An antimicrobial peptide and its application in juvenile spat farming

Abstract

The application provides an antibacterial peptide and application thereof in juvenile shellfish culture, and the amino acid sequence of the antibacterial peptide is SEQ ID NO:2. The antibacterial peptide screened by the application is derived from bivalve shellfish, has less toxic and side effects on the shellfish, is suitable for playing a role in the shellfish, and has specificity and pertinence to pathogenic bacteria in the shellfish culture water. The amino acid sequence of the antibacterial peptide is optimized, has strong bacteriostatic activity, high stability, and high expression efficiency in yeast. After high expression of the antibacterial peptide, the antibacterial peptide is prepared into microcapsules with a particle size suitable for filtering and eating of the juvenile shellfish, so that the problems of the prior art, such as direct spraying into the culture water to cause the juvenile shellfish to be unable to eat, and low expression efficiency of microalgae 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 spat farming. Background Art

[0002] Bivalves, represented by the Manila clam, play a vital role in my country's aquaculture. They not only provide high-quality protein but also, through their filter-feeding nature, play a key role in promoting nutrient cycling in water bodies and maintaining ecological balance. According to the latest Fisheries Statistical Yearbook, my country's annual production of Manila clam reached over 3 million tons in 2023. Globally, Manila clam aquaculture production in countries and regions including the Philippines, South Korea, Japan, Thailand, and Vietnam totals over 4 million tons.

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

[0004] In production, antibiotics are often dissolved in concentrations tens to hundreds of times the oral dose for animals and then sprayed directly into the water used to culture juvenile spat to suppress pathogenic microorganisms. However, this is largely ineffective because filter-feeding shellfish cannot ingest dissolved antibiotics in the water. Furthermore, excessive use can easily lead to drug resistance in pathogenic bacteria.

[0005] Antimicrobial peptides are a class of small peptides found widely in animals, plants, and microorganisms. As a crucial component of the innate immune system, they rapidly kill bacteria by disrupting pathogen cell membranes. They are also less likely to induce drug resistance, demonstrating significant potential as an alternative to antibiotics. In aquaculture, antimicrobial peptides not only directly inhibit pathogens but also serve as dietary additives, improving the growth performance of aquatic animals, enhancing immune function, and maintaining a balanced intestinal flora.

[0006] Although there have been many reports on the application of antimicrobial peptides in aquaculture, there are few reports on filter-feeding bivalves represented by Manila clams. The only reports are that heterologous antimicrobial peptides such as insects are directly dissolved in the culture water of juvenile scallops or expressed in bait microalgae and then fed to juvenile mussels, 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 filtered particles of bivalve juveniles is 4-10μm, which cannot be directly dissolved in the aquaculture water and ingested by the 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 present invention aims to provide an antimicrobial peptide and its use in juvenile spat aquaculture. The provided antimicrobial peptide is derived from bivalve molluscs, undergoes amino acid optimization, and exhibits specific inhibitory effects against bivalve pathogens. The present invention also involves highly soluble expression of the antimicrobial peptide in Pichia pastoris. The antimicrobial peptide is then spray-dried into microcapsules of a particle size suitable for filter feeding by bivalve juvenile spats, and then fed to the juvenile spats.

[0008] The present invention first provides an antimicrobial peptide isolated 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, wherein a nucleic acid segment encoding the antimicrobial peptide is inserted into the recombinant expression vector.

[0011] In another aspect, the present invention provides a recombinant engineered strain carrying the above-mentioned recombinant expression vector;

[0012] As specifically described in the examples, 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 octenylsuccinate, carboxymethyl cellulose and recombinantly expressed antimicrobial peptide powder, stir and dissolve, and then spray-dry.

[0017] The present invention also provides a method for cultivating bivalve juveniles, which comprises feeding the microcapsules.

[0018] The antimicrobial peptides screened and obtained by the present invention are derived from bivalve molluscs, have minimal toxic side effects on them, are suitable for functioning within bivalve molluscs, and are specific and targeted against pathogens in bivalve aquaculture water. The provided antimicrobial peptides have an optimized amino acid sequence, exhibit strong antimicrobial activity, high stability, and high expression efficiency in yeast. After high expression, the antimicrobial peptides can be prepared into microcapsules of a particle size suitable for filter feeding by juvenile mussels. This overcomes the existing problem of direct application into aquaculture water, which prevents mussels from ingesting the peptides, and also overcomes the low expression efficiency and low antimicrobial peptide content in microalgae. 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 : 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 soaking, microcapsules, and antimicrobial peptide microcapsules on the growth of Ruditapes philippinarum during the attachment period and the survival rate after Vibrio challenge, including the antimicrobial peptide microcapsule 0 mg / g group (CK), antimicrobial peptide soaking group (Core not pelletised), group without antimicrobial peptide microcapsules (Wall granulation), and antimicrobial peptide microcapsule group (Core wall granulation).

[0024] Figure 6 : 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 : Effects of antimicrobial peptide microcapsules on intestinal microorganisms of Ruditapes philippinarum.

[0027] Figure 9 : Effects of antimicrobial peptide microcapsules on the transcriptome of Ruditapes philippinarum. DETAILED DESCRIPTION

[0028] The present invention isolates an antimicrobial peptide from the Manila clam and optimizes its length, charge, hydrophobicity, stability and antimicrobial activity through antimicrobial peptide design. The antimicrobial peptide is then highly expressed in a soluble form in Pichia pastoris to obtain an antimicrobial peptide that specifically and significantly inhibits pathogenic microorganisms of bivalve molluscs. The antimicrobial peptide is then prepared into microcapsules with a particle size suitable for bivalve filter feeding using spray drying technology, thereby reducing microbial diseases during juvenile mollusc culture.

[0029] The present invention is described in detail below with reference to the embodiments and accompanying drawings.

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

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

[0032] MLKLTGYIMHNLCFLFLCVLMVDSIPLRGRVDIIEKRIGVVESRMNSDNELAREMFLSLQQNFEDLNSSMINLKNQMCPIMDQCGVKEDQSDSEKNVEILQSSRVMMTLGLKREKQWVRDQVKKLFTQMGEHSDRLDQVTKMESDAYIEINKACMVMSSQQ KEIEALKETRTNLLNALISKNEEKYDYTCPPDWKKYSSFCYLFSEDIMKFDEARAYCKLLGATVADFKNQAENDVIADLPKVLRFAWIGYSDEEKEGTWISERTGQPASFTNFADGQPSGGIHQNCAVLHFYNGKWHDWTCHSGGKWHVVCKKDATIEFKL. like Figure 1 As shown, a peptide segment with potential antibacterial activity was initially obtained from the above amino acid sequence, KWRLIAIRKRLLKAI. Furthermore, this series was optimized to improve antibacterial efficacy 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 is positively charged (containing lysine [K] and arginine [R]), and the length is controlled between 15 and 25 amino acids (using efficient expression). Subsequently, the positive charge of the peptide is increased. The optimization principle is: in order 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 position 14 with "R" to obtain KWRLIAIRKRLLKRI. Then, hydrophobic amino acids (such as leucine [L], isoleucine [I], and valine [V]) are added. The specific operation is to replace the "I" at positions 5 and 7 with "L" to obtain KWRLLALRKRLLKRI. Finally, the stability of the peptide to proteases is enhanced, and residues are introduced reasonably. The specific operation is to replace the "A" at position 6 with "W" to obtain KWRLLWIRKRLLKRI (SEQ ID NO: 2).

[0034] The nucleotide sequence encoding the antimicrobial peptide KWRLLWIRKRLLKRI (SEQ ID NO: 2) was optimized for yeast-preferred codons to yield the nucleotide sequence AAGTGGAGACTGTTATGGATTAGGAAGAGACTGCTAAAGCGTATA (SEQ ID NO: 3). Primers with enzyme cleavage sites were designed to recombinantly construct the antimicrobial peptide nucleic acid sequence into the pPICZ vector. The sequence was then electroporated into the Pichia pastoris GS115 strain and plated onto YPDS plates containing Zeocin to screen for positive strains. After sequencing confirmed the sequence of the positive strains, positive clones were selected and cultured in methanol induction medium (BMMY) at 28°C and 250 rpm for 30 hours to a concentration of 1 OD. Induction was then performed with 1% methanol for 5 days. Dextrin (500 g / L) was added to the antimicrobial peptide expression yeast solution as a carrier and dried at 60°C to produce an antimicrobial peptide powder.

[0035] The antimicrobial peptides prepared by recombinant expression were used to test their inhibitory effects on several common pathogens in juvenile spat aquaculture water. Figure 2 As shown, insect antimicrobial peptides and unoptimized Philippine clam antimicrobial peptides were used as controls. The optimized antimicrobial peptides of the present invention exhibited strong inhibitory effects against Vibrio parahaemolyticus, Vibrio alginolyticus, and Vibrio splendidus. However, the unoptimized Philippine clam antimicrobial peptides only inhibited Vibrio parahaemolyticus and Vibrio splendidus, and their inhibitory effect on Vibrio splendidus was lower than that of the optimized peptides. Furthermore, the insect antimicrobial peptides only inhibited Vibrio parahaemolyticus, and their inhibitory effect was slightly weaker than that of the Philippine clam antimicrobial peptides.

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

[0038] The antimicrobial peptide microcapsules prepared in this example were fed to planktonic spat of Ruditapes philippinarum at a density of 362 g of planktonic spat per pond (50 cubic meters of water), a salinity of 20-22 ppt, and a water temperature of 28.5-30.6°C. Four treatment groups were set: 0 mg / g (antimicrobial peptide microcapsules / spa), 0.07 mg / g, 0.14 mg / g, and 0.21 mg / g, with three ponds (n=3) for each concentration. The water was changed every two days, and the mass of clams in each pond was recorded. At the end (after the onset of attachment), samples were collected from each pond for counting and shell length measurement.

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

[0040] Example 2: Preparation of antibacterial peptide microcapsules

[0041] The antibacterial peptide microcapsules were prepared according to the method of Example 1, and microcapsules without antibacterial peptide were also prepared: 200 g of sodium octenyl succinate starch and 50 g of carboxymethyl cellulose were added to 1.3 L of water, stirred and dissolved, and then spray dried. The inlet air temperature of the spray dryer was 168°C, and the outlet air temperature was 88°C. The final microcapsules were collected and stored for later use.

[0042] The antibacterial peptide microcapsules 0 mg / g group (CK), antibacterial peptide soaking group (Core not pelletised), microcapsules without antibacterial peptide group (Wall granulation), and antibacterial peptide microcapsules group (Core wall granulation) were set up to feed the juvenile Ruditapes philippinarum: 2 g of juvenile shell per barrel (35 L of water), with the initial specifications of the juvenile shell being 7.125 ± 0.06 mm in length and 67 ± 0.94 mg in weight. The salinity was 25-30 ppt, and the water temperature was 25-30°C. There were 4 barrels for each treatment (n = 4), and the weight of each barrel was recorded every 7 days. After 17 days of cultivation, the samples were weighed and the shell length was measured.

[0043] The results are shown in Table 1 Figure 5 Compared with the control group, the soaking group and the microcapsules without antibacterial peptide group, the antibacterial peptide microcapsules significantly improved the total weight and individual weight Figure 5 A and Figure 5 B). After the end of the cultivation, the survival rate of the Ruditapes philippinarum was found to be antibacterial peptide microcapsules > antibacterial peptide soaking group > control group, microcapsules without antibacterial peptide group Figure 5 E). These results show that only the microcapsules containing the antibacterial peptide can promote the growth and survival of Ruditapes philippinarum, and the direct soaking of the antibacterial peptide or the microcapsules without the antibacterial peptide have no effect.

[0044] Example 3: Preparation of antibacterial peptide microcapsules

[0045] The antibacterial peptide microcapsules were prepared according to the method of Example 1.

[0046] The conditions for feeding the Manila clam with the antibacterial peptide microcapsules are as follows: density: 2 g of juvenile clam per barrel (35 L of water body), size: shell length 1.34±0.15 mm, weight 0.72±0.21 mg, salinity: 25-30 ppt, water temperature: 20-25℃, setting: 0 mg / g (antibacterial peptide microcapsules / clam), 0.1 mg / g, 0.5 mg / g, 1 mg / g, 3 mg / g, a total of 5 groups, 4 barrels (n=4) for each group (concentration), the weight of each group of Manila clam is recorded every 7 days, and the weight is recorded after 17 days.

[0047] The results show that 0.1 mg / g of antibacterial peptide microcapsules can improve the survival rate of Manila clam infected with Vibrio( Figure 6 E). 0.1 mg / g of antibacterial peptide microcapsules can significantly improve CAT and SOD activities, and reduce MDA content, thereby improving the antioxidant capacity of juvenile clam; feeding of antibacterial peptide microcapsules can improve ACP, AKP and LZM activities( Figure 7 ).

[0048] Further analysis of the intestinal microorganisms of the Manila clam shows that the Chao1 and Shannon indexes of the intestinal microorganisms after feeding of the antibacterial peptide microcapsules are significantly higher than those of the control group (p<0.05), and the PCOA analysis shows that there is a significant difference in the community composition between the two groups; the dominant bacteria at the phylum level of the two groups are Proteobacteria, Firmicutes and Bacteroidota, among which the abundance of Proteobacteria in the control group is higher than that in the antibacterial peptide microcapsule group, and the abundance of Firmicutes is higher in the antibacterial peptide microcapsule group; among them, Tenacibaculumn is a kind of Proteobacteria, which is widely considered as a pathogenic bacterium in aquatic animals, and the results show that vibrio and waddia, which are pathogenic bacteria, appear in the control group, both of which belong to the phylum of Proteobacteria, while Lysinibacillus, Solibacillus_silvestris and other potential probiotics in aquatic animals are more common in the antibacterial peptide microcapsule group. In summary, the antibacterial peptide may inhibit the pathogenic bacteria in the intestine of the Manila clam, and at the same time increase the probiotics in the intestine to promote growth and improve immunity( Figure 8 ).

[0049] Further analysis of the transcriptome of Manila clams 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 immune recognition gene (PGRP-SC2) in the intestine were upregulated, reducing the abundance of harmful bacteria in the intestine and thereby increasing the abundance of beneficial bacteria ( Figure 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 can be prepared into microcapsules with a particle size suitable for juvenile spat filter feeding. This not only overcomes the existing problem of directly spraying them into the aquaculture water, which makes it impossible for juvenile spat to ingest them, but also overcomes the problems of low microalgae expression efficiency and low antimicrobial peptide content.

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 segment encoding the antimicrobial peptide according to claim 1.

4. A recombinant engineering strain, characterized in that The recombinant engineered 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 products against Vibrio parahaemolyticus, Vibrio alginolyticus or Vibrio splendens.

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, wherein The preparation method of the microcapsule is as follows: sodium starch octenylsuccinate, carboxymethyl cellulose and recombinantly expressed antimicrobial peptide powder are added with water, stirred and dissolved, and then spray-dried.

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

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