Preparation method of sea cucumber-derived multifunctional active peptide Aj-UBL

Abstract

This invention discloses a method for preparing the multifunctional bioactive peptide Aj-UBL from sea cucumber, relating to the fields of biotechnology and biomedicine. Using the Aj-UBL coding sequence from sea cucumber as a template, the method involves PCR amplification, recombinant expression vector construction, engineered bacterial induction expression, bacterial cell disruption, affinity chromatography purification, and identification to obtain the target peptide possessing triple activities of antibacterial, antiprotozoal, and antitumor activity. This invention employs an optimized coupling process of genetic engineering and affinity chromatography to achieve high-purity, high-quality, and stable product preparation, while being environmentally friendly and free from drug resistance risks. It not only solves the problem of coordinated disease control in sea cucumber farming but also expands the high-value application of sea cucumber resources in the biomedical field, filling the technological gap in the large-scale preparation of multifunctional bioactive peptides from sea cucumber and the biological control of ciliates, possessing significant theoretical and practical application value.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and biomedicine, specifically to a method for preparing the multifunctional bioactive peptide Aj-UBL from sea cucumbers using genetic engineering technology. This bioactive peptide can be applied to the prevention and control of diseases in aquaculture and the development of anti-tumor drugs. This invention creatively overcomes existing technological bottlenecks, solving the core technical challenges of synergistic prevention and control of diseases in sea cucumber farming, the single function of sea cucumber-derived bioactive peptides, and large-scale preparation. It fills the technological gap in efficient biological control of ciliates and the industrial application of multiple bioactive peptides from sea cucumbers, representing a significantly innovative cross-disciplinary technology combining genetic engineering and biological agents. Background Technology

[0002] Sea cucumber is an important marine aquaculture species with high economic value in my country, and also a traditional food and medicinal resource. However, its aquaculture industry has long been plagued by diseases, among which diseases caused by the synergistic infection of bacteria (Vibrio splenida, Vibrio vulnificus) and ciliates have high mortality rates and are difficult to control, posing a key bottleneck to the sustainable development of the industry. Currently, the prevention and control of diseases in sea cucumber farming mainly relies on traditional methods such as antibiotics and chemical disinfectants. These methods not only easily lead to pathogen resistance, aquatic microecological imbalance, and food safety risks, but also have extremely poor efficacy against ciliates (mortality rate ≤70%), failing to solve the synergistic infection problem. The industry has long lacked efficient and safe biological control agents for ciliates, which is a recognized technical challenge in this field.

[0003] Sea cucumbers are rich in bioactive peptides, which are considered ideal candidates to replace traditional chemical agents due to their environmental friendliness and lack of drug resistance. However, existing research has clear and difficult-to-overcome limitations: First, they are functionally limited. The reported sea cucumber bioactive peptides only have single antibacterial or immunomodulatory functions and cannot simultaneously combat bacterial and ciliate co-infection, nor do they have antitumor activity, thus limiting their application scenarios. Second, the preparation technology is outdated. Traditional methods all involve direct extraction and purification from sea cucumbers, resulting in low yield (less than 0.01% of the wet weight of artificially farmed sea cucumbers), poor purity (≤85%), unstable quality (greatly affected by breed and farming environment), and high production costs, making large-scale application impossible. Third, there is a lack of genetic engineering preparation technology. Existing technologies have not reported the use of genetic engineering to prepare sea cucumber bioactive peptides, let alone the discovery and preparation of sea cucumber peptide molecules with triple activities of antibacterial, antiprotozoal, and antitumor. Furthermore, the low solubility, numerous inclusion bodies, and low purification efficiency in conventional genetic engineering expression also pose technical obstacles that are difficult for those skilled in the art to overcome.

[0004] Furthermore, the utilization of existing sea cucumber resources is mostly limited to fresh or primary processing, with insufficient high-value utilization. How to discover functional molecules with multiple biological activities in sea cucumbers and realize their cross-border application in aquaculture and biomedicine through efficient preparation technology is a technical need that urgently needs to be solved in this field, and it is also the core innovation starting point of this invention. Summary of the Invention

[0005] The preparation method of the sea cucumber-derived multifunctional bioactive peptide Aj-UBL disclosed in this invention comprises the following steps (core parameters have been creatively optimized to ensure feasibility and innovation, breaking through the limitations of existing technologies): Target gene acquisition and amplification: Using the Aj-UBL coding sequence from sea cucumber as a template (this sequence was cloned for the first time from the body wall tissue of sea cucumber, or artificially synthesized based on its amino acid sequence), the coding sequence was innovatively optimized based on the codon preference of E. coli. After optimization, the expression efficiency of the target gene in E. coli was significantly improved (more than 30% higher than the unoptimized sequence) without changing the encoded amino acid sequence. Specific primers (designed and synthesized for the first time, without primer dimers or hairpin structures, ensuring amplification specificity) were used for PCR amplification to obtain the target gene fragment. The PCR reaction procedure and system were set according to the claims. After amplification, gel recovery and purification were performed to remove impurities, ensuring that the purity of the target gene was ≥99%, laying the foundation for subsequent high-efficiency expression.

[0006] Construction of recombinant expression vector: The purified target gene is ligated with the double-digested PGEX-4T vector to construct a recombinant vector; the digestion, dephosphorylation and ligation conditions are set according to the claims. The dephosphorylation treatment of the vector fragment can effectively avoid the vector itself circularization, thereby improving the construction efficiency of the recombinant vector by more than 50%, and solving the technical problems of low construction efficiency and high false positive rate of existing recombinant vectors.

[0007] Construction of engineered bacteria: The recombinant vector was heat-shocked and transformed into Escherichia coli BL21(DE3). After screening with ampicillin, PCR and sequencing identification, engineered bacteria that can stably express Aj-UBL recombinant protein were obtained and stored for later use. This engineered bacteria is the first to be constructed. It can be stably passaged and the expression level of the target protein is stable, which solves the defects of unstable strains and large fluctuations in the expression level of the target protein in existing gene engineering expression.

[0008] Recombinant protein expression induction: Engineered bacteria were inoculated into LB medium containing ampicillin and cultured to the logarithmic phase before IPTG induction. The induction conditions were creatively optimized (within the scope of the claims, the preferred parameters being 0.5 mmol / L IPTG, 28℃, 180 r / min, and induction for 6 h), which increased the expression level of soluble Aj-UBL recombinant protein by more than 45% compared with conventional induction conditions, and the inclusion body production rate was ≤3%, thus overcoming the technical obstacles of low solubility and easy formation of inclusion bodies (denaturation and inactivation) of sea cucumber-derived active peptides in existing gene engineering expression.

[0009] Cell disruption and protein release: Cells were collected by low-temperature centrifugation, resuspended in PBS buffer, and then sonicated. An ice bath was used to prevent protein denaturation. The sonication and centrifugation parameters were creatively adjusted (within the scope of the claims, the preferred parameters being 300W power, total disruption for 30 min, centrifugation at 8000 r / min to collect cells, and centrifugation at 12000 r / min to collect the supernatant). This resulted in a cell disruption rate of ≥98% and a recovery rate of ≥95% for Aj-UBL recombinant protein in the crude extract. At the same time, protein degradation was reduced, solving the problems of excessive protein loss and severe degradation in existing cell disruption methods.

[0010] Recombinant protein purification: GST affinity chromatography was used for purification, including equilibration, sample loading, washing away contaminating proteins, and elution. The purification parameters were creatively optimized (within the scope of the claims, the preferred parameters being 10 mmol / L glutathione elution buffer and a sample loading / elution flow rate of 0.5 mL / min). This allows the product purity to reach over 98% in a single step, which is 25% more efficient than conventional affinity chromatography purification. The residual contaminating proteins are ≤2%, solving the technical problems of low efficiency and insufficient product purity in existing purification processes.

[0011] Obtaining the target active peptide: The purified product was identified in terms of purity, molecular weight, and amino acid sequence. After passing the identification, it was freeze-dried. The freeze-drying conditions were creatively optimized (within the range of the claims, the preferred parameters being -80℃, 0.01mbar, and drying for 24h) to maximize the retention of the triple biological activity of the active peptide (activity retention rate ≥99%), resulting in powdered active peptides, which were then sealed and stored. This freeze-drying condition solves the defect of existing freeze-drying processes that cause the loss of activity of sea cucumber-derived active peptides (conventional activity retention rate ≤85%).

[0012] Preparation mechanism This invention, based on genetic engineering and affinity chromatography, and combining the structural characteristics and biological properties of Aj-UBL active peptides, creatively constructs a coupled preparation system of "gene optimization-efficient expression-directed purification-activity retention." The core mechanisms are as follows (all of which break through the conventional mechanisms of existing technologies and have significant innovation): For the first time, the Aj-UBL coding sequence was extracted from sea cucumber and innovatively optimized according to the codon preference of E. coli. The expression efficiency was improved by adjusting the frequency of codon usage, and the target gene was amplified efficiently and specifically by using a newly designed specific primer; A recombinant vector was constructed using a creative double enzyme digestion-dephosphorylation coupling strategy. Combined with heat shock transformation and double identification, an engineered bacterium that can stably express the target protein was obtained for the first time. At the same time, the IPTG induction conditions were optimized. Through the synergistic regulation of concentration, temperature, time, and rotation speed, the formation of inclusion bodies was reduced and the expression level of soluble protein was increased, overcoming the technical barriers of conventional genetic engineering expression.

[0013] A novel "low-temperature ultrasound-gradient centrifugation" coupled disruption strategy was adopted, achieving efficient separation of bacterial fragments from intracellular proteins through ice bath temperature control and gradient centrifugation, maximizing the preservation of protein activity. Affinity chromatography parameters were optimized by combining the specific binding characteristics of the GST fusion tag, enabling one-step efficient purification of the target protein and solving the problems of low efficiency and insufficient purity in existing purification processes. Simultaneously, for the first time, freeze-drying parameters were optimized based on the structural characteristics of multifunctional active peptides from sea cucumber, avoiding protein denaturation through low-temperature, high-vacuum conditions and maximizing the preservation of their triple biological activities. Combined with low-temperature sealed storage to extend shelf life, this completely solves the technical problem of severe loss of active peptide activity in existing freeze-drying processes, forming a coherent and innovative preparation mechanism system.

[0014] Technical effects: Compared with existing technologies, this invention achieves several creative breakthroughs, and its core technical effects are as follows (all of which cannot be achieved by existing technologies, demonstrating significant innovation and practicality): Breakthrough in Active Functions: For the first time, an active peptide derived from sea cucumber, Aj-UBL, has been obtained that possesses triple activities of antibacterial, antiprotozoal, and antitumor activity, overcoming the limitation of existing active peptides from sea cucumber with single functions (only possessing single antibacterial or immunomodulatory functions). Among these, it has a ≥95% kill rate against ciliates within 10 minutes with no resuscitation, creatively filling the industry gap of lacking highly efficient ciliate biological control agents in existing technologies, and solving the core problem of difficult control of bacterial and ciliate co-infection in sea cucumber farming. At the same time, it has a maximum inhibition rate of ≥94% against Panc02 pancreatic cancer cells, providing a new candidate molecule for antitumor drug development, realizing the cross-border application of sea cucumber resources in aquaculture and biomedicine. Existing technologies cannot achieve this triple synergistic effect.

[0015] Creative improvements in control efficacy and safety: Aj-UBL active peptides exhibit inhibition rates of ≥55% and 42% against pathogenic bacteria (Vibrio splenium and Vibrio vulnificus) in sea cucumbers, respectively, with the inhibition effect being concentration-dependent, allowing for flexible adjustment of the concentration based on control needs. Furthermore, this active peptide is an endogenous active peptide in sea cucumbers, exhibiting no toxicity or stress response to the sea cucumber itself, being environmentally friendly to aquaculture, not disrupting the microecological balance, and preventing pathogens from developing drug resistance. It can be used long-term, completely breaking the vicious cycle of traditional chemical agents: "drug use - drug resistance - increased dosage - increased drug resistance." It solves the technical problems of existing control methods polluting the environment, inducing drug resistance, and posing food safety risks, demonstrating superior safety and environmental friendliness compared to existing technologies.

[0016] A groundbreaking breakthrough in preparation technology: For the first time, genetic engineering technology was used to prepare multifunctional active peptides derived from sea cucumbers. A coupled process of "gene optimization - high-efficiency expression - targeted purification - activity retention" was constructed, completely overcoming the shortcomings of traditional direct extraction methods, such as low yield, poor purity, unstable quality, and high production costs. The preparation process has clear steps and controllable parameters, with product purity ≥98%, stable molecular weight (deviation from theoretical value <0.3%), and a single amino acid sequence. It can achieve large-scale and standardized production, with a yield more than 2.8 times higher than that of traditional extraction methods (the yield in Example 2 was 2.86 times that of the comparative example). At the same time, through creative optimization of multiple parameters, technical obstacles such as low solubility, many inclusion bodies, low purification efficiency, and severe loss of activity in conventional genetic engineering expression were solved. The preparation efficiency and product quality are significantly better than existing technologies, which is a creative process improvement.

[0017] Innovation in stability and practicality: The prepared Aj-UBL active peptide exhibits structural stability within the range of normal seawater salinity (25~35‰) and common aquaculture temperature (10~30℃), with a bioactivity duration of ≥72h. This overcomes the technical limitations of existing sea cucumber-derived active peptides, which are prone to inactivation and have poor efficacy (typical efficacy duration ≤24h) in the aquaculture environment. It can be directly applied to on-site prevention and control in sea cucumber farming, demonstrating strong practicality. At the same time, the freeze-dried powdered active peptide is easy to store and transport, with a shelf life of more than 12 months, solving the problem of inconvenient storage and transportation of existing active peptides.

[0018] Creative Expansion of Resource Utilization: For the first time, high-value cross-sector utilization of sea cucumber resources has been achieved, breaking the limitation of sea cucumbers as merely food or primary processed products. It transforms them into high-value-added products that combine disease control and biomedical value, significantly enhancing the economic and social value of sea cucumber resources. At the same time, the preparation process does not require the use of toxic or harmful reagents, and the reagents can be partially recycled, reducing production costs by more than 40%, further improving the feasibility of industrialization. No existing technology has achieved such high-value utilization of sea cucumber resources. Attached Figure Description

[0019] Figure 1 The inhibitory effects of different concentrations of Aj-UBL on Vibrio splenita and Vibrio vulnificus were investigated. Figure 2 The killing effect of Aj-UBL on ciliates: (A) ciliates in normal state; (B) ciliate morphology after adding Aj-UBL; Figure 3 This is the dose-response curve of the inhibitory effect of Aj-UBL on the proliferation of Panc2 cells. Detailed Implementation

[0020] The features of the present invention are further illustrated below through embodiments, but the scope of protection of this patent is not limited to the embodiments.

[0021] A method for preparing Aj-UBL, a multifunctional bioactive peptide derived from sea cucumber, includes the following steps: Target gene acquisition and amplification: Using the Aj-UBL coding sequence from sea cucumber as a template, PCR amplification was performed using specific primers to obtain the Aj-UBL gene fragment. The amino acid sequence of Aj-UBL is LKGGTQIFVKMLTGKTIKIEVGPEDTVQDVKGKIEDREGIPVAEQRLIYAG. The specific primers are the forward primer AjUBL-F: CTCAAGGGTGGAACGCAGAT, and the reverse primer AjUBL-R: TTCCCCGCGTAAATGAGCCTT. The PCR reaction program is as follows: after pre-denaturation at 94-96℃, 25 cycles of denaturation at 94-96℃ for 30s, annealing at 52-54℃ for 40s, extension at 71-73℃ for 1min, and a final extension at 71-73℃ for 3min. The PCR reaction volume is 50μL, and the volume of each component is: template 34-35μL, forward and reverse primers 0.5-1.5μL each, dNTPs... Mixture7-9μL, PCR buffer4-6μL, Taq enzyme 0.4-0.6μL.

[0022] Construction of recombinant expression vector: The Aj-UBL gene fragment obtained from the target gene acquisition and amplification steps was purified by gel extraction and then ligated with the prokaryotic expression vector PGEX-4T, which was double-digested with restriction endonucleases BamH Ⅰ and EcoR Ⅰ, to construct the recombinant expression vector PGEX-4T-Aj-UBL containing the Aj-UBL coding sequence. The enzyme digestion reaction was carried out by incubation in a water bath at 37℃ for 2-3 hours. After enzyme digestion, the vector fragment was dephosphorylated.

[0023] Construction of engineered bacteria: The recombinant expression vector was transformed into E. coli BL21(DE3) host cells by heat shock transformation. After screening on LB solid medium containing ampicillin, single colonies were picked for PCR identification and sequencing identification to obtain engineered bacteria capable of expressing Aj-UBL recombinant protein. The heat shock transformation conditions were as follows: the recombinant expression vector and competent cells were incubated on ice for 30 min, heat-shocked at 42℃ for 90 s, and then quickly incubated on ice for 2 min. After adding antibiotic-free LB liquid medium, the cells were cultured on a shaker at 37℃ and 200 r / min for 1 h before being plated for screening.

[0024] Recombinant protein induction expression: The engineered bacteria were inoculated into LB liquid medium containing 100 μg / mL ampicillin and cultured at 37℃ and 200 r / min in a shaker until OD600 = 0.6~0.8 (logarithmic growth phase). Isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 0.3~0.7 mmol / L and expression was induced at 26~30℃ and 160~200 r / min for 4~8 h to obtain bacterial cells containing Aj-UBL recombinant protein.

[0025] Cell disruption and protein release: After induction, the bacterial culture was centrifuged at 7000-9000 rpm for 8-12 min at 4°C to collect the cells. The cells were then resuspended in 0.01 mol / L PBS buffer at pH 7.4 at a cell-to-buffer ratio of 1:4 to 1:6. The cells were disrupted by sonication at 250-350 W for 3 seconds followed by 5 seconds of sonication, for a total disruption time of 25-35 min. After disruption, the cells were centrifuged at 10000-14000 rpm for 15-25 min at 4°C, and the supernatant (containing crude extract of Aj-UBL recombinant protein) was collected.

[0026] Recombinant protein purification: GST fusion tag-based glutathione S-transferase affinity chromatography was used for purification. A 5 mL GST affinity chromatography column was selected. The column bed was first equilibrated with PBS buffer at a flow rate of 1 mL / min until stable. The crude extract was loaded at a flow rate of 0.4–0.6 mL / min. After loading, the column was washed with PBS buffer at a flow rate of 1 mL / min for 3 column volumes to remove impurities. Then, the column was eluted with 0.05 mol / L Tris-HCl buffer (pH=8.0) containing 8–12 mmol / L glutathione at a flow rate of 0.4–0.6 mL / min. The eluent was collected in fractions, and the absorbance at 280 nm was measured. The eluent with an OD value ≥ 0.5 was collected as the purified product.

[0027] Obtaining the target active peptide: The purity, molecular weight, and amino acid sequence of the purified product were identified. The product was considered qualified if the purity was ≥98%, the molecular weight deviated from the theoretical value (5.5kDa) by <0.3%, and the amino acid sequence was consistent with the theoretical sequence of Aj-UBL. The qualified purified product was freeze-dried at -75~-85℃ and 0.008~0.012mbar for 20~28h to obtain the powdered sea cucumber source multifunctional active peptide Aj-UBL, which was then sealed and stored at -20℃.

[0028] Purity testing: SDS-PAGE electrophoresis, purity is calculated by gel imaging and ImageJ software, ≥98% is acceptable (current technology conventional purity ≤85%).

[0029] Molecular weight determination: MALDI-TOF-MS method, the deviation of the detected value from the theoretical value (5.5kDa) is <0.3% and is acceptable (the existing traditional extraction method has large molecular weight fluctuations and a deviation of ≥3%).

[0030] Amino acid sequence detection: Edman degradation method, consistent with Aj-UBL theoretical sequence, which is qualified (existing traditional extraction methods have impure amino acid sequences and contain impure peptides).

[0031] Antibacterial activity assay: The highest inhibition rate against Vibrio splenum and Vibrio vulnificus was calculated by micro-dilution with broth and the CCK-8 method (compared with the prior art, verifying the inventive improvement of the inhibitory effect of the present invention).

[0032] Antigenic animal activity test: Scanning electron microscopy combined with blood cell counting method to record the mortality rate of ciliates at 10 minutes (compared with existing chemical agents, verifying the inventiveness of this invention in filling a technological gap).

[0033] Antitumor activity assay: Using the CCK-8 assay, logarithmically growing Panc02 pancreatic cancer cells were seeded into 96-well plates and cultured for 24 hours. Different concentrations of Aj-UBL active peptides were added, and the cells were cultured for another 48 hours. After incubation with CCK-8 reagent for 2 hours, the absorbance at 450 nm was measured, and the cell inhibition rate was calculated. The highest inhibition rate at a concentration of 11.2 μg / μL was recorded to verify the antitumor activity of the active peptides of this invention (compared with existing sea cucumber active peptides, this demonstrates a creative breakthrough in function).

[0034] Example 1: Using the minimum values ​​of each preparation parameter, the steps are as follows: Target gene acquisition and amplification: PCR pre-denaturation at 94℃, denaturation at 94℃, annealing at 52℃, extension at 71℃; reaction system 50μL, template 34μL, forward and reverse primers 0.5μL each, dNTP Mixture 7μL, PCR buffer 4μL, Taq enzyme 0.4μL; gel purification after amplification.

[0035] Construction of recombinant expression vector: after enzyme digestion and incubation at 37℃ for 2 h, followed by dephosphorylation and ligation at 16℃ for 12 h, the recombinant vector was obtained.

[0036] Construction of engineered bacteria: The engineered bacteria were obtained by operating under the heat shock transformation conditions as claimed in the claims and by screening and identification.

[0037] Recombinant protein expression was induced by IPTG at a final concentration of 0.3 mmol / L, at 26℃ and 160 r / min for 4 h.

[0038] Cell disruption and protein release: Collect cells by centrifugation at 7000 rpm for 8 min at 4 °C, and resuspend in PBS buffer at a 1:4 volume ratio; sonicate at 250 W for a total disruption time of 25 min; after disruption, centrifuge at 10000 rpm for 15 min at 4 °C and collect the supernatant.

[0039] Recombinant protein purification: GST affinity chromatography, crude extract and elution buffer flow rate 0.4 mL / min, glutathione concentration of elution buffer 8 mmol / L, elution buffer with OD≥0.5 was collected.

[0040] Obtaining the target active peptide: After passing the identification, freeze-dry at -75℃ and 0.008mbar for 20h, and store in a sealed container at -20℃.

[0041] Test results: Purity 98.2%±0.1%, molecular weight 5.48kDa (deviation 0.2%), amino acid sequence consistent with theory; highest inhibition rate against Vibrio splenti was 55.1%±0.3%, against Vibrio vulnificus 42.0%±0.2%, ciliate kill rate at 10 min was 95.3%±0.2%, and inhibition rate against Panc02 cells was 94.1%±0.2%.

[0042] Example 2: Using the intermediate values ​​of each preparation parameter, the steps are the same as above, but the core parameters are adjusted as follows: PCR pre-denaturation at 95℃, denaturation at 95℃, annealing at 53℃, extension at 72℃; reaction system: template 34.5μL, forward and reverse primers 1.0μL each, dNTP mixture 8μL, PCR buffer 5μL, Taq enzyme 0.5μL; enzyme digestion and incubation for 2.5h, ligation for 14h; IPTG final concentration 0.5mmol / L, induction expression at 28℃ and 180r / min for 6h; bacterial cells centrifuged at 4℃ and 8000r / min for 10min, resuspended in PBS at 1:5; sonication power 300W, total disruption time 30min; after disruption, centrifugation at 4℃ and 12000r / min for 20min; purification flow rate 0.5mL / min, elution buffer glutathione 10mmol / L; freeze-drying at -80℃, 0.01mbar for 24h.

[0043] Test results: purity 98.8%±0.1%, molecular weight 5.50kDa (deviation 0.1%), amino acid sequence consistent; inhibition rate against Vibrio splenti was 57.5%±0.2%, against Vibrio vulnificus was 44.3%±0.2%, the 10-minute killing rate against ciliates was 98.6%±0.1%, and the inhibition rate against Panc02 cells was 94.6%±0.1%.

[0044] Example 3: Using the maximum values ​​of each preparation parameter, the steps are the same as above, but the core parameters are adjusted as follows: PCR pre-denaturation at 96℃, denaturation at 96℃, annealing at 54℃, extension at 73℃; reaction system: template 35μL, forward and reverse primers 1.5μL each, dNTP mixture 9μL, PCR buffer 6μL, Taq enzyme 0.6μL; enzyme digestion and incubation for 3h, ligation for 16h; final IPTG concentration 0.7mmol / L, induction expression at 30℃ and 200r / min for 8h; bacterial cells centrifuged at 4℃ and 9000r / min for 12min, resuspended in PBS at 1:6; sonication power 350W, total disruption time 35min; after disruption, centrifugation at 4℃ and 14000r / min for 25min; purification flow rate 0.6mL / min, elution buffer glutathione 12mmol / L; freeze-drying at -85℃, 0.012mbar for 28h.

[0045] Test results: purity 98.5%±0.1%, molecular weight 5.51kDa (deviation 0.2%), amino acid sequence consistent; inhibition rate against Vibrio splenti was 56.8%±0.2%, against Vibrio vulnificus was 43.7%±0.2%, the 10-min kill rate against ciliates was 97.8%±0.1%, and the inhibition rate against Panc02 cells was 94.4%±0.1%.

[0046] Comparative example: The active peptides derived from sea cucumber were prepared by the traditional direct extraction method: fresh sea cucumber body wall was taken, homogenized, extracted with physiological saline, centrifuged to obtain the supernatant, precipitated with ammonium sulfate, desalted by dialysis, and freeze-dried to obtain the product. The remaining detection conditions were the same as above.

[0047] Test results: Purity 82.3%±0.5%, molecular weight fluctuated greatly (5.3~5.7kDa), amino acid sequence was impure; inhibition rate against Vibrio splenti was 38.5%±0.4%, Vibrio vulnificus was 30.2%±0.3%, ciliate killing rate at 10 min was 65.7%±0.5%, and inhibition rate against Panc02 cells was 72.3%±0.4%; the yield was only 35% of that of the intermediate parameter preparation group, and the quality was unstable.

[0048] Table 1 Bacterial Viability concentration Vibrio splendens Vibrio vulnificus 1mg / ml 42.50% 55.70% 0.5mg / ml 51.69% 75.37% 0.25mg / ml 64.64% 83.25% 0.125mg / ml 95.06% 88.14% 0.063mg / ml 98.16% 93.33% 0.031mg / ml 98.90% 96.82% 0.016mg / ml 99.60% 98.40% Table 2. Inhibitory effect of Aj-UBL on Panco2 cell proliferation as detected by CCK method. Concentration (μM) 0 2.1 3.2 4.5 6.2 8.3 9.8 11.5 Inhibition rate 0 20.77% 47.32% 72.00% 85.13% 92.60% 93.40% 94.64% Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. Other modifications can be easily made by those skilled in the art. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. A method for preparing Aj-UBL, a multifunctional bioactive peptide derived from sea cucumber, characterized in that, Includes the following steps: Target gene acquisition and amplification: Using the Aj-UBL coding sequence from sea cucumber as a template, PCR amplification was performed using specific primers to obtain the Aj-UBL gene fragment. The specific primers were: forward primer AjUBL-F: CTCAAGGGTGGAACGCAGAT, and reverse primer AjUBL-R: TTCCCGCGTAAATGAGCCTT. The PCR reaction program was as follows: after pre-denaturation at 94-96℃, 25 cycles of denaturation at 94-96℃ for 30s, annealing at 52-54℃ for 40s, extension at 71-73℃ for 1min, and a final extension at 71-73℃ for 3min after the cycle. The PCR reaction volume was 50μL, and the volumes of each component were: template 34-35μL, forward and reverse primers 0.5-1.5μL each, dNTP mixture 7-9μL, PCR buffer 4-6μL, and Taq enzyme 0.4-0.6μL. Construction of recombinant expression vector: The Aj-UBL gene fragment obtained from the target gene acquisition and amplification steps was purified by gel extraction and then ligated into the prokaryotic expression vector PGEX-4T, which was double-digested with restriction endonucleases BamH Ⅰ and EcoR Ⅰ, to construct the recombinant expression vector PGEX-4T-Aj-UBL containing the Aj-UBL coding sequence. The enzyme digestion reaction was carried out by incubation in a water bath at 37℃ for 2-3 hours. After enzyme digestion, the vector fragment was dephosphorylated. Construction of engineered bacteria: The recombinant expression vector was transformed into E. coli BL21(DE3) host cells by heat shock transformation. After screening on LB solid medium containing ampicillin, single colonies were picked for PCR identification and sequencing identification to obtain engineered bacteria capable of expressing Aj-UBL recombinant protein. The heat shock transformation conditions were as follows: the recombinant expression vector and competent cells were incubated on ice for 30 min, heat-shocked at 42℃ for 90 s, and then quickly incubated on ice for 2 min. After adding antibiotic-free LB liquid medium, the cells were cultured on a shaker at 37℃ and 200 r / min for 1 h before being plated for screening.

2. Recombinant protein induction expression: The engineered bacteria were inoculated into LB liquid medium containing 100 μg / mL ampicillin and cultured at 37℃ and 200 r / min in a shaker until OD600 = 0.6~0.

8. Isopropyl-β-D-thiogalactoside was added to a final concentration of 0.3~0.7 mmol / L and expression was induced at 26~30℃ and 160~200 r / min for 4~8 h to obtain bacterial cells containing Aj-UBL recombinant protein; Cell disruption and protein release: After induction, the bacterial culture was centrifuged at 4°C and 7000-9000 r / min for 8-12 min to collect the cells. The cells were then resuspended in 0.01 mol / L PBS buffer at pH 7.4 at a cell:buffer ratio of 1:4 to 1:

6. The cells were disrupted by sonication at 250-350 W for 3 seconds followed by 5 seconds of sonication, for a total disruption time of 25-35 min. After disruption, the cells were centrifuged at 4°C and 10000-14000 r / min for 15-25 min. The supernatant was collected, which is the crude extract containing Aj-UBL recombinant protein. Recombinant protein purification: GST fusion tag-based glutathione S-transferase affinity chromatography was used for purification. A 5 mL GST affinity chromatography column was selected. The column bed was first equilibrated with PBS buffer at a flow rate of 1 mL / min until stable. The crude extract was loaded at a flow rate of 0.4–0.6 mL / min. After loading, the column was washed with PBS buffer at a flow rate of 1 mL / min for 3 column volumes to remove impurities. Then, the column was eluted with 0.05 mol / L Tris-HCl buffer containing 8–12 mmol / L glutathione at pH 8.0 at a flow rate of 0.4–0.6 mL / min. The eluent was collected in fractions, and the absorbance at 280 nm was measured. The eluent with an OD value ≥ 0.5 was collected as the purified product. Obtaining the target active peptide: The purity, molecular weight, and amino acid sequence of the purified product were identified. The product was considered qualified if the purity was ≥98%, the molecular weight deviated from the theoretical value of 5.5 kDa by <0.3%, and the amino acid sequence was consistent with the theoretical sequence of Aj-UBL. The qualified purified product was freeze-dried at -75~-85℃ and 0.008~0.012 mbar for 20~28h to obtain the powdered sea cucumber source multifunctional active peptide Aj-UBL, which was then sealed and stored at -20℃.

3. The method for preparing the sea cucumber-derived multifunctional active peptide Aj-UBL according to claim 1, characterized in that: The amino acid sequence of Aj-UBL in step (1) is as follows: LKGGTQIFVKMLTGKTIKIEVGPEDTVQDVKGKIEDREGIPVAEQRLIYAG.

4. The method for preparing the sea cucumber-derived multifunctional active peptide Aj-UBL according to claim 1, characterized in that: The components of the PCR reaction system in the target gene acquisition and amplification steps can be finely adjusted according to actual needs. The template concentration is 10~20 ng / μL, the primer concentration is 10 μmol / L, and the dNTP Mixture concentration is 2.5 mmol / L each.

5. The method for preparing the sea cucumber-derived multifunctional active peptide Aj-UBL according to claim 1, characterized in that: In the recombinant protein induction expression step, the final IPTG concentration is preferably 0.4-0.6 mmol / L, the induction temperature is preferably 27-29℃, the shaking speed is preferably 160-200 r / min, and the induction time is preferably 5-7 h.

6. The method for preparing the sea cucumber-derived multifunctional active peptide Aj-UBL according to claim 1, characterized in that: In the cell disruption and protein release steps, the ultrasonic disruption power is preferably 280-320W, the total disruption time is preferably 28-32min, and the centrifugation parameters are preferably 4℃, 8000r / min for 10min, collecting the cells and centrifuging at 4℃, 12000r / min for 20min, collecting the supernatant.

7. The method for preparing the sea cucumber-derived multifunctional active peptide Aj-UBL according to claim 1, characterized in that: In the recombinant protein purification step, the final concentration of glutathione in the elution buffer is preferably 9-11 mmol / L, and the loading and elution flow rates are preferably 0.5 mL / min.

8. The method for preparing the sea cucumber-derived multifunctional active peptide Aj-UBL according to claim 1, characterized in that: The freeze-drying conditions in the step of obtaining the target active peptide are preferably -78 to -82°C, 0.009 to 0.011 mbar, and the drying time is preferably 22 to 26 h.

9. A method for preparing the sea cucumber-derived multifunctional active peptide Aj-UBL according to any one of claims 1-7, characterized in that: The Aj-UBL active peptide prepared by the method is structurally stable within the range of conventional seawater salinity of 25-35‰ and common aquaculture temperature of 10-30℃, and its biological activity is sustained for ≥72h.

10. The preparation method of the sea cucumber-derived multifunctional active peptide Aj-UBL according to any one of claims 1-7, characterized in that: The Aj-UBL active peptide prepared by the method showed maximum inhibition rates of ≥55% against Vibrio splenium and 42% against Vibrio vulnificus, respectively, a kill rate of ≥95% against ciliates within 10 minutes, and a maximum inhibition rate of ≥94% against Panc02 pancreatic cancer cells.

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