Active polypeptide and application thereof in culture of tegillarca granosa

Abstract

The invention provides an active polypeptide and application thereof in culture of tegillarca granosa, and after amino acid optimization, the provided active polypeptide has a remarkable inhibition effect on common microorganisms such as vibrio and the like in aquaculture. The active polypeptide is further prepared into microcapsules with the particle size suitable for filter feeding of juvenile tegillarca granosa, and the microcapsules are fed to the tegillarca granosa, so that microbial diseases occurring in tegillarca granosa breeding are effectively reduced. The amino acid sequence of the polypeptide is SEQ ID NO: 2. The active polypeptide obtained by screening comes from pearl oyster, the amino acid sequence of the provided active polypeptide is optimized, and the active polypeptide has good antibacterial activity on vibrio harveyi. After high expression of the active polypeptide, the active polypeptide is prepared into the microcapsule with the particle size suitable for filter feeding of juvenile shellfish of tegillarca granosa, the growth of the tegillarca granosa is promoted, and diseases caused by vibrio harveyi are reduced.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of shellfish culture, and particularly relates to an active polypeptide and application thereof in mud clam culture. BACKGROUND

[0002] Mud clam is a kind of marine shellfish, also known as flower clam and blood clam. In some areas, it is also called tile ridge and hawa ridge because the shell surface radial rib looks like the ridge of ancient building roof tiles. Mud clam belongs to the phylum of mollusca, the class of bivalve, the order of septa, the family of clam and the genus of clam. Mud clam is one of the four major cultured shellfish in China, and is the most important marine aquaculture shellfish in the southeast coast of China. Artificial culture is carried out in Hebei, Shandong, Zhejiang, Fujian and Guangdong, and the yield is quite abundant. Mud clam meat is delicious, and can be eaten fresh or marinated with wine, and can also be made into dried products. The meat is ruby red with a golden thread-like color line on the edge, rich in protein, vitamins and various minerals such as calcium, iron, zinc, selenium and magnesium, which are beneficial to human health. Clam meat is rich in protein and vitamins, which is beneficial to human health. Clam blood is bright red and rich in nutrients. Clam shell can be used as medicine, which has the effects of removing blood clots and resolving phlegm accumulation.

[0003] During the culture of mud clam, various problems may be encountered, including deterioration of the culture environment, aging of the bottom material, degeneration of the germplasm, prevalence of bacterial diseases and influence of harmful algae. The deterioration of the culture environment is mainly caused by land source pollution and self-pollution of culture. The land source pollution sources include industrial pollution, agricultural pesticides and fertilizers and urban domestic sewage, which lead to excessive heavy metal content in the culture environment and are toxic to mud clam. The self-pollution of culture mainly refers to the large amount of sedimentation of mud clam excrement, dead shellfish and residual feed, which produces harmful substances through microbial decomposition and promotes the aging of the bottom material. In addition, long-term inbreeding leads to degeneration of the germplasm, which is manifested as low survival rate, slow growth rate, small size and decreased disease resistance. The prevalence of bacterial diseases and the proliferation of harmful algae also exacerbate the difficulties of mud clam culture.

[0004] Antibacterial peptide is a kind of polypeptide with broad biological activity, which has the functions of antibacterial, anti-inflammatory and immune regulation. It can kill bacteria, fungi and viruses, achieve antibacterial effect by destroying pathogen cell membrane or inhibiting its growth and reproduction, and reduce inflammation by interfering with inflammatory signaling pathways, which is important for the treatment of inflammatory diseases. In addition, active polypeptide can regulate immune function, affect immune response, promote cell proliferation and differentiation, accelerate wound healing and tissue regeneration, and has broad prospects in the field of wound repair. In aquaculture, active polypeptide is used for disease prevention and water purification, which can help fish and other aquatic organisms to resist diseases by inhibiting the growth of pathogens, reduce the bacterial infection rate of fish by adding to the feed, improve the growth rate and immunity, and improve the water quality by removing harmful microorganisms, which is of great significance to maintain the stability of the culture environment and improve the efficiency. Although active polypeptide has been reported to have many applications in aquaculture, its application in mud clam culture is rarely reported. SUMMARY

[0005] The present application aims to provide an active polypeptide and its application in cultivation of mud clams, the provided active polypeptide has significant inhibitory effect on microorganisms such as Vibrio commonly seen in aquaculture after being optimized by amino acids.

[0006] The present application first provides an active polypeptide, the amino acid sequence of which is KVYRRDLIEAHCRWCLYA (SEQ ID NO: 2).

[0007] A nucleic acid fragment for encoding the above active polypeptide has a specific sequence as follows: AAGGTCTACCGACGGGATTTGATAGAAGCCCATTGTCGCTGGTGCCTTTACGCG (SEQ ID NO: 3).

[0008] The present application further provides a recombinant expression vector, wherein the nucleic acid fragment for encoding the above active polypeptide is inserted into the recombinant expression vector.

[0009] The present application further provides a recombinant engineering strain, wherein the recombinant expression vector is carried. As a specific embodiment, the recombinant engineering strain is an Escherichia coli engineering strain.

[0010] The present application further provides application of the active polypeptide in preparation of antibacterial products. As a specific embodiment, the product is a microcapsule.

[0011] The present application further provides a microcapsule, wherein the active polypeptide is contained in the microcapsule. The microcapsule is prepared by mixing the active polypeptide with wall materials and then stirring and spray drying. As a specific embodiment, the wall material components are malt dextrin, gum arabic and β-cyclodextrin mixed in a ratio of 6:1:1.

[0012] The present application further provides a cultivation method of mud clam juveniles, wherein the microcapsule is fed.

[0013] The active polypeptide screened by the present application is derived from Pinctada martensii, the amino acid sequence of the provided active polypeptide is optimized, and the active polypeptide has good antibacterial property on Vibrio harveyi. The microcapsule prepared by the active polypeptide after high expression has a particle size suitable for mud clam juveniles to filter feed, and has a promoting effect on the growth of mud clams and reduces diseases caused by Vibrio harveyi. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 The antibacterial effect of each group of active peptides against Vibrio harveyi is shown in the figure, where a is the antibacterial effect of the optimized active peptides, b is the antibacterial effect of the active peptides before optimization, and c is the antibacterial effect of the original active peptides. Figure 2 : Particle size distribution diagram of active polypeptide microcapsules; Figure 3 Survival rate of mud clams after 7 days of culture; Figure 4 : Body size data of mud clams in each group; Figure 5 : MPO content in the bodies of mud clams in each group; Figure 6 : CAT content in the bodies of mud clams in each group; Figure 7 : MDA content in mud clams of each group; Figure 8 : SOD content in the bodies of mud clams in each group. Detailed Implementation

[0015] This invention designs and optimizes the length, charge, hydrophobicity, stability, and antibacterial activity of peptides isolated from pearl oysters. Then, through high-soluble expression in Escherichia coli, an active peptide with a significant inhibitory effect on Vibrio harveyi, the main pathogen causing damage to mud clams, is obtained. Subsequently, spray drying technology is used to prepare microcapsules with a particle size suitable for mud clam filter feeding, which can be used to reduce Vibrio harveyi disease during mud clam farming.

[0016] The present invention will now be described in detail with reference to the embodiments and accompanying drawings.

[0017] Example 1: Screening of active peptides and sequence optimization The original active polypeptide was obtained from the transcriptome of the pearl oyster *Pterocarpus martensii* through bioinformatics alignment, and its sequence is as follows (SEQ ID NO:1): TEKASLDRYMALDQPDDRVMCEYIWIDGTGEGIRSKCRTVDFEPKAAKELPVWNFDGSSTYQAEGSNSDMYLTPVALFNDPFRRRGKNKLVLCEVYKYNKKPAETNRRKTCKEVMDKAASELPWFGIEQEYTLLDNDGHPFGWPKN GYPGPQGPYYCGVGANKVYGRDIIEAHCRACLYAGVKICGCNAEVMPAQWEFQVGPCEGIDMGDHLWIGRYLLHRVAEDFGVIVSFDPKPMPGDWNGAGAHTNYSTKEMREEGGLKHIENAIEKMSKHHAKHIKAYDPNEGQDNA.

[0018] First, the peptide KVYGRDIIEAHCRACLYA, which may have antibacterial activity, was initially obtained from the above amino acid sequence. In order to improve the antibacterial effect and stability, the peptide was optimized.

[0019] The optimization principles are as follows: Ensure the amino acid sequence has an amphiphilic α-helix structure (possessing both hydrophilic and hydrophobic regions), the peptide carries a positive charge (containing lysine [K] and arginine [R]), and the length is controlled between 15-25 amino acids (for efficient expression). Subsequently, the positive charge of the peptide is increased. The optimization principle is: to enhance the positive charge and improve binding ability to the bacterial membrane, hydrophobic amino acids (such as A and G) are replaced with positively charged lysine (K) or arginine (R). Specifically, the "G" at position 4 is replaced with "R," resulting in KVYRRDIIEAHCRACLYA. Then, hydrophobic amino acids are added; specifically, the "I" at position 7 is replaced with "L," resulting in KVYRRDLIEAHCRACLYA. Finally, the peptide's stability against proteases is enhanced by appropriately introducing residues. Specifically, the "A" at position 14 is replaced with "W," resulting in KVYRRDLIEAHCRWCLYA (SEQ ID NO:2).

[0020] The encoding nucleotide sequence of the active polypeptide KVYRRDLIEAHCRWCLYA (SEQ ID NO:2) was obtained and optimized using yeast-preferred codons to yield the nucleotide sequence AAGGTCTACCGACGGGATTTGATAGAAGCCCATTGTCGCTGGTGCCTTTACGCG (SEQ ID NO:3). Primers containing enzyme cleavage sites were constructed to integrate the nucleic acid sequence of the active polypeptide into the pMD18T vector, which was then introduced into competent E. coli DH5α cells via electroporation. Positive clones were screened on selective agar plates containing kanamycin. These positive strains were inoculated into LB broth and cultured in a constant temperature shaking incubator at 37°C. When the bacteria reached the logarithmic growth phase, IPTG inducer was added. After the culture period, the bacterial suspension was collected, and the cell pellet was obtained by centrifugation. High-pressure cell disruption was used to break up the cells and release their internal components. Subsequently, the supernatant was separated from the cell debris by centrifugation at 12,000g to obtain a supernatant containing active peptides (cell walls were ruptured). To prepare the active peptide powder, dextrin (concentration of 500g / L) was added as a carrier to the yeast broth expressing the active peptides, and the mixture was dried at 60°C.

[0021] The above-mentioned bioactive peptide samples produced through recombinant technology were used to evaluate their inhibitory efficacy against Vibrio harveyi, the most harmful pathogen in the mud clam farming process. Original bioactive peptides from the Pinctada martensii oyster and unoptimized bioactive peptides were established as control groups. Figure 1 As shown, the inhibition zone assay results are highly consistent with the MIC quantitative data in Table 1. The optimized peptide (a) exhibits strong inhibitory activity against Vibrio harveyi, with an MIC value as low as 1 μg / mL, indicating that it can exert significant bactericidal / bacteriostatic effects at extremely low concentrations. While the unoptimized peptide (b) possesses some antibacterial basis, its antibacterial ability is relatively limited, only effective at higher concentrations. The original peptide (c) shows the weakest inhibitory activity, only inhibiting pathogen growth at the highest tested concentration.

[0022] The above results indicate that optimizing the peptide sequence through rational design can greatly enhance its antibacterial activity against Vibrio harveyi, and its efficacy far exceeds that of unoptimized and original peptides, showing good application prospects in the field of disease control in mud clam farming.

[0023] Table 1. Minimum inhibitory concentration (MIC) of the three peptides (μg / mL) Polypeptide Type / Concentration 64 32 16 8 4 2 1 0.5 Original Polypeptide - - - - - - - + Unoptimized Polypeptide - - - + + + + + Optimized Polypeptide + + + + + + + + Example 2: Preparation of active polypeptide microcapsules 1. Preparation of active polypeptide microcapsules Prepare 100 ml of active peptide suspension. Add a wall material composed of maltodextrin, gum arabic, and β-cyclodextrin in a 6:1:1 ratio (core material to wall material mass ratio 1:6). Stir for 30 min using a high-speed mixer to ensure complete dissolution of the wall material. Homogenize in a high-speed dispersion device at 12,000 rpm for 100 s. Set the inlet temperature of the spray dryer to 155 °C and the spray air channel diameter to 50 mm. After completion, collect the powder in the device; this is the microencapsulated product of the active peptides from Pinctada martensii.

[0024] 2. Determination of embedding rate A 200 mg microcapsule sample was placed in water and centrifuged at 5000 rpm for 10 min to rupture the microcapsule shells. After centrifugation, the supernatant was removed, the sample was inverted to remove residual water, and then transferred to a container containing 20 ml of anhydrous ethanol. The mixture was stirred with a magnetic stirrer for 5 min. The filtrate was then repeatedly filtered through filter paper, diluted, and adjusted to a fixed volume. The absorbance of the filtrate was measured using a spectrophotometer. The content of active peptides present in the outer layer of the microcapsules and the content of active peptides not embedded in the wall material were determined. The encapsulation rate of the active peptide microcapsules from *Pterocarpus martensii* was 81.35%.

[0025] 3. Particle size distribution Laser scattering particle size analysis was used to measure the microcapsules, and the particle size of the samples was characterized by weighted average particle size and particle size distribution range. 3g of *Pterocarpus martensii* active polypeptide microcapsules were weighed and placed in a feeding funnel. A drying test mode was selected, and the vibration control device of the equipment ensured that the sample entered the feeding funnel at a constant rate, thereby determining its particle size distribution range.

[0026] The particle size distribution of the microcapsules was evaluated using a Bettersize 2600 laser particle size analyzer. The results showed that the average particle size of the *Pterocarpus martensii* active polypeptide microcapsules was approximately 700 μm, exhibiting good particle size uniformity. Figure 2 .

[0027] Example 3: Application of active polypeptide microcapsules in mud clam farming Juvenile mud clams with intact shells, uniform body size, and good health were selected and randomly divided into four groups. They were reared in tanks with a sandy-mud bottom, a salinity of 21, and a water temperature of 20℃, and fed with an appropriate amount of diatoms. Three groups were supplemented daily with 50 mg / g, 100 mg / g, and 200 mg / g (low, medium, and high dose groups), respectively. The other group served as a blank control. All groups were treated with a concentration of 10... 6CFU / mL Vibrio harveyi was used to infect the mud clams. After 7 days, the survival rate of each group of mud clams was recorded. After 24 months of rearing, 10 mud clams of moderate size were selected from each group to measure their body length, height and weight. After the measurement, the mud clam meat was taken, mashed into a paste, and the contents of myeloperoxidase (MPO), catalase (CAT), malondialdehyde (MDA) and superoxide dismutase (SOD) in each group of mud clams were measured. Seven days after infection with Vibrio harveyi, the survival rate of mud clams in the control group was approximately 45%. Figure 3 After treatment with different doses of active polypeptide microcapsules, the survival rate of mud clams was significantly improved. As the treatment dose increased, the survival rate of mud clams showed an upward trend, but the survival rates of the high-dose group and the medium-dose group were similar.

[0028] After 24 months of cultivation, the body size of the mud clams can be observed. Figure 4 After intervention with active polypeptide microcapsules, the body size of mud clams significantly increased, with no significant difference between the high-dose and medium-dose groups, but significant differences between both groups and the low-dose group. This confirms that medium-dose active polypeptide microcapsules have a significant promoting effect on the growth of mud clams.

[0029] The contents of MPO, CAT, MDA and SOD in each group of mud clams are shown in the figure. Figures 5-8 As shown in the figure, the levels of CAT and SOD in the mud clams of the blank control group were significantly decreased, while the levels of MDA and MPO were significantly increased, indicating that Vibrio harveyi infection caused an inflammatory response in the mud clams, resulting in oxidative damage. After intervention with active peptide microcapsules, the levels of CAT and SOD in the mud clams were significantly increased (p<0.05), while the levels of MDA and MPO were significantly decreased (p<0.05). The intervention effects were better in the medium- and high-dose groups, indicating that active peptide microcapsules can reduce the oxidative stress caused by Vibrio harveyi infection in mud clams, and the antioxidant effects of the medium- and high-dose groups were better.

[0030] In summary, this invention provides an active polypeptide that inhibits Vibrio harveyi during the cultivation of mud clams. When made into microcapsules, the encapsulation rate is higher than 80%. Feeding mud clams with this polypeptide can improve their growth performance and reduce inflammatory damage caused by Vibrio harveyi infection, providing strong technical support for the healthy development of the mud clam farming industry.

Claims

1. An active polypeptide, characterized in that, The amino acid sequence of the active polypeptide is SEQ ID NO:

2.

2. A nucleic acid fragment, characterized in that, The nucleic acid fragment encodes the active polypeptide of claim 1.

3. The nucleic acid fragment of claim 2, wherein, The sequence of the nucleic acid fragment is SEQ ID NO:

3.

4. A recombinant expression vector, characterized in that, The recombinant expression vector has the nucleic acid fragment of claim 2 inserted therein.

5. A recombinant engineered strain, characterized in that, The recombinant engineering strain carries the recombinant expression vector of claim 4.

6. Use of the active polypeptide of claim 1 in the preparation of an antibacterial product.

7. A microcapsule characterized in that, The microcapsule contains the active polypeptide of claim 1.

8. The microcapsule as described in claim 7, characterized in that, The microcapsule is prepared by mixing the active polypeptide of claim 1 with wall material, stirring, and spray drying.

9. The microcapsule as described in claim 8, characterized in that, The wall material components are malt dextrin, gum arabic, and β-cyclodextrin.

10. A method for culturing of mud clam juveniles, characterized by, The method is to feed the microcapsule of claim 8 during the cultivation of the mud clam juvenile.

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