Frog follicle crude extract, VMO1 protein, and their extraction methods and uses

Abstract

The present invention belongs to the field of natural compounds, and specifically relates to a crude extract of frog follicles, the VMO1 protein, and their extraction methods and uses. The specific technical solution is as follows: A crude extract, wherein the crude extract is from frog follicles and does not contain egg grains. The preparation method includes: extracting the follicles of frogs, removing the egg grains under a sterile environment, adding a buffer solution, performing ultrasonic treatment and centrifugation, taking the supernatant, and filtering to obtain the crude extract. The crude extract obtained by the present invention has good maintained activity and can be further developed for subsequent uses. The crude extract obtained by the present invention can significantly reduce the surface tension, and at the same time has good antioxidant activity and good antibacterial ability, such as the ability to inhibit Pseudomonas aeruginosa. Since products derived from natural products have good biocompatibility, therefore, based on the above activities and functions, the crude extract provided by the present invention has good application prospects in multiple fields such as the field of cosmetics water retention, the field of food preservation, and the field of pharmaceuticals.

Description

Technical Field

[0001] The present invention belongs to the field of natural compounds, and particularly relates to a crude extract of frog follicles, VMO1 protein, and their extraction methods and uses. Background Art

[0002] Natural products refer to the components or their metabolites in plants, animals, and microorganisms, mainly including several categories such as polypeptides, alkaloids, hormones, and plant polyphenols. Due to their complex chemical structures and good biocompatibility, they have been widely used in the fields of food, medicine, health products, and cosmetics.

[0003] A foam nest, that is, a foamy follicle, is an important natural product in nature. As the largest reproductive structure in vertebrates, the foam nest of anuran amphibians provides a foamy outer shell during egg formation and a shelter for hatched larvae. Depending on the environment, the foam nest faces different environmental challenges, such as drying, extreme temperatures, ultraviolet radiation, microbial invasion, and insect attacks.

[0004] A surfactant is an activator that promotes surface activation of substances by reducing the activation energy of the surface of the reactants. Compared with chemical surfactants, biosurfactants have advantages such as better biocompatibility and environmental friendliness, and are expected to be applied to higher-demand fields such as food and cosmetics.

[0005] An antioxidant is a substance that can help capture and neutralize free radicals, thereby removing the damage of free radicals to the human body. Natural antioxidants have advantages such as strong activity and low toxicity, and can be used in the processing, production, and storage of food, and can also be used in fields such as anti-cancer, anti-inflammatory, and anti-aging, and can also be used for the prevention of some chronic diseases such as diabetes and cardiovascular diseases.

[0006] Antibiotic resistance is one of the greatest challenges in the 21st century. With the increasing understanding of the pathogenesis of bacteria and intercellular communication, researchers have discovered many potential strategies to develop new drugs for treating bacterial-mediated diseases. Among them, interfering with bacterial virulence and / or intercellular signaling pathways is a particularly remarkable method. Compared with traditional methods that rely on killing bacteria or preventing bacterial growth, this method is considered to impose less selective pressure on the development of bacterial resistance. Some natural bacteriostatic agents not only have a direct inhibitory effect but also have functional components that can reduce virulence. Specifically, the bacteriostatic agent does not inhibit bacterial growth, but can inhibit the production of virulence factors by bacteria, reduce virulence traits, or inhibit bacterial attachment to the host surface through upstream signaling pathways, etc., ultimately inhibiting bacterial invasion and colonization of the host.

[0007] In existing research, there have been no reports on the use of frog follicles as surfactants, antioxidants, and antibacterial agents (especially in terms of virulence inhibition). Moreover, in the existing techniques for extracting frog follicles, there are also defects such as low activity of the extract and extremely high viscosity, making it difficult to develop subsequent applications. Therefore, if a method for extracting frog follicles can be provided to overcome the above defects and develop its uses in more fields, it will have important scientific research and application values. Summary of the Invention

[0008] The object of the present invention is to provide a crude extract of frog follicles, VMO1 protein, and their extraction methods and uses.

[0009] To achieve the above object of the invention, the technical solution adopted by the present invention is: A crude extract, the crude extract is from frog follicles and does not contain egg grains.

[0010] Preferably, the follicles of the frog are extracted, the egg grains are removed under a sterile environment, a buffer solution is added, ultrasonic treatment and centrifugation are carried out, the supernatant is taken, and filtration is carried out to obtain the crude extract.

[0011] Preferably, the obtained material after removing the egg grains is freeze-dried and then a buffer solution is added.

[0012] Correspondingly, the application of the crude extract in reducing the surface tension.

[0013] Correspondingly, a surfactant containing the crude extract or prepared using the crude extract.

[0014] Correspondingly, the application of the crude extract in enhancing the antioxidant capacity.

[0015] Correspondingly, the application of the crude extract in antibacterial.

[0016] Preferably, the application of the crude extract in inhibiting the growth and / or viability and / or toxicity of Pseudomonas aeruginosa.

[0017] Correspondingly, cosmetics or their additives, foods, health products, and drugs containing the crude extract or prepared using the crude extract.

[0018] Correspondingly, a protein from frog follicles, the amino acid sequence of the protein is as shown in SEQ NO ID: 3.

[0019] The present invention has the following beneficial effects:

[0020] After removing egg grains, obtaining follicles, freeze-drying with a freeze dryer, dissolving in Tris-Hcl buffer, assisting dissolution with an ultrasonic machine, centrifuging to obtain the supernatant, and filtering, a crude extract of frog follicles was obtained. The crude extract of ordinary follicles is prone to inactivation, while the crude extract obtained by the method of the present invention maintains good activity and can be further developed for subsequent uses. At the same time, the freshly extracted crude extract has a very high viscosity, and the inventor failed to prepare foam liquid by other methods after many attempts. Finally, freeze-drying effectively solved this problem, and freeze-drying did not significantly affect the activity of the crude extract. The crude extract obtained by the present invention can significantly reduce the surface tension, and at the same time has good antioxidant activity and good antibacterial ability, such as the ability to inhibit Pseudomonas aeruginosa. Since natural product-derived products have good biocompatibility, based on the above activities and functions, the crude extract provided by the present invention has good application prospects in many fields such as the field of cosmetics water retention, the field of food preservation, and the field of pharmaceuticals.

[0021] The VMO1 protein expressed and purified in vitro by the present invention is a new type of protein and can be used as a biosurfactant. Compared with the RSN-2, Lv-ranaspumin proteins found in the foam nests of Leptodactylidae species and the Latherin protein found in horse sweat, the VMO1 protein shows a stronger ability to reduce the surface tension and has better application potential, including being used as a solubilizer, bactericide, and drug delivery system in the pharmaceutical industry according to its good foaming and defoaming effects and good disinfection and sterilization functions, being used in the pesticide industry according to its good suspending effect, and being used in the cosmetics field according to its good wetting ability and biocompatibility. Description of the Drawings

[0022] Figure 1 Schematic diagram of the effect control of the influence of the crude extract on the surface tension;

[0023] Figure 2 Schematic diagram of the effect control of the influence of the crude extract on the antioxidant activity measured by the FRAP method;

[0024] Figure 3 Schematic diagram of the effect control of the influence of the crude extract on the antioxidant activity measured by the ABTS method;

[0025] Figure 4 Schematic diagram of the effect of the crude extract on inhibiting Pseudomonas aeruginosa (protein circle hydrolysis);

[0026] Figure 5 Schematic diagram of the effect of the crude extract on inhibiting Pseudomonas aeruginosa (quantitative detection of bacteriochlorophyll and PA biofilm detection);

[0027] Figure 6 Schematic diagram of the structure of the pET-28a vector;

[0028] Figure 7Schematic diagram for the effect control of VMO1 protein on surface tension. Detailed implementation mode

[0029] The present invention provides a method for extracting a crude extract of frog follicles, which specifically includes the following steps: extracting the follicles of frogs, removing the egg grains under a sterile environment, freeze-drying the follicles without egg grains, dissolving them with Tris-HCl buffer solution, performing ultrasonic treatment and centrifugation, taking the supernatant, and filtering to obtain the crude extract.

[0030] The crude extract can be used to reduce surface tension, improve antioxidant capacity, and can also be used to inhibit the growth or toxicity of Pseudomonas aeruginosa.

[0031] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. If not specifically specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. The obtained data are all the averages obtained after at least 3 repetitions, and all the repetitions obtain valid data.

[0032] Example 1: Preparation of crude extract of frog follicles

[0033] Collect Rhacophorus dugritei from Xiling Snow Mountain, Chengdu City, Sichuan Province, collect follicles under a sterile environment, and remove the egg grains. Freeze-dry the follicles after removing the egg grains. The freeze-drying protectant is: none. The freeze-drying parameters are: VirTis Sentry 2.0 vacuum freeze-drying system: a product of SP Scientific Company, USA. Take 10 mL of the extraction solution in a 50 mL round-bottom centrifuge tube, pre-freeze it in a -20°C freezer for 12 h, and place it in the vacuum freeze-drying system (cold trap temperature -50°C, chamber pressure 105 psi) for freeze-drying for 48 h. Dissolve the freeze-dried follicles with Tris-HCI buffer solution with pH = 7.2, use an ordinary liquid ultrasonic assistor to ultrasonically treat the follicles, then centrifuge at a centrifugation parameter of 12000g for 10 minutes, take the supernatant, and filter it through a 0.22 μm microporous filter membrane to obtain the crude extract.

[0034] Using Tris-Hcl as the solvent, dilute the crude extract, and obtain crude extract foam liquids with different concentrations by gradient dilution for standby.

[0035] Example 2: Effect of crude extract of frog follicles on surface tension

[0036] In this example, the contact angle experiment is used to measure the surface tension by the sessile drop method, and the operation is as follows:

[0037] Plug in the instrument, turn on the computer, and double-click the application on the desktop to enter the main interface. Click the active image button in the upper right corner of the interface, and at this time, the image of the stage captured by the camera can be seen. Fix the injector or microsyringe above the stage, adjust the camera focal length to 0.7 times, and then rotate the knob behind the camera base to adjust the distance between the camera and the stage to make the image clearest. Add samples: Liquids can be drawn by rotating the sampling knob on the right side of the stage, or liquids can be extruded using a microsyringe. This embodiment is divided into 3 groups, including: (1) blank control group, set as: taking lysozyme at different concentrations as the blank control (dissolving lysozyme in acetate buffer, pH = 4.5); (2) BSA positive control group, set as: diluting the BSA solution into specific concentration gradients (dissolving BSA in phosphate buffer, pH = 7); (3) crude follicular extract group, set as: the foam liquid diluent is obtained by diluting the foam stock solution of tree frog (4 mg / mL) with Tri-HCL (pH = 7.0). Each group was respectively tested for the effect of the test substance on the surface tension of water at different concentrations. Each group was set with 3 replicates, and the results were averaged. The surface tension of water is 72 mN / m 2 。

[0038] When measuring the surface tension, the sample volume is when the liquid droplet is the largest. At this time, a clear large liquid bubble can be seen at the lower end of the injector from the active image. When the liquid droplet is about to drip but not dripping, click the freeze image button on the interface and save it. Enter the main interface of the pendant drop method program, and use the software to measure the surface tension value. The results are as Figure 1 shown in Table 1. Among them, the concentration of the crude extract foam liquid in Table 1 is the measured concentration of the protein in the foam liquid (rather than the diluted concentration of the foam liquid).

[0039] Table 1 Comparison table of surface tension results

[0040]

[0041]

[0042] The results show that lysozyme and BSA have no obvious effect on reducing the surface tension of liquids, while the crude extract foam liquid with a protein concentration above 0.11 mg / mL can significantly reduce the surface tension.

[0043] Example 3: Demonstration of the antioxidant effect of crude frog follicular extract

[0044] The total antioxidant capacity of the crude extract foam solution was tested using an antioxidant kit (FRAP method, ABTS method). The antioxidant test refers to comprehensively measuring various antioxidant substances and antioxidant enzymes in the test object to obtain the total antioxidant level of the test object. Crude extract foam solution samples with different concentration gradients were prepared according to the requirements of the kit. The wavelength of the microplate reader was adjusted to 734 nm, the working solution was prepared according to the requirements of the kit, and the extract / sample was mixed with the working solution according to the operation table of the kit. The absorbance value at 734 nm was measured within 10 minutes, and △A = A blank - A measurement.

[0045] The standard curve given by the kit is:

[0046] y = 0.7021x - 0.0012R 2 = 0.9985

[0047] Among them, x refers to the Trolox concentration (μmol / mL), and y is the absorbance difference △A.

[0048] The formula for calculating the total antioxidant capacity is:

[0049] Total antioxidant capacity (μmol Trolox / mg prot) = (△A + 0.0012) ÷ 0.7021 × V sample ÷ (V sample ÷ V total sample × C pr) = 1.424 × (△A + 0.0012) ÷ C pr

[0050] The results are as Figure 2 (FRAP), Figure 3 (ABTS) and shown in Tables 2 and 3.

[0051] Table 2 Comparison table of antioxidant activities of crude extracts tested by FRAP method

[0052]

[0053] Table 3 Comparison table of antioxidant activities of crude extracts tested by ABTS method

[0054]

[0055]

[0056] Due to the complexity of the environment where the tree frog foam nest is located, it means that the foam nest needs to have antioxidant capacity to maintain the long-term stability of the foam nest. In this study, we used a total antioxidant capacity kit to conduct an activity test on the crude extract of the tree frog foam nest. By measuring with the FRAP method and the ABTS method respectively, the foam solution showed certain antioxidant capacity, and the antioxidant capacity showed a certain protein concentration dependence.

[0057] Example 4: Demonstration of the antibacterial effect of the crude extract of frog follicles

[0058] Using Pseudomonas aeruginosa as the object to demonstrate the antibacterial effect of the crude follicular extract.

[0059] (1) Protein circle hydrolysis experiment

[0060] Pick a single colony of Pseudomonas aeruginosa PAO1 and inoculate it into 5 mL of LB broth medium. Incubate it in a constant temperature shaker at 37 °C and 220 rpm / min for 16 hours. Centrifuge to collect the bacterial cells and adjust them to OD 600 nm = 1 for standby. The PAO1 bacterial solution in this example is prepared here. The experimental plate is M9 solid medium containing 0.5% (w / v) skim milk powder. The preparation method of M9 solid medium is as follows: Dissolve 5 g of KH2PO4, 1 g of NaCl, and 15 g of Agar in 950 mL of deionized water and sterilize it by high-temperature and high-pressure. Prepare another 50 mL of sterile deionized water and heat it to about 60 °C to dissolve the skim milk powder. When the medium cools to 50 - 60 °C, add 10 mL of sterile NH4Cl, 1 mL of MgSO4, and 100 μL of CaCl2 to the medium, and at the same time inject the prepared skim milk powder solution. Divide it into multiple groups, and add foam solutions with different protein concentrations (0, 25, 50, 100, 200 μM) to each group. Shake well and pour the plates. Uniformly inoculate 3 points on the plate, and each point contains 2 μL of OD 600 nm = 1 bacterial solution. Set three replicates. After the bacterial solution dries, invert the plate and place it in a 37 °C incubator for 24 h. Measure the diameter (inner diameter) of the protein hydrolysis circle, take pictures, and record the experimental data.

[0061] The results are as Figure 4 shown. Figure 4 (Upper) are the culture photos of Pseudomonas aeruginosa under different foam solution concentrations, Figure 4 (Lower) are the diameters (unit: cm) of the protein hydrolysis rings formed by Pseudomonas aeruginosa under different foam solution concentrations. The results show that: compared with the obvious protein hydrolysis rings formed by untreated colonies, the addition of the foam solution group significantly reduced the production of extracellular proteases of Pseudomonas aeruginosa PAO1 in a dose-dependent manner and inhibited the growth of Pseudomonas aeruginosa.

[0062] (2) Bacteriochlorophyll quantitative detection experiment

[0063] Add 2 mL of LB broth medium containing 200 μg / mL concentration of foam solution to a 15 mL sterile centrifuge tube, inoculate the PAO1 bacterial solution with OD 600nm = 1 at an inoculation amount of 1%, set 6 biological replicates, and place it in a shaker at 37 °C and 180 r / min for 24 h. Collect the bacterial solution in each centrifuge tube, centrifuge at 12000 r / min for 2 mins, and collect the supernatant (about 2 mL) into a 5 mL centrifuge tube. After resuspending the bacterial cells with PBS, at OD600nm Measure the absorbance value. According to the ratio of supernatant: chloroform = 5:3 (v / v), add chloroform to the supernatant, and repeatedly invert and shake the centrifuge tube to extract bacteriochlorophyll. After the mixed liquid stands and separates, collect the lower chloroform extract into a 1.5 mL centrifuge tube. According to the ratio of chloroform extract: 0.2N HCl = 3:1 (v / v), add 0.2N HCl to the chloroform extract, and vigorously shake to extract bacteriochlorophyll into 0.2N HCl. After the mixed solution stands and separates, collect the upper HCl solution into a new 1.5 mL centrifuge tube and centrifuge at 12000 r / min for 1 min. Take 200 μL of the HCl solution obtained in the previous step into a 96-well plate, and use a microplate reader to detect the absorbance value of the solution at a wavelength of 525 nm. Data processing: Use the absorbance value of the HCl solution of each sample / the OD of the bacterial cells as the final quantitative detection result of bacteriochlorophyll. The results are as Figure 5 (left) shows that after treatment with the foam solution, the production of pyocyanin by Pseudomonas aeruginosa PAO1 decreased significantly.

[0064] (3) Quantitative detection of PA biofilm

[0065] Using a 96-well plate as the biofilm attachment matrix, add 200 μL of LB broth medium containing 200 μg / mL concentration of foam solution with bacterial cells to each well (inoculate the PAO1 bacterial solution with an OD 600nm = 1 at an inoculation amount of 1%), and set 6 biological replicates. Seal the 96-well plate with a sealing film and place it in a 37 °C incubator for static culture for 24 h. Then remove the bacterial solution in the 96-well plate, add 230 μL of PBS buffer, gently wash, repeat 2 times, and then let the plate air dry naturally. Add 220 μL of 0.1% crystal violet staining solution to each well, let it stand and stain for 20 mins, suck out the staining solution, then add 230 μL of PBS buffer, gently wash, repeat 2 times, and let the plate air dry naturally. Then add 230 μL of 95% ethanol solution to each well, let it stand for 10 mins, and gently pipette the liquid to fully dissolve the crystal violet. Take 200 μL of the above mixed solution and measure the absorbance value at a wavelength of 595 nm. The results are as Figure 5 (right) shows that after treatment with the foam solution, the biofilm of Pseudomonas aeruginosa decreased significantly.

[0066] Example 5: In vitro expression and functional demonstration of surfactant protein derived from frog follicles 1. Vitelline membrane outer layer protein 1 (VMO1) was identified from the proteome of the foam nest of Rhacophorus dugritei, which plays a role in reducing the surface tension of liquids in the foam nest. The VMO1 was aligned with the known protein sequences in the Uniprot database, and the results showed that it had low similarity (less than 60%) with the known proteins, indicating that it is a new type of protein. Therefore, the present invention further carried out in vitro expression and purification of VMO1. Gene synthesis and plasmid construction were performed on it, and the recombinant plasmid was obtained. The target protein sequence is shown as SEQ NO ID: 1, and the target amino acid sequence is shown as SEQ NO ID: 2.

[0067] The obtained recombinant plasmid was transferred into Escherichia coli competent BL21(DE3) cells. After heat shock at 42°C, it was spread on a plate containing 30 μg / mL kanamycin and cultured at 37°C. Then, single colonies were picked into a liquid medium containing 30 μg / mL kanamycin and cultured at 37°C until the OD value reached 0.6, and 0.5 mM inducer IPTG was added. Cultures were carried out overnight at 20°C and for 6 h at 37°C respectively, with the culture without the addition of the inducer as the negative control. After the culture was completed, centrifugation was carried out at 4000 rpm for 10 min, the supernatant was discarded, and the bacterial cells were collected. The collected bacterial cells were suspended in buffer A (1×PBS, pH = 7.4), and an ultrasonic disruptor was used to dissolve them sufficiently. The supernatant and precipitate were collected by centrifugation, and the precipitate was dissolved in buffer B (8M Urea, 50 mM Tris-HCl, 300 mM NaCl, pH = 8.0). Samples were prepared for the supernatant and precipitate proteins respectively.

[0068] Subsequently, scale-up expression was carried out: The bacterial liquid was cultured in a medium containing 30 μg / mL kanamycin. When the OD value reached 0.6, 0.5 mM inducer IPTG was added, and large-scale expression was carried out by culturing at 37°C for 6 h. The cell bacterial bodies were collected by centrifugation.

[0069] Protein purification: After the sample was collected and processed, affinity purification was carried out. After the induction ended, the crude protein was collected. The cell pellet was dissolved with buffer C (8M Urea, 50mM Tris, 300mM NaCl, 0.1% Triton X-100, pH 8.0), sonicated with a low-temperature ultra-high pressure cell disruptor, and then centrifuged to collect the supernatant crude protein. Take 5 mL of Ni-NTA and wash and equilibrate the Ni-NTA affinity column with 5 column volumes of Binding buffer. Incubate the crude protein with the equilibrated column packing on ice for 1 h, drain the flow-through, and collect the effluent. Wash and equilibrate the column with Binding buffer and Washing buffer in turn, and collect the effluent. Finally, elute with Elution buffer and collect the effluent. Treat the effluent fraction and the crude protein separately, prepare samples, and prepare for SDS-PAGE detection. Dialyze the purified fraction under the conditions: protein storage buffer 1×PBS, 20% Glycerol, 0.1% sarkosyl, 2 mM DTT, pH = 7.0. After dialysis, concentrate with PEG20000, filter with a 0.45 μm filter membrane, dispense 1 mL / tube, freeze-dry with a freeze dryer, and store at -80 °C.

[0070] Detection of target protein: SDS-PAGE detection and Western Blot verification were used. Treat the protein sample, prepare the sample, use a 12% separating gel, a 5% stacking gel, run the gel, and finally detect the molecular weight. Then treat the protein sample and prepare the sample. The stacking gel is 5% and the separating gel is 12%. The primary antibody is mouse anti-His tag, and the secondary antibody is goat anti-mouse. Use the tag antibody for verification.

[0071] Detection of surface activity of recombinant protein: Dissolve the purified VMO1 protein, and prepare different protein concentration gradients respectively. Test the surface tension of the solutions with different protein concentrations according to the method of Example 2.

[0072] 2. Experimental results

[0073] The outer vitelline membrane protein VMO1 gene from the foam nest of Rhacophorus dugritei was obtained. According to the sequence analysis results, the 1-19 AA signal peptide sequence was removed, the sequence was optimized for Escherichia coli, synthesized by gene synthesis and then subcloned into the pET-28a(+) vector. The restriction enzyme sites were selected as NdeI-XhoI, and the N-terminal HIS tag was fused to the vector. The HIS tag was used for protein detection and purification. The expressed amino acid sequence is shown in SEQ NO ID: 3.

[0074] The expression vector pET-28a carries a selectable C-terminal His tag and an N-terminal His / Thrombin / T7 protein tag. The pET-28a vector has a single multiple cloning site, such asFigure 6 As shown. The gene VMO1 was ligated to the expression vector pET-28a, further transformed into competent Escherichia coli BL21(DE3) cells, cultured, induced for expression, the cells were collected, and the protein was purified. The detection results showed that after purification of the fusion protein, an obvious band appeared at the position near the theoretical molecular weight by SDS-PAGE electrophoresis analysis.

[0075] To further confirm that the purified protein was the target protein, it was colored with a TMB color development kit and detected according to the Western Blot procedure. The results showed an obvious band at the corresponding position, indicating that this protein was the target protein. The results showed that the fusion protein was well expressed under the induction conditions of culturing overnight at 0.5 mM IPTG and 20 °C, culturing for 6 h at 37 °C, and centrifuging at 4000 rpm. After separation and purification, 6 mg of recombinant VMO1 freeze-dried protein with an amino acid sequence length of 185, a protein purity > 90%, and a protein size of approximately 19.8 KDa was obtained, and the protein powder was stored in a -80 °C refrigerator.

[0076] The results of the surface activity experiment of the recombinant protein are as Figure 7 shown. The results showed that compared with the blank control bovine serum albumin, the control group lysozyme and the foam solution, the VMO1 protein had a stronger ability to reduce the surface tension. It is a new protein with biological surface activity and has application potential in many fields such as the field of water retention in cosmetics, the field of food preservation, and the field of pharmaceuticals.

[0077] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations, variations, modifications, and substitutions made by those of ordinary skill in the art to the technical solutions of the present invention shall all fall within the protection scope determined by the claims of the present invention.

Claims

1. A protein from frog follicles, characterized in that: The amino acid sequence of the said protein is as shown in SEQ NO ID:

3.

2. Use of the protein according to claim 1 in reducing surface tension.

3. A surfactant comprising the protein according to claim 1 or prepared using the protein according to claim 1.

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