Preparation method and application of humanized III-type collagen

Abstract

The invention provides a preparation method and application of humanized III-type collagen, amino acid sequence modification is performed on the basis of natural III-type collagen, so that fermentation preparation, separation and purification are facilitated, and the collagen can effectively adapt to the in-vivo environment; a fermentation preparation method is optimized, a yeast expression system is utilized, and a methanol and sorbitol double induction means is used, so that the expression efficiency is remarkably improved; the collagen can promote repair after skin injury, especially has a relatively good protection effect on ultraviolet injury, and can resist oxidative stress and regulate secretion of inflammatory factors.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology research and development, and specifically provides a preparation method and application of humanized type III collagen. Background Art

[0002] Collagen is the main dermal component of human skin and has advantages such as biocompatibility and biodegradability. Its material-cell interaction is conducive to extracellular matrix (ECM) remodeling and tissue regeneration (see Arai Y, Choi B, Kim BJ, et al. Cryptic ligand on collagen matrix unveiled by MMP13 accelerates bone tissue regeneration via MMP13 / Integrinα3 / RUNX2 feedback loop. Acta Biomater. 2021; 125: 219–230.). The molecular composition of type III collagen is a homotrimer, in which the (Gly-XY)n tripeptide repeat sequence is the main feature of the helical region. Type III collagen is rich in blood vessels, intestines and skin, and is upregulated during the growth and development of organisms and wound healing (see Walimbe T, Calve S, Panitch A, et al. Incorporation of types I and III collagen in tunablehyaluronan hydrogels for vocal fold tissue engineering. Acta Biomater. 2019; 87: 97–107). During wound healing, type III collagen can induce inflammatory cells and fibroblasts to migrate to the wound site and promote connective tissue formation and repair (see Makuszewska M, Bonda T, M, et al. Expression of collagen type III in healing tympanic membrane. Int J Pediatr Otorhi. 2020; 136: 110196). Type III collagen is also a natural hemostatic substance that can induce platelet adhesion and aggregation to the bleeding site and activate some coagulation factors to cause coagulation cascade reaction (see Kuivaniemi H, Tromp G. Type III collagen (COL3A1): gene and protein structure, tissue distribution, and associated diseases. Gene. 2019; 707: 151–171). Studies have found that recombinant human type III collagen has a practical effect in promoting extracellular matrix remodeling, upregulating the synthesis of type I and type III collagen in the body, and alleviating skin photoaging caused by ultraviolet rays (see Wang J, Qiu H, Xu Y, et al. The biological effect of recombinant humanized collagen on damaged skin induced by UV-photoaging: an invivo study. Bioact Mater. 2022; 11: 154–165). Type III collagen has unique physiological functions and is widely used in food, beauty, medicine and other fields, and the market demand is increasing. At present, a variety of collagen-based medical devices have been approved for marketing, such as Matrix Wound Healing and Collagen Implant I.

[0003] Currently, collagen used in biomedicine, pharmaceutical applications and tissue engineering medical products is almost entirely of animal origin, with bovine collagen dominating in both quality and quantity (see Fertala A. Three decades of research on recombinant collagens: reinventing the wheel or developing new biomedical products? Bioengineering. 2020; 7(4): 155). Most collagen products on the market are extracted from mammalian tissues and organs, requiring a large amount of raw materials. In addition, safety aspects should be considered when using collagen from animal sources, including the risks of infectious viruses, pathogenicity and allergic reactions (see Fushimi H, Hiratsuka T, Okamura A, et al. Nishimura I. Recombinant collagen polypeptide as aversatile bone graft biomaterial. Communications Materials. 2020; 1(1): 871–8713), such as prions, HIV, foot-and-mouth disease virus, etc. (see Kotler ED, Marshall WS, Gareta EG. Sources of collagen for biomaterials in skin wound healing. Bioengineering. 2019; 6: 56). At the same time, religious sensitivity, purification and batch-to-batch differences are other limiting factors for natural collagen (see Liu D, Zhang X, Li T, et al. Extraction and characterization of acid- and pepsin-soluble collagens from the scales, skins and swim-bladders of grass carp (Ctenopharyngodon idella) Food Biosci. 2015; 9: 68–74).

[0004] The biosynthesis of collagen has been greatly developed in recent years. The expression of collagen by animal and plant cells, yeast and Escherichia coli systems and large-scale preparation by high-density fermentation technology is a promising option (see Xiang ZX, Gong JS, Li H, et al. Heterologous expression, fermentation strategies and molecular modification of collagen for versatile applications. Crit Rev Food Sci Nutr. 2023; 63(21): 5268-5289). Tang et al. used Escherichia coli as the host and carried out small-scale production of human-like collagen in a 10L bioreactor at 28°C and 0.1mM isopropyl-β-d-thiogalactopyranoside (IPTG) induction, and finally obtained a yield of 0.26g L-1. IPTG is toxic to the strain, so it was replaced by the expression strategy of lactose as an inducer with lower cost, and finally about 0.7 g L-1 collagen binding domain fusion protein was obtained in a 3L fermenter (see Fruchtl M, Sakon J, Beitle R. Alternate carbohydrate and nontraditional inducer leads to increased productivity of a collagen binding domain fusion protein via fed-batch fermentation. J Biotechnol. 2016; 226: 65–73). Escherichia coli is the most common prokaryotic host, but its poor post-translational modification ability, easy formation of inclusion bodies and endogenous pyrogens have always troubled researchers; and the animal and plant cell system is cumbersome to operate and requires strict culture conditions, and there is still a long way to go before industrial production.

[0005] Yeast is a safe and popular eukaryotic host for expressing pharmaceutical proteins. It not only has complete post-translational modification functions, but also avoids the formation of inclusion bodies and the production of endotoxins. Researchers have tried to use Pichia yeast fermentation to produce modified type III collagen (see Xiang ZX, Gong JS, Shi JH, et al. High-efficiency secretory expression and characterization of the recombinant type III human-like collagen in Pichia pastoris. Bioresour Bioprocess. 2022, 9 (1): 117). Therefore, the present invention is to develop a novel method for preparing type III collagen, modify the amino acid sequence of natural type III collagen, replace some amino acids, and then use a yeast system for biosynthesis, and optimize the fermentation conditions. The obtained type III collagen has good biological activity, which provides an effective way for the preparation and application of collagen. Summary of the invention

[0006] A first aspect of the present invention provides a type III collagen, characterized in that the amino acid sequence of the type III collagen is shown in SEQ ID NO:2.

[0007] The present invention modifies the partial sequence of the α1 chain of human wild-type type III collagen, replaces some hydrophobic residues with hydrophilic residues, and replaces some acidic amino acid residues with basic amino acid residues, thereby improving the water solubility of collagen and its bioavailability, and adapting to the weak alkaline environment in the body.

[0008] The second aspect of the present invention provides a method for preparing the type III collagen, characterized in that the method comprises: introducing a nucleotide sequence encoding the type III collagen into a yeast expression vector, fermenting and culturing the yeast, inducing with methanol and sorbitol, and harvesting the type III collagen.

[0009] Furthermore, the nucleotide sequence encoding the type III collagen is shown in SEQ ID NO:3.

[0010] Furthermore, the yeast is Pichia pastoris. Preferably, the Pichia pastoris is GS115.

[0011] Furthermore, the culture medium used in the culturing process includes: 15 g / L glycerol, 5 g / L mannitol, 20 g / L peptone, 8 g / L yeast powder, 0.5 g / L manganese sulfate, 0.5 g / L sodium molybdate dihydrate, 0.3 g / L magnesium sulfate, 0.2 g / L cobalt chloride, 0.06 g / L zinc sulfate, 0.05 g / L copper sulfate and 0.02 g / L chromium chloride.

[0012] Furthermore, the culturing step includes: using YPD medium to culture the yeast in a shaking incubator at 30°C and 220 r / min; inoculating the yeast into the medium at an inoculation rate of 5%, and culturing in a shaking incubator at 30°C and 260 r / min, and when OD600 reaches 4-6, adding 1.2% methanol and 0.5% sorbitol by volume to induce expression for 72 hours; after the induction, centrifuging at 5000 r / min for 10 min to collect the fermentation supernatant, and separating and purifying to obtain type III collagen.

[0013] The third aspect of the present invention provides a use of the type III collagen in the preparation of a product for protecting skin damage.

[0014] Furthermore, the damage is ultraviolet damage.

[0015] Furthermore, the product is a cosmetic.

[0016] Beneficial Effects

[0017] The present invention provides a preparation method and application of humanized type III collagen, which has the following beneficial effects:

[0018] (1) The sequence structure of natural type III collagen was modified to improve the hydrophilicity of the protein, facilitate fermentation preparation and separation and purification, and effectively adapt to the in vivo environment;

[0019] (2) The fermentation preparation method was optimized, and the yeast expression system was used, using methanol and sorbitol dual induction to significantly improve the expression efficiency;

[0020] (3) The collagen can promote skin repair after damage, especially has a good protective effect against ultraviolet damage, can resist oxidative stress, and regulate the secretion of inflammatory factors. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 : Collagen protein electrophoresis;

[0022] Figure 2 : Cell relative viability determination;

[0023] Figure 3 :Histological observation of animal skin;

[0024] Figure 4 : SOD level detection;

[0025] Figure 5 :MDA level detection;

[0026] Figure 6 :IL-1β level detection;

[0027] Figure 7 :IL-6 level detection. DETAILED DESCRIPTION

[0028] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents, biological materials, and detection kits are all commercially available unless otherwise specified.

[0029] Example 1 Collagen Molecular Design

[0030] Natural collagen is a large molecular weight insoluble fibrin protein with a large number of Gly-XY repeating sequences in its sequence. It is also subject to strict post-modification regulation and self-assembly process during synthesis, which makes it difficult to express collagen. Some studies have molecularly modified type III collagen, replaced hydrophobic amino acids other than Pro with hydrophilic amino acids, and expressed it using the Pichia pastoris system to achieve high collagen production.

[0031] This study is based on the partial sequence of the α1 chain of wild-type human type III collagen, whose amino acid sequence is shown in SEQ ID NO: 1 and the GenBank accession number is KAI4037253.1. The Gly-XY basic skeleton is retained, and some hydrophobic amino acids (such as Tyr, Leu, Ile, Val, His, etc.) except Pro are replaced with hydrophilic residues (such as Asn, Gln, Lys, Thr, etc.). In addition, some acidic amino acids (such as Asp and Glu) are replaced with basic amino acids (such as Lys and Arg), and the amino acid sequence is partially truncated at the end, which can improve the water solubility of collagen and its bioavailability, and adapt to the weak alkaline environment in the body. The amino acid sequence of type III collagen after modification is shown in SEQ ID NO: 2. After codon optimization, its nucleotide sequence is shown in SEQ ID NO: 3. After adding EcoR I and Not I restriction sites at both ends, gene synthesis is performed to obtain the target sequence.

[0032] Example 2 Preparation of collagen

[0033] In the present invention, Pichia pastoris is selected to express the target protein. Pichia pastoris is a methylotrophic yeast with low price, relatively short expression time, and co-translational and post-translational processing capabilities. Moreover, Pichia pastoris can be post-translationally modified, such as O and N-linked glycosylation and disulfide bond formation, to achieve correct folding, solubility, stability and appropriate biological activity. Therefore, the expression system can effectively express foreign proteins.

[0034] In the present invention, the target gene is amplified by PCR technology and restriction sites are introduced. After double digestion with EcoR I and Not I, it is connected to the expression vector pPIC9K, named pPIC9K-muCL; the vector is transformed into Escherichia coli DH5α and screened by kanamycin resistance. A single colony is picked for culture, and the recombinant plasmid pPIC9K-muCL is extracted using a plasmid extraction kit. The recombinant plasmid pPIC9K-muCL is linearized by Sac I endonuclease and electroporated into Pichia pastoris GS115, and positive clones are screened. The recombinant strains are named GS115-muCL.

[0035] The yeast culture medium is one of the key factors for improving the expression of the target protein. The present invention has been improved on the basis of the conventional yeast BMGY culture medium. In terms of carbon source, in addition to the original glycerol of the BMGY culture medium, mannitol is added to enrich the carbon source types; in terms of nitrogen source, peptone and yeast powder are still the main ones, but the content ratio of the two is adjusted; trace elements are added to promote yeast growth. After optimization, the culture medium includes: 15g / L glycerol, 5g / L mannitol, 20g / L peptone, 8g / L yeast powder, 0.5g / L manganese sulfate, 0.5g / L sodium molybdate dihydrate, 0.3g / L magnesium sulfate, 0.2g / L cobalt chloride, 0.06g / L zinc sulfate, 0.05g / L copper sulfate and 0.02g / L chromium chloride, and the balance is water.

[0036] Induced expression is also a key factor affecting the expression of the target protein. The most common way to induce expression in Pichia pastoris is to use methanol induction, but methanol has a certain toxic effect on cells. If the dosage is too large, it may kill the cells, and if the dosage is too small, it is difficult to induce expression. Therefore, using methanol induction alone is often difficult to achieve the purpose of efficient expression of exogenous proteins. In the Pichia pastoris expression system, one of the most famous carbon sources that can be used with methanol is sorbitol. Sorbitol does not induce or inhibit the AOX promoter, so using sorbitol instead of glycerol in the mixed substrate method can reduce cell growth rate and increase the formation rate of specific products. In the present invention, methanol and sorbitol are mixed for induction to increase the expression of the target protein. Preliminary experiments show that compared with the use of methanol alone, the protein expression can be increased by about 20-35%.

[0037] The GS115-muCL strain was cultured in a shaking incubator at 30°C and 220 r / min using YPD medium. Then, the GS115-muCL strain was inoculated into the modified culture medium at a 5% inoculation rate. The culture medium volume did not exceed 20% of the shake flask volume. The culture was cultured in a shaking incubator at 30°C and 260 r / min. When the OD600 reached 4-6, 1.2% methanol and 0.5% sorbitol were added to induce expression for 72 hours. After the induction, the fermentation supernatant was collected by centrifugation at 5000 r / min for 10 min. The target product was separated and purified by Ni2+ affinity chromatography. The target protein shown was named muCL. Protein electrophoresis was used for detection, as shown in Figure 1 As shown, a target protein with a molecular weight of about 25 KD appeared.

[0038] Example 3 Collagen promotes cell proliferation

[0039] In this section, human epidermal cell line HaCaT and fibroblast cell line BJ-5ta were used to study the physiological activity of collagen. In the experiment, commercially available type III recombinant collagen (purchased from Jiangsu Jiangshan Juyuan Biotechnology Co., Ltd., trade name Yue Lixin) was used as a control sample for the experiment. The type III recombinant collagen was recorded as CL protein. The specific steps are as follows:

[0040] Take the above cells in the exponential growth phase and transfer 1×10 5 Cells were seeded in 96-well plates and randomly divided into 3 groups, each with 3 replicates: muCL group, 50 ng / mL of muCL protein was added to each well; CL group, 50 ng / mL of CL protein was added to each well; control group, equal volume of culture medium was added to each well. After 48 h of culture, cell proliferation activity was determined by MTT assay. Take 10 μL of 12 mM MTT solution and incubate the cells at 37 °C for 4 h in the dark; pour out the culture medium and wash 3 times with PBS; dissolve the generated formazan salt with DMSO and measure its absorbance at 490 nm in a spectrophotometer.

[0041] Relative cell viability = (OD value of experimental well - OD value of blank control well) / (OD value of control well - OD value of blank control well), with the well with culture medium alone as the blank control well.

[0042] The results are as follows Figure 2 As shown, collagen can promote the proliferation of skin epidermal cells and fibroblasts, and the modified muCL has a stronger ability to promote cell proliferation, which is significantly improved compared with the control group, indicating that it has good application prospects in treating skin injuries, promoting skin repair, and maintaining normal physiological functions of skin tissue.

[0043] Example 4 Collagen against skin oxidative damage

[0044] 4.1 UV skin damage modeling and drug administration

[0045] The mice were irradiated with UVB using an ultraviolet phototherapy device. Forty Balb / c mice were randomly divided into four groups, with 10 mice in each group. The mice in the normal group (NC group) were only shaved and not irradiated; the other groups were irradiated with UVB every day, with an initial dose of 100 mJ / cm 2 , increase by 500mJ / cm per day 2 , up to 300mJ / cm 2 Then keep it unchanged for a total of 2 weeks.

[0046] After irradiation, the model groups were given medications respectively: muCL group: 100 mg / kg of recombinant muCL protein was applied to the back every day; CL group: 100 mg / kg of recombinant CL protein was applied to the back every day; control group: 100 mg / kg of normal saline was applied to the back every day. Each group was given medication for 6 weeks.

[0047] 4.2 Pathological observation

[0048] After the experiment, the experimental mice in each group were killed, and 1 cm × 1 cm skin of the irradiated area on the back was taken and fixed with 4% paraformaldehyde. Pathological sections were prepared after dehydration, embedding, and slicing. HE and Masson staining were performed, and the histopathological changes were observed under an optical microscope.

[0049] The results are as follows Figure 3 As shown, the collagen fibers in the dermis of the normal group were arranged neatly, and the skin structure was dense and sparse; the dermis of the control group was significantly thinned, the dermis collagen fibers were reduced, the arrangement was disordered, and the density distribution was uneven. The muCL and CL treatments can make the dermis thicker and more uniform in thickness, more complete in structure, and clearer in layers. In addition, the recovery of the muCL group was better, and the dermis fibers tended to be neatly arranged and the structure was complete. The above structure shows that the recombinant collagen provided in the present invention can improve the skin damage of mice caused by ultraviolet radiation and promote the recovery of skin physiological functions.

[0050] 4.3 Oxidative stress detection

[0051] UVB can indirectly damage skin cell DNA by stimulating oxidative stress, causing skin cell necrosis. Reducing skin oxidative stress is an effective way to prevent skin photoaging. Therefore, in this section, we detected oxidative stress-related indicators in skin tissue to investigate the protective and repair effects of collagen on the skin. Take an appropriate amount of back skin from each group of mice, mix it with physiological saline, grind it into a homogenate, centrifuge it to obtain the supernatant, and detect the SOD and MDA content in the skin according to the instructions of the kit (purchased from Beijing Solebow Technology Co., Ltd.).

[0052] like Figure 4 , Figure 5 As shown in the figure, compared with the NC group, the SOD level in the skin of the control group mice was significantly reduced, while the MDA level was significantly increased, indicating that the damage caused by oxidative stress to the skin was serious. After treatment, the SOD level was restored, and the MDA level decreased significantly, and the treatment effect of the muCL group was more obvious. It shows that the collagen provided in this application can effectively combat oxidative stress in skin tissue.

[0053] 4.4 Detection of inflammatory factor secretion

[0054] The skin tissue fluid of each group of mice was obtained (preparation method is the same as in Section 4.3), and the ELISA kit (purchased from Nanjing Jiancheng Technology Co., Ltd.) was used to detect the IL-1β and IL-6 content in the tissue fluid. Figure 6 , Figure 7 As shown in the figure, compared with the normal group, the levels of IL-1β and IL-6 in the skin of mice in the control group were significantly increased. After treatment, the levels of inflammatory factors in the treatment group were significantly reduced, and the levels of IL-1β and IL-6 in the muCL group were closer to the normal physiological state, indicating that the collagen can inhibit the secretion of inflammatory factors and resist skin tissue damage caused by ultraviolet rays.

[0055] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments may still be modified, or some or all of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A type III collagen, characterized in that: The amino acid sequence of the type III collagen is shown in SEQ ID NO:

2.

2. A method for preparing type III collagen as claimed in claim 1, characterized in that: The method comprises: introducing a nucleotide sequence encoding the type III collagen into a yeast expression vector, fermenting and culturing the yeast, inducing the yeast with methanol and sorbitol, and harvesting the type III collagen.

3. The method according to claim 2, characterized in that The nucleotide sequence encoding the type III collagen is shown in SEQ ID NO:

3.

4. The method according to claim 2, characterized in that: The yeast is Pichia pastoris.

5. The method according to claim 2, characterized in that: The culture medium used in the culture process includes: 15g / L glycerol, 5g / L mannitol, 20g / L peptone, 8g / L yeast powder, 0.5g / L manganese sulfate, 0.5g / L sodium molybdate dihydrate, 0.3g / L magnesium sulfate, 0.2g / L cobalt chloride, 0.06g / L zinc sulfate, 0.05g / L copper sulfate and 0.02g / L chromium chloride.

6. The method according to any one of claim 5, characterized in that: The culturing step comprises: using YPD culture medium to culture the yeast in a shaking platform at 30°C and 220 r / min; inoculating the yeast in the culture medium at an inoculation amount of 5%, culturing in a shaking platform at 30°C and 260 r / min, and when OD600 reaches 4-6, adding 1.2% methanol and 0.5% sorbitol by volume to induce expression for 72 hours; after the induction is completed, centrifuging at 5000 r / min for 10 minutes to collect the fermentation supernatant, and separating and purifying to obtain type III collagen.

7. Use of the type III collagen according to any one of claims 1 to 6 in the preparation of a product for protecting against skin damage.

8. The use according to claim 7, characterized in that: The damage is ultraviolet damage.

9. The use according to claim 7, characterized in that: The product is a cosmetic.

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