Recombinant XVII type collagen-thymosin beta4 fusion protein as well as preparation method and application thereof

Abstract

The invention discloses a recombinant XVII type collagen-thymosin beta4 fusion protein as well as a preparation method and application thereof, the recombinant XVII type collagen-thymosin beta4 fusion protein is a fragment of human XVII type collagen and a thymosin beta4 protein sequence, and the amino acid sequence of the recombinant XVII type collagen-thymosin beta4 fusion protein is shown as SEQ ID NO: 1. The recombinant XVII type collagen-thymosin beta4 fusion protein disclosed by the invention has good safety, can regulate and regulate intercellular signal transduction, and has good effects of promoting hair follicle cell proliferation and promoting hair regeneration.

Description

Technical Field

[0001] The present invention belongs to the technical field of synthetic biology, and in particular relates to a recombinant type XVII collagen-thymosin β4 fusion protein and a preparation method and application thereof. Background Art

[0002] Collagen is a fibrous protein composed of three peptide chains entangled with each other. It is widely present in the connective tissue of animals and is the most widely distributed functional protein in mammals. So far, collagen can be divided into fibrillar collagen and non-fibrillar collagen according to its structure and function. Common fibrillar collagens include type I, type II, type II, type V, type XI, etc.; non-fibrillar collagens include reticular collagen, anchoring collagen and transmembrane collagen, among which type XVII collagen (COL17) belongs to the transmembrane collagen among non-fibrillar collagens.

[0003] Type XVII collagen has a molecular weight of 180 kDa and is a transmembrane protein located in the basement membrane zone of the epidermis. It is encoded by the COL17A1 gene, with its C-terminus located in the extracellular matrix and its N-terminus located in the cytoplasm. COL17 is a protein produced in the deepest layers of the epidermis that prevents epidermal thickening in newborn mice by coordinating with the Wnt signaling pathway. This signaling pathway, among other things, controls the frequency of some cell divisions.

[0004] Type XVII collagen is also a component of hemidesmosomes in cells and plays an important role in the function of epithelial cells-basement membrane, including regulating the adhesion, separation and developmental differentiation of epithelial cells. Type XVII collagen promotes the proliferation and renewal of epidermal stem cells, promotes competitive regeneration of skin tissue, delays aging, and can also promote the maintenance of the stemness of hair follicle stem cells, regulate the differentiation of hair follicle stem cells, and regulate the proliferation of epidermal cells between hair follicles, prevent hair follicle aging, and has hair care effects.

[0005] Thymosin β4 (Tβ4) is a polypeptide composed of 43 amino acid residues, with a molecular weight of 4963Da and an isoelectric point of 5.1. It is highly conserved in mammals. Conformation studies have shown that thymosin β4 exists and functions in a single-chain form, with an internal repeat region between residues 31-43 and 18-30, and two highly helical regions. Thymosin β4 is isolated from the thymus and is also widely present in other organs and tissues, with the highest content in the spleen and lungs, followed by the kidneys, heart, brain, liver, and testes. In thymocytes, the mRNA content of thymosin β4 is 7 times higher than that of thymic stromal cells, and is expressed in all types of blood cells. Thymosin β4 can promote the growth and differentiation of hair follicle stem cells, as well as the production of matrix proteases, thereby promoting hair growth.

[0006] However, thymosin β4 has a small molecular weight, is difficult to express and purify, and type XVII collagen is present in very low levels in the human body. It is a non-extracellular secretory collagen with a long amino acid chain and a large molecular weight. It is very difficult to extract or synthesize and cannot be mass-produced. Therefore, such raw materials are rarely seen on the market. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a recombinant type XVII collagen-thymosin β4 fusion protein and its preparation method and application in view of the deficiencies of the above-mentioned prior art. The fusion protein has good safety, can regulate the periodic cycle of hair follicles, promote hair growth, and can also regulate the activity of hair follicle stem cells, achieve the purpose of regulating signal transduction between cells and promoting hair follicle proliferation, and has a good effect of promoting hair follicle cell proliferation and hair regeneration.

[0008] In order to solve the above technical problems, the technical solution adopted by the present invention is: a recombinant type XVII collagen-thymosin β4 fusion protein, characterized in that the recombinant type XVII collagen-thymosin β4 fusion protein is a fusion protein of a fragment of human type XVII collagen and thymosin β4 protein, and the amino acid sequence of the fusion protein is shown in SEQ ID NO:1.

[0009] Furthermore, the present invention provides a nucleic acid encoding the above-mentioned recombinant type XVII collagen-thymosin β4 fusion protein, characterized in that the sequence of the nucleic acid is shown in SEQ ID NO:2.

[0010] Furthermore, the present invention provides an expression vector comprising the above nucleic acid.

[0011] Furthermore, the present invention provides a host cell into which the above expression vector is introduced, characterized in that the host cell is Escherichia coli, yeast or Bacillus subtilis.

[0012] The above host cell is characterized in that the host cell is Pichia pastoris.

[0013] Furthermore, the present invention provides a method for preparing the above-mentioned recombinant type XVII collagen-thymosin β4 fusion protein, characterized in that it includes: inserting a nucleotide sequence encoding the amino acid sequence into an expression vector to obtain a recombinant plasmid vector, then introducing the recombinant plasmid vector into a host cell, and then culturing the host cell introduced with the recombinant plasmid vector, collecting and separating and purifying the cell culture to obtain the recombinant type XVII collagen-thymosin β4 fusion protein.

[0014] The above method is characterized in that the host cell secretes the recombinant type XVII collagen-thymosin β4 fusion protein into the culture medium.

[0015] The above method is characterized in that the separation and purification adopts one or a combination of salting out, ion exchange chromatography, affinity chromatography, hydrophobic chromatography, acid-base precipitation and membrane separation.

[0016] Furthermore, the present invention provides a use of the above-mentioned recombinant type XVII collagen-thymosin β4 fusion protein in the preparation of medicines, cosmetics, health products or medical devices for promoting hair follicle cell vitality and hair regeneration.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] 1. The present invention solves the problem of small molecular weight and difficult expression of thymosin β4 protein by fusing and repeating a fragment of human type XVII collagen with thymosin β4 protein. By fermentation, separation and purification, and biological activity analysis, a fusion protein with biological activity of promoting cell proliferation and hair follicle repair and regeneration is obtained.

[0019] 2. The recombinant type XVII collagen-thymosin β4 fusion protein of the present invention has good safety, can regulate the periodic cycle of hair follicles, promote hair growth, and can also regulate the activity of hair follicle stem cells to achieve the purpose of regulating signal transduction between cells and promoting hair follicle proliferation. It has a good effect of promoting hair follicle cell proliferation and promoting hair regeneration.

[0020] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a diagram of the construction process of the Pichia pastoris engineered bacteria expressing the recombinant type XVII collagen-thymosin β4 fusion protein of Example 1 of the present invention.

[0022] Figure 2 This is the electrophoresis diagram of protein expression of the recombinant type XVII collagen-thymosin β4 fusion protein of the present invention.

[0023] Figure 3 This is the protein electrophoresis diagram of the purified recombinant type XVII collagen-thymosin β4 fusion protein of the present invention.

[0024] Figure 4 The results are the results of the cell proliferation promotion test of the recombinant type XVII collagen-thymosin β4 fusion protein of the present invention.

[0025] Figure 5 This is the result of the recombinant type XVII collagen-thymosin β4 fusion protein of the present invention promoting the expression of Wnt10b and other related genes. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0027] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0028] The present invention provides a recombinant type XVII collagen-thymosin β4 fusion protein which can effectively promote the proliferation of hair follicle cells and promote hair regeneration. The fusion protein is a fusion protein of a fragment of human type XVII collagen and thymosin β4 protein. The amino acid sequence of the fusion protein is shown in SEQ ID NO: 1.

[0029] SEQ ID NO: 1:

[0030] MSDKPDMAEIEKFDKSKLKKTETQEKNPLPSKETIEQEKQAGESGPPGPPGAMGPPGPPGAPMSDKPDMAEIEKFDKSKLKKTETQEKNPLPSKETIEQEKQAG ESGPPGPPGAMGPPGPPGAPMSDKPDMAEIEKFDKSKLKKTETQEKNPLPSKETIEQEKQAGESGPPGPPGAMGPPGPPGAPMSDKPDMAEIEKFDKSKLKKTE TQEKNPLPSKETIEQEKQAGESGPPGPPGAMGPPGPPGAPMSDKPDMAEIEKFDKSKLKKTETQEKNPLPSKETIEQEKQAGESGPPPGPPGAMGPPGPPGAPMS DKPDMAEIEKFDKSKLKKTETQEKNPLPSKETIEQEKQAGESGPPGPPGAMGPPGPPGAPMSDKPDMAEIEKFDKSKLKKTETQEKNPLPSKETIEQEKQAGES.

[0031] Codons were optimized for host cell expression, and an XhoI restriction site was introduced at the 5' end of the sequence and an XbaI restriction site was introduced at the 3' end to facilitate later gene manipulation. After the above optimization, a nucleic acid encoding the recombinant type XVII collagen-thymosin β4 fusion protein was obtained, and the sequence is shown in SEQ ID NO:2.

[0032] SEQ ID NO:2:

[0033]

[0034] The present invention also provides an expression vector comprising the above nucleic acid, which may contain regulatory sequences (such as transcription and translation start and stop codons) that are specific to the type of host (e.g., bacteria, fungi, plants or animals) into which the vector is to be introduced, taking into account whether the vector is DNA-based or RNA-based. In a specific embodiment, the expression vector is pPiczαA.

[0035] The present invention also provides a host cell into which the above expression vector is introduced, wherein the host cell refers to a cell into which an exogenous nucleic acid has been introduced, including the offspring of such cells. Host cells include transformants and transformed cells, which include primary transformed cells and offspring derived therefrom, regardless of the number of passages. Offspring may not be completely identical to parent cells in terms of nucleic acid content, but may contain mutations. The host cell is selected from any one of Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, and Bacillus subtilis. In a specific embodiment, the host cell is Pichia pastoris X33.

[0036] The present invention also provides a method for preparing the above-mentioned recombinant type XVII collagen-thymosin β4 fusion protein, comprising: inserting the nucleotide sequence encoding the amino acid sequence into an expression vector to obtain a recombinant plasmid vector, then introducing the recombinant plasmid vector into a host cell, then culturing the host cell introduced with the recombinant plasmid vector, using the above-mentioned host cell to express the target protein, collecting and separating and purifying the cell culture to obtain the recombinant type XVII collagen-thymosin β4 fusion protein. The above-mentioned method is characterized in that the host cell secretes the recombinant type XVII collagen-thymosin β4 fusion protein into the culture medium.

[0037] The present invention does not impose any limitation on the expression mode, which can be determined as needed, such as induced expression. For induced expression, the inducer can be methanol or the like.

[0038] In a specific embodiment, the host cell is Pichia pastoris. After obtaining the genetically engineered Pichia pastoris, the specific culture conditions are as follows: the genetically engineered Pichia pastoris is inoculated into a YPD medium and cultured at 30°C and 220 rpm for 22 h to 24 h until the OD 600 =18~20 as the seed liquid for the upper tank. After the seed liquid is expanded, it is inoculated into a fermentation tank with an initial volume of 5L at a 10% inoculation rate. The culture temperature is 30°C and the pH is 5.0~6.0. When the glycerol is exhausted, glycerol fed-batch culture is started. After 22h, methanol is added for induction culture. The temperature in the initial stage of induction is 25°C~30°C and the pH is 5.0~6.0. The tank is released after 54h of induction.

[0039] The present application does not impose any limitation on the separation and purification method, which can be determined as needed, for example, one or a combination of salting out, ion exchange chromatography, affinity chromatography, hydrophobic chromatography, acid-base precipitation and membrane separation can be used.

[0040] Example 1: Expression of recombinant type XVII collagen-thymosin β4 fusion protein

[0041] The nucleotide sequence of the synthetic recombinant type XVII collagen-thymosin β4 fusion protein gene (the nucleotide sequence is shown in SEQID NO:2): According to the preference of Pichia pastoris codons, the gene was codon optimized without changing the amino acid sequence, and an XhoI restriction site was introduced at the 5' end of the sequence, and an XbaI restriction site was introduced at the 3' end. The optimized gene sequence was fully synthesized and ligated into the expression vector pPICZαA. Pichia pastoris X33 was used as the expression host bacteria, and the obtained pPICzaA-XVII-Tβ4 cloning plasmid was linearized by electroporation and transformed into X33. High-copy positive clones were selected by the bleomycin gradient method, and Pichia pastoris genetically engineered bacteria were obtained by culturing at 30.0°C for 72 hours. The brief construction process of the Pichia pastoris engineered bacteria is as follows: Figure 1 shown.

[0042] The genetically engineered Pichia pastoris obtained above was inoculated into YPD medium and cultured until OD 600 =21.3, 10% inoculation was added to a fermenter with an initial volume of 5 L, the culture temperature was 30 ° C, pH = 6, OD 600 The concentration of glycerol was controlled at 20% to 30%. When the glycerol was exhausted, glycerol feeding culture was started. When the wet weight of the bacteria reached 205 g / L or more, methanol was added for induction culture. The temperature during the induction stage was 25°C and the pH was 6.0. After 54 hours of induction, the tank was opened and the supernatant was collected by centrifugation. The protein expression was shown in the following table. Figure 2 shown.

[0043] Example 2: Purification of recombinant type XVII collagen-thymosin β4 fusion protein

[0044] The supernatant collected by centrifugation in Example 1 was filtered through a hollow fiber column and then ultrafiltered to 20% of the initial volume.

[0045] The concentrated supernatant was added evenly to 10 50 mL centrifuge tubes, and the pH was adjusted to 2-11 using hydrochloric acid solution and sodium hydroxide solution. The tubes were placed at 4°C for 1 hour and centrifuged at 8000 rpm for 10 minutes. The supernatant was subjected to SDS-PAGE. When pH = 2, protein electrophoresis showed the lowest impurity content and the highest target protein content.

[0046] Prepare the equilibrium buffer according to the isoelectric point of the protein: 20mmol / L PBS buffer (liquid A, pH 6.0), and use 20mmol / L PBS buffer + 1.0mol / L NaCl (liquid B, pH 6.0) as the eluent. Dilute the crude protein in the previous step with liquid A as the loading solution, and load it on a 5mLQ selfie XL ion exchange chromatography column after filtration. Equilibrate the column with equilibrium buffer before loading. After loading, rinse 3 column volumes with liquid A, and then perform gradient elution with liquid B at a flow rate of 2.5m L / min. Collect the eluted components and detect them with SDS-PAGE.

[0047] Ultrafiltration desalting: G25 desalting column desalting, that is, using 25mL G25 filler, the operation process is similar to the gel filtration chromatography steps, each time 6.5mL is loaded, and about 8mL is collected. Desalting can be completed 10 minutes after loading.

[0048] The protein was concentrated to 20% to 30% of the initial volume by ultrafiltration, and then pre-frozen in a -20°C refrigerator for 4 hours. It was then transferred to a vacuum freeze dryer for freeze-drying. The freeze-dried protein was collected after 48 hours and stored in a -80°C refrigerator for later use.

[0049] Example 3: Characterization of recombinant type XVII collagen-thymosin β4 fusion protein

[0050] The protein electrophoresis of the fermented and purified samples was detected by SDS-PAGE. The results were as follows: Figure 3 As shown, the predicted molecular weight is about 90kDa, which is different from the theoretical molecular weight. It is speculated that the protein is glycosylated. The molecular weight of the protein after enzyme cleavage is consistent with the theoretical molecular weight. The protein sequence coverage determined by mass spectrometry is 100%, and the electrophoretic purity is ≥95%.

[0051] Example 4: Proliferation experiment of human hair follicle papilla cells with recombinant type XVII collagen-thymosin β4 fusion protein

[0052] (1) Human dermal papilla cells (DPCs) in the logarithmic growth phase were collected and the serum components on the cell membrane were removed by trypsin digestion. The cell concentration was adjusted to 10 cells per well by cell counting. 3 ~10 4 Continue to culture overnight.

[0053] (2) According to the experimental design, the recombinant type XVII collagen-thymosin β4 fusion protein of the present invention was prepared into 0.05 mg / mL, 0.1 mg / mL, and 0.5 mg / mL respectively and filtered through a 0.22 μm sterile filter membrane for later use.

[0054] (3) The above three raw materials were added to each well of the 96-well plate prepared with cells according to the above concentrations, and a blank control group was set up. The cells were cultured for another 24 hours.

[0055] At the corresponding detection time point, take out the cells from the incubator, add 10 μL of CCK-8 reagent to each well, and incubate in the dark for 2 hours (the specific incubation time depends on the number of cells, and the linearity is generally optimal when the OD value is 0.8-1.5).

[0056] (4) Measure the light absorbance of each well at 450 nm on an ELISA reader, record the results, and draw a cell growth curve with time as the horizontal axis and absorbance as the vertical axis.

[0057] The cell proliferation conditions of each group are shown in Table 1. Under the concentration gradient of 0.05 mg / mL, 0.1 mg / mL, and 0.5 mg / mL, the proliferation rate of the three samples gradually increased with increasing concentration. When the concentration increased to above 0.5 mg / ml, the proliferation rate tended to be stable. Figure 4 This is the proliferation curve of DPC cells promoted by recombinant type XVII collagen-thymosin β4 fusion protein. Therefore, it can be concluded that the treatment of recombinant type XVII collagen-thymosin β4 fusion protein has no toxic effect on DPC cells and has a certain proliferation effect on DPC cells. DPCs are essential for the proliferation, upward migration, differentiation of hair follicle stem cell progenitor cells and the formation of other types of HF cells. In addition, studies have shown that when the number of DPCs reaches a certain level, DPCs can promote hair follicle growth and affect the size and shape of hair. Therefore, it is of great significance to study the proliferation of DPCs and gain a deeper understanding of the regulatory mechanisms that promote their proliferation.

[0058] In summary, the recombinant type XVII collagen-thymosin β4 fusion protein is a relatively safe substance. Under normal conditions of use, it will not cause harm or dangerous reactions to the human body and the environment and has no cytotoxicity.

[0059] Table 1: Proliferation rate of DPC cells promoted by recombinant type XVII collagen-thymosin β4 fusion protein (x±s, n=3)

[0060]

[0061] Example 5: Recombinant type XVII collagen-thymosin β4 fusion protein promotes hair follicle proliferation by regulating the Wnt-βcatenin signaling pathway

[0062] (1) According to the experimental design, the recombinant type XVII collagen-thymosin β4 fusion protein of the present invention was prepared into 0.05 mg / mL, 0.1 mg / mL, and 0.5 mg / mL respectively and filtered through a 0.22 μm sterile filter membrane for later use.

[0063] (2) The above three raw materials were added to each well of the 96-well plate prepared with cells according to the above concentrations, and a blank control group was set up. The cells were cultured for another 24 hours.

[0064] (3) Total cellular RNA was extracted from the collected cells, and quantitative PCR (qPCR) was performed in triplicate using the SYBR Green PCR premix kit and the 7500 real-time PCR system. The expression of Wnt10b, LEF1, β-catenin, and Axin2 genes related to the Wnt / β-catenin pathway was detected in DPCs. The Wnt / β-catenin signaling pathway has the function of regulating the hair growth cycle and promoting hair follicle differentiation. All steps were performed according to the manufacturer's protocol. -ΔΔCt This method uses β-actin as an endogenous control gene for data normalization.

[0065] The results are as follows Figure 5 As shown, recombinant type XVII collagen-thymosin β4 fusion protein can upregulate LEF1 (Lymphoid Enhancer Binding Factor 1, mainly involved in the typical Wnt / β-catenin signaling pathway) and β-catenin (β-catenin, a multifunctional protein, a key transcription factor in the Wnt / β-catenin signaling pathway) in DPC. The expression of Wnt10b and Axin2 genes was also significantly upregulated. The results showed that recombinant type XVII collagen-thymosin β4 fusion protein treatment can promote the expression of Wnt / β-catenin pathway genes in DPC.

[0066] In summary, the recombinant type XVII collagen-thymosin β4 fusion protein of the present invention has good safety, can regulate the periodic cycle of hair follicles and promote hair growth, and can also regulate the activity of hair follicle stem cells to achieve the purpose of regulating intercellular signal transduction and promoting hair follicle proliferation. It has a good effect of promoting hair follicle cell proliferation and promoting hair regeneration.

[0067] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent structural change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A recombinant type XVII collagen-thymosin β4 fusion protein, characterized in that: The recombinant type XVII collagen-thymosin β4 fusion protein is a fusion protein of a fragment of human type XVII collagen and thymosin β4 protein, and the amino acid sequence of the fusion protein is shown in SEQ ID NO:

1.

2. A nucleic acid encoding the recombinant type XVII collagen-thymosin β4 fusion protein according to claim 1, characterized in that: The sequence of the nucleic acid is shown in SEQ ID NO:

2.

3. An expression vector comprising the nucleic acid according to claim 2.

4. A host cell introduced with the expression vector according to claim 3, characterized in that: The host cell is Escherichia coli, yeast or Bacillus subtilis.

5. The host cell according to claim 4, characterized in that The host cell is Pichia pastoris.

6. A method for preparing the recombinant type XVII collagen-thymosin β4 fusion protein as claimed in claim 1, characterized in that: include: The nucleotide sequence encoding the amino acid sequence is inserted into an expression vector to obtain a recombinant plasmid vector, and then the recombinant plasmid vector is introduced into a host cell. The host cell introduced with the recombinant plasmid vector is cultured, and the cell culture is collected, separated and purified to obtain the recombinant type XVII collagen-thymosin β4 fusion protein.

7. The method according to claim 6, characterized in that The host cell secretes the recombinant type XVII collagen-thymosin β4 fusion protein into the culture medium.

8. The method according to claim 6, characterized in that The separation and purification adopts one or a combination of salting out, ion exchange chromatography, affinity chromatography, hydrophobic chromatography, acid-base precipitation and membrane separation.

9. Use of the recombinant type XVII collagen-thymosin β4 fusion protein as claimed in claim 1 in the preparation of medicines, cosmetics, health products or medical devices for promoting hair follicle cell vitality and hair regeneration.

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