Recombinant collagen as well as preparation method and application thereof

Abstract

The invention relates to the field of gene engineering, in particular to preparation and application of recombinant collagen. Collagen mutants are obtained through site-specific mutagenesis, mutants with good effects are screened to prepare the anti-wrinkle composition, the length, area and depth of skin wrinkles can be effectively reduced, the anti-wrinkle effective rate of the collagen Q160A mutant composition is 90%, the excellent rate is 30%, and the collagen Q160A mutant composition has good 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 has unique biological activity, inherent biomimetic advantages, high abundance in nature, and renewable properties. In recent years, with the rapid development of the global cosmetics market and the substantial improvement of scientific and technological research and development, natural collagen has attracted much attention as a functional raw material for cosmetics. Natural collagen has many advantages such as good biocompatibility, degradability and low antigenicity. However, its macromolecular characteristics of a three-dimensional triple helix structure make it difficult to directly penetrate the dense skin surface to achieve skin care effects. In order to improve 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 way to develop collagen-based beauty and skin care products. Collagen peptides can not only provide a matching amino acid source for the synthesis of collagen in the body, but the hydrophilic groups in their molecular structure also enhance the moisturizing properties of the skin. In addition, collagen peptides have good nutritional properties, UV resistance, 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 skin wound repair.

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

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

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

[0007] The culture medium for the activated lactic acid bacteria preparation is 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. They were all placed on a sterile operating table for ultraviolet sterilization for 30 min before inoculation.

[0009] The first object of the present invention is to provide an engineered bacterium containing a collagen recombinant vector, wherein a collagen mutant is introduced into the engineered bacterium 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 its sequence is shown in SEQ ID NO: 2, and a Usp45 signal peptide and a His tag are added.

[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 anti-wrinkle and wrinkle-removing effect.

[0021] The preferred increase percentage of the anti-wrinkle 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: 3-5.

[0025] A more preferred amino acid sequence of the mutated collagen is shown in SEQ ID NO:3.

[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 Biotechnology (Shanghai) Co., Ltd.

[0038] The preferred restriction 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 two restriction endonucleases, SnaB I and Eco81I, to expose the sticky ends at both ends of the vector. The gene fragment obtained by PCR amplification was also digested with SnaB I and Eco81 I and then recovered.

[0041] The gene fragment recovered after restriction 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 Lactococcus lactis NZ9000 competent bacteria 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 remainder 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 contains 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 methyl propanediol and 9 parts of 1,3-propylene glycol by weight.

[0050] The chelating agent is caprylhydroxamic acid.

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

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

[0053] S2, homogenizing the mixture obtained in S1 and cooling it, adding a chelating agent and collagen and stirring 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 anti-wrinkle composition, effectively reduces the length, area and depth of skin wrinkles, and has a good anti-wrinkle and wrinkle removal effect.

[0055] Beneficial effects of the present invention:

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

[0057] (2) By mutation and screening, protein mutants with enhanced anti-wrinkle and wrinkle-removing effects are obtained. The prepared composition effectively reduces the length, area and depth of skin wrinkles, has good anti-wrinkle and wrinkle-removing effects, and has good application prospects in anti-wrinkle and wrinkle-removing 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 nude mouse wrinkle length;

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

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

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

[0064] The collagen mature peptide was obtained through GenBank, and its gene sequence was obtained for codon optimization of lactic acid bacteria expression system, and primers with restriction sites were designed using Primer 5.0 software, and the restriction sites were SnaB I and Eco81 I. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the optimized collagen gene sequence was obtained by PCR. Its specific sequence is shown in SEQ ID NO.2.

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

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

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

[0068] According to the amino acid map, a group of amino acids that may affect the spatial structure of collagen were screened out and mutated. The variants were G160A, N197K, and Q396E. The nucleotide sequences of muG160A, muN197K, and muQ396E were obtained by site-directed mutagenesis. 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, and sequence verification after enzyme digestion identification, 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 at 30°C overnight, and the next day, 5% of the inoculum was inoculated into 100 mL of LB liquid medium (containing 5 μg mL -1 Erythromycin), 30℃, static culture until OD600=0.5, add 100μL 0.3M CuSO4 that has been sterilized by filtration in the clean bench, 30℃, static culture for 4h, and then culture at 16℃ for 24h.

[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 using 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 above wild-type and mutant collagens are successfully expressed in the recombinant strains with high purity.

[0072] Example 4 Preparation of anti-wrinkle composition

[0073] The raw materials of the anti-wrinkle composition include, by weight percentage, 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.

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

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

[0076] The chelating agent is caprylhydroxamic acid.

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

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

[0079] S2, homogenizing the mixture obtained in S1 and cooling it, adding a chelating agent and collagen and stirring to obtain the composition.

[0080] Example 5 Anti-wrinkle experiment in mouse model

[0081] The experimental animals were 6-8 week old male HR-1 nude mice. The UVB irradiation conditions were as follows: UVM-225D Mineralight with an emission wavelength of 302nm was used for UV irradiation. The test object was applied at the same time as the irradiation. The test period was 8 weeks.

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

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

[0084] Week 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 injected intraperitoneally, and the skin images of the test area were captured. The depth and length of skin wrinkles were analyzed using 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 4: The composition comprises type 3 collagen mutant.

[0091] Note: The control group did not receive any 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 It shows that the composition of the present invention can reduce the length of wrinkles on the naked model skin subjected to photo-oxidation, 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 It shows that the composition of the present invention can reduce the area of ​​skin wrinkles in the photo-oxidation nude mouse model, and compositions 2 and 4 are significantly better than composition 1 in 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-oxidative nude mouse model, and composition 2 is significantly superior to composition 1 in terms of the efficacy of reducing the depth of skin wrinkles.

[0101] The experimental results show that the anti-wrinkle effects of composition 2 and 4 are significantly better than those of composition 1. Composition 2 has the best anti-wrinkle effect. After the mice were smeared with the composition of the present invention, there were no adverse reactions, indicating that the composition of the present application is safe and has no side effects.

[0102] Example 6 Human anti-wrinkle and wrinkle removal 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 implement composition 1-4, use it once in the morning and evening after cleansing, and use it continuously for eight weeks.

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

[0110] Anti-wrinkle effect criteria:

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

[0112] Significant effect: Compared with before use, wrinkles are reduced by 60%-90%, which is a significant improvement.

[0113] Effective: Compared with before use, wrinkles 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, with no improvement.

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

[0116] Table 1 Anti-wrinkle effect

[0117] Composition 2 and Composition 4 have good wrinkle removal effects. After using the composition for 8 weeks, fine lines on the face are significantly smoothed, wrinkles are effectively reduced or alleviated, and the overall effect is better than that of Composition 1. The anti-wrinkle effectiveness of Composition 2 is 90%, and the excellent rate is 30%. The anti-wrinkle effectiveness of Composition 4 is 85%, and the excellent rate is 25%. No adverse conditions such as skin allergies and redness and swelling occurred in the subjects, indicating that the composition of the present invention is safe and has no side effects, and has broad application prospects in anti-wrinkle and wrinkle removal products.

[0118] The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A recombinant collagen, the amino acid sequence of which is shown in SEQ ID NO: 3 or 5.

2. The recombinant collagen according to claim 1, whose sequence is shown in SEQ ID NO:

3.

3. 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 in the culture medium; (3) Isolate and purify the target protein.

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

5. An anti-wrinkle composition comprising the recombinant collagen according to any one of claims 1-2 or the recombinant collagen prepared by the method according to any one of claims 3-4.

6. The composition according to claim 5, further comprising collagen, a thickener, a humectant, a chelating agent and deionized water.

7. The composition according to claim 6, 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.

8. A method for preparing a composition according to any one of claims 5 to 7, comprising the steps of: S1. After homogenizing the thickener and deionized water, add the humectant, stir and heat; S2, homogenizing the mixture obtained in S1 and cooling it, adding a chelating agent and collagen and stirring to obtain the composition.

9. Use of the recombinant collagen according to any one of claims 1 to 2, the anti-wrinkle composition according to any one of claims 5 to 7, and the anti-wrinkle composition prepared according to the method according to claim 8 in the preparation of anti-wrinkle products.

10. The use according to claim 9, wherein the product can effectively reduce one or more of the length, area and depth of skin wrinkles.

Images