A recombinant type XVII collagen fusion protein and its preparation method and application

Abstract

The present invention belongs to the field of biomedical technology, and specifically relates to a recombinant type XVII collagen fusion protein and its preparation method and application. The recombinant type XVII collagen fusion protein includes human epidermal growth factor, the transmembrane domain of collagen XVII, and a fusion protein obtained by recombinant elastin. The recombinant type XVII collagen fusion protein provided by the present invention can quickly target the epidermal growth factor receptor (EGFR) of epithelial cells between the epidermis and the dermis, so that the recombinant type XVII collagen fusion protein can better play a transmembrane role: compared with a single type XVII collagen, it has a stronger ability to activate other types of collagen. It can effectively promote the self-repair of the skin barrier after microneedle administration and shorten the cycle of skin self-repair.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and more specifically, to a recombinant type XVII collagen fusion protein and a preparation method and application thereof. Background Art

[0002] Collagen XVII, or type 17 collagen, is a transmembrane protein that plays a key role in maintaining skin firmness and elasticity. Unlike other types of collagen, type 17 collagen is mainly distributed in the area where the epidermis and dermis meet, namely the basement membrane zone, and is essential for maintaining the toughness and tensile strength of the skin. Studies have shown that the essence of aging is the decline of the function of the basement membrane zone, the bridge from the epidermis to the dermis, which leads to a decrease in the efficiency of clearing and renewing senescent cells, or even a loss of them. The core reason for the decline of the basement membrane zone is the loss of type 17 collagen.

[0003] Therefore, it is very important to supplement type 17 collagen. The molecular weight of natural type 17 collagen is about 150kD. When supplemented by means of facial masks, it is easy to form a water film on the epidermis and is difficult to be directly absorbed by the epidermis. Existing technologies often reduce the molecular weight of type XVII collagen to promote skin absorption, or directly absorb the complete type XVII collagen through the gastrointestinal tract by oral administration, or directly inject it between the epidermis and the dermis by means of microneedles.

[0004] However, the above-mentioned technical methods, such as reducing type 17 collagen: small molecule collagen molecular weight is generally 1-3kD to be easily absorbed, but type 17 collagen must contain a transmembrane structure to have the corresponding effect, and the minimum required is about 30kD. Therefore, it is difficult to reduce the molecular weight of type 17 collagen so that it can be absorbed by the epidermis; oral method: the amount of protein products absorbed by the gastrointestinal tract after oral administration is very small, and even less reaches the face after gastrointestinal absorption, so large doses of protein products are often required orally, which has a considerable burden on the body; microneedle injection: microneedle injection can reach between the epidermis and the dermis, playing a good filling role, but type 17 collagen is easily metabolized after injection and the duration is short, resulting in the user having to endure the injection pain again in a short period of time, low acceptance, and the epidermis is prone to infection after injection, which also makes this method less popular. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention proposes a recombinant type XVII collagen fusion protein and a preparation method and application thereof. The recombinant type XVII collagen fusion protein can effectively solve the technical problems of short action time of collagen XVII and long skin repair time.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] In a first aspect, a recombinant type XVII collagen fusion protein comprises human epidermal growth factor, collagen XVII transmembrane domain and elastin-like protein connected in sequence; the amino acid sequence of the fusion protein is shown in SEQ ID NO.1.

[0008] Furthermore, the amino acid sequence of the human epidermal growth factor is shown in SEQ ID NO.2; the amino acid sequence of the collagen XVII transmembrane domain is shown in SEQ ID NO.3; and the amino acid sequence of the elastin-like protein is shown in SEQ ID NO.4.

[0009] In the second aspect, a nucleotide sequence encoding the recombinant type XVII collagen fusion protein as described in the first aspect, wherein the nucleotide sequence is shown as SEQ ID NO.5.

[0010] In the third aspect, a recombinant expression vector comprises the nucleotide sequence described in the second aspect.

[0011] In a fourth aspect, an engineered Escherichia coli bacteria contains the recombinant expression vector described in the third aspect.

[0012] A fifth aspect is a method for preparing a recombinant type XVII collagen fusion protein, comprising the following steps:

[0013] (1) adding restriction endonuclease nucleotide sequences to the 5' and 3' ends of the nucleotide sequence shown in SEQ ID NO.5;

[0014] (2) inserting the sequence of step (1) into an expression vector, transforming into competent Escherichia coli cells, and screening monoclonal strains that highly express recombinant type XVII collagen fusion protein;

[0015] (3) The strain screened in step (2) is subjected to high-density fermentation expression and purification to obtain a recombinant type XVII collagen fusion protein.

[0016] Furthermore, the expression vector is selected from pET32a or pET28a.

[0017] In a sixth aspect, the use of the recombinant type XVII collagen fusion protein as described in the first aspect in cell proliferation promoting preparations, skin care products or cosmetics.

[0018] In a seventh aspect, the use of the recombinant type XVII collagen fusion protein prepared by the method described in the fifth aspect in cell proliferation promoting preparations, skin care products or cosmetics.

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

[0020] The recombinant type XVII collagen fusion protein provided by the present invention can quickly target the epidermal growth factor receptor (EGFR) of epithelial cells between the epidermis and the dermis, so that the recombinant type XVII collagen fusion protein can better play a transmembrane role: compared with a single type XVII collagen, it has a stronger ability to activate other types of collagen. It can effectively promote the self-repair of the skin barrier after microneedle administration and shorten the cycle of skin self-repair. The elastin-like protein fused according to the structural characteristics of the recombinant type XVII collagen of the present invention is temperature-sensitive, can form nanoparticles after epidermal administration, can be slowly released between the epidermis and the dermis, significantly prolongs the action time of collagen, and improves the compliance of users.

[0021] Cell experiments have shown that the recombinant type XVII collagen fusion protein provided by the present invention can effectively promote cell adhesion, migration and proliferation.

[0022] The recombinant type XVII collagen fusion protein constructed by the present invention has wide applications, and can be applied to cosmetic additives, skin beauty care and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the solutions in the present application or the prior art, a brief introduction is given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0024] Figure 1 Schematic diagram for the construction of the recombinant vector pET32a-egf-col17-elps;

[0025] Figure 2 This is the SDS-PAGE electrophoresis diagram for screening of EGF-Col17-ELPS high-expression strains, where lane 1 is the SDS-PAGE electrophoresis diagram of the supernatant of the E. coli empty host. Figures 2 to 8 The SDS-PAGE electrophoresis diagram of the expression supernatant of different clones;

[0026] Figure 3 The figure is a result of electrophoresis of purification of the high expression strain of the present invention, wherein lane 1 is the E. coli engineering cell; lane 2 is the supernatant after the E. coli engineering cell is crushed; lane 3 is the crude pure collection liquid; lane 4 is the collection liquid after nickel column purification; lane 5 is the collection liquid after molecular sieve sepharose G50 purification;

[0027] Figure 4 This is a bar graph showing the effects of different recombinant protein products on the proliferation of mouse embryonic fibroblasts (NIH / 3T3) at different times;

[0028] Figure 5 This is a bar graph showing the increase in type I collagen expression promoted by different recombinant proteins;

[0029] Figure 6 This is a bar graph showing the increase in type III collagen expression promoted by different recombinant proteins;

[0030] Figure 7 This is a bar graph showing the increase in the expression of type IV collagen promoted by different recombinant proteins;

[0031] Figure 8 This is a bar graph showing the increase in the expression of type VII collagen promoted by different recombinant proteins;

[0032] Fig. 9 This is a bar graph showing the increase in the expression of type ⅩⅦ collagen promoted by different recombinant proteins;

[0033] Fig.10 This is a bar graph showing the wound repair rate of rats with different recombinant proteins;

[0034] Fig.11 The following are the observation pictures of the morphological effects of different recombinant proteins on wound repair in rats. DETAILED DESCRIPTION

[0035] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0036] In order to enable those skilled in the art to better understand the solution of the present application, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the accompanying drawings.

[0037] Example 1 Construction of different types of recombinant protein expression plasmids

[0038] The recombinant protein plasmid pET32a-egf-col17-elps of the present invention is constructed; the recombinant protein expression plasmid pET32a-col17-elps containing only type XVII collagen and elastin-like protein is constructed; and the recombinant protein expression plasmid pET32a-eg-elps containing only human epidermal growth factor and elastin-like protein is constructed.

[0039] 1. Construction of expression plasmid encoding pET32a-egf-col17-elps protein

[0040] The expression vector pET32a (eg Figure 1), purchased from Invitrogen (Invitrogen Life Sciences, Inc., USA), according to Figure 1 Restriction sites EcoRI and HindIII were designed at relevant positions. The nucleotide sequence encoding the recombinant type XVII collagen fusion protein was obtained by artificial optimization of the preferred codons of Escherichia coli and obtained by artificial synthesis. The 5' and 3' ends of the synthesized nucleotide sequence contain restriction sites of EcoRI and HindIII, respectively. The nucleotide fragment of the recombinant type XVII collagen fusion protein will be inserted between the same restriction sites of pET32a to obtain the expression plasmid encoding the pET32a-col17 protein, wherein the amino acid sequence of the recombinant type XVII collagen fusion protein is as follows:

[0041] NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELRLNT

[0042] NAYSAGSVFGVPNNMASCSPTLHPGLSTSSVFGMQNNLAPSLTTLSHGTTTSTAYGVKK

[0043] NMPQSPAAVNTGVSTSAACTTSVQSDDLLHKDCKFLILEKDNTPAKKEMELLIMTKDSGK

[0044] VFTASPASIAATSFSEDTLKKEKQAAYNADSGLKAEANGDLKTVSTKGKTTTADIHSYGSS

[0045] GGGGSGGGGGVGGAGGGGPWGPAPAWCWLLGLLLTWLLLLGLLFGLIALAGNLRGSPGPK

[0046] GDMGSPGPKGDRGFPGTPGIPGPLGHPGPQGPKGQKGSVGDPGMEGPMGQRGREGPMGP

[0047] RGEAGPPGSGEKGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGPQGPPGPVGLQGLRGEVG

[0048] LPGVKGDVPGKGVPGKGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGV

[0049] PGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGFGVPGFG(SEQ ID NO.1)

[0050] The recombinant type XVII collagen fusion protein specifically comprises the following amino acid sequences connected in sequence: (1) the amino acid sequence of human epidermal growth factor is shown in SEQ ID NO.2; (2) the amino acid sequence of the transmembrane domain of recombinant type XVII collagen is shown in SEQ ID NO.3; (3) the amino acid sequence of elastin-like protein is shown in SEQ ID NO.4; wherein,

[0051] SEQ ID NO.2:

[0052] NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR;

[0053] SEQ ID NO.3:

[0054] LNTNAYSAGSVFGVPNNMASCSPTLHPGLSTSSVFGMQNNLAPSLTTLSHGTTTTSTAYG

[0055] VKKNMPQSPAAVNTGVSTSAACTTSVQSDDLLHKDCKFLILEKDNTPAKKEMELLIMTKD

[0056] SGKVFTASPASIAATSFSEDTLKKEKQAAYNADSGLKAEANGDLKTVSTKGKTTTADIHSY

[0057] GSSGGGGSGGGGGVGGAGGGPWGPAPAWCWLLGLLLTWLLLLGLLFGLIALAGNLRGSP

[0058] GPKGDMGSPGPKGDRGFPGTPGIPGPLGHPGPQGPKGQKGSVGDPGMEGPMGQRGREGP

[0059] MGPRGEAGPPGSGEKGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGPQGPPGPVGLQGLRGEVGLPGVKGD;

[0060] SEQ ID NO.4:

[0061] VPGKGVPGKGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVG VPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGFGVPGFG;

[0062] The nucleotide sequence of the recombinant type XVII collagen fusion protein is:

[0063]

[0064] 2. Construct pET32a-col17-elps containing only type XVII collagen and elastin-like protein

[0065] Its nucleotide sequence is as follows:

[0066] CTGAACACCAATGCATATAGCGCGGGCTCTGTGTTTGGCGTCCCGAACAATATGG

[0067] CGAGCTGCAGCCCGACCCTGCATCCGGGCCTGTCCACCTCGAGCAGCGTTTTCGGCATG

[0068] CAGAACAATCTGGCGCCGTCACTGACCACCCTGAGCCACGGTACCACCACCACCAGCA

[0069] CCGCGTACGGCGTTAAAAAAAATATGCCGCAGAGCCCGGCGGCCGTGAATACCGGCGT

[0070] GAGCACCAGCGCGGCATGCACCACTTCAGTGCAGAGCGATGATCTGCTGCATAAAGAT

[0071] TGCAAATTTCTGATCCTGGAAAAAGACAATACCCCGGCGAAAAAAGAAATGGAACTGC

[0072] TGATTATGACCAAAGATAGCGGCAAAGTGTTTACCGCGAGCCCGGCATCAATTGCCGC

[0073] CACCAGTTTCAGCGAAGATACTCTGAAAAAAGAAAAACAGGCTGCATACAACGCCGAT

[0074] AGCGGTCTGAAAGCGGAAGCGAATGGCGATCTGAAAACGGTCAGCACCAAAGGCAAA

[0075] ACCACCACCGCCGATATCCATAGCTATGGCAGCTCAGGTGGCGGCGGCTCGGGTGGTG

[0076] GCGGCGGCGTTGGCGGTGCGGGTGGCGGCCCGTGGGGTCCGGCGCCGGCGTGGTGCTG

[0077] GCTGCTTGGCCTGCTGCTGACCTGGCTGCTGCTGCTGGGCCTGCTGTTTGGCCTGATTG

[0078] CACTGGCCGGTAATCTGCGTGGCAGCCCGGGCCCGAAAGGGGATATGGGCTCCCCGGG

[0079] CCCGAAAGGTGATCGTGGCTTTCCGGGCACCCCGGGTATTCCGGGCCCGCTGGGCCAC

[0080] CCGGGCCCGCAGGGTCCGAAAGGCCAGAAGGGTAGCGTGGGTGATCCGGGCATGGAA

[0081] GGCCCGATGGGCCAGCGCGGCCGCGAAGGCCCGATGGGCCCGCGCGGTGAAGCGGGC

[0082] CCGCCGGGCAGCGGTGAAAAAGGCGAACGTGGTGCGGCGGGCGAACCGGGCCCGCAC

[0083] GGTCCGCCGGGTGTGCCGGGTTCTGTAGGCCCGAAAGGCAGTTCAGGTAGCCCGGGCC

[0084] CGCAGGGCCCGCCGGGTCCGGTGGGCCTGCAGGGCCTGCGCGGCGAAGTGGGCCTGCC

[0085] GGGCGTGAAAGGTGATGTGCCGGGTAAAGGCGTGCCGGGCAAAGGTGTACCGGGAGT

[0086] GGGCGTGCCGGGTGTCGGCGTTCCGGGTGTGGGCGTACCGGGTGTGGGCGTGCCGGGC

[0087] GTTGGCGTGCCTGGTGTTGGCGTACCGGGCGTGGGCGTTCCGGGCGTGGGTGTGCCGG

[0088] GCGTGGGTGTTCCGGGCGTGGGTGTCCCGGGCGTGGGTGTTCCGGGCGTAGGCGTTCC

[0089] TGGCGTGGGTGTTCCGGGTGTTGGCGTGCCGGGCGTGGGCGTACCTGGCGTTGGCGTG

[0090] CCGGGCTTCGGCGTTCCGGGCTTCGGCTAA(SEQ ID NO.6)。

[0091] 3. Construction of pET32a-egf-elps containing only human epidermal growth factor and elastin-like polypeptide

[0092] The nucleotide sequence is as follows:

[0093] AACTCAGACAGTGAATGCCCGCTGAGCCATGATGGCTATTGCCTGCATGATGGGGTTT

[0094] GCATGTATATTGAAGCGTTAGATAAATATGCGTGCAATTGCGTTGTTGGCTATATTGGC

[0095] GAACGTTGCCAGTATCGTGATCTGAAATGGTGGGAACTGCGCGTCCCGGGCAAAGGCG

[0096] TGCCGGGTAAAGGTGTGCCGGGCGTGGGCGTGCCTGGTGTTGGCGTGCCGGGCGTGGG

[0097] TGTGCCGGGCGTGGGCGTTCCGGGCGTGGGCGTGCCGGGCGTTGGCGTGCCGGGCGTT

[0098] GGTGTTCCGGGTGTGGGCGTGCCGGGTGTGGGTGTGCCGGGCGTGGGCGTGCCGGGTG

[0099] TGGGCGTGCCGGGCGTAGGCGTGCCGGGTGTGGGTGTTCCGGGCGTTGGCGTGCCGGG

[0100] CGTGGGTGTGCCGGGTGTTGGTGTGCCGGGCTTTGGCGTTCCGGGCTTTGGTTAA (SEQ ID NO. 7).

[0101] Example 2 Expression and purification of different recombinant type XVII collagen fusion proteins

[0102] (1) Screening of high-expression monoclonal strains: Recombinant expression vectors pET32a-egf-col17-elps, pET32a-col17-elps, and pET32a-egf-elps were extracted to a concentration range of 1 to 100 ng / μl, and single digestion was performed with salI restriction enzyme to remove the vectors that were not connected to the target fragment. 5 to 10 μl of plasmid was mixed with 100 μl of Escherichia coli BL21 (DE3) competent cells, placed on ice for 10 min, transferred to a 42°C water bath, placed for 60 to 90 seconds, and then immediately transferred to an ice bath for 2 minutes, 1 ml of culture medium containing ampicillin was added, and placed in a constant temperature shaking box. The shaking parameters were set as follows: temperature 37°C, speed 60 rpm, and time 60 minutes. Select monoclonal colonies with smooth edges and large individuals, place them in liquid culture medium containing 100μg / ml ampicillin, place them in a 30℃ incubator, and shake and culture them at 220rpm for 16-18h. Centrifuge at 10000rpm for 10min, resuspend the precipitate in 20ml PBS solution, and ultrasonically disrupt (parameters: 300W, ultrasonic for 2s, stop for 3s, total time 3min), centrifuge and take the supernatant for SDS-PAGE electrophoresis to analyze the protein expression of the supernatant. The results are shown in Figure 2 .

[0103] (2) High-density fermentation of high-expression strains: Select high-expression strains, inoculate at 5% (30L fermenter), and use low temperature for high-density fermentation. That is, control the temperature at 30°C, and when the bacterial count reaches half of the logarithmic phase (about 4 hours, OD 600nm When the pH value was about 15, lactose was added to induce the culture medium at a final concentration of 10 g / L for 6 hours. When the pH value began to rise, the fermentation was stopped.

[0104] (3) Preliminary purification of recombinant type XVII collagen fusion protein: The fermentation broth was centrifuged at 4000 rpm for 30 minutes, and the precipitate was collected and washed twice with PBS. The precipitate was resuspended in PBS and homogenized by a high-pressure homogenizer (parameters: 1200 bar, repeated three times), and the resuspended liquid was placed in a 2-8°C water bath for 30 minutes. Centrifuged under this temperature condition, the supernatant was taken, and filtered through 0.8+0.45μm. The clarified liquid was placed in a 40°C water bath for 30 minutes, and the precipitate was taken through the plate layer. After resuspending with an equal volume of PBS, it was placed in a 40°C water bath for 30 minutes, filtered through a plate filter press (maintaining the temperature at 40°C), and the filter cake was taken. After the filter cake was resuspended with an equal volume of bingding buffer, it was placed in a 2-8°C water bath again and stirred for 30 minutes, centrifuged to take the supernatant, and filtered through 0.8+0.45μm to obtain a crude pure product.

[0105] (4) Nickel column purification: After the nickel column is equilibrated with binding buffer (20 mM Pb, pH 7.4), the crude pure solution and filler are thoroughly mixed (maintaining room temperature below 30°C), washed with binding buffer, and eluted with elution buffer (20 mM Pb, pH 7.4, 300 mM imidazole) containing 300 mM imidazole.

[0106] (5) Molecular sieve purification: The nickel column elution sample is concentrated 10 times by ultrafiltration through a 10kD membrane package, and then subjected to molecular sieve sepharose G50 gel filtration chromatography to obtain the final product. The electrophoretic purity can reach 95% or above. Figure 3 .

[0107] (6) The methods of steps (3) to (5) are used to purify egf-col17-elps, col17-elps, and egf-elps, respectively, and the purity can reach 95% or above.

[0108] Example 3 Cell proliferation promotion experiment

[0109] (1) Coating: The egf-col17-elps, col17-elps, egf-elps purified in Example 2 and commercial egf (sigma, E9644, i.e., recombinant human epidermal growth factor), col17 (Wuhan Yipu, YHJ81301, i.e., recombinant human type 17A1 collagen) and elps (directly synthesized) protein samples (i.e., elastin-like protein) were diluted with protein coating solution to a protein concentration of 2 μg / ml, and then coated on a 96-well plate, with 6 replicates per group, and the coating was allowed to stand at 4°C for 24 hours. A total of 4 plates were coated (one plate each at different time points of 24h, 48h, 72h, and 96h);

[0110] (2) Cell inoculation: Human non-small cell lung cancer cells (A549) in a cell culture flask were washed three times with sterile PBS solution, digested and terminated with trypsin, centrifuged at 1000 rpm for 8 minutes, the supernatant was discarded, serum-free basal medium was added to resuspend and wash, and centrifuged again. Repeat three times to remove the serum in the cell suspension, count with a hemocytometer, add 3% low-serum medium, and dilute the cell number to 10 4 Pieces / ml. Add 100 μl to each well of a 96-well plate, i.e. 1000 cells per well;

[0111] (3) Cell culture: Place the 96-well plate in an incubator at 37°C, 5% CO2 and culture for 24 h, 48 h, 72 h, and 96 h respectively;

[0112] (4) Cell proliferation assay: Take out a 96-well plate every 24 h, add 10 μl of CCK-8 reagent to each well and continue culturing in the incubator for 3 h, and measure the absorbance (OD) at 450 nm;

[0113] (5) Data statistical analysis: SPSS statistics software was used to perform significance analysis on the data.

[0114] The test results are shown in Tables 1 to 4 and Figure 4 , from Tables 1 to 4 and Figure 4 It can be seen that:

[0115] After culturing A549 cells for 24 hours, the cell proliferation of each group of samples was basically the same, with no significant difference, which indicates that the cells may have just completely attached to the wall at this time.

[0116] After 48 hours of culture, it can be clearly found that the recombinant albumin containing egf, such as the egf group, the egf-col17-elps group of the present invention and the egf-elps group, are significantly different from the control group (PBS group) (P < 0.05), and the proliferation efficiency is better than that of the PBS control group.

[0117] After 72 hours of culture, the proliferation efficiency of the egf group, the egf-col17-elps group of the product of the present invention and the egf-elps group was also better than that of the PBS control group, and there were significant differences between the egf-col17-elps group of the product of the present invention and the egf group and the egf-elps group. The proliferation efficiency of the egf-col17-elps product of the present invention was better than that of the egf group and the egf-elps group.

[0118] After 96 hours of culture, there was no significant difference in the other groups, but the proliferation effect of the product EGF-Col17-ELPS of the present invention was still significantly better than that of the other groups;

[0119] This experiment shows that the product EGF-Col17-ELPS of the present invention not only has a similar effect of promoting cell proliferation as EGF, but also is superior to the single EGF product in terms of action efficiency and action time.

[0120] Table 1 OD450 of different protein samples coated with cck-8 for 24h nm Test results

[0121]

[0122] Table 2 OD450 of different protein samples coated with cck-8 for 48h nm Test results

[0123]

[0124] Table 3 OD450 of different protein samples coated with cck-8 for 72h nm Test results

[0125]

[0126] Table 4 OD450 of different protein samples coated with cck-8 for 96h nm Test results

[0127]

[0128] Example 4 Effect of recombinant type XVII collagen fusion protein on the expression of other types of collagen

[0129] HaCaT cells (human immortalized keratinocytes) were used as a cell model, and 30,000 cells were inoculated into a six-well plate per well, and cultured for 24 hours with DMEM high-glucose medium containing 10% fetal bovine serum to allow them to adhere to the wall. Protein products of each group were added to make their final concentrations 0, 6.25, 12.5, 25, 50, and 100 pmol / L, respectively. Total RNA was extracted by TRIZOL method, 1 μg of total RNA was taken for reverse transcription to synthesize cDNA, and PCR reaction was performed. The reaction conditions were: 94°C pre-denaturation for 5 min, 94°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 1 min, 35 cycles, and 72°C extension for 10 min.

[0130] ImageJ was used to analyze the brightness of PCR bands. The experimental results are shown in Figures 5 to 9And Table 5. It can be seen from the figure that when the recombinant type XVII collagen fusion protein egf-col17-elps of the present invention is 0-50pmol / L, the mRNA transcription level of different types of collagen in HaCaT cells increases with the increase of drug concentration. At 25-50pmol / L, the mRNA expression of different types of collagen is the highest, and then it shows a gentle trend with the increase of drug concentration.

[0131] As can be seen from Table 5, the recombinant type XVII collagen fusion protein EGF-Col17-ELPS of the present invention has a significantly better regeneration promoting effect on different types of collagen than other groups, including 2.836 times for type I, 5.389 times for type III, 2.364 times for type IV, 1.781 times for type VII, and 2.698 times for type XVII. This indicates that the present invention has a stronger ability to activate other types of collagen than single type XVII collagen.

[0132] Table 5 Regeneration effects of different groups of proteins on different types of collagen

[0133]

[0134]

[0135] Example 5 Effect of recombinant type XVII collagen fusion protein on wound healing in rats

[0136] Each group of protein samples was diluted to 200 μg / ml with PBS solution containing 3% mannitol, sterile filtered, added to the small holes of a polytetrafluoroethylene plate with a diameter of 16 mm, freeze-dried, and the frozen protein membrane was placed in a sterile culture dish and dried for use.

[0137] Six-week-old female SD rats were randomly divided into seven groups, with six rats in each group. All rats were intraperitoneally injected with 30 mg / ml sodium pentobarbital solution (1 ml / kg). After anesthesia, the rats were shaved on the back and thoroughly depilated with a depilatory cream to leave no hair residue on the skin surface. The back of the rat was fully cleaned with warm water using a cotton swab and dried, and then a 1 cm round hole punch was used to stamp the back of the rat to create a wound with a diameter of 16 mm on the back of the rat. Each rat was fed individually.

[0138] The prepared protein membrane was applied to the back of each rat and fixed with sterile gauze. The wound was photographed and recorded on the 4th, 7th, 10th and 16th day after surgery, and the wound area was compared with the initial wound area to obtain the wound healing rate. Wound healing rate = (initial wound area - postoperative wound area) / initial wound area * 100%.

[0139] The results are shown in Table 6 and Fig.10 7 days after surgery, all wounds were free of infection. Compared with the initial wounds, the wound healing rates of the egf group, egf-col-17-elps group and egf-elps group were significantly higher than those of the control group, col17 group, elps group and col17-elps group. The wound healing rate of the recombinant protein egf-col-17-elps group of the present invention was the highest at 70.25%.

[0140] Ten days after surgery, the EGF-Col-17-ELPS group had basically recovered, while the EGF and EGF-ELP groups had not yet lost their gamma. Fig.11 The wounds of the control group, col17 group, elps group, and col17-elps group had not yet healed; 16 days after surgery, the skin of rats in each group was basically repaired.

[0141] Table 6 Wound repair rate of different groups of proteins on rat skin

[0142]

[0143]

[0144] Obviously, the embodiments described above are only some embodiments of the present application, rather than all embodiments. The preferred embodiments of the present application are given in the accompanying drawings, but they do not limit the patent scope of the present application. The present application can be implemented in many different forms. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive. Although the present application is described in detail with reference to the aforementioned embodiments, for those skilled in the art, it is still possible to modify the technical solutions recorded in the aforementioned specific implementation methods, or to perform equivalent replacement of some of the technical features therein. Any equivalent structure made using the contents of the specification and drawings of this application, directly or indirectly used in other related technical fields, is similarly within the scope of patent protection of this application.

Claims

1. A recombinant type XVII collagen fusion protein, characterized in that: The fusion protein comprises human epidermal growth factor, collagen XVII transmembrane domain and elastin-like protein connected in sequence; the amino acid sequence of the fusion protein is shown in SEQ ID NO.

1.

2. The recombinant type XVII collagen fusion protein according to claim 1, characterized in that: The amino acid sequence of the human epidermal growth factor is shown in SEQ ID NO.2; the amino acid sequence of the collagen XVII transmembrane domain is shown in SEQ ID NO.3; and the amino acid sequence of the elastin-like protein is shown in SEQ ID NO.

4.

3. A nucleotide sequence encoding the recombinant type XVII collagen fusion protein according to claim 1 or 2, characterized in that: The nucleotide sequence is shown in SEQ ID NO.

5.

4. A recombinant expression vector, characterized in that: It comprises the nucleotide sequence according to claim 3.

5. An engineered Escherichia coli, characterized in that: Contains the recombinant expression vector according to claim 4.

6. The method for preparing the recombinant type XVII collagen fusion protein according to claim 1, characterized in that: The following steps are involved: (1) adding restriction endonuclease nucleotide sequences to the 5' and 3' ends of the nucleotide sequence shown in SEQ ID NO.5; (2) inserting the sequence of step (1) into an expression vector, transforming into competent E. coli cells, and screening monoclonal strains that highly express recombinant type XVII collagen fusion protein; (3) The strain screened in step (2) is subjected to high-density fermentation expression and purification to obtain a recombinant type XVII collagen fusion protein.

7. The method for preparing the recombinant type XVII collagen fusion protein according to claim 6, characterized in that: The expression vector is selected from pET32a or pET28a.

8. Use of the recombinant type XVII collagen fusion protein as claimed in claim 1 or 2 in the preparation of cell proliferation promoting preparations, skin care products or cosmetics.

9. Use of the recombinant type XVII collagen fusion protein prepared by the method according to claim 6 or 7 in the preparation of cell proliferation promoting preparations, skin care products or cosmetics.

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