Recombinant humanized type VII collagen, its expression vector and genetically engineered bacteria

Abstract

The present invention discloses a recombinant humanized type VII collagen, its expression vector and genetically engineered bacteria. In the present invention, functional short peptides binding to cell integrin in human type VII collagen are screened, and 10 tandem repeats are designed to respectively constitute recombinant humanized type VII collagens JYC701 and JYC702. After codon optimization, they are amplified and cloned into the pPIC9K vector. After enzymatic digestion and linearization, they are transformed into Pichia pastoris cells, and high-copy recombinants are screened through G418 resistance gradient to obtain Pichia pastoris genetically engineered bacteria capable of expressing recombinant humanized type VII collagens JYC701 and JYC702. The recombinant humanized type VII collagens JYC701 and JYC702 of the present invention exhibit excellent functions of promoting cell adhesion, cell proliferation and migration, have the activity of promoting the repair of tissues such as skin and enhancing the resistance of skin, and are suitable for preparing products such as skin care products and medical devices.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering and relates to a recombinant humanized type VII collagen, its expression vector and genetically engineered bacteria. Background Art

[0002] Type VII collagen is an important structural protein mainly present in the skin basement membrane and plays a key role in the skin, especially in terms of skin strength and elasticity. Type VII collagen forms anchoring fibrils at the dermo-epidermal junction, fixing the epidermis to the dermis, preventing the epidermis from separating from the dermis, providing additional mechanical support to the skin, and thus ensuring the skin's stability and tensile strength under external stress. In addition, it is also present in other tissues, such as the basement membrane regions of the oral mucosa, esophagus, colon, etc. When the skin is damaged, type VII collagen can promote wound healing and help restore tissue integrity. Studies have found that defects or mutations in type VII collagen are associated with various genetic diseases, the most common being dystrophic epidermolysis bullosa (DEB). DEB is a rare genetic skin disease. Mutations in the type VII collagen gene (Col7a1) in patients result in a reduction or absence of the number of anchoring fibrils. Since the epidermis and dermis cannot be firmly connected, the skin is extremely prone to separation, forming large areas of blisters and erosions. Due to the close association between type VII collagen and skin diseases, scientists are actively exploring its application in skin repair, using heterologous biosynthesis methods to produce recombinant type VII collagen for the development of therapeutic drugs or tissue engineering materials to promote wound healing or treat skin diseases. In addition, with age, the synthesis and function of type VII collagen also decline, which leads to a decrease in skin elasticity and anti-injury ability, manifested as skin aging, wrinkles and sagging. Therefore, type VII collagen is also an important target in anti-aging research.

[0003] Type VII collagen [α1(VII)]3 in the skin presents a long fibrous triple-stranded right-handed helix, which is composed of three identical α1(VII) chains wound around each other. Its structure can be divided into three main functional domains: the N-terminal globular domain (NC1 domain), the triple-helical domain, and the C-terminal globular domain (NC2 domain). Among them, the NC1 domain is rich in irregular secondary structures and is an important site for the interaction of type VII collagen with other extracellular matrix molecules or basement membrane components. For example, in the basement membrane, the NC1 domain can interact with laminin (especially laminin 332); the triple-helical domain is the most important structural domain of type VII collagen, with high tensile strength and stability, and is an important part of the formation of anchoring fibrils. Type VII collagen fixes the epidermis to the dermis through this structure to prevent skin separation, so it is crucial for maintaining the mechanical strength of the skin; while the NC2 domain may be related to the polymerization and stability of anchoring fibrils and plays a role in the formation of collagen fibrils. Studies have shown that the interaction between the NC2 domain and other type VII collagen molecules is crucial for the formation of a stable fiber network. Some functional modifications occur during the synthesis and functional regulation of type VII collagen, and these modifications may affect its role in the skin. For example, certain sites in type VII collagen are glycosylated, and in addition, the NC1 domain and NC2 domain are also cleaved by specific proteases after synthesis, and this process may regulate their localization and fiber formation in the basement membrane. Summary of the Invention

[0004] In the present invention, by screening the functional domains of human type VII collagen that bind to cell integrins, two functional peptide segments with potentially high activity are obtained, and they are tandemly repeated 10 times respectively to form recombinant humanized type VII collagen JYC701 and JYC702. After codon optimization, they are amplified and cloned into the pPIC9K vector, linearized by enzyme digestion, and then transformed into Pichia pastoris cells. High-copy recombinants are screened through a G418 resistance gradient, so as to obtain high-copy Pichia pastoris genetic engineering strains that can express recombinant humanized type VII collagen JYC701 and JYC702 respectively.

[0005] The technical solution of the present invention is as follows:

[0006] A functional short peptide, whose amino acid sequence has 100% identity with human type VII collagen and has a functional site for binding to cell integrins, and its amino acid sequence is as shown in SEQ ID No.1 or SEQ ID No.2.

[0007] Recombinant humanized type VII collagen JYC701 or JYC702, which is composed of 10 tandemly repeated functional short peptides with amino acid sequences shown in SEQ ID No.1 or SEQ ID No.2, and its amino acid sequence is shown in SEQ ID No.3 or SEQ ID No.4.

[0008] A gene encoding the above recombinant humanized type VII collagen JYC701 or JYC702, and its nucleotide sequence is shown in SEQ ID No.5 or SEQ ID No.6.

[0009] A recombinant expression vector, which is a recombinant expression vector containing the gene encoding the above recombinant humanized type VII collagen JYC701 or JYC702. In the specific embodiments of the present invention, the recombinant expression vectors pPIC9K-JYC701 or pPIC9K-JYC702 containing the gene encoding the above recombinant humanized type VII collagen JYC701 or JYC702 are taken as examples.

[0010] The construction method of the above recombinant expression vector is as follows:

[0011] Perform high-fidelity PCR amplification on the gene sequence encoding the recombinant humanized type VII collagen JYC701 or JYC702 shown in SEQ ID No.5 or SEQ ID No.6, and clone the target fragment purified by gel recovery seamlessly downstream of the α-mating factor secretion signal peptide of the pPIC9K empty vector. After verification by colony PCR and sequencing, enrich to obtain the recombinant expression vectors pPIC9K-JYC701 or pPIC9K-JYC702.

[0012] A Pichia pastoris genetic engineering bacterium for producing the above recombinant humanized type VII collagen JYC701 or JYC702, which contains the above recombinant expression vector pPIC9K-JYC701 or pPIC9K-JYC702.

[0013] The construction method of the above Pichia pastoris genetic engineering bacterium is as follows:

[0014] Digest the above recombinant expression vector pPIC9K-JYC701 or pPIC9K-JYC702 to linearize it and then transform it into Pichia pastoris GS115 competent cells. Select positive transformants and screen for high-copy recombinants through G418 resistance gradient to obtain a Pichia pastoris genetic engineering bacterium for producing recombinant humanized type VII collagen JYC701 or JYC702.

[0015] The preparation method of the above recombinant humanized type VII collagen JYC701 or JYC702 is as follows:

[0016] The Pichia pastoris genetic engineering bacteria for producing recombinant humanized type VII collagen JYC701 or JYC702 are inoculated into the BMMY induction medium, induced for expression with methanol, and the fermentation supernatant is purified to obtain high-purity recombinant humanized type VII collagen JYC701 or JYC702.

[0017] A composition comprising the above-mentioned recombinant humanized type VII collagen JYC701 or JYC702, and the composition includes but is not limited to skin care products, pharmaceutical compositions, etc.

[0018] An article comprising the above-mentioned recombinant humanized type VII collagen JYC701 or JYC702, and the article includes but is not limited to medical devices, biomaterials, tissue engineering products, etc.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] In the present invention, by screening the functional domains of human type VII collagen that bind to cell integrins, two functional short peptide sequences with potentially high activity are obtained, and they are respectively tandemly repeated 10 times to obtain recombinant humanized type VII collagen JYC701 and JYC702 with the functions of promoting cell adhesion, proliferation and migration. Cell experiments found that both recombinant humanized type VII collagen JYC701 and JYC702 showed excellent effects of promoting the adhesion, proliferation and migration of human fibroblasts and epidermal cells, and the effect of recombinant humanized type VII collagen JYC701 was better than that of JYC702. In addition, in the present invention, the coding gene sequences of recombinant humanized type VII collagen JYC701 and JYC702 are codon-optimized according to the codon preference of Pichia pastoris, recombinant expression vectors are respectively constructed, and they are respectively transferred into Pichia pastoris strains. High-copy recombinants are screened by G418 resistance gradient to obtain high-copy Pichia pastoris genetic engineering bacteria capable of respectively producing recombinant humanized type VII collagen JYC701 and JYC702, realizing the large-scale production of recombinant humanized type VII collagen JYC701 and JYC702. The recombinant humanized type VII collagen JYC701 and JYC702 of the present invention can be used to promote the repair of tissues such as the skin, enhance the resistance of the skin, and are suitable for preparing products such as skin care or medical devices. Description of the Drawings

[0021] Figure 1 Schematic diagram of the plasmid structures for expressing recombinant humanized type VII collagen JYC701 (a) and JYC702 (b).

[0022] Figure 2Electrophoresis diagrams of plasmids expressing recombinant humanized type VII collagen JYC701 and JYC702. In figure (a), lane 1: linearized fragment of pPIC9K empty plasmid; lane 2: linearized fragment of pPIC9K + recombinant humanized type VII collagen JYC701 plasmid. In figure (b), lane 1: linearized fragment of pPIC9K empty plasmid; lane 2: linearized fragment of pPIC9K + recombinant humanized type VII collagen JYC702 plasmid.

[0023] Figure 3 SDS-PAGE diagrams of the culture supernatant (induced with methanol for 72 hours) of Pichia pastoris genetic engineering bacteria for producing recombinant humanized type VII collagen JYC701 and JYC702. In figure (a), lanes 1 - 6: SDS-PAGE of the supernatant of high-copy strain JYC701, lane 7: fermentation supernatant of the blank control strain. In figure (b), lanes 1 - 5: SDS-PAGE of the supernatant of high-copy strain JYC702, lane 6: fermentation supernatant of the blank control strain.

[0024] Figure 4 Total ion current diagram of recombinant humanized type VII collagen JYC701.

[0025] Figure 5 Result diagram of molecular weight determination of recombinant humanized type VII collagen JYC701.

[0026] Figure 6 Result diagrams of the effects of recombinant humanized type VII collagen JYC701 and JYC702 on the proliferation of epidermal cells and fibroblasts.

[0027] Figure 7 Result diagrams of the effects of recombinant humanized type VII collagen JYC701 and JYC702 on the migration of epidermal cells and fibroblasts.

[0028] Figure 8 Result diagrams of the effects of recombinant humanized type VII collagen JYC701 and JYC702 on the adhesion of epidermal cells and fibroblasts. Detailed implementation manners

[0029] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions of the present invention will be elaborated in detail below through the preferred embodiments of the present invention. However, the following embodiments do not limit the protection scope of the present invention.

[0030] In the embodiments of the present invention, those not described in detail are completed by using conventional molecular biology experimental methods. Processes such as PCR, restriction enzyme digestion, seamless cloning, and codon optimization involved in the embodiments can be understood and easily implemented by those skilled in the art according to the product instructions or basic knowledge in the art, and thus will not be described in detail.

[0031] Example 1: Design and construction of high-copy Pichia pastoris genetic engineering bacteria of recombinant humanized type VII collagen JYC701 and JYC702

[0032] (1) Using bioinformatics and protein structure prediction database tools (NCBI, AlphaFold), functional short peptides that can bind to cell integrin were screened from human type VII collagen, and their amino acid sequences are shown in SEQ ID No.1 and SEQ ID No.2 respectively. After being tandemly repeated 10 times, they constitute recombinant humanized type VII collagen JYC701 and JYC702 respectively, and their amino acid sequences are shown in SEQ ID No.3 and SEQ ID No.4.

[0033] (2) The amino acid sequences of the recombinant humanized type VII collagen JYC701 and JYC702 obtained in step (1) were codon-optimized according to the codon preference of Pichia pastoris to obtain the nucleotide sequences of the optimized recombinant humanized type VII collagen JYC701 and JYC702, which are shown in SEQ ID No.5 and SEQ ID No.6 respectively.

[0034] (3) The nucleotide sequences in step (2) were entrusted to Shanghai Qingke Biotechnology Co., Ltd. for gene synthesis. The gene sequence of the recombinant humanized type VII collagen JYC701 shown in SEQ ID No.5 was amplified with primer P1 (nucleotide sequence shown in SEQ ID No.7) and primer P2 (nucleotide sequence shown in SEQ ID No.8), and the gene sequence of the recombinant humanized type VII collagen JYC702 shown in SEQ ID No.6 was amplified with primer P3 (nucleotide sequence shown in SEQ ID No.9) and primer P4 (nucleotide sequence shown in SEQ ID No.10). After the above high-fidelity PCR amplification products were purified by nucleic acid gel, they were seamlessly cloned downstream of the α-mating factor secretion signal peptide of the pPIC9K plasmid by Gibson assembly technology. After being verified correct by colony PCR and sequencing, the recombinant plasmids pPIC9K-JYC701 and pPIC9K-JYC702 were enriched. The structures of the recombinant plasmids pPIC9K-JYC701 and pPIC9K-JYC702 are as Figure 1 shown, and the plasmid electrophoresis pattern is as Figure 2As shown. Subsequently, the recombinant plasmids pPIC9K-JYC701 and pPIC9K-JYC702 were digested overnight at 37°C with SalI restriction endonuclease (purchased from NEB, and the specific operation was carried out according to the kit instructions). Then, a gel extraction kit (purchased from Sangon Biotech Co., Ltd.) was used to purify the linearized recombinant expression plasmids, which were then electrotransformed into competent Pichia pastoris GS115 cells. The electrotransformed cells were activated and cultured in fresh YPD medium for 2 - 3 hours, and then spread on YNB selection medium and incubated upside down in a 30°C incubator for 2 - 3 days until transformants grew. First, the transformant monoclonal colonies were verified by colony PCR. The verified transformants were respectively spread on screening plates containing 1 g / L, 2 g / L, 3 g / L, and 4 g / L G418. After 2 - 4 days of culture, the transformants with better growth on the high-concentration G418 plates were picked for purification and rescreening verification. After genomic extraction and correct verification of the transformants obtained from the rescreening, they were stored in glycerol.

[0035] (4) The obtained high-copy Pichia pastoris genetic engineering strains were inoculated into 30 mL of BMGY medium (this medium consists of 20 g / L peptone, 10 g / L yeast extract, 100 mL of 10×YNB, 100 mL of 10× potassium phosphate buffer pH 6.0, 100 mL of 10× glycerol, and the balance is water), cultured for 16 - 20 hours, and then inoculated into 30 mL of BMMY medium (this medium consists of 20 g / L peptone, 10 g / L yeast extract, 100 mL of 10×YNB, 100 mL of 10× potassium phosphate buffer pH 6.0, and the balance is water) at an inoculum size with an initial OD 600 of 1 for culture. Every 24 hours, 300 μL of filter-sterilized methanol was supplemented. After 72 hours, 2 mL of the sample was centrifuged to collect the supernatant. 500 μL of the supernatant was concentrated in an ultrafiltration tube with a size of 10 kDa to obtain approximately 50 μL of concentrated solution. 24 μL of the concentrated solution was mixed evenly with 6 μL of 5×loading buffer and then treated in boiling water for 10 - 15 min. After centrifugation for 15 min, 10 - 15 μL of the supernatant was loaded onto SDS-PAGE to complete the qualitative analysis of recombinant humanized type VII collagen JYC701 and JYC702. The results of SDS-PAGE detection are shown in Figure 3 , and it can be seen from Figure 3 that high-copy Pichia pastoris genetic engineering strains capable of successfully expressing recombinant humanized type VII collagen JYC701 and JYC702 were obtained.

[0036] By recovering the target protein bands at the corresponding cleavage positions, digesting them with trypsin, detecting the peptides after digestion by liquid chromatography-mass spectrometry, and comparing the detected peptides by database building.

[0037] Figure 4 This is the total ion chromatogram of the peptides after trypsin digestion of pure recombinant humanized type VII collagen JYC701. It can be seen from the figure that the peptides after JYC701 digestion all belong to the regions of the human type VII collagen sequence selected during design, and the peptide coverage rate is 100%, which proves that the peptides in this target band all originate from the collagen designed in the present invention, and JYC701 was successfully and efficiently secreted extracellularly in Pichia pastoris.

[0038] Figure 5 This is the result graph of mass spectrometry for determining the molecular weight. It can be seen from the figure that the actual molecular weight of recombinant humanized type VII collagen JYC701 is 28.6 kDa, which is consistent with the theoretical molecular weight.

[0039] Example 2 Detection of the cell proliferation promoting activities of recombinant humanized type VII collagens JYC701 and JYC702

[0040] The MTT method was used to detect the ability of recombinant humanized type VII collagens JYC701 and JYC702 to promote cell proliferation, with human epidermal cells and human fibroblasts used as experimental cells respectively. The results are as Figure 6 shown. The blank group was a control experiment using only cell culture medium without adding any components. It can be seen that recombinant humanized type VII collagens JYC701 and JYC702 at 10 ppm, 100 ppm, and 1000 ppm all showed excellent cell proliferation promoting activities, and with the increase in the concentrations of recombinant humanized type VII collagens JYC701 and JYC702, their promoting effects were enhanced. In addition, compared with recombinant humanized type VII collagen JYC702, recombinant humanized type VII collagen JYC701 showed more excellent cell proliferation promoting activity.

[0041] Example 3 Detection of the cell migration promoting activities of recombinant humanized type VII collagens JYC701 and JYC702

[0042] The cell scratch method was used to measure cell migration. Human epidermal cells and human fibroblasts were used as experimental cells respectively to detect the ability of recombinant humanized type VII collagens JYC701 and JYC702 to promote cell migration. The results are as Figure 7As shown, the blank group is a control experiment in which no components are added and only cell culture medium is used for culturing. It can be seen that recombinant humanized type VII collagen JYC701 and JYC702 at 10 ppm, 100 ppm, and 1000 ppm all exhibit excellent cell migration-promoting activities, and as the concentrations of recombinant humanized type VII collagen JYC701 and JYC702 increase, their promoting effects are enhanced. In addition, compared with recombinant humanized type VII collagen JYC702, recombinant humanized type VII collagen JYC701 exhibits more excellent cell migration-promoting activity.

[0043] Example 4 Detection of the Cell Adhesion-Promoting Activities of Recombinant Humanized Type VII Collagen JYC701 and JYC702

[0044] Using the centrifugation method, human epidermal cells and human fibroblasts were used as experimental cells to detect the ability of recombinant humanized type VII collagen JYC701 and JYC702 to promote cell adhesion. The results are as Figure 8 shown, the blank group is a control experiment in which no components are added and only cell culture medium is used for culturing. It can be seen that recombinant humanized type VII collagen JYC701 and JYC702 at 10 ppm, 100 ppm, and 1000 ppm all exhibit excellent cell adhesion-promoting activities, and as the concentrations of recombinant humanized type VII collagen JYC701 and JYC702 increase, their promoting effects are enhanced. In addition, compared with recombinant humanized type VII collagen JYC702, recombinant humanized type VII collagen JYC701 exhibits more excellent cell adhesion-promoting activity.

Claims

1. A functional short peptide, characterized in that, The amino acid sequence is as shown in SEQ ID No.1 or SEQ ID No.

2.

2. Recombinant humanized type VII collagen JYC701 or JYC702, characterized in that, It is composed of 10 tandemly repeated functional short peptides with the amino acid sequence as shown in SEQ ID No.1 or SEQ ID No.2, and its amino acid sequence is as shown in SEQ ID No.3 or SEQ ID No.

4.

3. A gene encoding the recombinant humanized type VII collagen JYC701 or JYC702 according to claim 2, characterized in that, Its nucleotide sequence is as shown in SEQ ID No.5 or SEQ ID No.

6.

4. A recombinant expression vector, characterized in that, It is a recombinant expression vector containing the gene encoding recombinant humanized type VII collagen JYC701 or JYC702 as described in claim 3.

5. The recombinant expression vector according to claim 4, wherein It is the recombinant expression vector pPIC9K-JYC701 or pPIC9K-JYC702 containing the gene encoding recombinant humanized type VII collagen JYC701 or JYC702 as described in claim 3, and is constructed by the following steps: Perform high-fidelity PCR amplification on the gene sequence encoding recombinant humanized type VII collagen JYC701 or JYC702 as shown in SEQ ID No.5 or SEQ ID No.6, and clone the target fragment after gel recovery and purification seamlessly downstream of the α-mating factor secretion signal peptide of the pPIC9K empty vector. After verification by colony PCR and sequencing and being correct, enrich it to obtain the recombinant expression vector pPIC9K-JYC701 or pPIC9K-JYC702.

6. A Pichia pastoris genetic engineering bacterium for producing the recombinant humanized type VII collagen JYC701 or JYC702 as claimed in claim 2, characterized in that, It contains the recombinant expression vector pPIC9K-JYC701 or pPIC9K-JYC702 as described in claim 5.

7. The construction method of the Pichia pastoris genetic engineering bacterium according to claim 6, characterized in that, The steps are as follows: Linearize the recombinant expression vector pPIC9K-JYC701 or pPIC9K-JYC702 as described in claim 5 by enzyme digestion and transform it into the competent cells of Pichia pastoris GS115. Select positive transformants and screen for high-copy recombinants through G418 resistance gradient to obtain the Pichia pastoris genetic engineering bacteria for producing recombinant humanized type VII collagen JYC701 or JYC702.

8. The preparation method of the recombinant humanized type VII collagen JYC701 or JYC702 according to claim 2, characterized in that, The steps are as follows: Inoculate the Pichia pastoris genetic engineering bacteria for producing recombinant humanized type VII collagen JYC701 or JYC702 as described in claim 6 into the BMMY induction medium, induce expression with methanol, and purify the fermentation supernatant to obtain high-purity recombinant humanized type VII collagen JYC701 or JYC702.

9. A composition, characterized in that, It contains the recombinant humanized type VII collagen JYC701 or JYC702 as described in claim 2.

10. The composition according to claim 9, characterized in that, The described composition is a skin care product or a pharmaceutical composition.

11. An article, characterized in that, It contains the recombinant humanized type VII collagen JYC701 or JYC702 as described in claim 2.

12. The article according to claim 11, characterized in that, The described product is a medical device.

13. The article according to claim 11, wherein The described product is a biomaterial.

14. The article according to claim 11, characterized in that, The described product is a tissue engineering product.

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