Recombinant humanized VII type collagen as well as expression vector and genetically engineered bacterium thereof

Abstract

The invention discloses a recombinant humanized VII type collagen as well as an expression vector and a genetically engineered bacterium thereof. The preparation method comprises the following steps: screening functional oligopeptides combined with cell integrin in human VII type collagen, designing 10 times of tandem repetition to respectively form recombinant humanized VII type collagen JYC701 and JYC702, amplifying and cloning the recombinant humanized VII type collagen JYC701 and JYC702 into a pPIC9K carrier after codon optimization, transforming the recombinant humanized VII type collagen JYC701 and JYC702 into pichia pastoris cells after enzyme digestion linearization, and screening the recombinant humanized VII type collagen JYC701 and JYC702. And high-copy recombinants are screened through G418 resistance gradient, so that the pichia pastoris genetically engineered bacteria capable of expressing the recombinant humanized VII type collagen JYC701 and JYC702 are obtained. The recombinant humanized VII type collagen JYC701 and JYC702 show excellent functions of promoting cell adhesion, cell proliferation and migration, have the activity of promoting repair of tissues such as skin and improving resistance of the skin, and are suitable for preparing products such as skin care products and medical instruments.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering and relates to a recombinant humanized type VII collagen and an expression vector and genetic engineering bacteria thereof. Background Art

[0002] Type VII collagen is an important structural protein that is mainly found in the basement membrane of the skin. It plays a key role in the skin, especially in terms of the strength and elasticity of the skin. Type VII collagen forms anchoring fibrils at the dermal-epidermal junction, fixes the epidermis to the dermis, prevents the epidermis from separating from the dermis, and provides additional mechanical support to the skin, thereby ensuring that the skin maintains stability and tensile strength under external stress. In addition, it is also present in other tissues, such as the basement membrane area 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 a variety of genetic diseases, the most common of which is dystrophic epidermolysis bullosa (DEB). DEB is a rare genetic skin disease in which mutations in the type VII collagen gene (Col7a1) in patients lead to a reduction or loss of anchoring fibers. Since the epidermis and dermis cannot be firmly connected, the skin is very likely to separate, forming large areas of blisters and erosions. Due to the close relationship 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 will also decline, which will lead to a decrease in the skin's elasticity and damage resistance, manifested as skin aging, wrinkles and sagging, etc. 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 fiber-like three-strand right-handed helix, which is composed of three identical α1(VII) chains. Its structure can be divided into three main functional domains: N-terminal globular domain (NC1 domain), triple-helical domain (Triple-helical domain) and C-terminal globular domain (NC2 domain). Among them, the NC1 domain is rich in irregular secondary structure and is an important site for the interaction between type VII collagen and 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 domain of type VII collagen, which has high tensile strength and stability and is an important part of the formation of anchoring fibers. Type VII collagen fixes the epidermis to the dermis through this structure to prevent skin separation, and is therefore essential for maintaining the mechanical strength of the skin; the NC2 domain may be related to the polymerization and stability of anchoring fibers and plays a role in the formation of collagen fibers. Studies have shown that the interaction between the NC2 domain and other type VII collagen molecules is essential for the formation of a stable fiber network. Type VII collagen will undergo some functional modifications during its synthesis and functional regulation, which may affect its role in the skin. For example, certain sites in type VII collagen will be glycosylated. In addition, the NC1 domain and NC2 domain will also be cleaved by specific proteases after synthesis. This process may regulate its localization in the basement membrane and fiber formation. Summary of the invention

[0004] The present invention obtains two functional peptide segments with potential high activity by screening the functional domains binding to cell integrins in human type VII collagen, and repeats them in series 10 times to respectively constitute recombinant humanized type VII collagen JYC701 and JYC702, amplify and clone them into a pPIC9K vector after codon optimization, linearize them after enzyme digestion, and transform them into Pichia pastoris cells, and screen high-copy recombinants through a G418 resistance gradient, so as to obtain high-copy Pichia pastoris genetic engineering strains that can respectively express the recombinant humanized type VII collagen JYC701 and JYC702.

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

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

[0007] The recombinant humanized type VII collagen JYC701 or JYC702 is composed of 10 tandemly repeated functional short peptides as 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] The nucleotide sequence of the gene encoding the recombinant humanized type VII collagen JYC701 or JYC702 is shown as 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 recombinant humanized type VII collagen JYC701 or JYC702. In a specific embodiment of the present invention, the recombinant expression vector pPIC9K-JYC701 or pPIC9K-JYC702 containing the gene encoding the recombinant humanized type VII collagen JYC701 or JYC702 is taken as an example.

[0010] The method for constructing the above-mentioned recombinant expression vector comprises the following specific steps:

[0011] The gene sequence encoding the recombinant humanized type VII collagen JYC701 or JYC702 shown in SEQ ID No.5 or SEQ ID No.6 was amplified by high-fidelity PCR, and the target fragment after gel recovery and purification was seamlessly cloned into the downstream of the α-mating factor secretion signal peptide of the pPIC9K empty vector, and enriched after verification by colony PCR and sequencing to obtain the recombinant expression vector pPIC9K-JYC701 or pPIC9K-JYC702.

[0012] The genetically engineered Pichia pastoris bacteria for producing the above-mentioned recombinant humanized type VII collagen JYC701 or JYC702 contains the above-mentioned recombinant expression vector pPIC9K-JYC701 or pPIC9K-JYC702.

[0013] The construction method of the above-mentioned Pichia pastoris genetically engineered bacteria comprises the following steps:

[0014] The above-mentioned recombinant expression vector pPIC9K-JYC701 or pPIC9K-JYC702 was linearized by enzyme digestion and then transformed into Pichia pastoris GS115 competent cells, positive transformants were selected, and high-copy recombinants were screened by G418 resistance gradient to obtain Pichia pastoris genetically engineered bacteria that produce recombinant humanized type VII collagen JYC701 or JYC702.

[0015] The preparation method of the above-mentioned recombinant humanized type VII collagen JYC701 or JYC702 comprises the following steps:

[0016] The genetically engineered Pichia pastoris producing the recombinant humanized type VII collagen JYC701 or JYC702 is inoculated into BMMY induction medium, induced to express 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, wherein the composition includes but is not limited to skin care products, pharmaceutical compositions, etc.

[0018] A product comprising the above-mentioned recombinant humanized type VII collagen JYC701 or JYC702, wherein the product 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] The present invention obtains two functional short peptide sequences with potential high activity by screening the functional domains binding to cell integrins in human type VII collagen, and repeats them in series 10 times to obtain recombinant humanized type VII collagen JYC701 and JYC702 with the function of promoting cell adhesion, proliferation and migration. Cell experiments have found that both recombinant humanized type VII collagen JYC701 and JYC702 show excellent effects in promoting the adhesion, proliferation and migration of human fibroblasts and epidermal cells, and the efficacy of recombinant humanized type VII collagen JYC701 is better than that of JYC702. In addition, the present invention optimizes the coding gene sequences of recombinant humanized type VII collagen JYC701 and JYC702 according to the codon preference of Pichia pastoris, constructs recombinant expression vectors respectively, and transfers them into Pichia pastoris strains respectively, and screens high-copy recombinants through G418 resistance gradient to obtain high-copy Pichia pastoris genetic engineering bacteria capable of producing recombinant humanized type VII collagen JYC701 and JYC702 respectively, thereby realizing 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 skin, improve the resistance of skin, and are suitable for preparing products such as skin care or medical devices. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0022] Figure 2The electrophoresis diagram of plasmids expressing recombinant humanized type VII collagen JYC701 and JYC702, wherein (a) Lane 1: pPIC9K empty plasmid linearized fragment; Lane 2: pPIC9K+recombinant humanized type VII collagen JYC701 plasmid linearized fragment; (b) Lane 1: pPIC9K empty plasmid linearized fragment; Lane 2: pPIC9K+recombinant humanized type VII collagen JYC702 plasmid linearized fragment.

[0023] Figure 3 The SDS-PAGE diagram of the supernatant of the culture medium of Pichia pastoris genetically engineered bacteria for producing recombinant humanized type VII collagen JYC701 and JYC702 (methanol induced for 72 hours), wherein (a) Lanes 1 to 6: SDS-PAGE of the supernatant of the high-copy strain JYC701, Lane 7: the fermentation supernatant of the blank control strain; (b) Lanes 1 to 5: SDS-PAGE of the supernatant of the high-copy strain JYC702, Lane 6: the fermentation supernatant of the blank control strain.

[0024] Figure 4 This is the total ion current of recombinant humanized type VII collagen JYC701.

[0025] Figure 5 This is a graph showing the molecular weight determination results of recombinant humanized type VII collagen JYC701.

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

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

[0028] Figure 8 This figure shows the effects of recombinant humanized type VII collagen JYC701 and JYC702 on the adhesion of epidermal cells and fibroblasts. DETAILED DESCRIPTION

[0029] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is described in detail below through the preferred embodiments of the present invention, but the following embodiments do not limit the protection scope of the present invention.

[0030] In the embodiments of the present invention, conventional molecular biological experimental methods are used without further explanation. The processes such as PCR, enzyme cutting, seamless cloning, codon optimization, etc. involved in the embodiments are understandable and easily implemented by those skilled in the art based on the product instructions or basic knowledge in the field, and therefore will not be described in detail.

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

[0032] (1) Bioinformatics and protein structure prediction database tools (NCBI, AlphaFold) were used to screen functional short peptides that can bind to cell integrins from human type VII collagen. The amino acid sequences of these short peptides are shown in SEQ ID No. 1 and SEQ ID No. 2, respectively. After 10 tandem repetitions, they constituted recombinant humanized type VII collagen JYC701 and JYC702, respectively. The amino acid sequences of these short peptides are shown in SEQ ID No. 3 and SEQ ID No. 4, respectively.

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

[0034] (3) The nucleotide sequence in step (2) was 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 using primers P1 (nucleotide sequence as shown in SEQ ID No. 7) and primers P2 (nucleotide sequence as shown in SEQ ID No. 8). The gene sequence of the recombinant humanized type VII collagen JYC702 shown in SEQ ID No. 6 was amplified using primers P3 (nucleotide sequence as shown in SEQ ID No. 9) and primers P4 (nucleotide sequence as shown in SEQ ID No. 10). The high-fidelity PCR amplification products were purified by nucleic acid gel and seamlessly cloned into the downstream of the α-mating factor secretion signal peptide of the pPIC9K plasmid using the Gibson assembly technology. After verification 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 follows: Figure 1 The plasmid electrophoresis diagram is shown in Figure 2As shown. Then, the recombinant plasmids pPIC9K-JYC701 and pPIC9K-JYC702 were digested with SalI restriction endonuclease (purchased from NEB, and the specific operation was carried out according to the kit instructions) at 37°C overnight, and then the linearized recombinant expression plasmids were purified using a gel recovery kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.) and then electroporated into Pichia pastoris GS115 competent cells. The electroporated cells were activated and cultured with fresh YPD medium for 2-3 hours, and then spread on YNB selection medium and placed in a 30°C incubator for inverted culture for 2-3 days to grow transformants. First, the transformant single clone was verified by colony PCR, and the verified correct transformants were spread on 1g / L, 2g / L, 3g / L and 4g / L After 2-4 days of cultivation on the G418 screening plate, the transformants that grew well on the high-concentration G418 plate were picked for purification and re-screening verification. The genome of the re-screened transformants was extracted and verified to be correct and then stored in glycerol.

[0035] (4) The obtained high-copy Pichia genetically engineered strain was inoculated into 30 mL of BMGY medium (the medium consisted of 20 g / L peptone, 10 g / L yeast extract, 10× YNB 100 mL, 10× potassium phosphate buffer pH 6.0 100 mL, 10× glycerol 100 mL, and the balance was water), cultured for 16-20 hours, and then the initial OD was calculated. 600 The inoculum size was 1 and inoculated into 30 mL BMMY medium (the medium consisted of 20 g / L peptone, 10 g / L yeast extract, 10×YNB 100 mL, 10× potassium phosphate buffer pH 6.0 100 mL, and the balance was water) for cultivation. 300 μL of filtered and sterilized methanol was added every 24 hours. After 72 hours, 2 mL of sample was centrifuged to collect the supernatant. 500 μL of supernatant was concentrated in an ultrafiltration tube with a size of 10 kDa to obtain about 50 μL of concentrated solution. 24 μL of concentrated solution and 6 μL of 5×loading buffer were mixed evenly and placed in boiling water for 10-15 minutes. After centrifugation for 15 minutes, 10-15 μL of supernatant was taken and loaded into 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 ,from Figure 3 It can be seen that a high-copy Pichia pastoris genetic engineering strain that can successfully express recombinant humanized type VII collagen JYC701 and JYC702 was obtained.

[0036] The target protein bands at the corresponding cutting positions were recovered and digested with trypsin, and the peptides after digestion were detected by LC-MS, and the detected peptides were compared with the library.

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

[0038] Figure 5 This is a graph showing the molecular weight determination results by mass spectrometry. As can be seen from the figure, the actual molecular weight of the recombinant humanized type VII collagen JYC701 is 28.6 kDa, which is consistent with the theoretical molecular weight.

[0039] Example 2 Detection of cell proliferation promoting activity of recombinant humanized type VII collagen JYC701 and JYC702

[0040] The MTT method was used to detect the ability of recombinant humanized type VII collagen JYC701 and JYC702 to promote cell proliferation using human epidermal cells and human fibroblasts as experimental cells. Figure 6 As shown, the blank group is a control experiment in which no ingredients are added and only cell culture medium is used for culture. It can be seen that 10ppm, 100ppm, and 1000ppm of recombinant humanized type VII collagen JYC701 and JYC702 all exhibit excellent cell proliferation promoting activity, and as the concentration of recombinant humanized type VII collagen JYC701 and JYC702 increases, the promoting effect increases. In addition, compared with recombinant humanized type VII collagen JYC702, recombinant humanized type VII collagen JYC701 exhibits more excellent cell proliferation promoting activity.

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

[0042] The cell scratch method was used to measure cell migration, and 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 migration. Figure 7As shown, the blank group is a control experiment in which no ingredients are added and only cell culture medium is used for culture. It can be seen that 10ppm, 100ppm and 1000ppm of recombinant humanized type VII collagen JYC701 and JYC702 all exhibit excellent cell migration promoting activity, and as the concentration of recombinant humanized type VII collagen JYC701 and JYC702 increases, the promoting effect increases. 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 cell adhesion promoting activity of recombinant humanized type VII collagen JYC701 and JYC702

[0044] The centrifugation method was used to detect the ability of recombinant humanized type VII collagen JYC701 and JYC702 to promote cell adhesion using human epidermal cells and human fibroblasts as experimental cells. Figure 8 As shown, the blank group is a control experiment in which no ingredients are added and only cell culture medium is used for culture. It can be seen that 10ppm, 100ppm and 1000ppm of recombinant humanized type VII collagen JYC701 and JYC702 all exhibit excellent cell adhesion promoting activity, and as the concentration of recombinant humanized type VII collagen JYC701 and JYC702 increases, the promoting effect increases. 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 shown as 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 as 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.

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

6.

4. A recombinant expression vector, characterized in that: A recombinant expression vector containing the gene encoding the recombinant humanized type VII collagen JYC701 or JYC702 according to claim 3.

5. The recombinant expression vector according to claim 4, characterized in that The recombinant expression vector pPIC9K-JYC701 or pPIC9K-JYC702 containing the gene encoding the recombinant humanized type VII collagen JYC701 or JYC702 according to claim 3 is constructed by the following steps: The gene sequence encoding the recombinant humanized type VII collagen JYC701 or JYC702 shown in SEQ ID No.5 or SEQ ID No.6 was amplified by high-fidelity PCR, and the target fragment after gel recovery and purification was seamlessly cloned into the downstream of the α-mating factor secretion signal peptide of the pPIC9K empty vector, and enriched after verification by colony PCR and sequencing to obtain the recombinant expression vector pPIC9K-JYC701 or pPIC9K-JYC702.

6. The genetically engineered Pichia pastoris for producing the recombinant humanized type VII collagen JYC701 or JYC702 according to claim 2, characterized in that: Contains the recombinant expression vector pPIC9K-JYC701 or pPIC9K-JYC702 according to claim 5.

7. The method for constructing a genetically engineered Pichia pastoris according to claim 6, characterized in that: Here are the steps: The recombinant expression vector pPIC9K-JYC701 or pPIC9K-JYC702 according to claim 5 is linearized and transformed into Pichia pastoris GS115 competent cells, positive transformants are selected, and high-copy recombinants are screened by G418 resistance gradient to obtain Pichia pastoris genetically engineered bacteria that produce recombinant humanized type VII collagen JYC701 or JYC702.

8. The method for preparing the recombinant humanized type VII collagen JYC701 or JYC702 according to claim 2, characterized in that: Here are the steps: The genetically engineered Pichia pastoris producing the recombinant humanized type VII collagen JYC701 or JYC702 according to claim 6 is inoculated into a BMMY induction medium, induced to express with methanol, and the fermentation supernatant is purified to obtain a high-purity recombinant humanized type VII collagen JYC701 or JYC702.

9. A composition or product, characterized in that It comprises the recombinant humanized type VII collagen JYC701 or JYC702 according to claim 2.

10. The composition or product according to claim 9, characterized in that The composition is a skin care product or a pharmaceutical composition, and the product is a medical device, a biomaterial or a tissue engineering product.

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