High-activity recombinant human XVII type collagen as well as preparation method and application thereof

Abstract

The invention discloses a high-activity recombinant human XVII type collagen as well as a preparation method and application thereof, and belongs to the technical field of bioengineering. The recombinant human XVII type collagen comprises 138 amino acids (SEQ ID NO: 1) of an XVII type collagen functional area and 15 amino acids (SEQ ID NO: 2) of a cell binding structural domain of I type collagen, and the amino acid sequences of the recombinant human XVII type collagen are further mutated to obtain the high-activity recombinant human XVII type collagen. The invention also provides an expression vector construction method of the protein and a preparation method of the recombinant human XVII type collagen. The theoretical molecular weight of the protein is small and is 14.6 kDa, and cytological experiments show that the protein has high biological activity for promoting cell migration, proliferation and adhesion and can be used for research, development and application of medical instruments and biological skin care products.

Description

Technical Field

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

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

[0003] The collagen in the human body is divided into fibrous collagen and non-fibrous collagen. Type XVII collagen (COL17, also known as BP180) is a transmembrane non-fibrous collagen, a homogeneous trimer composed of three identical α1 (XVII) chains. The α1 chain of type XVII collagen is composed of 1497 amino acids, with a single-chain molecular weight of 180kDa, 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 hemidesmosomes and achieves skin anti-aging effects by affecting the stemness and proliferation of ESCs. In addition, type XVII collagen prevents hair follicle aging by maintaining the stemness of hair follicle stem cells, regulating the differentiation of hair follicle stem cells, and regulating the proliferation of epidermal cells between hair follicles. Therefore, type XVII collagen is of great significance in preventing hair loss and scalp care.

[0004] The content of type XVII collagen in humans and animals is extremely low, and its extraction is extremely difficult and impossible. Due to the lack of commercial type XVII collagen products from humans or other animals on the market, the application of human type XVII collagen is limited. Industrial mass production of full-length transmembrane type XVII collagen is almost impossible to achieve due to cost and expression reasons. Based on the active domain of type XVII collagen, the recombinant type XVII collagen with biological properties is rationally designed, and its efficient expression in industrial strains is achieved, which will effectively promote the application of type XVII collagen in skin care products and medical fields, and 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 a preparation method and application of the recombinant human type XVII collagen.

[0006] In order to achieve the purpose of the present invention, the inventors conducted in-depth research and optimized the sequence of human type XVII collagen, selected 138 amino acids in the active region of human type XVII collagen, and obtained the recombinant human type XVII collagen as shown in SEQ ID NO: 1. The recombinant human type XVII collagen has a theoretical molecular weight of 13.2 kDa, can be efficiently expressed in the Escherichia coli prokaryotic expression system, and has good biological functional activities such as promoting cell proliferation, cell adhesion and migration.

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

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

[0009] Preferably, the phenylalanine at position 20 and the proline at position 113 of the amino acid sequence shown in SEQ ID NO:3 are mutated into aspartic acid to obtain recombinant human type XVII collagen, whose amino acid sequence is shown in SEQ ID NO:4; or the glutamic acid at position 68 and the histidine at position 128 of the amino acid sequence shown in SEQ ID NO:3 are mutated into aspartic acid to obtain recombinant human type XVII collagen, whose amino acid sequence is shown in SEQ ID NO:5.

[0010] It should be noted that the present invention provides a recombinant human type XVII collagen, which also includes an amino acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 4 as a repeating unit, which is repeated 1-5 times, preferably 1-3 times.

[0011] In order to increase the expression level of the above collagen and facilitate protein purification, a 6xHis-SUMO tag can be added to the N-terminus of the recombinant human type XVII collagen for fusion expression, and the 6xHis-SUMO expression gene can be 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 recombinant human type XVII collagen.

[0013] The present invention also provides an expression vector of the above gene, including but not limited to pET28a, pET32a, pPIC9K.pCDNA3.1, etc., preferably pET28a vector.

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

[0015] The present invention also provides a method for preparing the recombinant human type XVII collagen, comprising the steps of 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 has excellent biological activities such as promoting cell proliferation, adhesion and cell scratch healing, and can be used in wound healing, scar repair and other aspects.

[0017] Recombinant human type XVII collagen is used to prepare biological skin care products, medical materials, medicines, health care products, etc. Among them, the biological skin care products are made into essences, creams, facial masks, etc. The medical materials can be made into external dressings, etc.

[0018] Compared with the prior art, the recombinant collagen of the present invention has the following beneficial effects: The present invention provides a recombinant collagen with a small molecular weight and easy expression, and has multiple biological functions. The amino acid sequence of the recombinant human type XVII collagen of the present invention is derived from human type XVII and type I collagen, and the active domain sequence of human type XVII is combined with the type I collagen cell binding domain, which has high biocompatibility, and further constructs the RGD domain, further improving its multiple biological activities such as promoting cell adhesion, promoting cell scratch healing, and promoting cell proliferation activity. And it has a small molecular weight, is easy to express efficiently, and is suitable for industrial production and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solution of the embodiment of the present invention, the following briefly introduces the drawings required for describing the embodiment: Figure 1 This is the electrophoresis diagram of the expression and purification of recombinant human type XVII collagen.

[0020] Figure 2 This is a graph showing the results of a cell proliferation experiment.

[0021] Figure 3 This is a graph showing the results of a cell adhesion experiment.

[0022] Figure 4 This is an electron micrograph of the cell scratch healing experiment.

[0023] Figure 5 This is the result of cell scratch healing experiment. DETAILED DESCRIPTION

[0024] In order to better understand the present invention, the present invention is further described below by way of examples. 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, and therefore, the present invention is not limited to the specific embodiments of the following disclosure.

[0025] In the embodiments of the present invention, conventional molecular biological experimental methods are used unless otherwise specified. The processes such as PCR, enzyme digestion, ligation, and codon optimization involved in the embodiments are all understandable and easily implemented by those skilled in the art based on the product instructions or basic knowledge in the field, so they are not described in detail. Example 1

[0026] Design of amino acid sequence of recombinant human type XVII collagen The amino acid sequence of the original human type XVII collagen in the present invention comes from the UniProt database, Collagen alpha-1 (XVII) chain, 1497aa, UniProtKB / Swiss-Prot: Q9UMD9.

[0027] The recombinant human type XVII collagen of the present invention is obtained by selecting 138 amino acids, namely, positions 567-620 (54aa), positions 641-646 (6aa), positions 683-715 (33aa) and positions 1438-1482 (45aa), from the functional domain of type XVII collagen to obtain the recombinant human type XVII collagen (abbreviated as COL17-1) of SEQ ID NO: 1. The theoretical molecular weight of the recombinant type XVII collagen is 13.2kDa. In theory, the smaller the molecular weight of the protein, the easier it is to express in a heterologous system, but its function is closely related to the amino acid sequence. The functional activity of the small molecule recombinant type XVII collagen will be verified later.

[0028] The amino acids 772-786 in type I collagen (SEQ ID NO:2: GPQGIAGQRGVVGLP) are the cell binding domains of type I collagen, and the amino acid sequence of the recombinant type XVII collagen obtained by recombinant is 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. In order 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 to obtain a recombinant human type XVII collagen (referred to as COL17-2) containing two RGD domains (underlined in SEQ ID NO:4). The amino acid glutamic acid at position 68 and the histidine at position 128 were mutated to aspartic acid to obtain a recombinant human type XVII collagen (referred to as COL17-3) containing two RGD domains (underlined in SEQ ID NO:5).

[0029] It should be noted that the amino acid sequence of the recombinant human type XVII collagen described in the present invention may also be 1-5 repetitions of the amino acid sequence in SEQ ID NO:4 or SEQ ID NO:5.

[0030] SEQ ID NO:1 GSPGPKGDMGSPGPKGDRGFPGTPGIPGPLGHPGPQGPKGQKGSVGDPGMEGPMGEKGERGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPPGQKGEMGTPPGKGDRGPAGPPGHPGPPGPRGHKGEKGDKGDQ SEQ ID NO:2 GPQGIAGQRGVVGLP SEQ ID NO:3 GSPGPKGDMGSPGPKGDRG F PGTPGIPGPLGHPGPQGPKGQKGSVGDPGMEGPMGEKGERGLQGLRG E VGLPGVKGDKGPMGPPGPKGDQGEKGPPGQKGEMGTPPGPKGDRG P AGPPGHPGPPGPRG H KGEKGDKGDQGPQGIAGQRGVVGLP SEQ ID NO:4 GSPGPKGDMGSPGPKGD RGD PGTPGIPGPLGHPGPQGPKGQKGSVGDPGMEGPMGEKGERGLQGLRGEVGLPGVKGDKGPMGPPPGPKGDQGEKGPPGQKGEMGTPPGKGD RGD AGPPGHPGPPGPRGHKGEKGDKGDQGPQGIAGQRGVVGLP SEQ ID NO:5 GSPGPKGDMGSPGPKGDRGFPGTPGIPGPLGHPGPQGPKGQKGSVGDPGMEGPMGEKGERGLQGL RGD VGLPGVKGDKGPMGPPGPKGDQGEKGPPGQKGEMGTPPGPKGDRGPAGPPGHPGPPGP RGD KGEKGDKGDQGPQGIAGQRGVVGLP Example 2

[0031] Construction and expression of recombinant human type XVII-I collagen expression vector In order to fully illustrate the advantages of recombinant human type XVII-I collagen, the expression, purification and biological activity of recombinant human type XVII collagen (SEQ ID NO: 1, referred to as COL17-1), recombinant type XVII-I collagen (SEQ ID NO: 4, referred to as COL17-2) and recombinant type XVII-I collagen (SEQ ID NO: 5, referred to as COL17-3) were also studied in the examples of the present invention. Since the methods used by the three are the same, the recombinant human type XVII collagen is used as an example for description in the examples. The expression and purification processes of proteins with different sequence lengths are the same, and the present invention preferentially uses the sequence in SEQ ID NO: 5 for description.

[0032] 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 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. In order to increase the expression of the protein and facilitate protein purification, a 6xHis-SUMO tag can be added to the N-terminus of the recombinant human type XVII collagen for fusion expression. After sequence synthesis, the 6xHis-SUMO expression gene can be fused to the 5' end of the type XVII collagen gene by overlap extension PCR to form a 6xHis-SUMO-XVII expression gene. This DNA fragment was cloned and amplified by PCR, and Nde I and Xho I restriction sites were added by primers during amplification. The target fragment was inserted into the pET28a vector by restriction digestion and ligation. The recombinant vector was cloned and screened and sequenced to obtain the pET28a- 6xHis - SUMO - XVII recombinant expression vector.

[0033] The pET28a-6xHis-SUMO-XVII plasmid was transformed into the Escherichia coli expression strain BL21 (DE3), and single clones were obtained by kanamycin resistance screening. Single clones were picked and cultured overnight in 5 mL 2xYT medium. The clones were inoculated at a ratio of 1:100 and cultured at 37°C with shaking until the OD600 was 0.6-0.8. The culture temperature was lowered to 20°C and IPTG was added to a final concentration of 0.2 mM. 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

[0034] Purification of recombinant human type XVII collagen (1) Bacterial lysis: Resuspend the cells in 50 mM phosphate (pH 8.0 containing 300-500 mM sodium chloride and 10-20 mM imidazole) at a ratio of 1:15, and lyse the cells using a homogenizer in a cold water environment. Repeat the lysis cycle 3 times. The target protein is mainly in the lysis supernatant ( Figure 1 , Lane 5). After lysis, the supernatant was collected by centrifugation or filtration.

[0035] (2) The supernatant from the previous step was purified using a Ni-NTA column, and the target protein was eluted from the column using a 250 mM imidazole solution to obtain the target protein solution ( Figure 1 , lane 6).

[0036] (3) Dialysis and Ulp-1 digestion: Add 1 / 50000 mass ratio of Ulp1 enzyme to the protein solution in the previous step, mix well and put into a dialysis bag with a molecular weight cutoff of 3kDa, and dialyze in 50mM phosphate without imidazole to remove imidazole. Change the dialysate every 8 hours, and dialyze 3 times in total. After the enzyme digestion tag and target protein separation ( Figure 1 , Lane 7), dialysis needs to be performed at 2-8°C.

[0037] (4) Reverse nickel column purification to remove the label: The protein solution from the previous enzymatic digestion and dialyzation is passed through the nickel column again. The tagged protein will bind to the nickel column, while the target protein will flow through. The flow-through solution can be collected to obtain the target protein with higher purity ( Figure 1 , lane 8).

[0038] (5) Dialysis and desalination: The protein solution obtained from the previous purification step was re-loaded into a dialysis bag with a molecular weight cutoff of 3 kDa and dialyzed for desalination in 10 mM phosphate (pH 8.0). The dialysate was replaced every 8 hours. The protein solution was harvested after the dialysate was replaced three times.

[0039] (6) Purification by anion exchange chromatography: The protein solution obtained in the previous step is subjected to anion exchange chromatography to remove impurity proteins and nucleic acids to obtain high-purity recombinant human type XVII collagen ( Figure 1 , lane 9). Example 4

[0040] Evaluation of the cell proliferation activity of recombinant human collagen XVII BALB / c 3T3 cells (mouse embryonic fibroblasts) in good logarithmic growth phase were selected, digested with 0.25% trypsin, centrifuged and resuspended in DMEM medium containing 10% serum and counted; BALB / c 3T3 cells were inoculated in 96-well culture plates at 4000 cells per well, and blank controls without cells were set up at the same time. After culturing for 16 hours at 37°C and 5% CO2, DMEM medium containing 2% serum was replaced, and culture was continued for 16 hours until the cells adhered well. The original culture medium was aspirated, and 500μg / ml of the recombinant type XVII collagen of the present invention was added. The cell control group was a DMEM medium containing 2% serum, and culture was continued for 48 hours. 4 hours before the end of the culture, 5g / L of MTT solution was added, and the culture was terminated after continuing to culture for 4 hours; the liquid in the well was aspirated and discarded, washed once with PBS, DMSO was added to dissolve the crystals, and the absorbance value of each well was measured at a wavelength of 570nm.

[0041] Cell proliferation rate = (absorbance value of the assay well - absorbance value of the blank control group) / (absorbance value of the cell control group - absorbance value of the blank control group) × 100%.

[0042] In this example, the control group was commercially available recombinant type XVII collagen, which was added to the cell culture medium at 500 μg / ml, with 3 parallel samples in each group. Figure 2 As shown, the experimental results show that under the condition of a concentration of 500 μg / ml, the positive control group and recombinant COL17-1, recombinant COL17-2 and recombinant COL17-3 have a proliferation rate of 156.7%, 153.7% and 170.2% on BALB / c 3T3 cells, respectively; 153.8% of the commercially available recombinant type XVII collagen, indicating that the recombinant XVII collagen prepared by the present invention can significantly promote the proliferation of fibroblasts and has biological activity to promote cell proliferation. By comparison, it is not difficult to find 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

[0043] Evaluation of the cell adhesion promoting activity of recombinant human collagen XVII The prepared recombinant type XVII collagen was diluted with PBS and added to a 96-well cell culture plate at a final concentration of 500 μg / ml. 100 μL of protein solution was added to each well and placed at 37°C for 16 h. PBS was added to the control wells. BALB / 3T3 cells were digested with 0.25% trypsin, counted, and 5×10 4 Cells were cultured in a 37°C, 5% CO2 incubator for 2 hours. PBS was washed three times to remove non-adherent cells, and then 200 μL DMEM culture medium was added; 5 g / L MTT solution was added, and the cells were incubated in a 37°C, 5% CO2 cell incubator for 2 hours before removal. The absorbance values ​​of the 96-well plate at 450 nm and 630 nm were read with an ELISA reader, and the absorbance was measured at 450 nm with 630 nm as the reference wavelength, and the measurement results were recorded.

[0044] Cell adhesion promotion rate = (450nm absorbance of the experimental group - 450nm absorbance of the negative control group) / 450nm absorbance of the negative control group × 100%.

[0045] 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. Figure 3As shown, the results show that at a concentration of 500 μg / ml, the cell adhesion promoting rates of recombinant COL17-1, recombinant COL17-2 and recombinant COL17-3 on BALB / c 3T3 cells are 29.53%, 25.96% and 31.70%, respectively; the cell adhesion promoting rate of commercially available recombinant type XVII collagen is 24.28%. The data show that the recombinant XVII collagen prepared by the present invention has good cell adhesion promoting activity, and the ability of recombinant human XVII collagen (recombinant COL17-3) to promote cell adhesion is better than that of other recombinant type XVII collagens (recombinant COL17-1 and recombinant COL17-2). Example 6

[0046] Evaluation of the cell migration-promoting activity of recombinant human collagen XVII Take the recombinant human type XVII collagen of the present invention and the commercially available recombinant type XVII collagen of the reference substance, dilute the sample to 0.5 mg / mL with DMEM 2% serum medium, pH 7.0-7.4. Culture and passage BALB / c 3T3 cells normally. Inoculate the cells in good condition into a 6-well plate, and place 5×10 5 Inoculate 2 mL of cell suspension per well and culture for 24 hours. Use a 200 μl pipette tip perpendicular to the surface of the 6-well plate to make a scratch perpendicular to the bottom line of the 6-well plate in the monolayer of cells. Try to keep the width of the scratch consistent during the process. Wash with PBS three times to remove free cells and cell fragments caused by the scratch. Add protein solution diluted with 2% serum DMEM culture medium to the wells and continue to culture in a 37°C, 5% CO2 incubator. Take samples and take photos at 0h and 24h. Figure 4 Image J software was used to obtain the initial scratch area and the area of ​​the blank area without cells, and the migration rate was calculated. The migration rate = (initial scratch area - blank area after cell migration / initial scratch area) * 100%. The experimental results are shown in Figure 5 The migration rates of recombinant COL17-1, recombinant COL17-2 and recombinant COL17-3 at a protein concentration of 500 μg / ml were 35.05%, 31.44% and 37.50% respectively; the migration rate of commercially available recombinant type XVII collagen was 30.82%. The data show 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.

[0047] In summary, according to the results of Examples 4-6, the recombinant human XVII collagen of the present invention has good cytological activity. The recombinant human XVII collagen (recombinant COL17-3) obtained by further fusing the cell binding domain of type I collagen and mutating the RGD structure threshold has better cytological activity.

[0048] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection 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 contains 1-5 repeating units, and the amino acid sequence of the recombinant human type XVII collagen repeating unit is shown in SEQ ID NO:

1.

2. The recombinant human type XVII collagen according to claim 1, characterized in that: The amino acid sequence of the recombinant human type XVII collagen repeating unit also includes SEQ ID NO:2; the amino acid sequence of the recombinant human type XVII collagen repeating unit is shown in SEQ ID NO:

3.

3. The recombinant human type XVII collagen according to claim 2, characterized in that: The amino acid sequence of the recombinant human type XVII collagen repeating unit as claimed in claim 2 is subjected to single-point mutation or multi-point mutation, wherein the mutation is as follows: (1) Phenylalanine at position 20 mutated to aspartic acid; (2) Glutamic acid at position 68 mutated to aspartic acid; (3) Proline at position 113 mutated to aspartic acid; (4) The histidine at position 128 was mutated to aspartic acid.

4. The recombinant human type XVII collagen according to claim 3, characterized in that: The recombinant human type XVII collagen is obtained by mutating the amino acids at positions 20 and 113 into aspartic acid, and its amino acid sequence is shown in SEQ ID NO:4; or by mutating the amino acids at positions 68 and 128 into aspartic acid, and its amino acid sequence is shown in SEQ ID NO:

5.

5. A gene encoding the recombinant human type XVII collagen according to any one of claims 1 to 5.

6. A recombinant vector, characterized in that: A gene comprising the recombinant human type XVII collagen as claimed in claim 5.

7. The recombinant vector according to claim 6, characterized in that The vector is any one of pET28a, pET32a, pPIC9K, and pCDNA3.

1.

8. A recombinant strain, characterized in that Contains the gene according to claim 5, or contains the recombinant vector according to any one of claims 6-7.

9. The method for preparing recombinant human type XVII collagen according to any one of claims 1 to 4, characterized in that: The method comprises the following steps: fusing the 6xHis-SUMO expression gene to the 5' end of the gene according to claim 5 to obtain the 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 the recombinant strain and inducing expression, collecting protein, and purifying it.

10. Use of the recombinant human type XVII collagen according to any one of claims 1 to 4 in the preparation of biological skin care products, medical materials, medicines and health care products.

Images