Preparation method and application of umbilical cord extract

Abstract

The invention relates to the technical field of animal umbilical cord extraction, in particular to a preparation method and application of an animal umbilical cord extract. The trypsin mutant is adopted to prepare the umbilical cord extract, the enzymolysis efficiency is improved, the skin repairing effect of the obtained umbilical cord extract is remarkably superior to that of a common enzymolysis product, and in repairing of a mouse burn model, the recovery speed of mouse skin is 2 days earlier than that of a common umbilical cord extract. The umbilical cord extract prepared by the method provided by the invention is expected to be widely applied to wound or skin repair.

Description

Technical Field

[0001] The invention relates to the technical field of animal umbilical cord extraction, and in particular to a preparation method and application of an animal umbilical cord extract. Background Art

[0002] A large number of studies have shown that animal umbilical cord proteins, umbilical cord peptides, umbilical cord extracts, and umbilical cord extracts all have good effects in repairing tissue damage, promoting metabolism, delaying skin aging, regulating the body's immunity, anti-cancer, and liver protection. They also have excellent antioxidant activity and are often used in clinical practice to treat oxidation-induced diseases. Their antioxidant activity is mainly regulated by protein.

[0003] Umbilical cord extract contains umbilical cord stem cell exosomes, which contain natural ingredients such as small molecule peptides, free amino acids, vitamin A group, organic acids, inorganic acids, etc. that are indispensable for healthy skin, as well as up to hundreds of active nutrients and signal substances required for regulating cell growth. It plays a key role in comprehensively conditioning the skin, improving skin quality, and preventing aging. It can quickly repair the skin barrier and restore a youthful, healthy and energetic state.

[0004] At present, extracts from animal tissues are usually prepared by enzymatic hydrolysis. For example, the invention patent with the publication number CN105420325B discloses a method for preparing umbilical cord polypeptides by one-step enzymatic hydrolysis using trypsin. The invention patent with the publication number CN101773522B discloses a two-step enzymatic hydrolysis method for preparing porcine intestinal mucosa extracts, which first uses trypsin to enzymatically hydrolyze the porcine intestinal mucosa, and then uses papain to enzymatically hydrolyze the enzymatic hydrolysis product.

[0005] However, the extracts prepared by the above methods have defects such as low extraction rate and poor extract effect. Summary of the invention

[0006] The first aspect of the present invention provides a method for preparing an umbilical cord extract. In order to overcome the problems of low enzymatic hydrolysis efficiency and complicated process in the prior art, a mutant of trypsin is used to enzymatically hydrolyze the animal umbilical cord. The specific steps include:

[0007] (1) Take the animal umbilical cord tissue and homogenize it;

[0008] (2) adding an aqueous solution of trypsin or its mutant to the homogenate obtained in step (1) to prepare a mixed solution, which is then stirred, centrifuged, and the supernatant is collected;

[0009] (3) Inactivation of microorganisms and trypsin: inactivating the supernatant obtained in step (2) and collecting the inactivated supernatant;

[0010] (4) Filtration and ultrafiltration of the extract: filtering the supernatant obtained in step (3) to remove pathogens;

[0011] (5) ultrafiltration of the filtrate obtained in step (4), wherein the ultrafiltration conditions are: inlet pressure 0.5 to 1.2 MPa, outlet pressure -0.5 to -1.2 MPa, and temperature 4 to 15°C;

[0012] (6) Freeze-drying and storage of the extract: Freeze the filtrate obtained in step (5) to -40°C to -45°C within 3-6 hours and vacuum dry it.

[0013] Preferred animal umbilical cord sources include pigs, cattle, sheep, etc.

[0014] The preferred trypsin can be from any commercially available product, and the more preferred wild-type amino acid GenBank ID of the trypsin is: 1AVW_A, which contains 223 amino acids.

[0015] A more preferred amino acid sequence is shown in SEQ ID NO: 1, and a specific sequence is

[0016]

[0017] The preferred nucleotide sequence is shown in SEQ ID NO: 2, and its specific sequence is:

[0018] ;

[0019] More preferably, the trypsin is a mutant, which can be selected from one or more of N31K, S92D, and S161L;

[0020] The amino acid sequence of the preferred mutant N31K is shown in SEQ ID NO: 3, and its specific sequence is MHPLLILAFVGAAVAFPSDDDDKIVGGYTC K ENSVPYQVS

[0021] LNAGYHFCGG SLINDQWVVS AAHCYQYHIQ

[0022] VRLGEYNIDV LEGGEQFIDA SKIIRHPKYS SWTLDNDILL IKLSTPAVIN

[0023] ARVSTLLLPS ACASAGTECL

[0024] ISGWGNTLSS GVNYPDLLQC LVAPLLSHAD CEASYPGQIT NNMICAGFLE

[0025] GGKDSCQGDS GGPVACNGQL

[0026] QGIVSWGYGC AQKGKPGVYT KVCNYVDWIQ ETIAANS;

[0027] The amino acid sequence of the preferred mutant N31K / S92D is shown in SEQ ID NO:4, and its specific sequence is

[0028] MHPLLILAFVGAAVAFPSDDDDKIVGGYTC K ENSVPYQVS LNAGYHFCGG

[0029] SLINDQWVVS AAHCYQYHIQ

[0030] VRLGEYNIDV LEGGEQFIDA S D IIRHPKYS SWTLDNDILL IKLSTPAVIN

[0031] ARVSTLLLPS ACASAGTECL

[0032] ISGWGNTLSS GVNYPDLLQC LVAPLLSHAD CEASYPGQIT NNMICAGFLE

[0033] GGKDSCQGDS GGPVACNGQL

[0034] QGIVSWGYGC AQKGKPGVYT KVCNYVDWIQ ET IAANS;

[0035] The amino acid sequence of the preferred mutant N31K / S161L is shown in SEQ ID NO:5, and its specific sequence is

[0036] MHPLLILAFVGAAVAFPSDDDDKIVGGYTC K ENSVPYQVS LNAGYHFCGG

[0037] SLINDQWVVS AAHCYQYHIQ

[0038] VRLGEYNIDV LEGGEQFIDA SNIIRHPKYS SWTLDNDILL IKLSTPAVIN

[0039] ARVSTLLLPS ACASAGTECL

[0040] I SGWGNTLSS GVNYPDLLQC L VAPLLSHAD CEASYPGQIT NNMICAGLE

[0041] GGKDSCQGDS GGPVACNGQL

[0042] QGIVSWGYGC AQKGKPGVYT KVCNYVDWIQ ETIAANS

[0043] The preferred concentration of trypsin or variants is 5-20 g / L, more preferably 10-20 g / L.

[0044] Preferably, in step (1), the umbilical cord tissue is cleaned with water before homogenization. More preferably, the cleaned umbilical cord tissue is soaked in iodine tincture and then deiodinated with alcohol after removal.

[0045] Preferably, the centrifugation in step (2) is performed at 5000-10000 r / min for 10-15 min.

[0046] Preferably, inactivate in a water bath at 60°C-85°C for 30-40 minutes;

[0047] Another aspect of the present invention provides an umbilical cord extract obtained by the above method. The extract obtained by the method has high activity and strong stability and has a wide range of application scenarios.

[0048] Another aspect of the present invention provides an application of the umbilical cord extract. Experiments on mouse models have shown that the umbilical cord extract has a good effect on skin wound healing and is expected to be widely used in skin repair and scar removal products. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Western electrophoresis of enzyme mutants. Band 1 is the N31K / S92D enzyme mutant, and band 2 is the N31K / S161L enzyme mutant.

[0050] Figure 2 Enzyme activity detection diagram. 1-6 are the enzyme activities of trypsin WT and mutants N31K, S92D, S161 L, N31K / S92D, and N31K / S161L, respectively.

[0051] Figure 3 Healing rate of burn skin in mice in different treatment groups.

[0052] Figure 4 HE staining of skin tissue. Histological observation results of mouse scald skin in different treatment groups at 5d and 11d. A: blank group; B: extract 1 group; C: extract 2 group; D: extract 3 group. DETAILED DESCRIPTION

[0053] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited thereto.

[0054] In the following examples, various processes and methods not described in detail are conventional methods known in the art. The sources, trade names and components of the reagents used are indicated when they first appear, and the same reagents used thereafter are the same as the first indicated unless otherwise specified; the reagents and materials involved are all obtained from commercial sources unless otherwise specified.

[0055] Example 1 Preparation of trypsin mutants

[0056] Screening of trypsin mutants In order to improve the activity of wild-type trypsin (GenBank: 1AVW_A, amino acid sequence SEQ ID NO: 1, encoding nucleotide sequence SEQ ID NO: 2), the inventors screened a large number of mutations of amino acids near the active site of the enzyme by directed evolution technology.

[0057] Design PCR primers as follows:

[0058] F: GGC AATTCT AAATTGTGGGCGGCTATACCT (underlined is the restriction endonuclease EcoRI recognition site, SEQ ID NO: 6);

[0059] R: ATA GCGGCCGC AGCAGACCATTGCCGCCAAT (the underline indicates the recognition site of restriction endonuclease NotI, SEQ ID NO: 7).

[0060] Trypsin gene (SEQ ID NO: 2) was used as a template and PCR amplification was performed using the above primers using GeneMorph II random mutation PCR kit (Stratagene). The PCR product was recovered from gel, digested with EcoRI and NotI, and ligated with the pET21a vector digested with the same enzymes, transformed into Escherichia coli BL21 (DE3), spread on LB+Amp plates, and cultured inverted at 37°C. After transformants appeared, they were picked up one by one with a toothpick into a 96-well plate, 150 μL of LB+Amp medium containing 0.1 mM IPTG was added to each well, and the cells were cultured at 37°C and 220 rpm for about 6 h. The supernatant was discarded after centrifugation, and the cells were resuspended in buffer and repeatedly frozen and thawed to obtain Escherichia coli cell lysate containing trypsin.

[0061] Take out 50 μL of lysate to two new 96-well plates, measure the trypsin activity and protein content at 37°C, and calculate the specific activity of different mutants.

[0062] The experimental results showed that the mutation sites at which the enzyme activity was significantly improved at 37°C were N31K, S92D or S161L.

[0063] On this basis, a trypsin mutant comprising the above-mentioned combination of one or two mutation sites among N31K, S92D or S161L was obtained.

[0064] Example 2 Construction of recombinant strains

[0065] The above enzyme mutant nucleotides were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd., and the wt and its variants were successfully cloned into the plasmid pET-30a(+), and 6 His tags were added to the N-terminus of the gene during the construction of the expression plasmid. The successfully constructed recombinant plasmids were transformed into Escherichia coli BL21(DE3) cells and recorded as E. coli BL21(DE3) / pET-30a(+)-WT, E. coli BL21(DE3) / pET-30a(+)-mutN31K, E. coli BL21(DE3) / pET-30a(+)-mutS92D, E. coli BL21(DE3) / pET-30a(+)-mutS161L, E. coli BL21(DE3) / pET-30a(+)-mutN31K / S92D, E. coli BL21(DE3) / pET-30a(+)-mutN31K / S161L. Pick a single recombinant colony, culture it in a culture medium, perform colony PCR identification, perform restriction enzyme digestion identification, sequence verification, and select wild-type and mutant engineered bacteria for preservation.

[0066] Example 3 Expression and enzyme activity determination of trypsin

[0067] Three strains of each type were selected and cultured in liquid LB medium (containing 5 μg / mL erythromycin), and cultured at 30°C overnight. The next day, 5% inoculation was inoculated into 100mL LB liquid medium (containing 5 μg / mL erythromycin), and cultured at 30°C until OD600 = 0.5. 100 μL of 0.3M CuSO4 was added to the clean bench, and cultured at 30°C for 4 hours, and then cultured at 16°C for 24 hours. 90 μL of the supernatant of the recombinant strain cell wall liquid was taken, mixed with 30 μL of 4×Protein Loading Buffer, and boiled at 100°C for 10 minutes. Centrifuged at 4°C, 12000rpm for 5 minutes, collected the sample treatment liquid, and purified by ion exchange chromatography.

[0068] Specifically, 10.0 mL of trypsin and its mutant concentrate was applied to a HiTrap Q HP anion column pre-equilibrated with 10 mmol / L Tris-HCl (pH 8.0), and then linear gradient elution was performed with 10 mmol / L Tris-HCl (pH 8.0) containing 1 mol / L NaCl. The enzyme activity was detected by colorimetry, and the purity of the gradient eluted protein solution was detected by SDS-PAGE gel electrophoresis.

[0069] like Figure 1 As shown, SDS-PAGE was performed on the double mutant trypsin, and a clear and bright band of about 23.4Kda was obtained, indicating that the above mutants were successfully expressed and had a high purity.

[0070] like Figure 2 As shown, WT, N31K, S92D, S161L, N31K / S92D, N31K / S161L (corresponding to Figure 2 The average extracellular enzyme activities of the enzyme types were 225.6U / mL, 255.1U / mL, 245.3U / mL, 276.8U / mL, 332.7U / mL and 305.2U / mL respectively.

[0071] Depend on Figure 2 It can be seen that the enzyme activities of mutants N31K and S161L were significantly improved compared with the wild type (P<0.05), and the enzyme activities of mutants N31K / S92D and N31K / S161L were significantly improved compared with the wild type (P<0.01).

[0072] * indicates P<0.05, ** indicates P<0.01.

[0073] Wild-type trypsin and N31K / S92D and N31K / S161L enzyme mutants with higher enzyme activity were selected for the preparation of animal umbilical cord extracts.

[0074] Example 4 Preparation of umbilical cord extract

[0075] (1) Animal tissue processing: Pig umbilical cord tissue was collected and cleaned with sterile pure water. The umbilical cord tissue was cut into 1 cm pieces with sterile scissors under sterile conditions. 3 The blocks were transferred to iodine tincture and soaked for 2 min, then taken out and deiodinated with 75% by volume alcohol, rinsed three times with sterile pure water, and mechanically homogenized under sterile conditions.

[0076] (2) Enzymatic hydrolysis of the extract: Add 4 times the volume of 25 g / L aqueous solution of trypsin or its mutant to the tissue homogenate obtained in step (1), and then place it in a blender. After stirring for 24 hours, add an equal volume of sterile pure water and mix well. Centrifuge at 8000 r / min for 10 min, collect the supernatant and store at 5°C.

[0077] (3) Inactivation of microorganisms and trypsin: The supernatant from step (2) was inactivated in a 65°C water bath for 30 minutes, and the inactivated supernatant was collected.

[0078] (4) Filtration and sterilization of the extract: The supernatant after inactivation in step (3) was sequentially filtered through filters with pore sizes of 100 μm, 50 μm, 20 μm, 100 nm, 50 nm, and 20 nm to remove pathogens. The filtering conditions were an inlet pressure of 0.9 MPa and a temperature of 8°C, and the filtrate was collected.

[0079] (5) Ultrafiltration of the extract: The filtrate collected in step (4) is subjected to positive and negative pressures to filter membranes with molecular weights of 50,000 Daltons and 5,000 Daltons, respectively. Cytokines are those with molecular weights less than 50,000 Daltons and greater than 5,000 Daltons, and complex polypeptides are those with molecular weights less than 5,000 Daltons. The filtrate is collected separately under the following filtering conditions: inlet pressure 1.0 MPa, outlet pressure -1.0 MPa, temperature 5°C, and low temperature storage.

[0080] The umbilical cord extract prepared using wild-type trypsin is extract 1, the umbilical cord extract prepared using the enzyme mutant N31K / S92D is extract 2, and the umbilical cord extract prepared using the trypsin enzyme mutant A31T / K92N is extract 3.

[0081] Example 5 Construction of mouse burn model and study of the effects of umbilical cord extract

[0082] Forty C57 mice aged 6-8 weeks were selected and randomly divided into four groups (blank group, extract group 1, extract group 2, and extract group 3), with 10 C57 mice in each group. After depilation with 6% sodium sulfide dilution, a homemade burn and scald instrument was used, the temperature was adjusted to 100°C, and a uniform iron was fixed on the depilated area on the back of the mice for 5 seconds. Before incision, 5% chloral hydrate was used for anesthesia. Different drugs were applied to the burn and scald wounds on the back every day. Photos were taken on the 3rd, 5th, 7th, 9th, 11th, 13th, and 15th days, and the burn area and healing rate were analyzed.

[0083] The healing rate of burns and scalds = (original area - unhealed wound area) / original area.

[0084] The results are shown in Figure 3 . The healing rate of burns and scalds in the extract 2-3 group was close to 100% 9 days after injury, and the healing rate of burns and scalds in the blank group was close to 100% 13 days after injury. The burns and scalds in the extract 1 group were completely healed about 11 days after injury. The healing rate of the extract 1 group was significantly different from that of the blank group (P<0.05). The healing time of the extract 2-3 group was about 9 days. Compared with the blank group, the healing rate of burns and scalds in the healing process was significantly greater than that of the blank group (P<0.01), and there was also a significant difference with the extract 1 group (P<0.05), and the complete healing time was about 2 days earlier.

[0085] Samples were collected on the 5th and 11th days, and the burned mice were killed by cervical dislocation. The animal ethics protection law was strictly followed during the operation. The mice were disinfected in 75% ethanol solution for 60 seconds and then transferred to a clean bench. The burn wound on the back of the mouse was separated with microsurgical scissors, about 0.3 cm away from the edge of the burn wound. The free wound edge skin graft was transferred to a surgical dissection microscope and trimmed flat and regular. The skin grafts were fixed with polyformaldehyde for 24 hours, dehydrated with gradient concentrations of ethanol (70%, 80%, 90%, 95% and 100%), and embedded into tissue wax blocks after transparent wax immersion.

[0086] HE staining: Bake the paraffin sections in an oven at 60°C for 1 hour, then place the sections in xylene I for 20 minutes, xylene II for 20 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, 75% ethanol for 5 minutes, and wash with tap water for 10 seconds. Slices were dyed in hematoxylin solution for 4 minutes, washed with tap water for 10 seconds, differentiated with differentiation solution for 5 seconds, washed with tap water for 10 seconds, blued with blue solution, and rinsed with running water for 10 seconds. Slices were dehydrated in 85% and 95% gradient ethanol for 5 minutes each, and stained in eosin solution for 5 minutes. Slices were placed in anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, anhydrous ethanol III for 5 minutes, xylene I for 5 minutes, xylene II for 5 minutes, and sealed with neutral gum.

[0087] Depend on Figure 4 It can be seen that in the blank group, the damaged tissues were loosely arranged at 5 days after scalding, with a large amount of powder-stained edema fluid between tissues, obvious tissue structure and cell edema (HE, 300x), and a certain amount of new capillaries and fibroblasts were visible in the damaged tissues at 11 days after scalding (HE, 300x);

[0088] In the extract group 1, at 5 days after scalding, the damaged tissue was loosely arranged, with pink-stained edema fluid and infiltrating inflammatory cells between tissues, and obvious tissue structure and cell edema (HE, 300x); at 11 days after scalding, abundant new capillaries, fibroblasts and infiltrating inflammatory cells were observed in the damaged tissue (HE, 300x);

[0089] In extract group 2, at 5 days after scalding, the damaged tissue was loosely arranged, with a small amount of pink-stained edema fluid and infiltrating inflammatory cells between tissues (HE, 300x); at 11 days after scalding, a large number of capillaries and fibroblasts were visible, and the granulation tissue was well-developed and healthy (HE, 300x).

[0090] In extract group 3, at 5 days after scalding, the damaged tissues were loosely arranged, with a small amount of edema fluid and infiltrating inflammatory cells between tissues (HE, 300x); at 11 days after scalding, a large number of capillaries and fibroblasts were visible, and the granulation tissue was well-developed and healthy (HE, 300x).

[0091] That is, the healing condition and histological score of mouse burn skin in extract groups 2-3 were better than those in the blank group and extract group 1 (P<0.05).

[0092] Therefore, the present application uses the above-mentioned enzyme mutants to prepare umbilical cord extracts, which not only simplifies the preparation process and effectively improves the enzymatic hydrolysis efficiency, but also further the umbilical cord extracts prepared have very obvious effects on the repair of burn tissues, and are significantly better than the extracts obtained by conventional methods, and are expected to be used in skin repair products.

[0093] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims.

Claims

1. A method for preparing an umbilical cord extract, characterized in that: A trypsin mutant is used to enzymatically hydrolyze pig umbilical cord tissue, wherein the mutant is a wild-type trypsin with one or more mutations of N31 K, S92D or S161 L, wherein the amino acid sequence of the wild-type trypsin is shown in SEQ ID NO.

1.

2. The preparation method according to claim 1, characterized in that: The mutant is N31 K / S92D or N31 K / S161 L.

3. The preparation method according to claim 1, characterized in that: The amino acid sequence of the mutant is shown in SEQ ID NO.

4.

4. The preparation method according to claim 1, characterized in that: The amino acid sequence of the mutant is shown in SEQ ID NO.

5.

5. The preparation method according to claim 1, characterized in that: The preparation method comprises the following steps: (1) Take the animal umbilical cord tissue and homogenize it; (2) adding an aqueous solution of trypsin or its mutant to the homogenate obtained in step (1) to prepare a mixed solution, which is then stirred, centrifuged, and the supernatant is collected; (3) Inactivation of microorganisms and trypsin: inactivating the supernatant obtained in step (2) and collecting the inactivated supernatant; (4) Filtration of the extract: Filter the supernatant obtained in step (3) to remove pathogens; (5) ultrafiltration of the filtrate obtained in step (4), wherein the ultrafiltration conditions are: inlet pressure 0.5 to 1.2 MPa, outlet pressure -0.5 to -1.2 MPa, and temperature 4 to 15°C; (6) Freeze-drying and storage of the extract: Freeze the ultrafiltration filtrate obtained in step (5) to -40°C to -45°C within 3-6 hours and vacuum dry it.

6. The preparation method according to claim 5, characterized in that: The animal umbilical cord tissue is derived from pigs, cattle or sheep.

7. The preparation method according to claim 5, characterized in that: The inactivation step is to inactivate in a water bath at 60°C-85°C for 30-40 minutes.

8. The preparation method according to claim 5, characterized in that: The concentration of trypsin or its mutant is 10-20 g / L.

9. The umbilical cord extract prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the umbilical cord extract according to claim 9 in preparing a skin repair preparation.

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