A highly active recombinant human type XVII collagen and its preparation method and application

Abstract

The present invention discloses a highly active recombinant human type XVII collagen, its preparation method and application, belonging to the field of bioengineering technology. The recombinant human type XVII collagen of the present invention comprises 138 amino acids (SEQ ID NO: 1) of the functional region of type XVII collagen and 15 amino acids (SEQ ID NO: 2) of the cell-binding domain of type I collagen, and further mutates its amino acid sequence to obtain a highly active recombinant human type XVII collagen. The present invention also provides a method for constructing an expression vector of the protein and a method for preparing recombinant human type XVII collagen; the theoretical molecular weight of the protein is relatively small, being 14.6 kDa, and cytological experiments show that the protein has high biological activities of promoting cell migration, proliferation and adhesion, and can be used for the research and development and application of medical devices and biological skin care products.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and relates to recombinant human type XVII collagen and its preparation method and application. Background Art

[0002] Collagen is widely distributed in human skin, blood vessels, bones, tendons, etc., and is the main matrix and scaffold of these tissues. Collagen is the most abundant protein in mammals, accounting for 25% - 35% of the total protein, and plays a very important physiological function in the body.

[0003] Collagen in the human body is divided into fibrillar collagen and non-fibrillar collagen. Type XVII collagen (COL17, also known as BP180) is a transmembrane non-fibrillar collagen, a homogeneous trimer composed of three identical α1(XVII) chains. The α1 chain of type XVII collagen consists of 1497 amino acids, and the single-chain molecular weight reaches 180 kDa, also known as BP180. Structurally, type XVII collagen is divided into three major structural regions: intracellular, transmembrane, and extracellular. Its N-terminus is located in the cytoplasm, and its C-terminus is located in the extracellular matrix. It is a key component of the basement membrane hemidesmosome, and achieves the effect of skin anti-aging by affecting the stemness and proliferation ability of ESCs. In addition, type XVII collagen plays a role in preventing hair follicle aging by maintaining the stemness of hair follicle stem cells, regulating the differentiation of hair follicle stem cells, and controlling the proliferation of epidermal cells between hair follicles. Therefore, type XVII collagen is of great significance in anti-hair loss and scalp care.

[0004] The content of type XVII collagen in the human and animal bodies is extremely low, and the extraction difficulty is extremely high and cannot be achieved. Due to the lack of commercial type XVII collagen products from human or other animal sources in the market, the application of human type XVII collagen is limited. Industrial mass production of full-length transmembrane type XVII collagen is almost impossible due to cost and expression level reasons. Rational design of recombinant type XVII collagen with biological properties based on the active domain of type XVII collagen and achieving its high expression in industrial strains will effectively promote the application of type XVII collagen in the fields of skin care products and medicine, which is of great significance. Summary of the Invention

[0005] The purpose of the present invention is to provide a highly active recombinant human type XVII collagen and the preparation method and application of the recombinant human type XVII collagen.

[0006] To achieve the invention object of the present invention, after in-depth research, the inventor optimized the human-derived type XVII collagen sequence, selected 138 amino acids in the active region of human-derived type XVII collagen, and obtained the recombinant human-derived type XVII collagen shown in SEQ ID NO:1. The theoretical molecular weight of this recombinant human-derived type XVII collagen is 13.2 kDa, which can be highly expressed in the Escherichia coli prokaryotic expression system, and has good biological function activities such as promoting cell proliferation, cell adhesion and migration.

[0007] To further improve the biological activity of the above recombinant human-derived type XVII collagen, the amino acid sequence shown in SEQ ID NO:1 was recombined with 15 amino acids (SEQ ID NO:2) in the cell-binding domain of human type I collagen. The amino acid sequence of the obtained recombinant human-derived type XVII collagen is shown in SEQ ID NO:3. The theoretical molecular weight of this protein is relatively small, being 14.6 kDa.

[0008] The Arg-Gly-Asp (RGD) domain (motif) is an important recognition site of integrin and can bind to at least 8 different integrins. Further, one or more amino acid residues at the 20th phenylalanine, 68th glutamate, 113th proline, and 128th histidine positions in the amino acid sequence shown in SEQ ID NO:3 were mutated into aspartic acid to construct the RGD domain, resulting in recombinant human-derived type XVII collagen.

[0009] Preferably, the 20th phenylalanine and 113th proline in the amino acid sequence shown in SEQ ID NO:3 were mutated into aspartic acid to obtain recombinant human-derived type XVII collagen, and its amino acid sequence is shown in SEQ ID NO:4; or the 68th glutamate and 128th histidine in the amino acid sequence shown in SEQ ID NO:3 were mutated into aspartic acid to obtain recombinant human-derived type XVII collagen, and its amino acid sequence is shown in SEQ ID NO:5.

[0010] It should be noted that the present invention provides a recombinant human-derived type XVII collagen, which also includes using the amino acid sequences shown in SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:4 as repeat units, with 1 - 5 repeats, preferably 1 - 3 repeats.

[0011] To increase the expression level of the above-mentioned collagen and facilitate protein purification, a 6xHis-SUMO tag can also be added to the N-terminus of recombinant human type XVII collagen for fusion expression. The 6xHis-SUMO expression gene is fused to the 5' end of the type XVII collagen gene to form a 6xHis-SUMO-XVII expression gene.

[0012] The present invention also provides a gene encoding the above-mentioned recombinant human type XVII collagen.

[0013] The present invention also provides an expression vector for the above-mentioned gene. The vector includes, but is not limited to, pET28a, pET32a, pPIC9K, pCDNA3.1, etc., and preferably the pET28a vector.

[0014] The present invention also provides a recombinant strain, which is a host cell transformed or transfected with the above-mentioned expression vector. The host cells include, but are not limited to, bacterial cells such as Escherichia coli, fungal cells such as yeast, plant cells such as tobacco cells, and animal cells such as CHO cells. Preferably Escherichia coli cells.

[0015] The present invention also provides a method for preparing the above-mentioned recombinant human type XVII collagen, including culturing and inducing expression of the recombinant strain, and purifying the recombinant human type XVII collagen.

[0016] Cell experiments have confirmed that the recombinant type XVII collagen provided by the present invention all has excellent biological activities such as promoting cell proliferation, adhesion, and cell scratch healing, and can be used in aspects such as wound healing and scar repair.

[0017] The recombinant human type XVII collagen is applied to the preparation of biological skin care products, medical materials, drugs, etc. Among them, the biological skin care products are made into essence, cream, facial mask stickers, etc. The medical materials can be made into external application patches, etc.

[0018] Compared with the prior art, the recombinant collagen of the present invention has the following beneficial effects:

[0019] The present invention provides a recombinant collagen with a small molecular weight, easy to express, and having multiple biological functions. The amino acid sequence of the recombinant human type XVII collagen of the present invention comes from human type XVII and type I collagen, combining the active domain sequence of human type XVII and the cell-binding domain of type I collagen, with high biocompatibility. Further constructing an RGD domain further improves its multiple biological activities such as promoting cell adhesion, cell scratch healing ability, and cell proliferation activity. And it has a small molecular weight, is easy to express efficiently, and is suitable for industrial production and application. Description of the Drawings

[0020] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the attached drawings required for the description of the embodiments:

[0021] Figure 1 It is the electrophoresis diagram of the expression and purification of recombinant human type XVII collagen.

[0022] Figure 2 It is the result diagram of the cell proliferation experiment.

[0023] Figure 3 It is the result diagram of the cell adhesion experiment.

[0024] Figure 4 It is the electron microscopy diagram of the cell scratch healing experiment.

[0025] Figure 5 It is the result diagram of the cell scratch healing experiment. Detailed implementation manners

[0026] In order to better understand the present invention, the following further illustrates the present invention through embodiments. In order to facilitate a full understanding of the present invention, many specific details are set forth in the following description; however, the present invention may also be implemented in other ways different from those described herein. Therefore, the present invention is not limited to the limitations of the specific embodiments disclosed in the following specification.

[0027] In the embodiments of the present invention, unless otherwise specified, all are completed by using conventional molecular biology experimental methods. The processes such as PCR, restriction enzyme digestion, ligation, and codon optimization involved in the embodiments can be understood and easily implemented by those skilled in the art according to the product specifications or basic knowledge in the art, and thus will not be elaborated further. Embodiment 1

[0028] Design of the amino acid sequence of recombinant human type XVII collagen

[0029] In the present invention, the amino acid sequence of the original human type XVII collagen comes from the UniProt database, Collagen alpha-1(XVII) chain, 1497aa, UniProtKB / Swiss-Prot: Q9UMD9.

[0030] The recombinant human type XVII collagen of the present invention is composed of a total of 138 amino acids selected at positions 567-620 (54aa), 641-646 (6aa), 683-715 (33aa), and 1438-1482 (45aa) on the functional domain of type XVII collagen, and the recombinant human type XVII collagen of SEQ ID NO:1 (abbreviation: COL17-1) is recombinantly obtained. The theoretical molecular weight of this recombinant type XVII collagen is 13.2 kDa. Theoretically, the smaller the molecular weight of a protein, the easier it is to express in a heterologous expression system, but its function is closely related to the amino acid sequence. Subsequently, the functional activity of this small molecule recombinant type XVII collagen will be verified.

[0031] The amino acids at positions 772-786 in type I collagen (SEQ ID NO:2: GPQGIAGQRGVVGLP) are the cell-binding domain of type I collagen, and the amino acid sequence of the recombinant type XVII collagen recombinantly obtained therefrom is as shown in SEQ ID NO:3. The Arg-Gly-Asp (RGD) domain (motif) is an important recognition site for integrins and can bind to at least 8 different integrins. To improve the functional activity of the above-mentioned small molecule recombinant type XVII collagen, the phenylalanine at position 20 and the proline at position 113 were mutated to aspartic acid, and a recombinant human type XVII collagen containing 2 RGD domains (underlined in SEQ ID NO:4) (abbreviation: COL17-2) was obtained. The glutamic acid at position 68 and the histidine at position 128 were mutated to aspartic acid, and a recombinant human type XVII collagen containing 2 RGD domains (underlined in SEQ ID NO:5) (abbreviation: COL17-3) was obtained.

[0032] It should be noted that the amino acid sequence of the recombinant human type XVII collagen described in the present invention can also be 1-5 repeats of the amino acid sequence in SEQ ID NO:4 or SEQ ID NO:5.

[0033] SEQ ID NO:1

[0034] GSPGPKGDMGSPGPKGDRGFPGTPGIPGPLGHPGPQGPKGQKGSVGDPGMEGPMGEKGERGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPPGQKGEMGTPGPKGDRGPAGPPGHPGPPGPRGHKGEKGDKGDQ

[0035] SEQ ID NO:2

[0036] GPQGIAGQRGVVGLP

[0037] SEQ ID NO:3

[0038] GSPGPKGDMGSPGPKGDRG F PGTPGIPGPLGHPGPQGPKGQKGSVGDPGMEGPMGEKGERGLQGLRG E VGLPGVKGDKGPMGPPGPKGDQGEKGPPGQKGEMGTPGPKGDRG P AGPPGHPGPPGPRG H KGEKGDKGDQGPQGIAGQRGVVGLP

[0039] SEQ ID NO:4

[0040] GSPGPKGDMGSPGPKGD RGD PGTPGIPGPLGHPGPQGPKGQKGSVGDPGMEGPMGEKGERGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPPGQKGEMGTPGPKGD RGD AGPPGHPGPPGPRGHKGEKGDKGDQGPQGIAGQRGVVGLP

[0041] SEQ ID NO:5

[0042] GSPGPKGDMGSPGPKGDRGFPGTPGIPGPLGHPGPQGPKGQKGSVGDPGMEGPMGEKGERGLQGL RGD VGLPGVKGDKGPMGPPGPKGDQGEKGPPGQKGEMGTPGPKGDRGPAGPPGHPGPPGP RGD KGEKGDKGDQGPQGIAGQRGVVGLP Example 2

[0043] Construction and Expression of Recombinant Human Collagen XVII-I Expression Vector

[0044] To fully illustrate the advantages of recombinant human type XVII-I collagen, in the examples of the present invention, the expression, purification, and biological activity studies of recombinant human type XVII collagen (SEQ ID NO:1, abbreviated as COL17-1), recombinant type XVII-I collagen (SEQ ID NO:4, abbreviated as COL17-2), and recombinant type XVII-I collagen (SEQ ID NO:5, abbreviated as COL17-3) were also carried out. Since the methods used for the three are the same, the example describes recombinant human type XVII collagen as an example. The expression and purification processes of proteins with different sequence lengths are the same, and the present invention preferably uses the sequence in SEQ ID NO:5 for illustration.

[0045] Based on the amino acid sequence of recombinant human type XVII collagen (SEQ ID NO:5), a gene sequence encoding recombinant human type XVII collagen was designed and synthesized. Different expression hosts can be optimized for codon preference accordingly to improve the expression sequence of the recombinant protein in the host cell. After the sequence design optimization was completed, it was handed over to Tianjin Zhonghe Gene Technology Co., Ltd. for synthesis. To increase the protein expression level and facilitate protein purification, a 6xHis-SUMO tag can be added to the N-terminus of recombinant human type XVII collagen for fusion expression. After the sequence was synthesized, the 6xHis-SUMO expression gene can be fused to the 5' end of the type XVII collagen gene through overlap extension PCR to form the 6xHis-SUMO-XVII expression gene. This DNA fragment was cloned and amplified by PCR. During amplification, Nde I and Xho I restriction enzyme cleavage sites were added through primers, and the target fragment was inserted into the pET28a vector through enzyme digestion and ligation. After the recombinant vector was screened by cloning and verified by sequencing, the pET28a-6xHis-SUMO-XVII recombinant expression vector was obtained.

[0046] The pET28a-6xHis-SUMO-XVII plasmid was transformed into the Escherichia coli expression strain BL21(DE3). Monoclonal colonies were obtained by screening with kanamycin resistance. A monoclonal colony was picked and cultured overnight in 5 mL of 2xYT medium. It was inoculated at a ratio of 1:100 and cultured with shaking at 37 °C until the OD600 reached 0.6 - 0.8. Then the culture temperature was reduced to 20 °C and IPTG was added to a final concentration of 0.2 mM, and the culture was continued for 16 hours to induce protein expression ( Figure 1 , lane 3), and the cells were collected by centrifugation for the next purification step. Example 3

[0047] Purification of recombinant human type XVII collagen

[0048] (1)Cell lysis: Resuspend the cells at a ratio of 1:15 in 50 mM phosphate (pH 8.0 containing 300 - 500 mM sodium chloride and 10 - 20 mM imidazole), and lyse the cells with a homogenizer in a cold water environment. Lyse the cells in cycles three times. The target protein is mainly in the lysate supernatant ( Figure 1 , lane 5). After lysis, collect the supernatant by centrifugation or filtration.

[0049] (2)Purify the lysate supernatant from the previous step using a Ni-NTA column, and elute the target protein from the column with a 250 mM imidazole concentration solution to obtain the target protein solution ( Figure 1 , lane 6).

[0050] (3)Dialysis and Ulp-1 digestion: Add Ulp1 enzyme at a mass ratio of 1 / 50000 to the protein solution from the previous step, mix well, and place it in a dialysis bag with a molecular weight cut-off of 3 kDa. Dialyze against 50 mM phosphate without imidazole to remove imidazole. Replace the dialysis fluid every 8 hours for a total of three times. After digesting the tag, the tag and the target protein are separated ( Figure 1 , lane 7). Dialysis needs to be carried out at 2 - 8 °C.

[0051] (4)Purify and remove the tag by reverse nickel column: Pass the protein solution after digestion and dialysis from the previous step through the nickel column again. The tagged protein will bind to the nickel column, while the target protein will pass through. Collect the flow-through to obtain the target protein with higher purity ( Figure 1 , lane 8).

[0052] (5)Dialysis for desalting: Place the protein solution obtained from the previous purification step back into a dialysis bag with a molecular weight cut-off of 3 kDa and dialyze against 10 mM phosphate (pH 8.0) to remove salts. Replace the dialysis fluid every 8 hours during dialysis, and harvest the protein solution after replacing the dialysis fluid three times.

[0053] (6)Purify by anion exchange chromatography; Use anion exchange chromatography to remove impurity proteins and nucleic acids from the protein solution obtained from the previous step to obtain highly pure recombinant human type XVII collagen ( Figure 1 , lane 9). Example 4

[0054] Evaluation of the cell proliferation promoting activity of recombinant human type XVII collagen

[0055] Select BALB / c 3T3 cells (mouse embryonic fibroblasts) in good condition in the logarithmic growth phase, digest them with 0.25% trypsin, resuspend them in DMEM medium containing 10% serum after centrifugation and count; inoculate BALB / c 3T3 cells into a 96-well culture plate at 4000 cells per well, and at the same time set a blank control without adding cells. After culturing in an environment of 37°C and 5% CO2 for 16 hours, change to DMEM medium containing 2% serum and continue to culture for 16 h until the cells adhere well. Aspirate the original medium, add the recombinant type XVII collagen in the present invention at 500 μg / ml, and the cell control group is DMEM medium containing 2% serum, and continue to culture for 48 h. 4 h before the end of the culture, add 5 g / L MTT solution, continue to culture for 4 h and then terminate the culture; aspirate the liquid in the wells, wash once with PBS, add DMSO to dissolve the crystals, and measure the absorbance value of each well at a wavelength of 570 nm.

[0056] Promoting cell proliferation rate = (absorbance value of the measurement well - absorbance value of the blank control group) / (absorbance value of the cell control group - absorbance value of the blank control group) × 100%.

[0057] In this example, the control group was a commercially available recombinant type XVII collagen, which was added to the cell culture medium at 500 μg / ml, and there were 3 parallel samples in each group. As Figure 2 shown, the experimental results show that under the condition of a concentration of 500 μg / ml, the promoting proliferation rates of the positive control group, recombinant COL17-1, recombinant COL17-2 and recombinant COL17-3 on BALB / c 3T3 cells were 156.7%, 153.7% and 170.2% respectively; that of the commercially available recombinant type XVII collagen was 153.8%, indicating that the recombinant type XVII collagen prepared by the present invention can significantly promote the proliferation of fibroblasts and has the biological activity of promoting cell proliferation. It is not difficult to find by comparison that the ability of recombinant COL17-3 to promote cell proliferation is better than that of other recombinant type XVII collagens (recombinant COL17-1 and recombinant COL17-2). Example 5

[0058] Evaluation of the cell adhesion promoting activity of recombinant human-derived type XVII collagen

[0059] After diluting the prepared recombinant type XVII collagen with PBS, add it to a 96-well cell culture plate at a final concentration of 500 μg / ml respectively, add 100 μL of the protein solution to each well, place it at 37°C for 16 h, and add PBS to the control wells. Digest BALB / 3T3 cells with 0.25% trypsin, count, and add 5×10 4Cells were cultured in a 37°C, 5% CO2 incubator for 2 h. They were washed three times with PBS to remove non-adherent cells, and then 200 μL of DMEM medium was added. A 5 g / L MTT solution was added, and after incubation in a 37°C, 5% CO2 cell incubator for 2 h, the samples were taken out. The absorbance values of the 96-well plate were read at 450 nm and 630 nm using a microplate reader. With 630 nm as the reference wavelength, the absorbance was measured at 450 nm, and the measurement results were recorded.

[0060] Cell adhesion promotion rate = (absorbance at 450 nm in the experimental group - absorbance at 450 nm in the negative control group) / absorbance at 450 nm in the negative control group × 100%.

[0061] In this example, the control group was a commercially available recombinant type XVII collagen sample solution, which was added to the cell culture medium at 500 μg / ml, with 3 parallel samples in each group. The experimental results are as Figure 3 shown. The results indicate that under the condition of a concentration of 500 μg / ml, the cell adhesion promotion rates of recombinant COL17-1, recombinant COL17-2, and recombinant COL17-3 on BALB / c 3T3 cells were 29.53%, 25.96%, and 31.70% respectively; the cell adhesion promotion rate of the commercially available recombinant type XVII collagen was 24.28%. The data show that the recombinant type XVII collagen prepared by the present invention has good cell adhesion promotion activity, and the ability of recombinant human-derived type XVII collagen (recombinant COL17-3) to promote cell adhesion is superior to that of other recombinant type XVII collagens (recombinant COL17-1 and recombinant COL17-2). Example 6

[0062] Evaluation of the cell migration promotion activity of recombinant human-derived type XVII collagen

[0063] The recombinant human-derived type XVII collagen of the present invention and the control, commercially available recombinant type XVII collagen, were taken and diluted to 0.5 mg / mL with DMEM 2% serum medium, with a pH of 7.0 - 7.4. BALB / c 3T3 cells were cultured and passaged normally. Cells in good condition were seeded into a 6-well plate, and 2 mL of cell suspension was inoculated at 5×10 5 cells / well. They were cultured for 24 h. A 200 μl pipette tip was used to make a scratch perpendicular to the surface of the 6-well plate in the confluent monolayer cells, ensuring that the scratch width was as consistent as possible during the process. The cells were washed three times with PBS to remove the free cells and cell debris caused by the scratch, and a protein solution diluted with 2% serum DMEM medium was added to the wells. They were then continued to be cultured in a 37°C, 5% CO2 incubator. Samples were taken and photographed at 0 h and 24 h, as Figure 4。The initial scratch area and the area data of the cell-free blank area were obtained using Image J software, and the migration rate was calculated. Migration rate = (Initial scratch area - Blank area after cell migration / Initial scratch area) * 100%. The experimental results are as Figure 5 shown. Under the condition that the protein concentration was 500 μg / ml, the migration rates of recombinant COL17-1, recombinant COL17-2, and recombinant COL17-3 were 35.05%, 31.44%, and 37.50% respectively; the migration rate of commercially available recombinant type XVII collagen was 30.82%. The data indicate that the recombinant type XVII collagen in the present invention has good biological activity in promoting cell migration, and recombinant COL17-3 is superior to the other two recombinant type XVII collagens (recombinant COL17-1 and recombinant COL17-2) in the present invention.

[0064] In summary, according to the results of Examples 4-6, it shows that the recombinant human-derived type XVII collagen of the present invention has good cytological activity. Further, by fusing the cell-binding domain of type I collagen and mutating to construct the RGD structural threshold, the constructed recombinant human-derived type XVII collagen (recombinant COL17-3) has better cytological activity.

[0065] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

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

1.

2. A recombinant human type XVII collagen, characterized in that, The amino acid sequence of the recombinant human type XVII collagen is as shown in SEQ ID NO:4 or SEQ ID NO:

5.

3. A gene encoding the recombinant human type XVII collagen according to any one of claims 1-2.

4. A recombinant vector, characterized in that, A gene containing the recombinant human type XVII collagen according to claim 3.

5. The recombinant vector according to claim 4, wherein The vector is any one of pET28a, pET32a, pPIC9K, pCDNA3.

1.

6. A recombinant strain, characterized in that, Containing the gene according to claim 3, or containing the recombinant vector according to any one of claims 4-5.

7. The preparation method of the recombinant human type XVII collagen according to any one of claims 1-2, characterized in that, Comprising the following steps: fusing the 6xHis-SUMO expression gene to the 5' end of the gene according to claim 3 to obtain an expression gene, inserting the expression gene into a vector to obtain a recombinant vector, transforming the recombinant vector into a host cell to obtain a recombinant strain, culturing and inducing the expression of the recombinant strain, collecting the protein, and purifying it.

8. Use of the recombinant human type XVII collagen according to any one of claims 1-2 in the preparation of biological skin care products, medical materials, drugs for wound healing or scar repair.

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