Recombinant III-type collagen with high thermal stability and high transdermal absorption and application thereof

Abstract

The invention discloses a recombinant III-type collagen with high thermal stability and high transdermal absorption and application thereof, and belongs to the technical field of genetic engineering medicines.A core amino acid sequence of secretory peptide is inserted into the upstream of a collagen active fragment gene, and a purified tag is added to the downstream of the collagen active fragment gene; carrying out protein expression and purification on the gene sequence in host bacteria; the amino acid sequence of the collagen active fragment is as shown in SEQ ID NO.3; the core amino acid sequence of the secretory peptide is as shown in SEQ ID NO. 2. The amino acid sequence of the recombinant III-type collagen with high thermal stability and high transdermal absorption is as shown in SEQ ID NO. 1. Through the mode, the recombinant III type collagen containing the secretory peptide overcomes the difficulty in absorbability of natural collagen, shows a higher transdermal absorption effect, and has high thermal stability.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering drugs, and in particular to a recombinant type III collagen with high thermal stability and high transdermal absorption and its application. Background Art

[0002] Collagen is the most abundant protein in the human body. It is widely used in many industries such as medicine, beauty and food. Collagen is also the most widely distributed and most prominent fibrous functional protein in animals, accounting for about 25-30% of the total protein content in the human body. According to relevant reports, collagen is a family of proteins with rich types. There are up to 28 types of collagen in the human body, of which type I, type II and type III collagen are the most studied, accounting for about 60%-80% of the total human collagen. Type III collagen plays a regulatory role in the structural organization and functional regulation of the extracellular matrix, and participates in many physiological activities in the body, such as structural support, cell differentiation and adhesion, inflammatory-related diseases and certain other diseases.

[0003] Traditional natural collagen is often used in various industries such as cosmetics, medical materials and food processing due to its physical and chemical properties such as biocompatibility and biodegradability. However, due to its large relative molecular mass, it is not easily absorbed by the human body. Therefore, how to improve the absorption characteristics of collagen has become one of the hot spots and difficulties that need to be overcome.

[0004] Compared with natural collagen, which has disadvantages such as large relative molecular weight, immunogenicity, complex composition and difficulty in purification, recombinant collagen overcomes these defects, greatly improves the utilization efficiency of collagen, and effectively ensures the biosafety of collagen products using collagen as raw material.

[0005] The idea of ​​increasing the secretion and expression level of foreign proteins by constructing a secretory peptide core sequence upstream of the gene has been widely used since it was proposed in the 1990s. Its mechanism of action is that during the synthesis of secretory proteins, signal peptides guide secretory proteins to reach and pass through the endoplasmic reticulum to promote the further secretion of the protein into the extracellular space.

[0006] The present invention further discovered in experiments that secretory peptides have the property of promoting transdermal absorption, which is helpful for the later development of collagen products using collagen as raw material.

[0007] Based on this, the present invention designs a recombinant type III collagen with high thermal stability and high transdermal absorption and its application to solve the above problems. Summary of the invention

[0008] In view of the above-mentioned shortcomings of the prior art, the present invention provides a recombinant type III collagen with high thermal stability and high transdermal absorption and its application.

[0009] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0010] The amino acid sequence of the recombinant type III collagen with high thermal stability and high transdermal absorption is shown in SEQ ID NO.1.

[0011] In order to better achieve the purpose of the present invention, the present invention also provides a method for preparing the highly thermally stable and highly transdermal recombinant type III collagen, inserting the core amino acid sequence of the secretory peptide upstream of the collagen active fragment gene, and adding a purification tag downstream of the collagen active fragment gene; then expressing and purifying the gene sequence in a host bacteria;

[0012] Wherein, the amino acid sequence of the collagen active fragment is shown in SEQ ID NO.3;

[0013] The core amino acid sequence of the secretory peptide is shown in SEQ ID NO.2.

[0014] Furthermore, the tag is a His-tag, and its amino acid sequence is shown in SEQ ID NO.4.

[0015] Furthermore, Escherichia coli is used as a host bacteria.

[0016] In order to better achieve the purpose of the present invention, the present invention also provides a coding gene for encoding the collagen active fragment in the preparation method of the highly thermally stable and highly transdermal absorbable recombinant type III collagen, and its nucleotide sequence is shown in SEQ ID NO.5.

[0017] In order to better achieve the purpose of the present invention, the present invention also provides an expression vector, which constructs a recombinant plasmid secretory peptide-collagen-pET-28a by connecting the collagen active fragment gene containing the core amino acid sequence of the secretory peptide to the pET-28a vector.

[0018] In order to better achieve the purpose of the present invention, the present invention also provides a strain for expressing the highly thermally stable and highly transdermal recombinant type III collagen, and transforming the expression vector into Escherichia coli competent cells.

[0019] In order to better achieve the purpose of the present invention, the present invention also provides a method for expressing the highly heat-stable and highly transdermal recombinant type III collagen. The strain is inoculated into LB culture medium, IPTG is added to induce expression at 37°C for 3.8 to 4.2 hours, the supernatant is collected, and the recombinant type III collagen is obtained after purification.

[0020] In order to better achieve the purpose of the present invention, the present invention also provides the use of the recombinant type III collagen in the preparation of biological medicines / cosmetics.

[0021] In order to better achieve the purpose of the present invention, the present invention also provides the use of the recombinant type III collagen in the preparation of a product having the function of promoting transdermal absorption or withstanding a high temperature of at least 97°C.

[0022] Compared with the prior art, the present invention has the following beneficial effects: 1. Through the high transdermal permeability experiment, the present invention verifies that the permeability of collagen containing secretory peptides is significantly higher than that of collagen without secretory peptides at the same time, and the difference between the two is significant. The recombinant type III collagen of the present invention overcomes the difficulty of natural collagen in absorption and shows a higher transdermal absorption effect. It can be used to prepare biological medicines / cosmetics with the function of promoting transdermal absorption.

[0023] 2. The present invention verified through cytotoxicity experiments that 24 hours after adding recombinant type III collagen, when the protein concentration was below 0.2 mg / mL, HaCaT cells grew normally, which was level 1 cytotoxicity and had a slight effect on cells.

[0024] 3. The present invention verifies through cell scratch experiments that at 24 hours, recombinant type III collagen has a relatively obvious migration-promoting effect on HaCaT cells. It can be used in the preparation of injury repair treatment, anti-wrinkle and other products.

[0025] 4. The present invention verifies through thermal stability experiments that when the temperature gradually rises, the recombinant type III collagen still has no gelation or floccules, and still maintains the basic size under SDS-PAGE electrophoresis without degradation, indicating that the recombinant type III collagen prepared by the present invention has high thermal stability. It can be used to prepare biological drugs / cosmetics that can withstand at least 97°C high temperature. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0027] Figure 1 This is the SDS-PAGE protein electrophoresis of the recombinant type III collagen containing secretory peptides after purification by Ni column affinity chromatography.

[0028] Figure 2 This is the SDS-PAGE protein electrophoresis diagram of recombinant type III collagen containing secretory peptides after purification by ion exchange column.

[0029] Figure 3 Representative results of collagen permeability experiment in female rat skin.

[0030] Figure 4 Representative results of collagen permeability experiment in male rat skin.

[0031] Figure 5 Representative results of cytotoxicity experiments.

[0032] Figure 6 Representative results of a cell scratch assay using recombinant type III collagen containing secreted peptides.

[0033] Figure 7 Comparison of the appearance of recombinant type III collagen before and after incubation at different temperatures.

[0034] Figure 8 The SDS-PAGE protein electrophoresis diagram of recombinant type III collagen after incubation at different temperatures. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] The main materials involved in the embodiments of the present invention are as follows:

[0037] Vector plasmid: pET-28a, host bacteria: Escherichia coli BL21 (DE3), Ni-NTA Agarose (affinity chromatography medium).

[0038] Buffers used for Ni-NTA Agarose affinity chromatography: washing buffer: 20mmol / L Tris-HCl, 0.25mol / LNaCl, 10mmol / L imidazole (pH 8.0); elution buffer A: 20mmol / L Tris-HCl, 0.25mol / LNaCl, 50mmol / L imidazole (pH 8.0); elution buffer B: 20mmol / L Tris-HCl, 0.25mol / L NaCl, 250mmol / L imidazole (pH 7.2); elution buffer C: 20mmol / L Tris-HCl, 0.25mol / L NaCl, 500mmol / L imidazole (pH 7.2).

[0039] Buffer used for ion exchange column: Equilibration buffer: 10 mmol / L NaAC (pH 4.5); Elution buffer D: 1 mol / L NaCl, 10 mmol / L NaAC (pH 4.5).

[0040] Example 1: The preparation method of recombinant type III collagen is as follows:

[0041] First, a peptide segment was selected from the amino acid sequence of human type III collagen (this peptide segment is located at positions 258 to 328 of human type III collagen) as the collagen active fragment, and its amino acid sequence is shown in SEQ ID NO.3;

[0042] SEQ ID NO.3:

[0043] GFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRP GLPGAAGARGNDGS

[0044] Its nucleotide sequence is shown in SEQ ID NO.5;

[0045] SEQ ID NO.5:

[0046] GGCTTTCCTGGTATGAAAGGCCATCGCGGCTTTGATGGTCGTAATGGCGAAAAGGTGAAACCGGCGCACCGGGCCTGAAAGGTGAAAATGGCCTGCCGGGTGAAAATGGTGCACCGGGCCCGATGGGCCCGCGTGGTGCACCTGGTGAACGTGGCCGTCCGGGCCTGCCTGGTGCTGCAGGTGCTCGTGGCAATGATGGATCC

[0047] Then, a core amino acid sequence of a secretory peptide consisting of 20 amino acids was inserted upstream of the gene to improve its transdermal absorption performance; and a tag 8×His-tag was added downstream of the gene to facilitate purification;

[0048] The core amino acid sequence of the secretory peptide is shown in SEQ ID NO.2;

[0049] SEQ ID NO.2: MDVFKKGFSIADEGVVAAVE

[0050] The amino acid sequence of the His-tag is shown in SEQ ID NO.4;

[0051] SEQ ID NO.4: HHHHHHHH

[0052] Afterwards, this sequence was expressed and purified using E. coli as a host bacterium;

[0053] The amino acid sequence of recombinant type III collagen is shown in SEQ ID NO.1;

[0054] SEQ ID NO.1:

[0055] MDVFKKGFSIADEGVVAAVEGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGE NGAPGPMGPRGAPGERGRPGLPGAAGARGNDGSHHHHHHHH

[0056] Example 2: Based on Example 1, the expression and purification method of recombinant type III collagen containing secretory peptide is as follows:

[0057] Step (1), preparation of recombinant type III collagen expression strain: the expression vector secretory peptide-collagen-pET-28a was transformed into Escherichia coli BL21 (DE3) competent cells, and the transformation conditions were: ice bath for 5 minutes, heat shock in a 42°C water bath for 50 seconds, ice bath again for 2 minutes, coating with kanamycin-containing LB plates, inverted at 37°C, and grown overnight.

[0058] Step (2), induced expression of recombinant type III collagen: the strain obtained in step (1) was inoculated into 100 mL of LB medium containing 50 μg / mL kanamycin, cultured at 37°C and 240 rpm, and after the bacterial solution became turbid, it was transferred to 1000 mL of LB medium for further culture. 600 =1.2, IPTG was added to a final concentration of 0.25mmol / L, and the expression was induced at 37℃ for 3.8-4.2h, and the cells were collected by centrifugation at 4000rpm for 20min. The cells were resuspended in 10mmol / L imidazole solution, and the cells were broken by ultrasonication and centrifuged at 12000rpm for 1h. The supernatant and precipitate were kept separately for subsequent SDS-PAGE electrophoresis.

[0059] Step (3), the supernatant obtained by the centrifugation of the bacterial cells obtained in step (2) was purified by Ni-NTA Agarose affinity chromatography, and the specific steps were as follows: the supernatant separated after centrifugation was added with 2 mL of Ni-NTA Agarose for binding, and after the binding was completed, 150 mL of washing buffer was used for three times to wash the impurities, and 50 mL of elution buffer A, 50 mL of elution buffer B, and 50 mL of elution buffer C were used to elute the target protein respectively. The purity of the purified collagen was identified by SDS-PAGE. The results are as follows Figure 1As shown, it can be clearly seen that the protein after two purifications is mainly concentrated in the elution buffers of 50mmol / L, 250mmol / L and 500mmol / L imidazole.

[0060] Step (4), collect the eluted protein solution, use a 3kD dialysis bag to place the protein solution in a 25mmol / LNaCl, 10mmol / L NaAC (pH 4.5) solution for overnight dialyzing, 12000rpm, 4℃ refrigerated centrifugation for 1h, load the centrifugal supernatant onto a 2Q column + 3SP series ion exchange column for purification, set up the program, and use a linear gradient of equilibrium buffer and elution buffer D for protein purification. After SDS-PAGE, collect the target protein and dialyze it again overnight into 10mmol / LPBS pH7.2, 0.15mol / L NaCl. Use a 3kD protein concentrator tube at 3400rpm to concentrate to 10mL. The results are as follows Figure 2 As shown, it can be clearly shown that the target protein is mainly concentrated in tubes 12 to 19, and there is no obvious target protein in the flow-through solution. Recombinant type III collagen with a single band was obtained through separation and purification.

[0061] Example 3: Experiment on high skin permeability of recombinant type III collagen.

[0062] Step (1), preparation of mouse skin: prepare 3 female (male) mice aged 6-8 weeks, kill them, peel them along the abdomen, remove the hair on the surface and the fat layer on the back, and clean them with 0.9% NaCl. Cut them into the size corresponding to the supply pool mouth, cut one mouse skin into two pieces, and store them at 4°C.

[0063] Step (2), specific operation steps of the transdermal experiment: 10mmol / L PBS pH7.2, 0.15mol / L NaCl buffer solution is added to the receiving pool in advance, and then the treated mouse skin is placed between the supply pool and the receiving pool, and clamped with a spring clamp, with the receiving protein side facing the supply pool and the permeable protein side facing the receiving pool, heated in a water bath to 32°C, and the stirring speed is 100rpm. The supply pools are named No. 1 to No. 6, respectively. Among them, the mouse skins clamped by No. 1 and No. 4, No. 2 and No. 5, No. 3 and No. 6 are all from the same mouse skin. Recombinant type III collagen containing secretory peptides and recombinant type III collagen without secretory peptides adjusted to 2mg / mL in advance are added to the supply pools in a volume of 2mL respectively. Collagen containing secretory peptides is added to supply pools No. 1, No. 2, and No. 3, and collagen without secretory peptides is added to supply pools No. 4, No. 5, and No. 6. Then, 200μL of samples are taken at 2h, 4h, 6h, and 8h respectively and the corresponding buffer is supplemented.

[0064] Step (3), determination of collagen content: The present invention uses a BCA kit to determine the collagen content in the sample. The results are as follows Figure 3-4 As shown in the figure, the permeation results of the skin of female mice and male mice showed that the permeation of the two collagens gradually increased with time, but the permeation of the collagen containing secretory peptides was significantly higher than that of the collagen without secretory peptides at the same time, and the difference between the two was significant. Then, the percentage increase was calculated using the following formula at the same time:

[0065] Increased amount = (permeation amount of protein containing secretory peptide - permeation amount of protein without secretory peptide) / permeation amount of protein without secretory peptide * 100%.

[0066] The results of the transdermal test using the skin of female mice were: 69%, 34%, 34%, 34% and 34% at 2h, 4h, 6h and 8h respectively, while the results of the transdermal test using the skin of male mice were: 52%, 78%, 61% and 65% at 2h, 4h, 6h and 8h respectively.

[0067] Example 4: Cytotoxicity assay (MTT method).

[0068] Step (1), culturing human immortalized keratinocytes (HaCaT cells): After the HaCaT cells have grown completely, trypsinize the cells and count the cells. 1×10 5 / mL cell suspension was plated and incubated at 37°C, 5% CO 2 Culture in an incubator for 24 h.

[0069] Step (2), adding collagen: dilute 2.6 mg / mL recombinant type III collagen containing secretory peptide to 0.2 mg / mL using complete culture medium, and filter the recombinant type III collagen using a 0.22 μM filter. Use complete culture medium to dilute to 0.100, 0.050, 0.025, 0.013, and 0.006 mg / mL in a gradient manner. Discard the old culture medium and add 100 μL of recombinant type III collagen at different concentrations. Place at 37°C and 5% CO 2 After culturing in the incubator for 24 h, 20 μL of MTT was added, and the cells were taken out after further culturing for 4 h.

[0070] Step (3), termination of culture: discard the solution in the well, add 150 μL of dimethyl sulfoxide (DMSO), incubate, and measure the absorbance at 490 nm.

[0071] Step (4), calculation: cell viability (%) = [(absorbance of experimental group - absorbance of blank group) / (absorbance of control group - absorbance of blank group)] × 100%.

[0072] The results are as follows Figure 5As shown, the results showed that 24 hours after adding recombinant type III collagen, when the protein concentration was below 0.2 mg / mL, HaCaT cells grew normally, which was level 1 cytotoxicity.

[0073] Example 5: Cell scratch assay.

[0074] Step (1), take out the culture dish from the incubator and observe the cell morphology under an inverted microscope, gently aspirate the culture medium, and then aspirate PBS and add it to the culture dish.

[0075] Step (2), after mixing, absorb pancreatic enzyme and add it to the culture dish, mix and let it stand in the incubator for digestion, then immediately add 1 mL of culture medium to terminate digestion, mix and transfer to a centrifuge tube, and centrifuge at 1000 rpm.

[0076] Step (3), after centrifugation, discard the supernatant, add the culture medium and mix well, take out 50 μL of the cell suspension and mix it evenly with an equal proportion of trypan blue solution.

[0077] Step (4), add a cell counting plate for counting, then prepare a cell suspension, add it to a six-well cell plate, plate it, and after culturing in an incubator for 24 hours, scratch the vertical positioning line, keep the gun tip straight, and do not repeat.

[0078] Step (5), taking pictures with an inverted microscope and saving them, then taking pictures of the scratched area at 24h and 48h, and processing and compiling the data using Image J and Graphpad software.

[0079] The results are as follows Figure 6 As shown, the results showed that at 24 hours, recombinant type III collagen had a more obvious migration-promoting effect on HaCaT cells.

[0080] Example 6: Thermal stability experiment.

[0081] With reference to the "Pharmaceutical Industry Standard of the People's Republic of China for Recombinant Collagen", the recombinant type III collagen stock solution of Example 2 was dialyzed into a PBS solution, the concentration was determined, and a 10 mg / mL solution was prepared and placed at (57±0.5)°C, (67±0.5)°C, (77±0.5)°C, (87±0.5)°C, and (97±0.5)°C. After incubation for 4 hours, the solution was analyzed by naked eye observation and SDS-PAGE.

[0082] The results are as follows Figure 7-8As shown, it can be clearly observed that, compared with before incubation, when the temperature gradually increases, the recombinant type III collagen still has no gelation or floccules, no precipitation, and still maintains the basic size under SDS-PAGE electrophoresis without degradation. The results show that the recombinant type III collagen prepared by the present invention has high thermal stability.

[0083] The present invention verifies the high transdermal permeability of recombinant collagen containing secretory peptides by means of an in vitro diffusion cell method, and verifies the normal function of recombinant collagen by means of a cell scratch experiment. Compared with collagen without secretory peptides, recombinant type III collagen containing secretory peptides overcomes the poor absorption of natural collagen, shows a higher transdermal absorption effect, and the recombinant type III collagen has high thermal stability. Therefore, it can be used to prepare biological drugs / cosmetics that have the function of promoting transdermal absorption or withstand high temperatures of at least 97°C, such as facial fillers, dressings, freeze-dried fibers, gels and other products.

[0084] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. Such modifications or replacements will not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Highly thermally stable and highly transdermal recombinant type III collagen, characterized by: Its amino acid sequence is shown in SEQ ID NO.

1.

2. A method for preparing the recombinant type III collagen with high thermal stability and high transdermal absorption according to claim 1, characterized in that: Insert the core amino acid sequence of the secretory peptide upstream of the collagen active fragment gene, and add a purification tag downstream of the collagen active fragment gene; then express and purify the gene sequence in the host bacteria; Wherein, the amino acid sequence of the collagen active fragment is shown in SEQ ID NO.3; The core amino acid sequence of the secretory peptide is shown in SEQ ID NO.

2.

3. The method for preparing recombinant type III collagen with high thermal stability and high transdermal absorption according to claim 2, characterized in that: The tag is a His-tag, and its amino acid sequence is shown in SEQ ID NO.

4.

4. The method for preparing recombinant type III collagen with high thermal stability and high transdermal absorption according to claim 2, characterized in that: Escherichia coli was used as the host bacteria.

5. A coding gene, characterized in that The nucleotide sequence of the collagen active fragment used to encode the method for preparing the recombinant type III collagen with high thermal stability and high transdermal absorption as described in claim 2 is shown in SEQ ID NO.

5.

6. An expression vector, characterized in that: The recombinant plasmid secretory peptide-collagen-pET-28a was constructed by connecting the collagen active fragment gene containing the core amino acid sequence of the secretory peptide to the pET-28a vector.

7. A strain for expressing highly thermally stable and highly transdermal recombinant type III collagen, characterized in that: The expression vector according to claim 6 is transformed into Escherichia coli competent cells.

8. A method for expressing a recombinant type III collagen with high thermal stability and high transdermal absorption, characterized in that: The strain according to claim 7 is inoculated into LB medium, IPTG is added to induce expression at 37° C. for 3.8 to 4.2 hours, and the supernatant is collected and purified to obtain recombinant type III collagen.

9. Use of the recombinant type III collagen according to claim 1 in the preparation of biological medicines / cosmetics.

10. Use of the recombinant type III collagen according to claim 1 in preparing a product having a function of promoting transdermal absorption or withstanding a high temperature of at least 97°C.

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