Recombinant collagen, preparation method and application thereof

Abstract

The present invention relates to the field of genetic engineering, and in particular to the preparation and application of recombinant collagen. Collagen mutants are obtained through site-directed mutagenesis, and the most effective mutants are screened and prepared into anti-wrinkle compositions. These compositions can effectively reduce the length, area, and depth of skin wrinkles. The collagen Q160A mutant composition has an anti-wrinkle efficacy of 90% and an excellent efficacy of 30%, suggesting promising application prospects in anti-wrinkle products.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering, and in particular to the preparation and application of recombinant collagen. Background Art

[0002] Natural collagen possesses unique biological activity, inherent biomimetic advantages, and is naturally abundant and renewable. In recent years, with the rapid growth of the global cosmetics market and significant advancements in scientific research and development, natural collagen has garnered significant attention as a functional ingredient in cosmetics. Natural collagen boasts numerous advantages, including excellent biocompatibility, biodegradability, and low antigenicity. However, its large molecular weight, triple-helical structure makes it difficult to directly penetrate the dense surface layer of the skin to achieve skincare benefits. To enhance the bioavailability of natural collagen in the cosmetics industry, hydrolyzing high-molecular-weight natural collagen to obtain low-molecular-weight collagen peptides is an important approach for developing collagen-based beauty and skincare products. Collagen peptides not only provide a compatible amino acid source for collagen synthesis in the body, but their hydrophilic groups also enhance skin moisturizing properties. Furthermore, collagen peptides possess excellent nutritional properties, UV protection, and the ability to promote cell growth and proliferation. They play a key role in maintaining the skin's internal environment, delaying skin aging, and promoting wound repair.

[0003] However, skin collagen decreases with age, and the collagen content is lower in women of all ages. There is a direct relationship between skin collagen and dermal thickness. Skin aging is one of the most obvious characteristics of aging. Therefore, the demand for skin care products with wrinkle removal and anti-wrinkle functions is increasing. However, the wrinkle removal and anti-wrinkle products in the existing technology have defects such as insignificant wrinkle removal and anti-wrinkle effects and causing human allergies. Therefore, wrinkle removal and anti-wrinkle products with better effects and greater safety have huge application prospects. Summary of the Invention

[0004] In order to solve the above problems, the present invention adopts genetic engineering methods to obtain recombinant collagen, and introduces amino acid mutations to screen and obtain recombinant collagen with better wrinkle-removing and anti-wrinkle effects.

[0005] The Lactococcus lactis NZ9000 competent cells used in the present invention were used for plasmid construction, and the lactic acid bacteria starter used in the experiment was purchased from Beijing Chuanxiu Technology Co., Ltd.

[0006] The culture medium for screening recombinant bacteria was LA solid medium: 1% peptone, 0.5% yeast extract, 1% sodium chloride, 1.5% agar powder, at 37 °C for recombinant bacteria culture. Add 100 μg mL -1 of ampicillin.

[0007] The culture medium for the activated lactic acid bacteria preparation was liquid LB medium: 1 g of peptone, 0.5 g of yeast extract, 1 g of sodium chloride, and 100 mL of distilled water.

[0008] LB medium and sucrose medium were sterilized in an autoclave at 121°C for 30 min, and YPD medium was sterilized in an autoclave at 108°C for 20 min. Before inoculation, they were placed on a sterile operating table for ultraviolet sterilization for 30 min.

[0009] The first object of the present invention is to provide an engineered bacterium containing a collagen recombinant vector, wherein the engineered bacterium is introduced with a collagen mutant to construct a recombinant Lactococcus lactis capable of secreting collagen.

[0010] The amino acid sequence of the collagen is shown in SEQ ID NO. 1.

[0011] 1 GPPGEPGNPG SPGNQGQPGN KGSPGNPGQP GNEGQPGQPG QNGQPGEPGS NGPQGSQGNPGKNGQPGSPG SQGSPGNQGS

[0012] 81 PGQPGNPGQP GEQGKPGNQG PAGEPGNPGS PGNQGQPGNK GSPGNPGQPG NEGQPGQPGQNGQPGEPGSN GPQGSQGNPG

[0013] 161 KNGQPGSPGS QGSPGNQGSP GQPGNPGQPG EQGKPGNQGP AGEGNPGSP GNQGQPGNKGSPGNPGQPGN EGQPGQPGQN

[0014] 241 GQPGEPGSNG PQGSQGNPGK NGQPGSPGSQ GSPGNQGSPG QPGNPGQPGE QGKPGNQGPAGEPGNPGSPG NQGQPGNKGS

[0015] 321 PGNPGQPGNE GQPGQPGQNG QPGEPGSNGP QGSQGNPGKN GQPGSPGSQG SPGNQGSPGQPGNPGQPGEQ GKPGNQGPAG

[0016] 401 EPGNPGSPGN QGQPGNKGSP GNPGQPGNEG QPGQPGQNGQ PGEPGSNGPQ GSQGNPGKNGQPGSPGSQGS PGNQGSPGQP

[0017] 481 GNPGQPGEQG KPGNQGPAGE PGNPGSPGNQ GQPGNKGSPG NPGQPGNEGQPGQPGQNGQP GEPGSNGPQG SQGNPGKNGQ

[0018] 561 PGSPGSQGSP GNQGSPGQPG NPGQPGEQGK PGNQGPAGG.

[0019] Preferably, the collagen nucleotide is codon-optimized and has a Usp45 signal peptide and a His tag added thereto.

[0020] Preferably, a mutant of collagen is introduced into the engineered bacteria, which has one or more amino acid mutations, and can effectively reduce the length, area and depth of skin wrinkles, and has a good wrinkle removal and anti-wrinkle effect.

[0021] The preferred increase percentage of wrinkle removal and anti-wrinkle effect is 5% or more, or 10% or more, or 20% or more.

[0022] Preferred collagen mutation sites are one or more of G160A, N197K, and Q396E.

[0023] A more preferred mutation site is G160A.

[0024] The amino acid sequence of the preferred mutated collagen is shown in SEQ ID NO: 2-4.

[0025] A more preferred amino acid sequence of the mutated collagen is shown in SEQ ID NO: 2.

[0026] The more preferred amino acid sequence of mutant Q160A is

[0027] 1 GPPGEPGNPG SPGNQGQPGN KGSPGNPGQP GNEGQPGQPG QNGQPGEPGS NGPQGSQGNPGKNGQPGSPG SQGSPGNQGS

[0028] 81 PGQPGNPGQP GEQGKPGNQG PAGEPGNPGS PGNQGQPGNK GSPGNPGQPG NEGQPGQPGQNGQPGEPGSN GPQGSQGNPA

[0029] 161 KNGQPGSPGS QGSPGNQGSP GQPGNPGQPG EQGKPGNQGP AGEGNPGSP GNQGQPGNKGSPGNPGQPGN EGQPGQPGQN

[0030] 241 GQPGEPGSNG PQGSQGNPGK NGQPGSPGSQ GSPGNQGSPG QPGNPGQPGE QGKPGNQGPAGEPGNPGSPG NQGQPGNKGS

[0031] 321 PGNPGQPGNE GQPGQPGQNG QPGEPGSNGP QGSQGNPGKN GQPGSPGSQG SPGNQGSPGQPGNPGQPGEQ GKPGNQGPAG

[0032] 401 EPGNPGSPGN QGQPGNKGSP GNPGQPGNEG QPGQPGQNGQ PGEPGSNGPQ GSQGNPGKNGQPGSPGSQGS PGNQGSPGQP

[0033] 481 GNPGQPGEQG KPGNQGPAGE PGNPGSPGNQ GQPGNKGSPG NPGQPGNEGQPGQPGQNGQP GEPGSNGPQG SQGNPGKNGQ

[0034] 561 PGSPGSQGSP GNQGSPGQPG NPGQPGEQGK PGNQGPAGG

[0035] Another object of the present invention is to provide a method for constructing the above-mentioned engineered bacteria, which comprises the following steps:

[0036] (1) Design of collagen gene

[0037] The collagen gene sequence was obtained through GenBank, and the codons of the lactic acid bacteria expression system were optimized. The optimized collagen gene sequence was obtained by PCR, and the primers of the restriction site were designed using Primer 5.0 software. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0038] The preferred restriction enzyme cleavage sites are SnaB I and Eco81 I.

[0039] (2) PCR amplification of collagen gene and construction of recombinant plasmid

[0040] Using pPIC9K-collagen as a template, PCR amplification was performed to obtain the target collagen gene. The original plasmid 368-PGK was digested with restriction enzymes SnaB I and Eco81 I, exposing the sticky ends of the vector. The PCR-amplified gene fragment was also digested with SnaB I and Eco81 I and recovered.

[0041] The gene fragment recovered after enzyme digestion was mixed with the vector pHBM-368-PGK fragment and reacted at 16°C overnight using T4 DNA ligase.

[0042] The recombinant plasmid can be identified by conventional methods, such as gel electrophoresis or sequencing after PCR amplification.

[0043] (3) Screening of positive transformants

[0044] The ligation product was transformed conventionally using competent Lactococcus lactis NZ9000 as the host, spread on LB solid medium containing ampicillin, cultured at 37°C overnight, and a single recombinant colony was picked.

[0045] The third object of the present invention is to provide a collagen composition and a preparation method. The preferred recombinant collagen composition comprises the following components, by weight percentage: 5-20% of the collagen, 0.5-2% of a thickener, 10-20% of a moisturizer, 1-5% of a chelating agent and the balance of deionized water.

[0046] Preferably, the composition comprises 5-10% of the collagen, 0.5-1% of a thickener, 15-20% of a moisturizer, 1-3% of a chelating agent and the balance of deionized water.

[0047] More preferably, it comprises 9.8% of the collagen, 0.6% of a thickener, 18% of a moisturizer, 2% of a chelating agent, and the balance of deionized water.

[0048] The thickener is carbomer (CAS No.: 9007-20-9).

[0049] The moisturizing agent consists of 9 parts of methylpropanediol and 9 parts of 1,3-propylene glycol by weight.

[0050] The chelating agent is octanoylhydroxamic acid.

[0051] The preparation method of the wrinkle-removing and anti-wrinkle composition comprises the following steps:

[0052] S1. After homogenizing the thickener and deionized water, add the humectant, stir and heat;

[0053] S2. The mixture obtained in S1 is mixed and homogenized, and the temperature is lowered. A chelating agent and collagen are added and stirred to obtain the composition.

[0054] The fourth object of the present invention is to provide an application of the collagen composition, which efficiently expresses collagen through genetically engineered bacteria, prepares the collagen into a corresponding wrinkle-removing and anti-wrinkle composition, effectively reduces the length, area and depth of skin wrinkles, and has a good wrinkle-removing and anti-wrinkle effect.

[0055] Beneficial effects of the present invention:

[0056] (1) Expressing collagen through genetic engineering is simple, efficient and high purity;

[0057] (2) Through mutation and screening, protein mutants with enhanced wrinkle removal and anti-wrinkle effects were obtained. The prepared composition effectively reduced the length, area and depth of skin wrinkles, had good wrinkle removal and anti-wrinkle effects, and had good application prospects in wrinkle removal and anti-wrinkle products. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 : Schematic diagram of collagen expression vector pHBM-368-PGK-Collagen;

[0059] Figure 2 : Collagen SDS-PAGE image;

[0060] Figure 3 :Statistical graph of wrinkle length in nude mice;

[0061] Figure 4 :Statistical graph of wrinkle area in nude mice;

[0062] Figure 5 : Statistical diagram of wrinkle depth in nude mice. DETAILED DESCRIPTION

[0063] Example 1 Design of collagen gene and construction of recombinant cloning vector

[0064] The mature collagen peptide was obtained from GenBank, and its gene sequence was codon-optimized for the lactic acid bacteria expression system. Primer 5.0 software was used to design primers with restriction sites for SnaB I and Eco81 I. Primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the optimized collagen gene sequence was obtained by PCR.

[0065] Using pPIC9K-Collagen as a template, PCR amplification was performed to obtain the target gene. The original plasmid 368-PGK was digested with restriction enzymes SnaBI and Eco81 I, exposing the sticky ends of the vector. The gene fragment amplified by PCR was also digested with SnaBI and Eco81 I and recovered.

[0066] The gene fragment recovered after enzyme digestion was mixed with the vector pHBM-368-PGK fragment and reacted overnight at 16°C using T4 DNA ligase. Figure 1 shown.

[0067] Example 2 Construction of collagen mutants and recombinant strains

[0068] Based on the amino acid map, a group of amino acids that may affect the spatial structure of collagen were screened and mutated. The variants were G160A, N197K, and Q396E. The nucleotide sequences of muG160A, muN197K, and muQ396E were obtained by site-directed mutagenesis. The wt and its mutants were successfully cloned into the plasmid pHBM-368-PGK, respectively. The successfully constructed recombinant plasmids were transformed into Lactococcus lactis NZ9000 competent cells and recorded as Lab-pHBM-368-PGK-Collagen-WT (abbreviated as WT), Lab-pHBM-368-PGK-Collagen-muG160A (abbreviated as G160A), Lab-pHBM-368-PGK-Collagen-muN197K (abbreviated as N197K), and Lab-pHBM-368-PGK-Collagen-muQ396E (abbreviated as Q396E). Pick a single recombinant colony, culture it in culture medium, perform colony PCR identification, enzyme digestion identification and sequencing verification, and select wild-type and mutant engineered bacteria for preservation.

[0069] Example 3 Collagen expression by recombinant engineered bacteria

[0070] Three strains of each type were selected and cultured in liquid LB medium (containing 5 μg mL -1 The culture was cultured in erythromycin) at 30℃ overnight, and the next day, 5% inoculum was inoculated into 100 mL LB liquid medium (containing 5 μg mL -1 Erythromycin) was cultured at 30°C and incubated until OD600 was about 0.5. Then, 100 μL of 0.3 M CuSO4 that had been sterilized by filtration was added to the clean bench and incubated at 30°C for 4 h. The culture was then incubated at 16°C for 24 h.

[0071] Take 90 μL of the supernatant of the recombinant strain cell wall liquid, filter the supernatant through a filter membrane, separate and purify it with Sephadex G100 gel column chromatography, and then freeze-dry to obtain the finished product. Take an appropriate amount of sample for SDS-PAGE electrophoresis, the results are as follows Figure 2 It can be seen that the size of the expressed proteins in the four samples is about 55 KDa, and the bands are clear and bright, indicating that the wild-type and mutant collagen proteins mentioned above are successfully expressed in the recombinant strains with high purity.

[0072] Example 4 Preparation of wrinkle-removing and anti-wrinkle composition

[0073] The raw materials of the wrinkle-removing and anti-wrinkle composition include, by weight percentage, 9.8% of the collagen, 0.6% of a thickener, 18% of a moisturizing agent, 2% of a chelating agent, and the balance of deionized water.

[0074] The thickener is carbomer (CAS No.: 9007-20-9).

[0075] The moisturizing agent consists of 9 parts of methylpropylene glycol and 9 parts of 1,3-propylene glycol in parts by weight.

[0076] The chelating agent is octanoylhydroxamic acid.

[0077] The preparation method of the wrinkle-removing and anti-wrinkle composition comprises the following steps:

[0078] S1. After homogenizing the thickener and deionized water, add the humectant, stir and heat;

[0079] S2. The mixture obtained in S1 is mixed and homogenized, and the temperature is lowered. A chelating agent and collagen are added and stirred to obtain the composition.

[0080] Example 5 Wrinkle removal and anti-wrinkle experiment in mouse model

[0081] Male HR-1 nude mice aged 6-8 weeks were used for the experiment. UVB irradiation conditions were as follows: UVM-225D Mineralight lamps with an emission wavelength of 302 nm were used for the experiment. The test substance was applied simultaneously with the irradiation. The experimental period was 8 weeks.

[0082] Week 1: 60 mJ / cm 2 , lasting 200s, 3 times a week (a minimal erythema dose);

[0083] Weeks 2-3: 120 mJ / cm 2 , lasting 400s, 3 times a week;

[0084] Weeks 4-5: 180 mJ / cm 2 , lasting 600s, 3 times a week;

[0085] Weeks 6-8: 240 mJ / cm 2 , lasting 800s, 3 times a week;

[0086] On the second day after the experiment, the animals were anesthetized with 0.7% sodium pentobarbital intraperitoneally, and images of the test area skin were captured. The depth and length of skin wrinkles were analyzed using a Skin-Visiometer-VL 650.

[0087] Composition group 1: the composition comprises wild-type collagen;

[0088] Composition group 2: the composition comprises type 1 collagen mutant;

[0089] Composition group 3: the composition comprises type 2 collagen mutant;

[0090] Composition group 4: the composition contains type 3 collagen mutant.

[0091] Note: The control group received no treatment.

[0092] The model group was treated with the same composition as the composition group except that the composition did not contain collagen.

[0093] Mutant 1 is G160A, mutant 2 is N197K, and mutant 3 is Q396E.

[0094] The experimental results are shown in Figure 3-5 .

[0095] Figure 3 Note: Compared with the control group, **P<0.01; compared with the model group, #P<0.05, ##P<0.01; and compared with composition 1, *P<0.05 for composition 2 and composition 4.

[0096] Figure 3 The results show that the compositions of the present invention can reduce the length of wrinkles on the naked skin model subjected to photooxidation, and compositions 2 and 4 are significantly better than composition 1 in reducing the length of skin wrinkles.

[0097] Figure 4 Note: Compared with the control group, **P < 0.01; compared with the model group, #P < 0.05, ##P < 0.01; and compared with composition 1, *P < 0.05 for composition 2 and composition 4.

[0098] Figure 4 This indicates that the composition of the present invention can reduce the area of skin wrinkles in a photo-oxidation nude mouse model, and compositions 2 and 4 are significantly better than composition 1 in terms of the efficacy of reducing the area of skin wrinkles.

[0099] Figure 5Note: Compared with the control group, **P<0.01; compared with the model group, #P<0.05, ##P<0.01; and compared with composition 1, *P<0.05.

[0100] Figure 5 This indicates that the composition of the present invention can reduce the depth of skin wrinkles in a photo-oxidation nude mouse model, and composition 2 is significantly better than composition 1 in reducing the depth of skin wrinkles.

[0101] The experimental results showed that Compositions 2 and 4 were significantly more effective in removing wrinkles than Composition 1. Composition 2 showed the best wrinkle-removing and anti-wrinkle effect. Furthermore, no adverse reactions were observed in mice after application of the compositions of the present invention, demonstrating that the compositions of the present application are safe and have no side effects.

[0102] Example 6 Human body wrinkle removal and anti-wrinkle experiment

[0103] 80 healthy women aged 30-40 years old, with no history of trauma, skin disease, infectious disease, or long-term medication, were selected and randomly divided into 4 groups, with 20 people in each group.

[0104] Gender: Female.

[0105] Age: 30-40 years old.

[0106] Treatment and usage

[0107] Treatment course: Four weeks is one course of treatment.

[0108] Usage: Use the application formula of the present invention to prepare compositions 1-4, apply once in the morning and evening after cleansing, for eight consecutive weeks.

[0109] The facial forehead wrinkles before use were used as the control group, and the facial forehead wrinkles after eight weeks of continuous use were used as the sample group.

[0110] Criteria for judging wrinkle removal and anti-wrinkle effects:

[0111] Excellent results: Compared with before use, wrinkles have been reduced by more than 90%, a significant improvement.

[0112] Significantly effective: Compared with before use, wrinkles are reduced by 60%-90%, a significant improvement.

[0113] Effective: Compared with before use, wrinkles have subsided by 20-70%, but a few wrinkles still exist, which has been improved.

[0114] Ineffective: Wrinkles were reduced by less than 20% compared to before use, indicating no improvement.

[0115] The wrinkle removal and anti-wrinkle results are shown in the table below:

[0116] Table 1 Anti-wrinkle effect

[0117]

[0118] Compositions 2 and 4 demonstrated excellent wrinkle-removing effects. After eight weeks of use, facial fine lines were significantly smoothed, and wrinkles were effectively reduced or alleviated, demonstrating overall superiority over Composition 1. Composition 2 achieved an effective rate of 90% for wrinkle removal, with an excellent rate of 30%. Composition 4 achieved an effective rate of 85%, with an excellent rate of 25%. No subjects experienced adverse reactions such as skin allergies or redness, demonstrating the safety and side effects of the compositions of the present invention, which hold broad application prospects in wrinkle-removing and anti-wrinkle products.

[0119] The preferred embodiments of the present invention have been described in detail above. It should be understood that numerous modifications and variations based on the concepts of the present invention are possible without inventive effort by those skilled in the art. Therefore, any technical solution that can be derived by one skilled in the art through logical analysis, reasoning, or limited experimentation based on the concepts of the present invention and the prior art should be within the scope of protection defined by the claims.

Claims

1. A recombinant collagen, the amino acid sequence of which is shown in SEQ ID NO:

2.

2. The method for preparing collagen according to claim 1, which is obtained by genetic engineering means, comprises the following steps: (1) Introducing the collagen gene into a plasmid vector to obtain a fusion plasmid; (2) Introducing the fusion plasmid into the host expression system and expanding the culture medium; (3) Isolate and purify the target protein.

3. The preparation method according to claim 2, wherein the host expression system is any one of yeast, lactic acid bacteria, and Escherichia coli.

4. An anti-wrinkle composition comprising the collagen shown in SEQ ID NO: 2, a thickener, a moisturizing agent, a chelating agent, and deionized water, wherein the thickener is carbomer, the moisturizing agent is composed of 9 parts by weight of methylpropanediol and 9 parts by weight of 1,3-propylene glycol, and the chelating agent is caprylhydroxamic acid.

5. The composition according to claim 4, wherein the contents of the components are as follows by weight: 9.8% collagen, 0.6% thickener, 18% moisturizer, 2% chelating agent and the balance deionized water.

6. A method for preparing the composition according to any one of claims 4 to 5, comprising the steps of: S1. After homogenizing the thickener and deionized water, add the humectant, stir and heat; S2. The mixture obtained in S1 is mixed and homogenized, and the temperature is lowered. A chelating agent and collagen are added and stirred to obtain the composition.

7. Use of the recombinant collagen according to any one of claims 1 to 2, the anti-wrinkle composition according to any one of claims 4 to 5, and the anti-wrinkle composition prepared according to the method according to claim 6 in the preparation of anti-wrinkle products.

8. The use according to claim 7, wherein the product can effectively reduce one or more of the length, area and depth of skin wrinkles.

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