Ovum follicle nest extract of polypedates leucomystax, rk_xp4 protein, and extraction method and use thereof

Abstract

This invention belongs to the field of biomedicine, specifically relating to an extract from the follicular nests of the black-webbed tree frog, the RK_XP4 protein, its extraction method, and its uses. The specific technical solution is: an extract derived from the follicular nests of the black-webbed tree frog. This invention provides a novel extract from the follicular nests of the black-webbed tree frog, which effectively reduces the motility of *Pseudomonas aeruginosa*, exhibiting a devirulence-reducing effect. Based on the follicular nest extract, this invention further expresses and purifies the RK_XP4 protein in vitro, a novel protein that also effectively reduces the motility of *Pseudomonas aeruginosa*.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to extracts of the follicles of the black-webbed tree frog, RK_XP4 protein, their extraction methods, and uses. Background Technology

[0002] The long-term widespread use and abuse of antibiotics has led to a continuous worsening of the global bacterial resistance problem, with the emergence of multidrug-resistant and pan-drug-resistant bacteria posing a serious threat to human public health. *Pseudomonas aeruginosa* has long been a model organism for microbiological research, particularly valuable in areas such as gene expression, antibiotic resistance, virulence factors, and biofilm formation. This bacterium possesses high genomic variability, metabolic flexibility, and phenotypic diversity, enabling it to adapt to various environments, existing as a harmless saprophytic bacterium or as an opportunistic human pathogen. *Pseudomonas aeruginosa* can cause acute and chronic infections in immunocompromised patients, including bloodstream infections, urinary tract infections, eye infections, and soft tissue infections, especially chronic lung infections commonly seen in patients with cystic fibrosis. Its inherent antibiotic resistance and tolerance, coupled with its ability to rapidly adapt to infectious environments, make it a significant challenge in clinical treatment.

[0003] Natural products refer to the components or metabolites of plants, animals, and microorganisms, mainly including polypeptides, alkaloids, hormones, and plant polyphenols. Through long-term evolution, natural products have formed coexistence or antagonistic relationships with microorganisms, often exhibiting higher targeting capabilities and good biocompatibility, making them ideal resources for screening highly active and safe antiviral molecules.

[0004] The foam nests of tailless amphibians are a unique type of natural protein foam structure. When exposed to the complex microbial environment of the wild for a long time, they can effectively protect the embryos from ultraviolet rays, pathogens, and predators. They are a unique biological resource for exploring new antimicrobial active substances.

[0005] Therefore, if new active proteins with antiviral activity can be obtained by screening tree frog foam nests, it will be of great significance for enriching the antiviral theory system and expanding the sources of anti-infective drugs. Summary of the Invention

[0006] The purpose of this invention is to provide extracts of black-webbed tree frog follicles, RK_XP4 protein, their extraction methods, and uses.

[0007] To achieve the above-mentioned objectives, the technical solution adopted by the present invention is: an extract, said extract being derived from the follicular nest (foam nest) of the black-webbed tree frog.

[0008] Accordingly, the method for preparing the extract involves extracting the follicular nests of the black-webbed tree frog and freeze-drying the follicular nests; dissolving the freeze-dried follicular nest powder with a buffer solution to obtain follicular nest fluid; and enriching the proteins in the black-webbed tree frog follicular nests using an ammonium sulfate fractionation precipitation method to obtain the extract.

[0009] Accordingly, the extract, or the extract prepared using the method, has non-disease treatment or diagnostic applications in terms of antiviral activity. Preferably, the extract is used to inhibit the virulence of *Pseudomonas aeruginosa*, particularly its motility.

[0010] Accordingly, the extract, or the extract prepared using the method, has applications in anti-oxidation for non-disease treatment or diagnosis.

[0011] Accordingly, a protein RK_XP4 from the follicular nest of the black-webbed tree frog, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0012] Accordingly, the protein RK_XP4 has applications in non-disease treatment or diagnosis, including its antibacterial and antiviral properties, particularly its ability to inhibit the motility of Pseudomonas aeruginosa.

[0013] Accordingly, pharmaceuticals, cosmetics, food, or health products containing the extract or the extract or protein RK_XP4 prepared by the method described herein.

[0014] Accordingly, pharmaceuticals, cosmetics, food or health products prepared using the extract or the extract or the protein RK_XP4 prepared by the method.

[0015] The present invention has the following beneficial effects:

[0016] Motility is a key biological basis for *Pseudomonas aeruginosa* to complete infection, colonization, spread, and pathogenesis. *P. aeruginosa* relies on flagella and type IV pili to drive swimming, swarming, and twitching movements, all of which jointly determine its initial adhesion and colonization on the host's airway, wounds, mucous membranes, and the surfaces of medical implants. Motility directly regulates the initiation and maturation of biofilm formation, a prerequisite for bacteria to build drug-resistant biofilms and evade host immunity and antibiotic killing. Simultaneously, motility facilitates bacterial invasion across tissues and spread to deeper tissues, exacerbating lung infections, burn wound infections, urinary tract infections, and bacteremia. Therefore, inhibiting the motility of *P. aeruginosa* can block its colonization, biofilm formation, and systemic spread at the source, making it a highly effective and safe target for anti-infective intervention.

[0017] This invention provides a method for obtaining an extract from the foam nest of the black-webbed tree frog. The extract effectively reduces the motility of *Pseudomonas aeruginosa*, exhibiting a devirulence-reducing effect. Based on the foam nest extract, this invention further expresses and purifies the RK_XP4 protein in vitro, a novel protein that also effectively reduces the motility of *Pseudomonas aeruginosa*. Therefore, both the black-webbed tree frog foam nest extract and the RK_XP4 protein provided by this invention possess good antiviral and anti-infective capabilities. Attached Figure Description

[0018] Figure 1 A schematic diagram of the inhibitory effect (MIC) experiment of extract from the foam nest of the black-webbed tree frog on the growth of Pseudomonas aeruginosa;

[0019] Figure 2 A schematic diagram illustrating the effect of extracts from the foam nests of the black-webbed tree frog on the motility of Pseudomonas aeruginosa.

[0020] Figure 3 A schematic diagram illustrating the antioxidant capacity of extracts from the foam nests of the black-webbed tree frog.

[0021] Figure 4 The HPLC fractional chromatogram of the extract from the foamy nest of the black-webbed tree frog;

[0022] Figure 5 A schematic diagram showing the effect of different fractions of the HPLC extract of the black-webbed tree frog's foam nest on the motility of Pseudomonas aeruginosa.

[0023] Figure 6 Image of pET-32a-RK_XP4 plasmid;

[0024] Figure 7 Figures showing the SDS-PAGE and Western Blot results of the RK_XP4 protein;

[0025] Figure 8 This is a schematic diagram illustrating the antibacterial effect of the RK_XP4 protein against Pseudomonas aeruginosa.

[0026] Figure 9 A schematic diagram illustrating the effect of RK_XP4 protein on the motility of Pseudomonas aeruginosa.

[0027] Figure 10 Molecular sieve separation diagram for Trx tags;

[0028] Figure 11 A schematic diagram illustrating the effect of the Trx tag on the motility of Pseudomonas aeruginosa.

[0029] Figure 12 This is a schematic diagram illustrating the antioxidant capacity of the RK_XP4 protein. Detailed Implementation

[0030] I. This invention provides a method for obtaining extracts from the follicles of the black-webbed tree frog, specifically including the following steps:

[0031] (1) Extract the follicle nests of the black-webbed tree frog, remove the eggs under sterile conditions, and freeze-dry the follicle nests;

[0032] (2) Dissolve the lyophilized follicle powder in 50mM Tris-HCl and 50mM NaCl (pH=7.8) buffer to obtain follicle fluid;

[0033] (3) Protein enrichment in the follicles of the black-webbed tree frog was achieved using ammonium sulfate fractionation precipitation: the supernatant was collected by centrifugation at 12,000 rpm for 20-30 min at a low temperature, such as 4°C. Solid ammonium sulfate was slowly added to the supernatant under continuous stirring until the solution reached 100% saturation. The solution was then allowed to stand at 4°C for 2 h and centrifuged again at 12,000 rpm for 15 min. The supernatant was discarded, the precipitate was collected, and resuspended in a 50 mM Tris-HCl and 50 mM NaCl (pH 7.8) buffer solution.

[0034] (4) Dialyze the resuspended extract at 4°C, changing the buffer solution three times with a volume greater than 100 times to remove ammonium sulfate. Store the obtained protein solution at -80°C for later use.

[0035] The extract can reduce the virulence of Pseudomonas aeruginosa and inhibit its motility.

[0036] Second, the present invention further extracted a protein RK_XP4 from the extract of the black-webbed tree frog follicle nest.

[0037] The amino acid sequence of the protein RK_XP4 is GPLISITYKCGVPHKLRVNCGRPAITHDECVKQNCCFDDSVPETIWCFYPLKSQSIVCKPDGHRFDCGYPGIPEKDCTDRGCCYDDSVPRVVWCYQPEVTSVEHHCNDVIPENRVDCGQPHISSEDCRQKECCYDSSVAGVPWCFKPEIKKVIG* (SEQ ID NO: 1). The protein RK_XP4 can inhibit the activity, virulence, toxicity, and motility of Pseudomonas aeruginosa.

[0038] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. All obtained data are average values ​​obtained after at least three repetitions, and each repetition yields valid data.

[0039] Example 1: Preparation of extract from the follicles of the black-webbed tree frog

[0040] Black-webbed tree frogs from Xishuangbanna Dai Autonomous Prefecture, Yunnan Province, were collected from follicular nests under sterile conditions, and the eggs were removed. The follicles after egg removal were freeze-dried; no freeze-drying protectant was used. Freeze-drying parameters were as follows: VirTis Sentry 2.0 vacuum freeze-drying system (SP Scientific, USA). 10 mL of the extract was placed in a 50 mL round-bottom centrifuge tube, pre-frozen at -20°C for 12 h, and then freeze-dried in the vacuum freeze-drying system (cold trap temperature -50°C, chamber pressure 105 psi) for 48 h.

[0041] The lyophilized follicles were dissolved in 50 mM Tris-HCl and 50 mM NaCl (pH=7.8) buffer. Proteins were initially enriched using ammonium sulfate fractionation: the solution was centrifuged at 12,000 × g for 20 min at 4 °C, and the supernatant was collected. Solid ammonium sulfate was slowly added under continuous stirring until the solution reached 100% saturation. After standing at 4 °C for 2 h, the solution was centrifuged again. The supernatant was discarded, and the precipitate was collected and resuspended in 50 mM Tris-HCl and 50 mM NaCl (pH=7.8) buffer. The resuspended extract was dialyzed at 4 °C, with the buffer volume changed three times (at least 100 times) to remove ammonium sulfate. The resulting protein solution was stored at -80 °C for later use.

[0042] Example 2: Effects of frog follicle nest extract

[0043] (I) Effects of frog follicle nest extract on Pseudomonas aeruginosa

[0044] 1. Minimum inhibitory concentration (MIC) and growth curve method

[0045] The minimum inhibitory concentration (MIC) of frog follicle nest extract against *Pseudomonas aeruginosa* was determined using the micro-broth dilution method. *P. aeruginosa* was cultured in LB broth at 37°C with shaking at 200 rpm until the logarithmic growth phase, and then diluted with fresh LB broth to a final concentration of approximately 1 × 10⁻⁶. 5 CFU / mL. Frog follicle nest extract was serially diluted twofold according to a specific concentration gradient and added to 96-well plates. An equal volume of bacterial culture was added to each well, bringing the final volume to 200 μL. All other conditions remained the same, with an equal volume of buffer added instead of frog follicle nest extract as a negative control. After incubating the 96-well plates at 37°C for 24 h, the OD was measured using a microplate reader. 600 value.

[0046] To assess the effect of the test compound on the growth kinetics of Pseudomonas aeruginosa, the logarithmic-phase bacterial culture was diluted to the initial OD.600 ≈0.05. Different concentrations of frog follicle nest extract were added to the treatment groups, while the control group received an equal volume of buffer. The culture system was incubated at 37℃ and 200 rpm with shaking, and samples were taken every 1 hour to measure OD. 600 Value, continuously monitored for 24 hours, in OD 600 Plot the growth curve over time.

[0047] The results are as follows Figure 1 As shown. Figure 1 The control group refers to the negative control. The results showed that the crude extract of the black-webbed tree frog foam nest had no significant inhibitory effect on the growth and reproduction of Pseudomonas aeruginosa PAO1 at different concentration gradients.

[0048] 2. The effect of frog follicle nest extract on the motility of Pseudomonas aeruginosa was detected by phenotypic experiments.

[0049] Twitching Motility: Add 10g of agar to 1L LB broth (final agar concentration 1%), autoclave, and after slight cooling, add 50μg / mL of the frog follicle ovary extract and pour into plates. After incubating the plates at room temperature overnight, use a 10μL pipette to aspirate 2μL of OD. 600nm A Pseudomonas aeruginosa bacterial suspension with a concentration of 1 was carefully inserted to the bottom of the culture medium. The suspension was then slowly aspirated, avoiding contact with the surface of the medium. After the suspension dried, the plate was placed upright in a 37°C incubator and incubated for 24 hours. The surface medium was removed, leaving an empty plate. 0.1% crystal violet solution was added to the empty plate, and the plate was stained for 30 minutes. The crystal violet solution was then removed, and the plate was rinsed with PBS. The diameter of the movement range was photographed and recorded.

[0050] Swarming Motility: Add 5 g of agar (final agar concentration of 0.5%) to 800 mL of deionized water, autoclave, and immediately add 200 mL of 5×M8 solution, 10 mL of filtered sterile 20% glucose solution (w / v), and 1 mL of filtered sterile 1 M MgSO4 solution. When the culture medium cools to about 60°C, add 25 mL of 20% casaminoacids solution (w / v), mix well, and then add 50 μg / mL of the frog follicle nest extract. Pour the mixture into plates. After the plates have been incubated at room temperature overnight, use a 10 μL pipette to aspirate 4 μL of OD. 600nm A bacterial suspension with a concentration of 1 was inoculated onto the surface of the culture medium. The plate was placed upright in a 37°C incubator and incubated for 24 hours. Photos were taken and the diameter of the movement range was measured.

[0051] Swimming Motility: Add 3 g of agar to 800 mL of deionized water and autoclave. Immediately add 200 mL of 5×M8 solution, 10 mL of filtered sterile 20% glucose solution (w / v), and 1 mL of filtered sterile 1 M MgSO4 solution. When the culture medium cools to approximately 60°C, add 25 mL of 20% casamino acids solution (w / v), mix well, and then add 50 μg / mL of the frog follicle ovary extract. Pour the mixture into plates. After incubating the plates at room temperature overnight, use a 10 μL pipette to aspirate 2 μL of the extract. 600nm =1 bacterial suspension was inoculated in the middle of the culture medium. After the bacterial suspension dried, the plate was placed upright in a 37℃ incubator and incubated for 24 hours. The diameter of the movement range was photographed and measured.

[0052] The results are as follows Figure 2 As shown. Figure 2 The control in this study refers to the negative control (set up in the same way as the negative control in the MIC experiment). Results showed that frog follicle nest extract at a concentration of 50 μg / mL significantly inhibited the motility of *Pseudomonas aeruginosa*, especially its swarming activity.

[0053] (II) Antioxidant capacity of frog follicle nest extract

[0054] The antioxidant activity of frog follicle nest extract was determined using the ABTS free radical scavenging assay antioxidant kit. Following the manufacturer's instructions, the ABTS stock solution was mixed with potassium persulfate solution and allowed to stand at room temperature in the dark for 12–16 h to generate a stable ABTS free radical working solution. Appropriate amounts of black-webbed tree frog follicle nest protein samples were taken, and different protein concentration groups (1, 2, 4, 8 mg / mL) were established. The diluted ABTS working solution was added, mixed well, and reacted at room temperature in the dark for 30 min. Simultaneously, the microplate reader was preheated for 30 min, and the absorbance was measured at 734 nm. An equal volume of 50 mM Tris-HCl buffer (containing 50 mM NaCl, pH=7.8) served as a blank control.

[0055] ABTS free radical scavenging rate (%) = (A blank - A assay) ÷ A blank × 100%.

[0056] The results are as follows Figure 3 As shown in the figure. The results indicate that the foam nest of the black-webbed tree frog possesses highly efficient antioxidant capacity, significantly scavenging free radicals and inhibiting oxidative stress, demonstrating good potential for antioxidant applications. This antioxidant activity can synergistically enhance its antiviral function, reduce infection-related oxidative damage, and improve the protein's application value in biomedicine, wound care, and functional materials.

[0057] Example 3: Obtaining and Demonstrating the Effects of Proteins from Frog Ovary Extracts

[0058] (a) Protein Acquisition

[0059] 1. The crude extract of the follicular nests obtained in Example 1 was fractionated using preparative high-performance liquid chromatography (HPLC) and separated into 18 fractions based on peak shape. Fractional fraction acquisition method: Using the follicular nest extract of the black-webbed tree frog obtained in Example 1 as raw material, the protein exhibiting resistance to *Pseudomonas aeruginosa* virulence was separated and purified by HPLC. 10g of lyophilized tree frog follicular nest powder was dissolved in 10mL of 50mM Tris-HCl, 50mM NaCl (pH=7.8) buffer, filtered through a 0.22µm filter membrane, and the collected filtrate was loaded onto a preparative HPLC C8 column. Gradient elution was performed using an elution system consisting of water (containing 0.1% trifluoroacetic acid, phase A): acetonitrile (containing 0.1% trifluoroacetic acid, phase B) at a flow rate of 18mL / min, and protein concentration was detected at 280nm. Each peak was collected to detect activity, and the fractions were lyophilized for later use.

[0060] The specific elution procedure includes: equilibrating the column under initial conditions (Phase A 95%, Phase B 5%), and injecting the sample after the baseline stabilizes. A linear gradient is applied from 0 to 28 min, with the Phase A proportion decreasing linearly from 95% to 68% (Phase B simultaneously increasing from 5% to 32%). From 28 to 38 min, the linear gradient continues, with the Phase A proportion decreasing linearly from 68% to 45% (Phase B simultaneously increasing from 32% to 55%). From 38 to 45 min, the Phase A proportion decreases linearly from 45% to 20% (Phase B simultaneously increasing from 55% to 80%). From 45 to 47 min, the Phase A proportion decreases to 0% (Phase B proportion increases to 100%), and this condition is maintained for 3 to 5 column volumes of elution to remove strongly retained impurities. Results are as follows: Figure 4 As shown.

[0061] After lyophilization, each component was dissolved in ultrapure water, and the protein concentration of each component was measured using NanoDrop. The protein concentration was then uniformly diluted to 10 μg / mL with ultrapure water, and the effect of each fraction on the swarming motility of *Pseudomonas aeruginosa* was examined according to the method in Example 2. The results are as follows... Figure 5 As shown (the control group is a blank control without the addition of any components).

[0062] The results showed that segments 3, 8, and 18 significantly inhibited the clustering movement of *Pseudomonas aeruginosa*. Specifically, segment 3 corresponded to the collection of substances from the peak at minutes 7.8–9.2; segment 8 to the collection of substances from the peak at minutes 16.4–18.4; and segment 18 to the collection of substances from the peak at minutes 38.2–45. Further experiments were conducted on segment 3.

[0063] 2. In vitro expression to obtain protein RK_XP4

[0064] The protein corresponding to segment 3 was named RK_XP4. Alignment of RK_XP4 with known protein sequences in the Uniprot database showed low similarity (highest similarity was 51.2%), suggesting it is a novel protein.

[0065] The protein, recombinantly expressed in vitro via E. coli prokaryotic cells, was purified by Ni-NTA affinity chromatography. SDS-PAGE showed successful expression and high purity of the RK_XP4 protein. Western blotting results confirmed correct expression of the target protein, meeting the requirements for subsequent functional validation and activity studies. Results are as follows... Figure 6 , 7 As shown ( Figure 7 In the diagram, because RK_XP4 has the Trx tag, it is named "Trx-XP4".

[0066] The specific method for in vitro expression and purification of RK_XP4 according to this invention includes: gene synthesis and plasmid construction, and obtaining recombinant plasmids (such as...). Figure 6 (As shown). The amino acid sequence of RK_XP4 is shown in SEQ NO ID: 1, and the amino acid sequence of the tagged RK_XP4 is shown in SEQ ID NO: 2.

[0067] SEQ ID NO: 2:

[0068] MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMHHHHHHSSGLEVLFQGPLISITYKCGV PHKLRVNCGRPAITHDECVKQNCCFDDSVPETIWCFYPLKSQSIVCKPDGHRFDCGYPGIPEKDCTDRGCCYDDSVPRVVWCYQPEVTSVEHHCNDVIPENRVDCGQPHISSEDCRQKECCYDSSVAGVPWCFKPEIKKVIG*.

[0069] The RK_XP4 protein coding sequence was obtained from the oviduct transcriptome data of the black-webbed tree frog. After codon optimization, it was cloned into a prokaryotic expression vector and transformed into Escherichia coli BL21 (DE3) competent cells. Positive clones were selected and cultured in LB medium containing 50 μg / mL ampicillin at 37°C with shaking at 200 rpm until the bacterial culture reached OD. 600≈ 0.6, add 500 nM IPTG to induce expression, and continue culturing at 16℃ for 20 hours. After induction, centrifuge at 3800 rpm / min for 15 minutes to collect the bacterial cells. Add buffer A (1×PBS, pH=7.4) to the collected bacterial cells for resuscitation, and use an ultrasonic homogenizer to fully dissolve them. Centrifuge to collect the supernatant and precipitate. Dissolve the precipitate in PBS buffer (containing 150 Mm NaCl, pH=7.4), and prepare samples from the supernatant and precipitate proteins separately.

[0070] Protein purification: After sample collection and processing, affinity purification was performed. The supernatant was collected by centrifugation to obtain crude protein. 5 mL of Ni-NTA was used to wash and equilibrate the Ni-NTA affinity column with 5 column volumes of binding buffer. The crude protein and equilibrated column packing were incubated on ice for 1 h, the flow-through was drained, and the eluent was collected. The column was then washed sequentially with binding buffer and washing buffer, and the eluent was collected. Finally, elution buffer was used, and the eluent was collected. The eluent fraction and crude protein were processed separately for SDS-PAGE analysis. The purified fraction was dialyzed under the following conditions: protein preservation buffer 1× PBS buffer (containing 150 M NaCl, pH=7.4). After dialysis, the sample was filtered through a 0.45 μm filter and aliquoted into 1 mL / tube containers, stored at -80℃.

[0071] Target protein detection: SDS-PAGE and Western blotting were used for detection and validation. Protein sample processing, preparation, 12% separating gel, 5% stacking gel, gel running, and final molecular weight determination were performed. The protein sample was then processed and prepared. A 5% stacking gel and a 12% separating gel were used. The primary antibody was a mouse anti-His tag, and the secondary antibody was a goat anti-mouse antibody. Validation was performed using the tag antibody.

[0072] (II) Effects of RK_XP4 protein (including tag) on ​​the clustering and movement of Pseudomonas aeruginosa

[0073] 1. The effect of RK_XP4 protein on the swarming ability of *Pseudomonas aeruginosa* was detected according to the method in Example 2. The results are as follows: Figure 8 , 9 As shown.

[0074] The results showed that the recombinant RK_XP4 protein significantly reduced the motility range of bacteria at concentrations of 5 μg / mL, 10 μg / mL and 20 μg / mL, demonstrating that the RK_XP4 protein can effectively inhibit the motility of Pseudomonas aeruginosa clusters.

[0075] 2. The effect of Trx tags on the clustering and movement of Pseudomonas aeruginosa

[0076] To further eliminate interference from the Trx tag on protein activity, RK_XP4 protein was digested with 3C Protease, and the digested sample was purified using an AKTA purification system. 10 mg of the digested protein mixture was filtered through a 0.22 µm filter membrane, and the filtrate was loaded onto a Sephadex G-75 column pre-equilibrated with the same buffer. Gradient elution with PBS buffer (pH=7.4) was performed at a flow rate of 0.3 mL / min, and protein concentration was detected at 280 nm. The protein was then lyophilized, concentrated, and its concentration was determined. Figure 10 As shown, the SDS-PAGE results indicate that 16–18.5 mL of the effluent sample was pure Trx tag.

[0077] The effect of Trx tags on the swarming movement of Pseudomonas aeruginosa was detected using the method described in Example 2. The results are as follows: Figure 11 As shown in the figure. The results indicate that the Trx tag did not inhibit the activity of Pseudomonas aeruginosa PAO1 cluster motility, further demonstrating that the RK_XP4 protein does indeed have the ability to inhibit Pseudomonas aeruginosa cluster motility.

[0078] (III) Antioxidant capacity of RK_XP4 protein

[0079] The antioxidant capacity of RK_XP4 protein was measured according to the method in Example 2, and the results are as follows: Figure 12 As shown in the figure. The results indicate that the RK_XP4 protein has good antioxidant capacity.

[0080] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Any modifications, alterations, substitutions, or variations made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention shall fall within the protection scope defined by the claims of the present invention.

Claims

1. An extract, characterized in that: The extract was obtained from the egg follicles of the black-webbed tree frog.

2. The method for the preparation of the extract according to claim 1, characterized by the fact that: Extract follicular nests from the black-webbed tree frog and freeze-dry the follicular nests; dissolve the freeze-dried follicular nest powder in buffer solution to obtain follicular nest fluid; The protein in the follicles of the black-webbed tree frog was enriched using ammonium sulfate fractionation precipitation to obtain the extract.

3. The use of the extract of claim 1 or the extract prepared by the method of claim 2 in antibacterial non-disease treatment or diagnostic applications.

4. Use according to claim 3, characterized in that: The application of the extract in inhibiting the motility of Pseudomonas aeruginosa.

5. The use of the extract of claim 1 or the extract prepared by the method of claim 2 in non-disease treatment or diagnosis with antioxidant properties.

6. A protein RK_XP4 from the ovarian follicular nest of the black webbed tree frog, characterized by: The amino acid sequence of the protein RK_XP4 is shown in SEQ ID NO:

1.

7. The use of the protein RK_XP4 of claim 6 in antibacterial non-disease treatment or diagnosis.

8. The use of the protein RK_XP4 of claim 6 in non-disease treatment or diagnosis for antioxidant effects.

9. A pharmaceutical, cosmetic, food, or health product comprising the extract of claim 1 or the extract prepared by the method of claim 2 or the protein RK_XP4 of claim 6.

10. Pharmaceuticals, cosmetics, food or health products prepared using the extract of claim 1 or the extract prepared by the method of claim 2 or the protein RK_XP4 of claim 6.

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