[0020] Figure 9 The schematic diagram for detecting the influence of antibacterial peptide SMGAP on cell toxicity. DETAILED DESCRIPTION

[0021] In order to make the technical problems solved by the present application clearer, the present application is further described below in combination with examples and drawings. The specific examples described herein are only used to explain the present application and do not limit the present application.

[0022] The antibacterial peptide SMGAP is located on the lipid droplet, and the protein sequence alignment analysis shows that the antibacterial peptide is coincident with the N-terminal of 3-phosphoglyceraldehyde dehydrogenase in the body of Scophthalmus maximus, and thus is a GAPDH-related antibacterial peptide. The present application first identifies the antibacterial peptide SMGAP on the lipid droplet through proteomics technology, and uses gene function technology to perform recombinant expression in cells, studies the bactericidal effect thereof in cells, and co-incubates with Edwardsiella piscicida infecting Scophthalmus maximus, and the bactericidal effect thereof shows that it has great significance in the research and application of actual novel antibacterial drugs.

[0023] Example 1

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

[0025] The lipid droplets were extracted from the body of Scophthalmus maximus (Yantai Development Zone Tianyuan Aquatic Products Co., Ltd.) which was attacked by injected Edwardsiella piscicida (CCTCC M208068) for 7 days, and a segment of amino acid sequence encoding antibacterial peptide SMGAP was screened through differential peptide segments, and the nucleotide sequence as shown in Figure 1 was obtained by comparing the protein sequence of GAPDH through BLAST technology.

[0026] Example 2

[0027] Construction of expression plasmid of antibacterial peptide SMGAP

[0028] As shown in Figure 2 , the nucleotide sequence of antibacterial peptide and fusion peptide was synthesized by GenScript Biotech Corporation and integrated into PCDH-FLAG plasmid, and the plasmid can express antibacterial peptide SMGAP and fusion peptide in cells.

[0029] High-concentration plasmid without endotoxin was extracted by using Qiaprep Spin Miniprep Kit of Qiagen, and then transfected by using ZETA life transfection reagent, 8ul transfection reagent and 8ug PCDH-SMGAP-fusion peptide-FLAG plasmid were added into 6-well plate with 1 million cells per well, and the expression was detected by using Western Blot after 48h of transfection, and the results are shown in Figure 3 , which shows that antibacterial peptide SMGAP has been successfully expressed.

[0030] Example 3

[0031] Detection of antibacterial activity of antibacterial peptide SMGAP

[0032] 0.75mg / ml of synthesized antibacterial peptide SMGAP was added into 200ul of LB medium, and the growth curve of bacteria was determined for 12 hours, the initial concentration of bacteria was 10 8 / ml, i.e. 0.1 OD, and the control group was 200ul of LB medium without antibacterial peptide SMGAP. The bacteria selected were Edwardsiella piscicida EIB202, Salmonella Typhimurium SL1344 and enterohemorrhagic Escherichia coli O157:H7 EDL933. As shown in Figures 4 to 6 , the antibacterial peptide SMGAP of the application can significantly inhibit the growth of the three strains.

[0033] Example 4

[0034] Intracellular verification of antibacterial function of antibacterial peptide SMGAP

[0035] The plasmid was transfected with ZETA life transfection reagent, 8ul transfection reagent and 8ug PCDH-SMGAP-FLAG plasmid were added to 6-well plates with 1 million RAW264.7 cells per well, and 48h after transfection, Edwardsiella piscicida EIB202, Salmonella Typhimurium SL1344, enterohemorrhagic Escherichia coli O157:H7 EDL933 were added at a ratio of MOI = 10:1, i.e. 10 7 bacteria were added, and 1ml of Opti-MEM medium was added to each well plate, and the infection was carried out in an incubator containing 5% CO2, with an infection temperature of 35℃. After 2 hours of infection, the cells were washed twice with PBS, then 1ml of Opti-MEM medium containing 1000ug / ml of gentamicin was added, and after 20 minutes of action, the cells were washed twice with PBS, and then 1ml of Opti-MEM medium containing 10ug / ml of gentamicin was added, and the action was carried out for 4 hours. After 4 hours, the cells were washed twice with PBS, 500ul of PBS containing 0.5% Triton was added to each well of cells to lyse the cells, the lysate was diluted 2-3 times, 5ul was dropped onto LB solid medium, the plate was inverted and placed in a 30℃ incubator for overnight culture, and then the number of colonies on the plate was counted by plate counting. The counting results are shown in Figure 7 Edwardsiella piscicida EIB202, Salmonella Typhimurium SL1344, enterohemorrhagic Escherichia coli O157:H7 EDL933 invading the cells were significantly killed after the cells expressed the antibacterial peptide SMGAP.

[0036] Example 5

[0037] Effect of antibacterial peptide SMGAP on cell proliferation and cytotoxicity

[0038] After adding 0.75mg / ml of SMGAP to 10 million RAW264.7 cells per well in a 96-well plate, 10ul of CCK8 reagent was added, and after incubation in a 37℃ incubator for 1 hour, the effect of SMGAP on cell proliferation was detected at absorbance A450nm. The results are shown in Figure 8 The absorbance of cells added with SMGAP did not change significantly, indicating that the antibacterial peptide SMGAP of the present application did not affect the cell proliferation ability.

[0039] After adding 0.75mg / ml SMGAP to 100,000 RAW264.7 cells per well in a 96-well plate, 20ul neutral red staining solution was added, and after incubation in a 37℃ incubator for 2 hours, the cells were washed twice with PBS, then 200ul / well neutral red lysis solution was added, and the cells were stained on a shaker for 10 minutes, then the effect of SMGAP on cytotoxicity was detected at absorbance A690nm. The results are shown in Table 1, with cells without the addition of SMGAP as the control group, and the absorbance of cells with the addition of SMGAP did not change significantly, indicating that the antibacterial peptide SMGAP of the present application does not affect cytotoxicity. Figure 9

[0040] The above describes specific embodiments of the present application and does not constitute a limitation on the scope of protection of the present application. Any modifications and variations made on the technical concept of the present application shall be included within the scope of protection of the present application.​

Claims

1. An antibacterial peptide SMGAP derived from Scophthalmus maximus, characterized in that, The amino acid sequence is shown as SEQ ID NO:

1.

2. A hybrid antimicrobial peptide derived from Scophthalmus maximus, characterized in that, The amino acid sequence is shown as SEQ ID NO:

2.

3. Use of the Scophthalmus maximus-derived antibacterial peptide SMGAP of claim 1 or the fusion antibacterial peptide of claim 2 for the preparation of an antibacterial product, characterized in that, In the antibacterial product, the antibacterial peptide SMGAP or the fusion antibacterial peptide is an active ingredient, the antibacterial product is an antibacterial product for inhibiting Edwardsiella tarda, Salmonella typhimurium and enterohemorrhagic Escherichia coli, the antibacterial product is an antibacterial product for aquatic products, and the antibacterial product is a feed additive, a veterinary drug or a preservative.

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