Antibacterial peptide and application thereof in juvenile mollusk culture

Abstract

The invention provides an antibacterial peptide and application thereof in juvenile mollusk culture. The amino acid sequence of the provided antibacterial peptide is SEQ ID NO: 2. The antibacterial peptide obtained through screening comes from bivalve shellfish, has small toxic and side effects on the bivalve shellfish, is suitable for playing a role in the bivalve shellfish, and has specificity and pertinence on bivalve shellfish culture water pathogenic bacteria. The amino acid sequence of the provided antibacterial peptide is optimized, the antibacterial activity is high, the stability is high, and the expression efficiency in yeast is high. After high expression of the antibacterial peptide, the microcapsule of which the particle size is suitable for filter feeding of the juvenile mollusks is prepared, so that the problem that the juvenile mollusks cannot feed due to direct splashing to culture water in the prior art is solved, and the problems of low microalgae expression efficiency and low antibacterial peptide content are solved.

Description

Technical Field

[0001] The invention belongs to the technical field of aquaculture, and particularly relates to an antimicrobial peptide and application thereof in juvenile shellfish culture. Background Art

[0002] Bivalve molluscs represented by the Manila clam play an important role in my country's aquaculture. They not only provide high-quality protein, but also play a key role in promoting nutrient circulation in water bodies and maintaining ecological balance with their filter-feeding characteristics. According to the latest Fisheries Statistical Yearbook, my country's annual production of Manila clams has reached more than 3 million tons in 2023. Globally, the total production of Manila clams in countries and regions including the Philippines, South Korea, Japan, Thailand and Vietnam is about 4 million tons.

[0003] The huge scale of aquaculture places higher demands on seed supply and quality. Factory-based seed cultivation has now become an important source of supply for Manila clam seed. In the process of factory-based seed cultivation, the growth of pathogenic microorganisms is one of the biggest threats to juvenile shellfish whose immune system has not yet fully developed, often leading to the annihilation of the shellfish. Moreover, this problem not only exists in the Manila clam seed cultivation process, but also has a wide impact on the entire bivalve seed cultivation industry. Studies have shown that pathogen infection causes nearly one million US dollars in economic losses to the global bivalve seed cultivation industry each year [E. Kunselman, K. Wiggin, RE Dine, et al. Microbial threats and sustainable solutions for molluscan aquaculture [J]. Sustainable Microbiology, 2019, 1 (1)], which seriously hinders the sustainable development of aquaculture. Therefore, there is an urgent need to develop effective antibacterial active substances to specifically control pathogenic microorganisms in shellfish culture ponds, improve the survival rate of Manila clam shellfish, and promote the sustainable development of aquaculture.

[0004] In production, antibiotics are often dissolved in dozens to hundreds of times the oral dose for animals and then sprayed into the water body of juvenile shellfish to inhibit pathogenic microorganisms. However, firstly, filter-feeding shellfish cannot ingest antibiotics dissolved in water, so the effect is minimal. Secondly, large-scale use can easily lead to drug resistance in pathogenic bacteria.

[0005] Antimicrobial peptides are a class of small molecule peptides widely found in animals, plants and microorganisms. As an important component of the innate immune system, they can quickly kill bacteria by destroying the cell membrane of pathogens and have the advantage of not easily inducing drug resistance, showing great potential in replacing antibiotics. In aquaculture, antimicrobial peptides can not only directly inhibit pathogenic microorganisms, but also be used as dietary additives to improve the growth performance of aquatic animals, enhance immune function, and maintain the balance of intestinal flora.

[0006] Although there have been many reports on the application of antimicrobial peptides in aquaculture, there are few reports on filter-feeding bivalve molluscs represented by the Philippine clam. The only reports are that insect-derived heterologous antimicrobial peptides are directly dissolved in the aquaculture water of scallop juveniles or expressed in bait microalgae and then fed to mussel juveniles, but the results are minimal [Uriarte, A. Farías, JC Castilla. Effect of antibiotic treatment during larval development of the Chilean scallop Argopecten purpuratus [J]. Aquacultural Engineering, 2001, 25 (3), 139-147; K. Wang, JL Chu, ZL Hu, et al. Using bait microalga as an oral delivery vehicle of antimicrobial peptide for controlling Vibrio infection in mussels. Fish & Shellfish Immunology, 2023, 136.]. The reasons are: first, antimicrobial peptides from other species may have limited effects in bivalve molluscs or may not be specific to shellfish pathogens; second, the size of the particles filtered by bivalve juveniles is 4-10μm, which can be directly dissolved in the aquaculture water and cannot be ingested by shellfish; third, although the particle size of expression systems such as microalgae is suitable, the expression efficiency is too low. Summary of the invention

[0007] The purpose of the present invention is to provide an antimicrobial peptide and its application in juvenile shellfish culture. The provided antimicrobial peptide is derived from bivalve shellfish itself, is amino acid optimized, and has a specific inhibitory effect on bivalve shellfish pathogens. The present invention also expresses the antimicrobial peptide in a soluble and high-quality manner in Pichia pastoris, and then uses spray drying technology to prepare the antimicrobial peptide into microcapsules with a particle size suitable for bivalve shellfish filtration feeding, and feeds the juvenile shellfish.

[0008] The invention firstly provides an antimicrobial peptide separated from Ruditapes philippinarum, whose amino acid sequence is KWRLLWIRKRLLKRI (SEQ ID NO: 2).

[0009] A specific sequence of the nucleic acid fragment encoding the above antimicrobial peptide is as follows: AAGTGGAGACTGTTATGGATTAGGAAGAGACTGCTAAAGCGTATA (SEQ ID NO: 3).

[0010] The present invention also provides a recombinant expression vector, in which a nucleic acid fragment encoding the antimicrobial peptide is inserted.

[0011] In another aspect, the present invention also provides a recombinant engineering strain, which carries the above-mentioned recombinant expression vector;

[0012] As a specific record of the embodiment, the recombinant engineering strain is a Pichia pastoris engineering strain.

[0013] The present invention also provides the use of the antimicrobial peptide in the preparation of antimicrobial products;

[0014] As a specific description of the embodiment, the product is a microcapsule.

[0015] Another aspect of the present invention further provides a microcapsule, wherein the microcapsule contains the recombinantly expressed antimicrobial peptide;

[0016] One preparation method of the microcapsule is to add water to sodium starch octenyl succinate, carboxymethyl cellulose and recombinantly expressed antimicrobial peptide powder, stir to dissolve, and then spray dry.

[0017] The invention also provides a method for cultivating bivalve juvenile oysters, wherein the method comprises feeding the microcapsules.

[0018] The antimicrobial peptides screened and obtained by the present invention are derived from bivalve molluscs, have less toxic and side effects on molluscs, are suitable for exerting effects in bivalve molluscs, and are specific and targeted to pathogenic bacteria in bivalve mollusc aquaculture water. The provided antimicrobial peptide amino acid sequence is optimized, has strong antibacterial activity, high stability, and high expression efficiency in yeast. After the antimicrobial peptide is highly expressed, it is prepared into microcapsules with a particle size suitable for juvenile shellfish to filter feed, which not only overcomes the problem of the prior art of directly sprinkling it into the aquaculture water body, resulting in the juvenile shellfish being unable to ingest it, but also overcomes the problem of low expression efficiency of microalgae and low content of antimicrobial peptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 : Schematic diagram of the original antimicrobial peptide sequence, optimization principles and the final sequence after optimization.

[0020] Figure 2 :The antibacterial activity of antimicrobial peptides against several common pathogens in bivalve aquaculture water.

[0021] Figure 3 : Preparation process of antimicrobial peptide microcapsules and morphology under scanning electron microscope.

[0022] Figure 4 :Effects of antimicrobial peptide microcapsules on the growth and survival of planktonic Ruditapes philippinarum.

[0023] Figure 5: Effects of antimicrobial peptide immersion, microcapsules, and antimicrobial peptide microcapsules on the growth of Ruditapes philippinarum during the attachment period and the survival rate after Vibrio challenge, including antimicrobial peptide microcapsule 0 mg / g group (CK), antimicrobial peptide immersion group (Core not pelletised), group without antimicrobial peptide microcapsules (Wall granulation), and antimicrobial peptide microcapsule group (Core wall granulation).

[0024] Figure 6 :The effect of antimicrobial peptide microcapsules on the survival rate of Philippine clams challenged with Vibrio philippinarum during the attachment period.

[0025] Figure 7 :The effect of antimicrobial peptide microcapsules on nonspecific immunity of Ruditapes philippinarum.

[0026] Figure 8 :The effect of antimicrobial peptide microcapsules on intestinal microorganisms of Ruditapes philippinarum.

[0027] Fig. 9 :The effect of antimicrobial peptide microcapsules on the gene transcriptome of Philippine clam. DETAILED DESCRIPTION

[0028] The invention separates an antimicrobial peptide from the Philippine clam, and optimizes its length, charge, hydrophobicity, stability and antimicrobial activity through antimicrobial peptide design; then, through Pichia pastoris soluble high expression, an antimicrobial peptide that specifically and significantly inhibits bivalve pathogenic microorganisms is obtained; then, the antimicrobial peptide is prepared into microcapsules with a particle size suitable for bivalve filter feeding by using spray drying technology, so as to reduce microbial diseases in the process of juvenile shellfish culture.

[0029] The present invention is described in detail below in conjunction with embodiments and drawings.

[0030] Example 1 Screening of antimicrobial peptides and sequence optimization

[0031] The original antimicrobial peptide was obtained from the transcriptome of Ruditapes philippinarum by biological information comparison, and its sequence is as follows (SEQ ID NO: 1):

[0032] MLKLTGYIMHNLCFLFLCVLMVDSIPLRGRVDIIEKRIGVVESRMNSDNELAREMFLSLQQNFEDLNSSMINLKNQMCPIMDQCGVKEDQSDSEKNVEILQSSRVMMTLGLKREKQWVRDQVKKLFTQMGEHSDRLDQVTKMESDAYIEINKACMVMSSQQ KEIEALKETRTNLLNALISKNEEKYDYTCPPDWKKYSSFCYLFSEDIMKFDEARAYCKLLGATVADFKNQAENDVIADLPKVLRFAWIGYSDEEKEGTWISERTGQPASFTNFADGQPSGGIHQNCAVLHFYNGKWHDWTCHSGGKWHVVCKKDATIEFKL. like Figure 1 As shown, firstly, a peptide segment with possible antibacterial activity was preliminarily obtained from the above amino acid sequence, and KWRLIAIRKRLLKAI was obtained. Further, the series was optimized to improve the antibacterial effect and stability.

[0033] The optimization principles are as follows: ensure that the amino acid sequence has an amphipathic α-helical structure (with both hydrophilic and hydrophobic regions), the peptide has a positive charge (containing lysine [K], arginine [R]), and the length is controlled at 15-25 amino acids (using efficient expression). Subsequently, the positive charge of the peptide is increased. The optimization principle is: to enhance the positive charge and improve the binding ability to the bacterial membrane, the hydrophobic amino acids (such as A) are replaced with positively charged lysine (K) or arginine (R). The specific operation is to replace the "A" at the 14th position with "R" to obtain KWRLIAIRKRLLKRI. Then increase the hydrophobic amino acids (such as leucine [L], isoleucine [I], valine [V]), and the specific operation is to replace the "I" at the 5th and 7th positions with "L" to obtain KWRLLALRKRLLKRI. Finally, enhance the stability of the peptide to proteases and introduce residues reasonably. The specific operation is to replace the "A" at position 6 with "W" to obtain KWRLLWIRKRLLKRI (SEQ ID NO: 2).

[0034] The coding nucleotide sequence of the antimicrobial peptide KWRLLWIRKRLLKRI (SEQ ID NO: 2) was optimized for yeast-preferred codons to obtain the nucleotide sequence AAGTGGAGACTGTTATGGATTAGGAAGAGACTGCTAAAGCGTATA (SEQ ID NO: 3), and primers with restriction sites were designed to recombinant the antimicrobial peptide nucleic acid sequence into the pPICZ vector, electrotransformed into the Pichia pastoris GS115 strain, and coated on a YPDS culture plate containing Zeocin to screen for positive strains. After sequencing confirmed that the sequence of the positive strain was correct, the positive clone was picked and cultured in a methanol induction medium (BMMY) at 28°C and 250rpm for 30 hours to a concentration of 1OD, and 1% methanol was added for induction for 5 days. Dextrin was added as a carrier (500g / L) to the antimicrobial peptide expression yeast solution and dried at 60°C to form an antimicrobial peptide powder.

[0035] The antimicrobial peptides prepared by the above recombinant expression were tested for their inhibitory effects on several common pathogens in juvenile spat culture water. Figure 2 As shown, insect antimicrobial peptides and antimicrobial peptides from the Philippine clam before optimization were set as controls. The optimized antimicrobial peptides of the present invention showed strong inhibitory effects on Vibrio parahaemolyticus, Vibrio alginolyticus and Vibrio splendidus; while the antimicrobial peptides from the Philippine clam before optimization only had inhibitory effects on Vibrio parahaemolyticus and Vibrio splendidus, and the inhibitory effect on Vibrio splendidus was lower than that of the optimized antimicrobial peptides. In addition, insect antimicrobial peptides only had inhibitory effects on Vibrio parahaemolyticus, and the effect was slightly weaker than that of the antimicrobial peptides from the Philippine clam.

[0036] according to Figure 3 The microcapsules were prepared according to the antimicrobial peptide microcapsule preparation process in the literature. The specific steps are as follows:

[0037] Take 40g sodium starch octenyl succinate, 10g carboxymethyl cellulose, 250g of the above antimicrobial peptide powder, add 1.3L water, stir to dissolve, and spray dry. The spray drying air inlet temperature is 168°C and the air outlet temperature is 88°C. Collect 250g of antimicrobial peptide microcapsules and store at room temperature for later use.

[0038] The antimicrobial peptide microcapsules prepared in this example were fed to planktonic spats of Ruditapes philippinarum: the density was 362 g planktonic spats / pond (50 cubic meters of water), the salinity was 20-22 ppt, and the water temperature was 28.5-30.6° C. Set: 0 mg / g (antimicrobial peptide microcapsules / spa), 0.07 mg / g, 0.14 mg / g, 0.21 mg / g, 4 treatment groups, 3 ponds (n=3) for each concentration, the water was changed every 2 days, the weight of the clams in each pond was recorded, and the samples were counted and the shell length was measured at the end (after the attachment began).

[0039] The results are as follows Figure 4After 17 days of feeding with antimicrobial peptide microcapsules, the weight of the 0.07 mg / g group increased by about 25% compared with the control group ( Figure 4 A), weight gain rate increased by 100% ( Figure 4 B). Further analysis of juvenile shell length, shell width and survival rate showed that 0.07 and 0.14 mg / g of antimicrobial peptide microcapsules significantly increased the survival rate of juvenile shellfish during the planktonic period ( Figure 4 E), but has little effect on shell length and shell width ( Figure 4 C and D).

[0040] Example 2: Preparation of antimicrobial peptide microcapsules

[0041] The preparation method of antimicrobial peptide microcapsules is the same as that in Example 1, and microcapsules without antimicrobial peptides are prepared at the same time: 200 g sodium starch octenyl succinate and 50 g carboxymethyl cellulose are added with 1.3 L of water, stirred to dissolve, and then spray dried. The spray drying air inlet temperature is 168°C and the air outlet temperature is 88°C. Finally, the microcapsules are collected and stored for later use.

[0042] A 0 mg / g antimicrobial peptide microcapsule group (CK), an antimicrobial peptide soaked group (Core not pelletised), a group without antimicrobial peptide microcapsule (Wall granulation), and an antimicrobial peptide microcapsule group (Core wall granulation) were set up. The juvenile Manila clam spat was fed with 2 g juvenile spat / bucket (35 L water body). The initial specifications of the juvenile spat were: shell length 7.125±0.06 mm, weight 67±0.94 mg, culture salinity 25-30 ppt, water temperature 25-30 ℃, 4 buckets (n=4) for each treatment, and the weight of each bucket was recorded every 7 days. Samples were taken for weighing and shell length measurement after 17 days of culture.

[0043] The results are as follows Figure 5 Compared with the control group, the soaking group and the group without antimicrobial peptide microcapsules, the antimicrobial peptide microcapsules significantly increased the total weight and single weight ( Figure 5 A and Figure 5 B). After the culture was completed, the Manila clam was challenged with poison and the survival rate was found to be the antimicrobial peptide microcapsule group > antimicrobial peptide soaking group > control group, and the group without antimicrobial peptide microcapsule ( Figure 5 E). These results indicate that only microcapsules containing antimicrobial peptides can promote the growth and survival of Manila clams, while microcapsules directly soaked in antimicrobial peptides or without antimicrobial peptides have no relevant effects.

[0044] Example 3: Preparation of antimicrobial peptide microcapsules

[0045] The preparation method of antimicrobial peptide microcapsules is the same as that in Example 1.

[0046] The conditions for feeding antimicrobial peptide microcapsules to Philippine clams are as follows: density: 2 g juvenile shellfish / bucket (35 L water body), specifications: shell length 1.34 ± 0.15 mm, weight 0.72 ± 0.21 mg, salinity: 25-30 ppt, water temperature: 20-25 ° C, setting: 0 mg / g (antimicrobial peptide microcapsules / shellfish), 0.1 mg / g, 0.5 mg / g, 1 mg / g, 3 mg / g, a total of 5 groups, each group (concentration) 4 buckets (n = 4), weigh and record the mass of each group of Philippine clams every 7 days, and take samples for weighing and measuring shell length after 17 days.

[0047] The results showed that 0.1 mg / g antimicrobial peptide microcapsules could improve the survival rate of Philippine clams infected with Vibrio philippinarum ( Figure 6 E). 0.1 mg / g antimicrobial peptide microcapsules significantly increased CAT and SOD activities, while reducing MDA content, thereby improving the antioxidant capacity of juvenile spat; feeding antimicrobial peptide microcapsules can increase ACP, AKP, and LZM activities ( Figure 7 ).

[0048] Further analysis of the intestinal microorganisms of the Philippine clam revealed that the Chao1 and Shannon indexes of the intestinal microorganisms after feeding with antimicrobial peptide microcapsules were significantly higher than those in the control group (p < 0.05), and PCOA analysis showed that there were significant differences in the community composition between the two groups; the dominant bacteria at the phylum level of the two groups were Proteobacteria, Firmicutes, and Bacteroidota, among which the abundance of Proteobacteria in the control group was higher than that in the antimicrobial peptide microcapsule group, while the abundance of Firmicutes was higher in the antimicrobial peptide microcapsule group; among them, Tenacibaculumn, as a type of Proteobacteria, is widely considered to be a pathogenic bacterium in aquatic animals. The results showed that pathogenic bacteria such as vibrio and waddia appeared in the control group, both of which belong to the Proteobacteria phylum, while the antimicrobial peptide microcapsule group was more likely to have Lysinibacillus, Solibacillus_silvestris, etc., which are considered to be potential probiotics in aquatic animals. In summary, antimicrobial peptides may promote growth and improve immunity by inhibiting pathogenic bacteria in the intestine of the Philippine clam and increasing probiotics in the intestine ( Figure 8 ).

[0049] Further analysis of the transcriptome of the Philippine clam revealed that the antimicrobial peptide microcapsule group and the control group did not cluster, and there were differences in gene expression; among them, the antimicrobial peptide microcapsule group upregulated 1806 genes and downregulated 3878 genes compared with the control group; KEGG enrichment found that the Vibrio cholerae infection signaling pathway in the antimicrobial peptide microcapsule group was significantly reduced compared with the control group; in addition, antimicrobial peptide microcapsules downregulated the expression of key pro-inflammatory and apoptotic genes after feeding, and inhibiting the expression of these genes may help clams avoid excessive inflammatory response in the early stage of Vibrio infection; at the same time, the expression of antimicrobial-related genes (COLEC12, gnbp1, IFI44, Clec4f, MBL, mll7730) and the regulation of immune recognition genes in the intestine (PGRP-SC2) were upregulated, reducing the abundance of harmful bacteria in the intestine, thereby increasing the abundance of beneficial bacteria ( Fig. 9 )

[0050] In summary, the antimicrobial peptides provided by the present invention can significantly antagonize pathogenic bacteria in juvenile spat breeding. After recombinant expression, they are prepared into microcapsules with a particle size suitable for juvenile spat filter feeding, which not only overcomes the problem of the prior art of directly spraying them into the aquaculture water body, resulting in the juvenile spat being unable to ingest them, but also overcomes the problems of low expression efficiency of microalgae and low content of antimicrobial peptides.

Claims

1. An antimicrobial peptide isolated from Ruditapes philippinarum, characterized in that: The amino acid sequence of the antimicrobial peptide is SEQ ID NO:

2.

2. A nucleic acid fragment, characterized in that The nucleic acid fragment encodes the antimicrobial peptide according to claim 1.

3. A recombinant expression vector, characterized in that: The recombinant expression vector is inserted with a nucleic acid fragment encoding the antimicrobial peptide according to claim 1.

4. A recombinant engineering strain, characterized in that: The recombinant engineering strain carries the recombinant expression vector according to claim 3.

5. The recombinant engineered strain according to claim 4, characterized in that The recombinant engineering strain is a Pichia pastoris engineering strain.

6. Use of the antimicrobial peptide according to claim 1 in the preparation of antimicrobial products.

7. The use according to claim 6, characterized in that The product is a microcapsule.

8. A microcapsule, characterized in that The microcapsule contains the antimicrobial peptide according to claim 1.

9. The microcapsule according to claim 8, characterized in that The preparation method of the microcapsule is to add water to sodium octenyl succinate starch, carboxymethyl cellulose and recombinantly expressed antimicrobial peptide powder, stir and dissolve them, and then spray dry them.

10. A method for cultivating bivalve juvenile shellfish, characterized in that: The method is to feed the microcapsules described in claim 8.

Images