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

Abstract

The present invention discloses a recombinant type III collagen with high thermal stability and high transdermal absorption and its application, belonging to the technical field of genetic engineering drugs. The core amino acid sequence of a secretion peptide is inserted upstream of the collagen active fragment gene, and a purification tag is added downstream of the collagen active fragment gene; then this gene sequence is subjected to protein expression and purification in a host bacterium; the amino acid sequence of the collagen active fragment is as shown in SEQ ID NO.3; the core amino acid sequence of the secretion peptide is as shown in SEQ ID NO.2. The amino acid sequence of the recombinant type III collagen with high thermal stability and high transdermal absorption is as shown in SEQ ID NO.1. By the above method, the recombinant type III collagen containing the secretion peptide of the present invention overcomes the poor 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 particularly relates to 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 multiple industries such as medicine, beauty, and food. Moreover, collagen is also the most widely distributed and 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, among which type I, type II, and type III collagen are more studied and account 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 in vivo physiological activities, such as structural support, cell differentiation and adhesion, inflammation-related diseases, and certain other diseases.

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

[0004] Compared with natural collagen, which has disadvantages such as relatively large 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 biological safety of collagen products using collagen as raw material.

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

[0006] The present invention further discovers in experiments that the secretion peptide has the property of promoting transdermal absorption, which is helpful for the development of collagen products using collagen as raw material in the later stage.

[0007] Based on this, the present invention designs 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 drawbacks of the prior art, the present invention provides recombinant type III collagen with high thermal stability and high transdermal absorption and its application.

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

[0010] Recombinant type III collagen with high thermal stability and high transdermal absorption, whose amino acid sequence is shown in SEQ ID NO.1.

[0011] To better achieve the object of the present invention, the present invention also provides a preparation method of the recombinant type III collagen with high thermal stability and high transdermal absorption. The core amino acid sequence of a secretion peptide is inserted upstream of the collagen active fragment gene, and a purification tag is added downstream of the collagen active fragment gene; then this gene sequence is subjected to protein expression and purification in a host bacterium;

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

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

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

[0015] Furthermore, Escherichia coli is used as the host bacterium.

[0016] To better achieve the object of the present invention, the present invention also provides a coding gene for coding the collagen active fragment in the preparation method of the recombinant type III collagen with high thermal stability and high transdermal absorption, and its nucleotide sequence is shown in SEQ ID NO.5.

[0017] To better achieve the object of the present invention, the present invention also provides an expression vector. By connecting the collagen active fragment gene containing the core amino acid sequence of the secretion peptide to the pET-28a vector, a recombinant plasmid secretion peptide - collagen - pET-28a is constructed.

[0018] To better achieve the object of the present invention, the present invention also provides a strain for expressing the recombinant type III collagen with high thermal stability and high transdermal absorption, and the above-mentioned expression vector is transformed into Escherichia coli competent cells.

[0019] To better achieve the object of the present invention, the present invention also provides an expression method of the recombinant type III collagen with high thermal stability and high transdermal absorption. The above-mentioned strain is inoculated into an LB medium, and IPTG is added at 37°C for culturing and inducing expression for 3.8 - 4.2 h, then the supernatant is collected and purified to obtain recombinant type III collagen.

[0020] To better achieve the object of the present invention, the present invention also provides the use of the recombinant type III collagen in the preparation of biopharmaceuticals / cosmetics.

[0021] To better achieve the object of the present invention, the present invention also provides the use of the recombinant type III collagen in the preparation of products having a function of promoting transdermal absorption or being resistant to at least 97 °C high temperature.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. Through high transdermal permeability experiments, the present invention verifies that the penetration amount of collagen containing a secretion peptide is significantly higher than that of collagen without a secretion peptide at the same time, and the difference between the two is significant. The recombinant type III collagen of the present invention overcomes the poor absorbability of natural collagen and shows a higher transdermal absorption effect. It can be used in the preparation of biopharmaceuticals / cosmetics having a function of promoting transdermal absorption.

[0023] 2. Through cytotoxicity experiments, the present invention verifies that after adding recombinant type III collagen for 24 hours, when the protein concentration is below 0.2 mg / mL, HaCaT cells grow normally, showing a grade 1 cytotoxicity and having a slight impact on the cells.

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

[0025] 4. Through thermal stability experiments, the present invention verifies that when the temperature gradually increases, the recombinant type III collagen still has no gelation or flocculation, and still maintains its 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 in the preparation of biopharmaceuticals / cosmetics resistant to at least 97 °C high temperature. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0027] Figure 1 It is the SDS-PAGE protein electrophoresis pattern of the recombinant type III collagen containing a secretion peptide after purification by Ni column affinity chromatography.

[0028] Figure 2 It is the SDS-PAGE protein electrophoresis pattern of the recombinant type III collagen containing a secretion peptide after purification by ion exchange column.

[0029] Figure 3 These are the representative results of the transdermal experiment of collagen in female mouse skin.

[0030] Figure 4 These are the representative results of the transdermal experiment of collagen in male mouse skin.

[0031] Figure 5 These are the representative results of the cytotoxicity experiment.

[0032] Figure 6 These are the representative results of the cell scratch experiment using recombinant type III collagen containing a secreted peptide.

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

[0034] Figure 8 These are the SDS-PAGE protein electrophoresis diagrams of recombinant type III collagen after incubation at different temperatures. Detailed implementation manners

[0035] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope 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 bacterium: Escherichia coli BL21(DE3), Ni-NTA Agarose (affinity chromatography medium).

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

[0039] Buffers used in the 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] GGCTTTCCTGGTATGAAAGGCCATCGCGGCTTTGATGGTCGTAATGGCGAAAAAGGTGAAACCGGCGCACCGGGCCTGAAAGGTGAAAATGGCCTGCCGGGTGAAAATGGTGCACCGGGCCCGATGGGCCCGCGTGGTGCACCTGGTGAACGTGGCCGTCCGGGCCTGCCTGGTGCTGCAGGTGCTCGTGGCAATGATGGATCC

[0047] After that, a core amino acid sequence of a 20 - amino - acid - composed secretory peptide was inserted upstream of its gene to improve its transdermal absorption performance; an 8×His - tag for purification was added downstream of its gene;

[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] Subsequently, this sequence was used as the host bacterium Escherichia coli for protein expression and purification;

[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: On the basis of Example 1, the method for expressing and purifying recombinant type III collagen containing a secretion peptide is as follows:

[0057] Step (1), Preparation of recombinant type III collagen expression strain: The expression vector secretion peptide - collagen - pET - 28a was transformed into Escherichia coli BL21(DE3) competent cells. The transformation conditions were: ice bath for 5 min, heat shock at 42 °C in a water bath for 50 s, ice bath again for 2 min, and then spread on an LB plate containing kanamycin, and incubated at 37 °C upside down 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, and cultured at 37 °C with 240 rpm. After the bacterial solution became turbid, it was transferred to 1000 mL of LB medium for further culture. When OD 600 = 1.2, IPTG was added with a final concentration of 0.25 mmol / L, and cultured at 37 °C for induction expression for 3.8 - 4.2 h, and then the cells were collected by centrifugation at 4000 rpm for 20 min. The cells were resuspended with 10 mmol / L imidazole solution, sonicated to break the cells, and centrifuged at 12000 rpm for 1 h. The supernatant and precipitate were respectively sampled for subsequent SDS - PAGE electrophoresis.

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

[0060] Step (4): Collect the eluted protein solution. Use a 3 kD dialysis bag to place the protein solution in a solution of 25 mmol / L NaCl and 10 mmol / L NaAC (pH 4.5) for overnight dialysis. Centrifuge at 12,000 rpm at 4 °C for 1 h. Load the centrifuged supernatant onto a 2Q column + 3SP tandem ion exchange column for purification. Set the program and use a linear gradient of the equilibration buffer and elution buffer D for protein purification. After SDS-PAGE, collect the target protein and dialyze it again overnight into 10 mmol / L PBS pH 7.2 and 0.15 mol / L NaCl. Concentrate it to 10 mL using a 3 kD protein concentrator tube at 3400 rpm. The results are as Figure 2 shown, which can clearly show 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 is obtained through separation and purification.

[0061] Example 3: High transdermal permeability experiment of recombinant type III collagen.

[0062] Step (1): Preparation of mouse skin: Prepare 3 female (similarly for male) mice at 6 - 8 weeks old. After sacrificing, peel them along the abdomen, then remove the surface hair and the back fat layer, and wash them clean with 0.9% NaCl. Cut them into the corresponding sizes of the supply pool openings, cut one mouse skin into two pieces, and store them at 4 °C.

[0063] Step (2): Specific operating steps of the transdermal experiment: Add 10 mmol / L PBS pH 7.2 and 0.15 mol / L NaCl buffer to the receiving pool in advance. Then place the treated mouse skin between the supply pool and the receiving pool, and clamp it with a spring clip. The protein-receiving side faces the supply pool, and the protein-permeating side faces the receiving pool. Heat it in a water bath to 32 °C, and the stirring speed is 100 rpm. Name the supply pools from No. 1 to No. 6. Among them, the mouse skins clamped between No. 1 and No. 4, No. 2 and No. 5, and No. 3 and No. 6 all come from the same mouse skin. Add the recombinant type III collagen containing the secretion peptide and the recombinant type III collagen without the secretion peptide, which have been adjusted to 2 mg / mL in advance, to the supply pools in a volume of 2 mL each. The collagen containing the secretion peptide is added to supply pools No. 1, No. 2, and No. 3, and the collagen without the secretion peptide is added to supply pools No. 4, No. 5, and No. 6. Then take 200 μL samples at 2 h, 4 h, 6 h, and 8 h and supplement the corresponding buffer.

[0064] Step (3), determination of collagen content: The present invention uses a BCA kit to determine the collagen content in the sampled specimens. The results are as Figures 3 - 4 shown. The transdermal results of female and male mouse skins show that as time increases, the permeation amounts of both types of collagen gradually increase. However, the permeation amount of collagen containing the secretion peptide is significantly higher than that of collagen without the secretion peptide at the same time point, and the difference between the two is significant. Then, the percentage increase is calculated using the following formula at the same time point:

[0065] Increase amount = (permeation amount of protein containing the secretion peptide - permeation amount of protein without the secretion peptide) / permeation amount of protein without the secretion peptide * 100%.

[0066] The results of the transdermal experiment using female mouse skin: at 2 h, 4 h, 6 h, and 8 h, the increases are 69%, 34%, 34%, and 34% respectively. The results of the transdermal experiment using male mouse skin: at 2 h, 4 h, 6 h, and 8 h, the increases are 52%, 78%, 61%, and 65% respectively.

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

[0068] Step (1), culture of human immortalized keratinocytes (HaCaT cells): After the growth of HaCaT cells is completed, the cells are digested with trypsin and counted. A cell suspension of 1×10 5 / mL is used for plating and cultured in an incubator at 37 °C and 5% CO2 for 24 h.

[0069] Step (2), addition of collagen: The recombinant type III collagen containing the secretion peptide at 2.6 mg / mL is diluted to 0.2 mg / mL with complete medium, and the recombinant type III collagen is filtered using a 0.22 μM filter. It is diluted to 0.100, 0.050, 0.025, 0.013, and 0.006 mg / mL in gradients with complete medium. Discard the old medium and add 100 μL of recombinant type III collagen with different concentrations, and culture in an incubator at 37 °C and 5% CO2 for 24 h. Then add 20 μL of MTT and continue to culture for 4 h before taking out.

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

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

[0072] The results are as Figure 5As shown, the results show that after adding recombinant type III collagen for 24 hours, when the protein concentration is below 0.2 mg / mL, HaCaT cells grow normally, showing a cytotoxicity level of 1.

[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 pipette PBS into the culture dish.

[0075] Step (2): After mixing, pipette trypsin into the culture dish, mix well, and let it stand in the incubator for digestion. Immediately add 1 mL of medium to terminate the digestion, mix well, transfer it to a centrifuge tube, and centrifuge at 1000 rpm.

[0076] Step (3): After centrifugation, discard the supernatant. Add medium and pipette to mix well. Take out 50 μL of cell suspension and mix it evenly with an equal volume of trypan blue solution.

[0077] Step (4): Add it to a cell counting chamber for counting. Then prepare a cell suspension, add it to a six-well cell plate for plating. After culturing in the incubator for 24 hours, make a scratch on the vertical positioning line, keep the pipette tip straight, and do not repeat.

[0078] Step (5): Take a photo and save it under an inverted microscope. Then take photos of the scratch at 24 hours and 48 hours, and use Image J and Graphpad software to process and comprehensively organize the data.

[0079] The results are as Figure 6 shown. The results show that at 24 hours, recombinant type III collagen has a relatively obvious effect on promoting the migration of HaCaT cells.

[0080] Example 6: Thermal stability experiment.

[0081] Referring to the "Recombinant Collagen Pharmaceutical Industry Standard of the People's Republic of China", dialyze the recombinant type III collagen stock solution in Example 2 into PBS solution, measure the concentration, and prepare it into a 10 mg / mL solution. Place it at (57 ± 0.5) °C, (67 ± 0.5) °C, (77 ± 0.5) °C, (87 ± 0.5) °C, (97 ± 0.5) °C, incubate for 4 hours, and then observe by naked eye and analyze by SDS-PAGE.

[0082] The results are as Figures 7 - 8As shown, it can be clearly observed that compared with before incubation, when the temperature gradually increases, recombinant type III collagen still shows no gelation or flocculation, does not produce precipitation, and still maintains its basic size under SDS-PAGE electrophoresis without degradation. The results indicate 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 a secretion peptide by means of an in vitro diffusion cell method, and supplements it with a cell scratch experiment to verify the normal function of the recombinant collagen. Compared with collagen without a secretion peptide, the recombinant type III collagen containing a secretion peptide overcomes the poor absorbability of natural collagen, shows a higher transdermal absorption effect, and this recombinant type III collagen has high thermal stability. Therefore, it can be used in the preparation of biopharmaceuticals / cosmetics with the function of promoting transdermal absorption or tolerating at least 97°C high temperature, such as products like facial fillers, dressings, freeze-dried fibers, and gels.

[0084] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these 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 It comprises a gene for encoding the active fragment of collagen in the method for preparing recombinant type III collagen with high thermal stability and high transdermal absorption as described in claim 2, and the nucleotide sequence of the active fragment of collagen is shown in SEQ ID NO.

5.

6. An expression vector, characterized in that: The invention comprises a gene encoding the highly heat-stable and highly transdermal-absorbable recombinant type III collagen as claimed in claim 1.

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 cosmetics.

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

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