Recombinant collagen as well as coding gene, recombinant vector, recombinant strain, preparation method and application of recombinant collagen and coding gene, recombinant vector and recombinant strain

Abstract

The invention belongs to the technical field of protein engineering and biology, and discloses a recombinant collagen, a coding gene thereof, a recombinant vector, a recombinant strain, a preparation method and application thereof. The amino acid sequence of the recombinant collagen is shown as SEQ ID NO. 1. The recombinant collagen is high in transdermal property, and has relatively good adhesion of fibroblasts and relatively good collagen metabolism capability of the fibroblasts. The collagen is widely applied to the fields of medical cosmetic plastic surgery, cosmetics and the like, and has a good application prospect.

Description

Technical Field

[0001] The present invention relates to the fields of protein engineering and biotechnology, and in particular, to a recombinant collagen and its coding gene, recombinant vector, recombinant strain, preparation method, and their applications. Background Art

[0002] Collagen is a crucial structural protein and is one of the most abundant and widely distributed proteins in mammals and even many organisms. It plays a core role in maintaining the structural support of the body, the stability and elasticity of tissues. Collagen is widely present in tissues such as bones, joints, muscles, skin, blood vessels, and corneas and is the main component of these tissues. With its unique triple-helix structure, collagen forms a molecular cable, strengthens tendons, and supports the membranes of the skin and internal organs. In bones and teeth, collagen binds to mineral grains to form hard bone tissue. In addition to structural support, collagen can also act as a signaling molecule, participating in regulating cell proliferation, differentiation, and migration, which is crucial for maintaining normal physiological functions and the overall health of the body. Recombinant collagen has wide applications in industries such as biomedicine, cosmetics, and food due to its advantages of low toxicity, low antigenicity, low immunogenicity, ability to guide cell regeneration, and good biocompatibility. In the field of medical aesthetics, collagen is also an important component for improving skin conditions and maintaining skin youthfulness and elasticity.

[0003] When collagen is applied to fields such as cosmetics, from the perspective of enhancing its biological activity, it is desired that collagen has good transdermal absorption performance. However, since collagen is a macromolecular bioactive substance, its own transdermal absorption ability is weak. Therefore, in previous studies, how to promote the transdermal absorption of collagen has become an important research hotspot. In recent years, with the extensive application of genetic engineering technology, researchers have created various types of recombinant collagen. Usually, short amino acid sequences from natural human collagen can be selected for splicing and / or repeated construction to obtain new recombinant collagen, and this type of recombinant collagen theoretically has advantages such as low immunogenicity and high biological activity.

[0004] However, in terms of designing short amino acid sequences as repeat units or splicing units, there is no prior art providing effective guidance to ensure that the designed recombinant collagen has good fibroblast adhesion and fibroblast collagen metabolism ability in addition to maintaining good biological activity and transdermal absorption ability. Summary of the Invention

[0005] The object of the present invention is to overcome the problem that existing recombinant collagen cannot take into account better fibroblast adhesion and fibroblast collagen metabolism ability while maintaining good biological activity and transdermal absorption ability, and to provide a recombinant collagen and its coding gene, recombinant vector, recombinant strain, preparation method and their applications. This recombinant collagen has high activity, good transdermal effect, and also takes into account better fibroblast adhesion and fibroblast collagen metabolism ability.

[0006] To achieve the above object, the first aspect of the present invention provides a recombinant collagen, which is a collagen with the amino acid sequence shown in SEQ ID NO. 1.

[0007] The second aspect of the present invention provides a gene encoding the recombinant collagen, which has a nucleotide sequence encoding the recombinant collagen as described above.

[0008] Preferably, the gene has the nucleotide sequence shown in SEQ ID NO. 2.

[0009] The third aspect of the present invention provides a recombinant vector, which contains the gene as described above.

[0010] Preferably, the expression vector of the recombinant vector is pPIC9K plasmid.

[0011] The fourth aspect of the present invention provides a recombinant strain, which contains the gene as described above or the recombinant vector as described above.

[0012] Preferably, the expression strain of the recombinant strain is yeast, more preferably Pichia pastoris strain GS115.

[0013] The fifth aspect of the present invention provides a preparation method of the recombinant collagen, which includes: inoculating the recombinant strain as described above into a fermentation medium for fermentation culture.

[0014] Preferably, the conditions of the fermentation culture include: temperature is 20 - 35 °C, rotation speed is 150 - 240 rpm, time is 60 - 80 h, and inoculum size is 1 - 15 vol%.

[0015] The sixth aspect of the present invention provides the application of at least one of the recombinant collagen as described above, the gene as described above, the recombinant vector as described above, the recombinant strain as described above and the recombinant collagen prepared by the method as described above in the preparation of collagen products.

[0016] Preferably, the collagen products are selected from at least one of skin products, products for enhancing fibroblast adhesion and products for enhancing fibroblast collagen metabolism ability.

[0017] Preferably, the collagen product is selected from any one of injectables, facial fillers, dressings, cosmetics, and tissue engineering materials.

[0018] Through the above technical solution, the present invention obtains the amino acid sequence of the natural human type III collagen alpha-1 chain protein ( GenBank : AGL34959.1) from the NCBI database, deeply mines and analyzes the biological information of this sequence, designs the hydrophilicity-hydrophobicity, charge distribution of the amino acid sequence and the protein spatial structure, so that the recombinant collagen of the present invention can achieve better fibroblast adhesion and fibroblast collagen metabolism ability while maintaining high biological activity. The amino acid sequence of the recombinant collagen of the present invention is shown in SEQ ID NO. 1, which ensures the realization of the biological function of the recombinant collagen and has the characteristics of high biocompatibility, good transdermal permeability, and low use risk.

[0019] The recombinant collagen provided by the present invention has broad application prospects in the fields of medical beauty and plastic surgery, cosmetics, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Protein detection result diagram of the recombinant collagen fermentation broth in Examples 1-3 SDS-PAGE wherein, Line1: Induced by S37 for 24 h, Line2 and Line4: Marker; Figure 2 Protein detection result diagram of the purified recombinant collagen in Examples 1-4 SDS-PAGE ; Figure 3 Fibroblast adhesion experiment diagram of the recombinant collagen in Example 2; Figure 4 Fibroblast collagen metabolism experiment diagram of the recombinant collagen in Example 3. DETAILED DESCRIPTION OF THE INVENTION

[0021] In the ranges disclosed herein, the endpoints and any values are not limited to the exact ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, between the endpoint values of each range, between the endpoint values of each range and a single point value, and between single point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0022] The first aspect of the present invention provides a recombinant collagen, and the recombinant collagen is a collagen with an amino acid sequence as shown in SEQ ID NO. 1.

[0023] In the second aspect of the present invention, there is provided a gene encoding recombinant collagen, which has a nucleotide sequence encoding the recombinant collagen as described above.

[0024] As is well known in the art, among the 20 different amino acids that make up proteins, except Met (ATG) or Trp (TGG) are each encoded by a single codon, and the other 18 amino acids are each encoded by 2 - 6 codons ( Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, 2nd Edition, 1989, see Appendix D on page 950). That is, due to the degeneracy of the genetic codons, there are usually more than one codon that determines an amino acid, and the substitution of the third nucleotide in the triplet codon often does not change the amino acid composition. Therefore, the nucleotide sequences of genes encoding the same protein can be different.

[0025] According to the present invention, preferably, the gene has the nucleotide sequence shown in SEQ ID NO. 2. In the present invention, the gene may only contain the sequence shown in SEQ ID NO. 2, or it may be a DNA molecule that takes the sequence shown in SEQ ID NO. 2 as the coding region and additionally adds other components. The other components may include any components required in the art for artificially synthesizing gene sequences and expressing them through expression vectors, such as promoters, enhancers, Kozak sequences, etc.

[0026] The amino acid sequence shown in SEQ ID NO. 1 as described above and the nucleotide sequence shown in SEQ ID NO. 2 as described above are shown in Table 1.

[0027] Table 1

[0028] The present invention improves the expression efficiency of recombinant collagen by adjusting the gene sequence. Specifically, by changing the synonymous codons in the gene to make it more in line with the codon preference of Pichia pastoris, rare codons are eliminated, and the translation efficiency is improved. The inventors have found that by adopting the above - preferred implementation manner, the expression level of the recombinant collagen provided by the present invention can be higher.

[0029] The nucleotide sequence provided by the present invention can generally be obtained by polymerase chain reaction (PCR) amplification, recombination, or artificial synthesis methods. Once the relevant nucleotide sequence is obtained, the relevant amino acid sequence can be obtained in large quantities by recombination methods. Usually, the obtained nucleotide sequence is cloned into a vector, then transferred into a genetically engineered bacterium, and then the relevant nucleotide sequence is separated from the proliferated host cells by conventional methods. The nucleotide sequence shown in SEQ ID NO. 2 of the present invention was entrusted to Jiangsu Saisuofei Biotechnology Co., Ltd. for synthesis.

[0030] In addition, the relevant nucleotide sequences can also be synthesized by known artificial chemical synthesis methods.

[0031] The third aspect of the present invention provides a recombinant vector, and the recombinant vector contains the gene as described above. In the present invention, the "vector" used in the recombinant vector can be various vectors known in the art, such as various commercially available plasmids, cosmids, phages, retroviruses, etc. The expression vector of the present invention is preferably the pPIC9K plasmid, and the recombinant plasmid pPIC9K-COL3-S37 is obtained.

[0032] The fourth aspect of the present invention provides a recombinant strain, and the recombinant strain contains the gene as described above or the recombinant vector as described above.

[0033] The host cell can be a prokaryotic cell or a eukaryotic cell. According to the present invention, preferably, the expression strain of the recombinant strain is yeast. More preferably, it is the Pichia pastoris strain GS115.

[0034] In the present invention, the recombinant vector can be transformed, transduced or transfected into a host cell (strain) by conventional methods in the art, such as chemical transformation by the calcium chloride method, high-voltage electroporation transformation.

[0035] In the present invention, the Pichia pastoris strain GS115 is used as the host bacterium, and the recombinant vector pPIC9K-COL3-S37 containing the recombinant collagen provided by the present invention is transformed into the GS115 host bacterium. The inventors have found through research that using the GS115 host bacterium has a higher ability to express recombinant collagen and has higher stability.

[0036] The fifth aspect of the present invention provides a method for preparing recombinant collagen, and the preparation method includes: inoculating the recombinant strain as described above into a fermentation medium for fermentation culture.

[0037] In the present invention, there are no particular limitations on the fermentation conditions of the recombinant strain, as long as the recombinant strain can be proliferated in large quantities through the fermentation process. Exemplarily, the fermentation process includes: randomly picking single colonies of the recombinant strain and inoculating them into an activation medium (the activation medium preferably contains: yeast extract 8 - 12 g / L, tryptone 15 - 25 g / L, glucose 15 - 25 g / L, agar powder 15 - 25 g / L), and performing activation culture by shaking culture at a rotation speed of 150 - 250 rpm at room temperature for 8 - 12 h; inoculating the strain after activation culture into a seed medium (the seed medium preferably contains: tryptone 15 - 25 g / L, yeast extract 5 - 15 g / L, YNB 10 - 15 g / L, glycerol 15 - 25 g / L, phosphate at pH 6.0 - 7.0, 80 - 120 mM) for seed culture, and after shaking culture at a rotation speed of 150 - 250 rpm at room temperature for 8 - 12 h, inoculating it into a fermentation medium (the fermentation medium preferably contains: tryptone 15 - 25 g / L, yeast extract 5 - 15 g / L, YNB 10 - 15 g / L) for induced expression, centrifuging the obtained fermentation broth, and collecting the supernatant to obtain a liquid containing recombinant collagen. The inventors have found through research that adopting the above fermentation process is beneficial to increasing the expression level of recombinant collagen.

[0038] The recombinant collagen provided by the present invention has a relatively low molecular weight, can more easily penetrate the stratum corneum of the skin, and thus be absorbed by the skin, more easily penetrate the skin barrier, and achieve a better transdermal effect.

[0039] The present invention also provides a protein preparation, which contains the recombinant collagen prepared by the method as described above.

[0040] In the present invention, the recombinant collagen can be made into corresponding protein preparations. Specifically, the protein preparations can exist in solid, semi - solid or liquid forms, and the protein preparations can contain excipients or additives for preparing the protein preparations, etc., which can be selected by those skilled in the art according to needs and will not be elaborated here.

[0041] In the present invention, the fermentation broth obtained by the above - mentioned preparation method can be directly used as a protein preparation, or the recombinant collagen can be obtained after separating and purifying the fermentation broth and used as a protein preparation. The separation and purification can adopt conventional protein separation methods in the art. That is to say, the recombinant collagen can be used in the form of whole cells of the recombinant strain, or in the form of a crude protein preparation or a purified protein preparation separated from the cells of the recombinant strain without purification. In the present invention, the protein preparation is purified and separated multiple times by using a conventional cation - exchange column lexcap SP 6FF in the art, and the detection effect of the purified protein is as Figure 2As shown. The inventor's research found that the protein preparation prepared by the above method has higher purity.

[0042] Based on the high stability, small molecular weight and high transdermal permeability of the recombinant collagen provided by the present invention, it has higher expression efficiency and good biocompatibility. The sixth aspect of the present invention provides the use of at least one of the recombinant collagen as described above, the gene as described above, the recombinant vector as described above, the recombinant strain as described above, and the recombinant collagen prepared by the method as described above in the preparation of collagen products. Preferably, the collagen product is selected from at least one of skin products, products for enhancing fibroblast adhesion, and products for enhancing fibroblast collagen metabolism ability.

[0043] In the present invention, the collagen product is selected from any one of injection agents, facial fillers, dressings, cosmetics, and tissue engineering materials.

[0044] On the premise of maintaining high biological activity, the recombinant collagen of the present invention achieves higher expression efficiency and high stability, can ensure the realization of the biological function of the recombinant collagen, and has the characteristics of high biocompatibility, good transdermal permeability, and low use risk, and has broad application prospects in the fields of medical beauty and plastic surgery, cosmetics, etc.

[0045] The present invention will be described in detail below through examples.

[0046] In the following examples, the pPIC9K plasmid was purchased from Saisuofei Biotechnology Co., Ltd.; type III small molecule collagen 02SC (control product, hereinafter referred to as 02SC) was purchased from Jiangsu Chuangjian Medical Technology Co., Ltd.; BSA (bovine serum albumin) was purchased from Solarbio; HDF cells were purchased from Guangdong Boxi Biology; HaCat cells were purchased from the Chinese Academy of Sciences. The remaining reagents and raw materials are all conventional commercially available products.

[0047] YPD medium: Tryptone 20 g / L, yeast extract 10 g / L, glucose 20 g / L, agar powder 20 g / L, autoclaved at 115°C for 30 min.

[0048] MD medium: Glucose 20 g / L, YNB 13.14 g / L, biotin 0.0004 g / L, agar powder 20 g / L, autoclaved at 115°C for 30 min.

[0049] BMMY medium: Tryptone 20 g / L, yeast extract 10 g / L, YNB 13.4 g / L, phosphate at pH 6.0 100 mM, autoclaved at 115°C for 30 min. When used, add the prepared 4X10 -5% biotin aqueous solution, 100 mM phosphate buffer (pH 6.0), 0.5% methanol.

[0050] BMGY medium: 20 g / L tryptone, 10 g / L yeast extract, 20 g / L glycerol, 13.4 g / L YNB (containing 10 g / L ammonium sulfate), autoclaved at 115 °C for 30 min. When in use, add the prepared 4×10 -5 % biotin aqueous solution, 100 mM phosphate buffer (pH 6.0).

[0051] Example 1-1 This example illustrates the design of the recombinant collagen sequence Obtain the amino acid sequence of the natural human type III collagen α1 chain protein (GenBank: AGL34959.1) through the NCBI database, and deeply mine and analyze the biological information of this sequence. For example, analyze the hydrophilicity and hydrophobicity and charge distribution of the amino acid sequence through the sequence amino acid analysis library peptides; use alphafold to predict the three-dimensional structure of recombinant collagen; use molecular dynamics software amber, etc. to perform molecular dynamics simulations on recombinant collagen to evaluate its structural stability. Finally, based on these analyses, the natural human type III collagen α1 chain is redesigned. The recombinant collagen achieves higher expression efficiency and high stability while maintaining high biological activity. The obtained amino acid sequence is the recombinant collagen (S37) shown in SEQ ID NO. 1. This sequence contains the biological active sites of the natural human type III collagen α1 chain and does not contain any exogenous amino acids such as tags. It is a recombinant humanized collagen, which ensures the realization of the biological function of recombinant collagen and has the characteristics of high biocompatibility and low use risk.

[0052] Example 1-2 This example illustrates the construction of a recombinant collagen expression strain According to the codon preference of Pichia pastoris, the amino acid sequence shown in SEQ ID NO. 1 in Example 1-1 was codon-optimized. The optimized coding gene nucleotide sequence is shown in SEQ ID NO. 2, named COL3-S37. The DNA sequence was commissioned to Jiangsu Saisuofei Biotechnology Co., Ltd. for synthesis and cloned into the Pichia pastoris expression vector pPIC9K to obtain the recombinant plasmid pPIC9K-COL3-S37. After linearizing the recombinant plasmid with Sac I enzyme, it was transformed into the GS115 host strain. After recovery, the bacterial solution was spread on the auxotrophic MD plate and statically cultured at 30 °C for 3 d to obtain single colonies.

[0053] Example 1-3 This example illustrates the induced expression of the recombinant collagen recombinant strain Randomly pick single colonies from the MD plates described in Examples 1-2 and inoculate them into YPD medium for activation. Incubate overnight at 30 °C with shaking at 220 rpm. Then transfer them to BMGY medium for seed culture at an inoculation amount of 10% (v / v), incubate overnight at 30 °C with shaking at 220 rpm, and then transfer them to BMMY medium for induced expression at an inoculation amount of 10% (v / v). Add 1% (w / v) methanol every 24 h for induction. After 72 h of induced culture, end the culture. Take the fermentation broth, centrifuge to collect the supernatant for electrophoresis detection. The electrophoresis detection results are as Figure 1 shown. The results show that recombinant collagen (S37) is expressed, and the molecular weight is less than 10 kDa, indicating that the recombinant collagen prepared in this example has a higher transdermal absorption rate than conventional macromolecular recombinant collagen.

[0054] Examples 1-4 This example illustrates the purification and preparation of recombinant collagen Centrifuge the fermentation broth containing recombinant collagen (S37) prepared in Examples 1-3, and take the supernatant as the feed solution. Clarify the feed solution with a 0.1 μm membrane package, and then concentrate the feed solution with a 1 kDa membrane package. Add buffer for top washing until the conductivity is 2.4 mS / cm, and adjust the pH to 4.0 to obtain the sample loading solution. The sample loading solution is purified multiple times through a cation exchange column lexcap SP 6FF to obtain a recombinant collagen (S37) sample. The electrophoresis results are as Figure 2 shown. There is only one clear protein band on the gel, indicating that the protein sample has a high purity, and the purity of the sample reaches over 90%. After the purified sample is replaced with PBS buffer and the protein concentration is determined by the BCA method, it is stored for later use.

[0055] Example 2 This example illustrates the fibroblast adhesion experiment of recombinant collagen On Day 0, dilute the recombinant collagen (S37) prepared in Examples 1-4 with PBS and coat it in a 96-well plate, and incubate overnight at 4 °C. On Day 1, discard the protein solution, and wash the wells twice with PBS, and discard the supernatant by flicking the plate. According to 3 E4 / well (3×10 4Add HDF-1 cells (number of cells per well), and culture them in an incubator at 37°C for 1 h. Observe the cell adhesion situation, and discard the cells by flicking the plate, then wash twice with PBS. Add CCK8 solution (a reagent for cell proliferation and toxicity detection) prepared with DMEM (Dulbecco's Modified Eagle Medium) basal medium, 100 μl / well (indicating adding 100 μl of CCK8 solution to each well); place at 37°C for 1 h, and read the value on an enzyme-linked immunosorbent assay (ELISA) reader. The reference substance (02SC) and the reference BSA (bovine serum albumin) adopt the same experimental method as the above recombinant collagen sample. After obtaining the OD value, plot the graph using Graphpad.

[0056] The experimental results are as Figure 3 shown. The recombinant collagen (S37) prepared by the present invention has an obvious effect of promoting cell adhesion compared with the reference substance (02SC), which is superior to the reference BSA (bovine serum albumin). And when the dilution concentration of S37 is 0.1 wt%, the effect of S37 in promoting cell adhesion is the strongest, and it has excellent effects in reducing skin laxity, reducing wrinkles, and maintaining skin elasticity.

[0057] Example 3 This example illustrates the fibroblast collagen metabolism experiment of recombinant collagen. Resuspend HDF cells (human dermal fibroblasts) in 10 vol% FBS + DMEM medium, and add them to a 6-well plate at 3E5 / well (3×10 5 cells / well). After the cells adhere to the wall, add a recombinant collagen (S37) sample diluted with 0.1 vol% FBS + DMEM medium to a concentration of 0.1 mg / mL. After mixing well, place it in an incubator at 37°C. After 24 h, extract RNA and reverse transcribe to obtain cDNA, and then perform qPCR experiments. The reference substance (02SC) adopts the same experimental method as the above recombinant collagen sample.

[0058] The experimental results are as Figure 4 shown. The recombinant collagen (S37) prepared by the present invention has an obvious effect of promoting the production of COL-1 (type I collagen) and COL-3 (type III collagen), and the effects are superior to those of the reference substance (02SC). Therefore, S37 has excellent advantages and effects in promoting skin elasticity, firmness, and anti-wrinkle efficacy.

[0059] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including any other suitable combination of each technical feature. These simple modifications and combinations should also be regarded as the content disclosed by the present invention and fall within the protection scope of the present invention.

Claims

1. A recombinant collagen, characterized in that: The recombinant collagen is a collagen with an amino acid sequence as shown in SEQ ID NO.

1.

2. A gene encoding recombinant collagen, characterized in that: The gene has a nucleotide sequence encoding the recombinant collagen according to claim 1.

3. The gene according to claim 2, characterized in that The gene has a nucleotide sequence shown in SEQ ID NO.

2.

4. A recombinant vector, characterized in that: The recombinant vector contains the gene according to claim 2 or 3.

5. A recombinant strain, characterized in that: The recombinant strain contains the gene according to claim 2 or 3 or the recombinant vector according to claim 4.

6. A method for preparing recombinant collagen, characterized in that: The preparation method comprises: inoculating the recombinant strain according to claim 5 into a fermentation medium for fermentation culture.

7. Use of at least one of the recombinant collagen according to claim 1, the gene according to claim 2 or 3, the recombinant vector according to claim 4, the recombinant strain according to claim 5, and the recombinant collagen prepared by the method according to claim 6 in the preparation of collagen products.

8. The use according to claim 7, characterized in that: The collagen product is selected from at least one of a skin product, a product for enhancing fibroblast adhesion, and a product for enhancing fibroblast collagen metabolism ability.

9. The use according to claim 7 or 8, characterized in that: The collagen product is selected from at least one of an injection, a facial filler, a dressing, a cosmetic, and a tissue engineering material.

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