Superconductor carrier protein Pro. STP and application thereof

Abstract

The invention discloses a superconductor carrier protein Pro. STP. The amino acid sequence of the superconductor carrier protein Pro. STP is shown as any one of SEQ ID NO.1-14. The invention also discloses a coding gene, an expression vector, host bacteria and application of the gene. The invention provides a superconductor carrier protein Pro. STP, biosynthesis thereof and a method for carrying protein in a non-fusion form. The superconductor carrier protein Pro. STP is stable in quality, low in cost and capable of carrying protein efficiently. The superconductor carrier protein Pro. STP synthesized by the method disclosed by the invention has good solubility. A prokaryotic system can be used for expression, the system is simple, the yield is high, the cost is low, and the gene has the biological function of carrying protein and can be used for molecular delivery of related products.

Description

Technical Field

[0001] The invention relates to a superconductor carrier protein Pro.STP and application thereof, belonging to the technical field of protein expression. Background Art

[0002] Cell-penetrating peptides (CPPs) are a class of short peptides that can cross cell membranes or tissue barriers, usually composed of 5 to 30 amino acids. Cell-penetrating peptides have unique structures and properties, and can transport biological macromolecules such as proteins, RNA, and DNA into cells through mechanisms such as endocytosis or direct penetration of cell membranes, thereby exerting their biological functions. Compared with other non-natural chemical molecules, cell-penetrating peptides are usually composed of natural amino acids, have good biocompatibility, low toxicity to cells, can achieve efficient molecular delivery at low concentrations, can be degraded after carrying proteins, and can be directly fused with biologically active molecules such as proteins and peptides to form fusion proteins with membrane-penetrating ability. Therefore, CPPs, as an efficient molecular delivery tool, have broad application prospects in biomedical research and drug development. With the in-depth study of its mechanism of action and structure, CPPs are expected to become an important tool for the treatment of various diseases in the future.

[0003] Frankel et al. first discovered in 1988 that the trans-transcription activator TAT from human immunodeficiency virus (HIV) HIV1 can effectively penetrate the cell membrane and enter the cell. Since then, various sources of transmembrane peptides have been discovered, such as Penetratin, which is derived from the 43rd to 58th amino acid residues of the Drosophila homeotic transcription factor ANTP, and the transcription factor VP22 from herpes simplex virus type 1 (HSV-1). There are also many artificially designed transmembrane peptides, such as the amphipathic transmembrane peptides Transportan, MPG, and Pep-1.

[0004] Morris et al. designed a 21-residue peptide vector Pep-1 (KETWWETWWTEWSQPKKKRKN), which consists of three domains: (1) a hydrophobic motif (KETWWETWWTEW) rich in five tryptophan residues, which can be used to efficiently target the cell membrane and form hydrophobic interactions with proteins; (2) a lysine-rich hydrophilic domain (KKKRKV) derived from the nuclear localization sequence (NLS) of the large T antigen of simian virus 40 (SV-40) to improve the intracellular delivery ability and solubility of the peptide vector; (3) a spacer domain (SQP) that separates the above two domains and contains a proline residue to enhance the flexibility and integrity of the hydrophobic and hydrophilic domains. Studies have shown that Pep-1 is independent of normal endocytosis and is rapidly localized to the nuclei of human HS-68, mouse NIH-3T3 fibroblasts or Cos cells in <10 minutes. In existing literature and patents, the function of cell-penetrating peptides for protein delivery is mainly achieved by fusing them with target proteins, while relatively few studies have been conducted on protein delivery in a non-fusion form. In addition, cell-penetrating peptides are usually composed of 5 to 30 amino acids and are mainly obtained by chemical synthesis. Although the chemical synthesis process is mature, it also faces the disadvantage of a sharp increase in production costs, making it difficult to mass-produce in industry. Summary of the invention

[0005] The purpose of the present invention is to provide a superconductor carrier protein Pro.STP.

[0006] The technical solution adopted by the present invention is:

[0007] A superconductor carrier protein Pro.STP, whose amino acid sequence is shown in any one of SEQ ID NO.1-14.

[0008] The coding gene of the above-mentioned superconductor carrier protein Pro.STP.

[0009] Preferably, the nucleotide sequence is shown in any one of SEQ ID NOs. 15-28.

[0010] The expression vector of the above-mentioned superconductor carrier protein Pro.STP.

[0011] The host bacteria of the above-mentioned superconductor carrier protein Pro.STP can be Escherichia coli BL21 (DE3), Escherichia coli Origami B (DE3), Escherichia coli Origami 2 (DE3), Escherichia coli JM109, Escherichia coli MG1655, Escherichia coli S17-1, Pichia pastoris, Saccharomyces cerevisiae or lactic acid bacteria.

[0012] The above-mentioned biosynthesis method of the superconductor carrier protein Pro.STP comprises the following steps:

[0013] (1) constructing the above-mentioned expression vector and transforming it into a host bacterium;

[0014] (2) Cultivating host bacteria and inducing protein expression;

[0015] (3) Purify the protein to obtain the above-mentioned superconductor carrier protein Pro.STP.

[0016] The above-mentioned superconductor carrier protein Pro.STP is used in the transmembrane transport of target proteins for non-medical purposes.

[0017] Preferably, the superconductor carrier protein Pro.STP is expressed by fusion with the target protein.

[0018] Preferably, the superconductor carrier protein Pro.STP is mixed with the target protein to form a complex, and then the target protein is transported across the membrane. Generally speaking, the superconductor carrier protein Pro.STP and the target protein can be dissolved in pure water or PBS respectively, and then Pro.STP and the target protein are simply mixed in a certain proportion, incubated at 4°C-25°C for 30min-2h to form a complex and achieve transmembrane transport. In order to achieve a good transport effect, the addition ratio of the superconductor carrier protein Pro.STP and the target protein is 1:0.1~20.

[0019] Preferably, the target protein is BAS, type III collagen or nonapeptide-1, and the molecular weights thereof are 66 kD, 13 kD and 1.2 kD, respectively.

[0020] Beneficial effects of the present invention:

[0021] The present invention provides a superconductor carrier protein Pro.STP, and a method for biosynthesis and protein delivery in a non-fusion form thereof, which has stable quality, low cost, and can efficiently deliver proteins. The superconductor carrier protein Pro.STP synthesized by the present invention has good solubility. It can be expressed by a prokaryotic system, has a simple system, high yield, low cost, has the biological function of a carrier protein, and can be used for products related to molecular delivery. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The plasmid map of the expression vector plasmid pET30a-intein-Pro.STP (1-13) of the present invention.

[0023] Figure 2 The plasmid map of the expression vector pET30a-Pro.STP-14 of the present invention.

[0024] Figure 3The SDS-PAGE gel image of the induced expression of Pro.STP1-13 of the present invention: lane BSA (0.5) is 0.5 mg / mL bovine serum albumin, lane CS is the electrophoresis image of the supernatant after Pro.STP and the intein are broken and separated and centrifuged after the broken precipitate EP is resuspended and incubated after engineering bacteria induction, and lane CP is the precipitate after centrifugation.

[0025] Figure 4 The SDS-PAGE gel diagram of the induced expression of Pro.STP14 of the present invention: the lane BSA (0.5) is 0.5 mg / mL bovine serum albumin, the lane Pro.STP14 is the electrophoresis diagram of the engineered bacteria induced expression and the purified protein.

[0026] Figure 5 The ability of Pro.STP to deliver BAS is observed by fluorescence microscopy of the present invention. Figure 5 A is a fluorescence image of Pro.STP-6 of the present invention carrying bovine serum albumin (BSA) penetrating pig skin; Figure 5 B is a fluorescence image of Pro.STP-7 of the present invention carrying bovine serum albumin (BSA) penetrating pig skin; Figure 5 C is a fluorescence image of bovine serum albumin (BSA) penetrating pig skin using pure water as a control.

[0027] Figure 6 The ability diagram of Pro.STP to deliver nonapeptide-1 observed by fluorescence microscopy of the present invention, Figure 6 A is a fluorescence image of Pro.STP-6 carrying nonapeptide-1 penetrating pig skin; Figure 6 B is a fluorescence image of Pro.STP-7 carrying nonapeptide-1 penetrating pig skin; Figure 6 C is a fluorescence image of nonapeptide-1 penetrating pig skin using pure water as a control.

[0028] Figure 7 The ability of Pro.STP to deliver type III collagen observed by fluorescence microscopy of the present invention is shown in the figure. Figure 7 A is a fluorescence image of Pro.STP-6 of the present invention carrying type III collagen penetrating pig skin; Figure 7 B is a fluorescence image of Pro.STP-13 of the present invention carrying type III collagen penetrating pig skin; Figure 7 C is a fluorescence image of Pro.STP-14 of the present invention carrying type III collagen penetrating pig skin;

[0029] Figure 7 D is a fluorescence image of Pro.STP-7 of the present invention carrying type III collagen penetrating pig skin; Figure 7 E is the fluorescence image of 0.5% glycyrrhizic acid-loaded type III collagen penetrating pig skin; Figure 7 F is the fluorescence image of type III collagen carried by 0.5% ethyl acetate penetrating pig skin;

[0030] Figure 7 G is a fluorescence image of type III collagen penetrating pig skin with pure water as control; Figure 7 H is the fluorescence image of type III collagen solidified by CA gel. DETAILED DESCRIPTION

[0031] The present invention is further illustrated by examples below, but is not intended to be limiting of the present invention. The specific materials used in the embodiments of the present invention and their sources are provided below. However, it should be understood that these are merely exemplary and are not intended to limit the present invention, and materials identical or similar to the types, models, qualities, properties or functions of the following reagents and instruments can be used to implement the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0032] Example 1: Preparation of superconductor carrier protein Pro.STP expression vector

[0033] (1) Gene design and synthesis

[0034] In the present invention, an expression vector of a superconductor carrier protein Pro.STP is constructed, and the amino acid sequence of the superconductor carrier protein Pro.STP is designed as shown in SEQ ID No.1-14, wherein the amino acid sequence shown in SEQ ID No.1 includes a capping peptide (AP) connected in sequence 4 , the cell-penetrating peptide pep-1, the amino acid sequence shown in SEQ ID No.2 includes a capping peptide (AP) connected in sequence 4 , penetrating peptide pep-1, spacer sequence PTPTP, penetrating peptide pep-1, SEQ ID No.3-6 is based on SEQ ID No.2 with the end-capping peptide (AP) 4 The amino acid sequence of the protein was replaced with GGGGS, EAAAK, ELYAVTGRGDSPASSAPIATS and (AP) 2 The amino acid sequence shown in SEQ ID No.7 includes the cell-penetrating peptide pep-1, the type IV collagen loop sequence DGIPGSAGE and the cell-penetrating peptide TAT (amino acid sequence YGRKKRRQRRR) connected in sequence; SEQ ID No.8-9 is based on SEQ ID No.7 with the addition of the terminal peptide (AP) 4 and (AP) 2The amino acid sequence shown in SEQ ID No. 10 includes the cell-penetrating peptide pep-1, the type IV collagen loop sequence GMKGLSGDRGDAG and the cell-penetrating peptide TAT (amino acid sequence YGRKKRRQRRR) connected in sequence; SEQ ID No. 11-12 is based on SEQ ID No. 10 with the addition of the terminal peptide (AP) 4 and (AP) 2 ; SEQ ID No.13 is based on SEQ ID No.6, with type III collagen sequence added to the C-terminus, and the cell-penetrating peptide and type III collagen are fused and expressed; SEQ ID No.14 is based on SEQ ID No.6, with 6*His purification tag and enterokinase cleavage site added to the front end, and the corresponding coding genes are shown in SEQ ID No.15-28. The end-capping peptide was designed into the primer, and the primer synthesis was handed over to Sangon Biotech (Shanghai) Co., Ltd., and the gene sequence synthesis was handed over to Beijing Qingke Biotechnology Co., Ltd.

[0035] Table 1: Primer design

[0036]

[0037]

[0038]

[0039] (2) Construction of expression vector

[0040] The above primers were used for PCR amplification, and the amplified products were recovered by gel agarose gel. The recovered products were subjected to blunt-end T4 ligation to obtain the corresponding plasmid.

[0041] Table 2: PCR amplification system

[0042] System components Ingredient volume Primer F 2.5μL Primer R 2.5μL 2xSuperNova PCR Mix(Dye) 25μL pET30a-PYL plasmid backbone / target gene sequence 1μL <![CDATA[ddH 2 The]]> To 50μL

[0043] After the PCR system was prepared, it was mixed and centrifuged. The PCR amplification conditions were as follows: the first stage was pre-denaturation at 98°C for 30s; the second stage was denaturation at 98°C for 10s, annealing at 50-72°C for 30s, extension at 72°C for 30s / kb, and 33 cycles; the third stage was final extension at 72°C for 2min. The PCR product was recovered using a universal DNA purification kit (Tiangen Biochemical Technology Co., Ltd.) and the operation steps in the product manual were followed.

[0044] Table 3: Gibson connection system

[0045]

[0046]

[0047] After mixing the above components on ice, place in a 50°C heat bath for 60 min. The obtained ligation product is stored on ice or at -20°C for subsequent competent cell transformation.

[0048] The ligation product was transformed into the host bacterium E. coli-DH5α by heat shock method, spread on LB culture resistance plate, incubated at 37°C overnight, randomly picked positive clones, cultured in LB liquid medium, 37°C, 220rpm overnight, and the plasmid was extracted using a plasmid rapid extraction kit to successfully construct plasmids pET30a-intein-Pro.STP (1-13) and pET30a-Pro.STP-14. The plasmid maps are shown in Figure 1 and Figure 2 .

[0049] (3) Construction of engineered bacteria

[0050] The recombinant expression plasmid obtained above was transferred into Escherichia coli competent cells BL 21 (DE3) by heat shock, and positive Escherichia coli genetically engineered bacteria were screened. The specific process was as follows: ① Take 5 μL of the recombinant expression plasmid and add it to 100 μL of Escherichia coli competent cells BL 21 (DE3), and let it stand on ice for 30 minutes; ② heat shock the mixture in a 42°C water bath for 90 seconds, and then quickly put it on ice for 2 minutes; ③ add 500 μL of non-resistant LB liquid culture medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride) to the mixture, and culture it at 37°C, 220rpm for 0.5 h; ④ take 200 μL of the bacterial solution and evenly spread it on an LB solid culture medium plate containing ampicillin resistance (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar, 50 μg / mL kana); ⑤ invert the plate and culture it in a 37°C constant temperature box for about 16 hours, until clearly visible colonies grow, and obtain the corresponding DE3-pET30a-Pro.STP-1~14 engineered bacteria.

[0051] Example 2: Induced expression and purification of engineered bacteria (DE3-pET30a-Pro.STP-1-13)

[0052] The single colony on the plate of Example 1 was placed in LB liquid medium containing kanamycin antibiotics, and cultured at 37°C, 220rpm for 10 hours, which was the primary seed solution. It was inoculated into a new LB medium at a 1% inoculum and cultured at 37°C overnight, which was the secondary seed solution. It was then inoculated into a new LB medium at a 5% inoculum and cultured at 37°C for 2 hours. IPTG was added at a final concentration of 0.5 mM and 18°C ​​for induction expression, and cultured for 20 hours. The bacteria were collected by centrifugation at 4000g and 4°C for 20 minutes.

[0053] Protein purification was carried out using our company's column-free purification method. The bacteria were resuspended to 20 OD / mL with lysis buffer 1 (20 mM Tris-HCl, 1 mM EDTA, 500 mM NaCl, pH 8.5), and 100X protease inhibitor PMSF was added. Then, the cells were disrupted by ultrasonication at 360W for 30 min (ultrasonication for 3 s and intervals for 6 s). 1 mL of the disrupted liquid was centrifuged at 4°C and 10000 g for 10 min to separate the supernatant (ES) and the precipitate (EP). After the EP was washed twice with lysis buffer, an equal volume of buffer 2 ((40 mM Bis-Tris, 2 mM EDTA was dissolved in PBS, pH 6.2) and resuspended, and placed at 28°C for 24 hours to allow the intein to fully self-cleave and the target protein Pro.STP fused with the intein to be cleaved. Then centrifuged at 4°C and 10000g for 10 minutes to separate the supernatant (CS) and the precipitate (CP). The target protein Pro.STP was located in the CS, and the CP was resuspended with an equal volume of buffer 2. Take 80μL CS and CP respectively, add 20μL 5X protein loading buffer, mix and heat in a boiling water bath for 10 minutes for SDS-PAGE running. The results are as follows: Figure 3 As shown, under induction, the constructed engineering bacteria DE3-pET30a-Pro.STP-1-13 all expressed the target protein; according to the grayscale analysis of the SDS-PAGE gel image, the expression levels of the target proteins in different engineering strains are shown in Table 4. It can be seen from Table 4 that the expression level of DE3-pET30a-Pro.STP-6 is 0.19 mg / mL, the expression level of DE3-pET30a-Pro.STP-7 is 0.21 mg / mL, and the expression level of DE3-pET30a-Pro.STP-13 is 0.18 mg / mL. The expression levels of DE3-pET30a-Pro.STP-6, DE3-pET30a-Pro.STP-7 and DE3-pET30a-Pro.STP-13 are higher than those of other engineered strains. The subsequent transdermal test description of the present invention mainly takes DE3-pET30a-Pro.STP-6, DE3-pET30a-Pro.STP-6 and DE3-pET30a-Pro.STP-13 as examples.

[0054] Table 4: Expression of target protein in different engineered strains

[0055]

[0056] The obtained supernatant CS was first removed from impurities using a 30kD ultrafiltration tube, and then concentrated using a 3kD ultrafiltration tube. 80μL of the protein solution from the removal and concentration process was taken, 20μL of 5X protein loading buffer was added, and the mixture was heated in a boiling water bath for 10 minutes for SDS-PAGE running. The results showed that the purity of the target protein obtained by the final 3k concentration was greater than 95%.

[0057] Example 3: Induced expression and purification of engineered bacteria (DE3-pET30a-Pro.STP-14)

[0058] The single colony on the plate of Example 1 was placed in LB liquid medium containing kanamycin antibiotics, and cultured at 37°C, 220rpm for 10 hours, which was the primary seed solution. It was inoculated into a new LB medium at a 1% inoculum and cultured at 37°C overnight, which was the secondary seed solution. It was then inoculated into a new LB medium at a 5% inoculum and cultured at 37°C for 2 hours. IPTG was added at a final concentration of 0.5 mM and 18°C ​​for induction expression, and cultured for 20 hours. The cells were collected by centrifugation at 4000g and 4°C for 20 minutes.

[0059] The cells were resuspended in lysis buffer (20 mM Tris-HCl, 1 mM EDTA, 500 mM NaCl, pH 8.5), 100X protease inhibitor PMSF was added, and then the cells were disrupted by ultrasonication at 360 W for 30 min (3 s for ultrasonication and 6 s for intervals). Then 1 mL of the disrupted liquid was centrifuged at 4 °C and 10000 g for 10 min, 80 μL of supernatant was taken, and the precipitate was resuspended in 1000 μL of lysis buffer.

[0060] The resulting broken liquid was centrifuged at 10000g and 4°C for 30min, the supernatant was collected, the Ni affinity column was washed with clean water, the column was balanced with buffer 1 (25mMTris, 200mM NaCl, pH8.0), the sample was loaded, the impurities were rinsed with a washing buffer containing 20mM imidazole (20mM imidazole, 25mM Tris, 200mM NaCl, pH8.0), the target protein was eluted with a solution containing 250mM imidazole (250mM imidazole, 25mM Tris, 200mM NaCl, pH8.0); the column was washed with a solution containing 1M imidazole, then washed with water, and finally filled with 20% ethanol.

[0061] After the protein solution was purified by Ni affinity column, the high concentration of imidazole was removed by desalting column, and then enterokinase was added to the protein solution, and the His tag was removed at 25°C overnight to collect Pro.STP-14. Take 80μL of purified Pro.STP-14, add 20μL of 5X protein loading buffer, mix and heat in boiling water bath for 10min for SDS-PAGE running. The results are as follows Figure 4 As shown, under induction, the constructed engineering bacteria DE3-pET30a-Pro.STP-14 expressed protein. According to the grayscale analysis of the SDS-PAGE gel image, the protein expression level of DE3-pET30a-Pro.STP-14 was 0.17 mg / mL.

[0062] The Pro.STP-14 collected above was subjected to liquid phase detection, instrument: Agilent 1260 high performance liquid chromatograph, chromatographic column: Agilent TC-C18 column (4.6mm×250mm, 5μm), column oven 35°C; mobile phase A: ultrapure water, mobile phase B: acetonitrile; flow rate 1.0mL / min, injection volume 20μL, detection wavelength 215nm, gradient elution, elution program: 0min, A: 95%, B: 5%; 2.5min, A: 95%, B: 5%; 10min, A: 40%, B: 60%; 12.5min, A: 40%, B: 60%; 12.6min, A: 95%, B: 5%; 15min, A: 95%, B: 5%. The liquid chromatogram has only a single significant peak, and the purity is greater than 99%.

[0063] Example 5: FITC labeling of target protein

[0064] (1) Prepare a protein sample to be cross-linked in 0.1 M sodium carbonate buffer (pH = 9) with a concentration of ≥ 2 mg / mL;

[0065] (2) Dissolve FITC in anhydrous DMSO to prepare a solution with a concentration of 1 mg / mL;

[0066] (3) Mix the protein solution and FITC solution so that the molar ratio of protein to FITC is 1:10 to 1:50. Gently stir the protein solution.

[0067] (4) After the required FITC is added, incubate the reaction solution at 4°C in the dark for 8 h;

[0068] (5) Add NH4Cl to a final concentration of 50 mM and terminate the reaction at 4°C for 2 h;

[0069] (6) Add an appropriate amount of 1×PBS to the above reaction solution, gently blow and mix, transfer the reaction solution to a spin-dried ultrafiltration tube, and centrifuge for 10-30 minutes. Discard the filtrate, add 1×PBS to the ultrafiltration tube to x mL, repeat the centrifugation and ultrafiltration operation 2-3 times until the ultrafiltrate in the collection tube is almost colorless and transparent, and the concentrated solution is the FITC-labeled protein;

[0070] (7) Store the conjugate at 4°C in the dark, add 0.05-0.2% Proclin 300 as a preservative. If the protein concentration is low (<1 mg / mL), add 0.1% BSA as a protein stabilizer) and store at 2-8°C in the dark.

[0071] Example 6: Preparation of pig skin for transdermal testing

[0072] Pig skin for transdermal testing was prepared in accordance with Appendix B of the Pharmaceutical Industry Standard of the People's Republic of China YY / T 0729.1-2009. Hairless pig skin was harvested from the abdominal area and stored in a refrigerator. Before use, the skin surface was cleaned with isopropyl alcohol and cut into strips measuring 38 mm × 467 mm in length, covered with a saline-soaked wipe to prevent the tissue from drying out. The pig skin was placed with the epidermis facing down, and the subcutaneous fat layer was carefully removed to expose the dermis. The treated skin strips were stored in gauze moistened with saline to retain moisture. Finally, the skin was trimmed to the desired size (150 mm × 150 mm) for testing.

[0073] Example 7: Transdermal experiment of superconductor carrier protein Pro.STP carrying bovine serum albumin BAS

[0074] The superconductor carrier protein Pro.STP was dissolved in pure water, and the superconductor carrier protein Pro.STP and FITC-labeled BSA (molecular weight 66 kD) were mixed and placed at 25° C. for 30 min. The experimental group and the blank control group were set according to Table 4.

[0075] Table 5: Experimental design

[0076] Superconductor transport protein a <![CDATA[BSA labeled with FITC b > Pure water total Experimental Group +17.5μL +12.5μL - 30μL Blank control group - +12.5μL +17.5μL 30μL

[0077] a The superconductor carrier proteins are Pro.STP-1-12 and Pro.STP-14, and the final concentration of the added proteins is 50 μM.

[0078] b BSA was added to a final concentration of 10 μM.

[0079] The mixed reagents were evenly dripped onto the prepared pigskin to ensure that the smear reagent formed a uniform film on the surface of the pigskin. After standing for 15 minutes in the dark, the experimental pigskin was cut and the experimental results of superconductor carrier protein carrying BSA transdermal were observed under a fluorescence microscope. The results are shown in Table 6. All Pro.STPs showed the biological function of carrying BSA transdermally relative to the blank control group, mainly using Pro.STP-6 and Pro.STP-7 as examples.

[0080] Table 6: Depth of BSA carried by different Pro.STPs penetrating into pig skin

[0081] Serial number Pro.STP Transdermal depth / μm 1 Pro.STP-1 70 2 Pro.STP-2 79 3 Pro.STP-3 68 4 Pro.STP-4 70 5 Pro.STP-5 75 6 Pro.STP-6 77 7 Pro.STP-7 292 8 Pro.STP-8 254 9 Pro.STP-9 298 10 Pro.STP-10 285 11 Pro.STP-11 264 12 Pro.STP-12 275 13 Pro.STP-14 82 14 Blank control 17

[0082] The transdermal results of Pro.STP-6 and Pro.STP-7 are as follows Figure 5 As shown, Figure 5 A is a fluorescence image of Pro.STP-6 of the present invention carrying bovine serum albumin (BSA) penetrating pig skin; Figure 5 B is a fluorescence image of Pro.STP-7 of the present invention carrying bovine serum albumin (BSA) penetrating pig skin; Figure 5 C is a fluorescence image of bovine serum albumin (BSA) penetrating pig skin using pure water as a control. The fluorescence detection depth of the blank control group was 17 μm, while that of Pro.STP-6 and Pro.STP-7 was 77 μm and 292 μm, respectively. Both Pro.STP-6 and Pro.STP-7 showed the biological function of the transported protein relative to the blank control. In the experimental results of transporting large molecular protein BSA, the large protein BSA in the blank control group only stayed on the surface of pig skin. The penetration depth of Pro.STP-1 composed of pep1 alone was 85μm, which was comparable to the effect of Pro.STP-6 composed of two pep-1s. Pro.STP-7 composed of pep-1 and TAT showed better ability. Pro.STP-7 could carry large molecular protein BSA through the epidermis of pig skin (the epidermis of pig skin is generally 30-120μm) and reach the dermis of pig skin (the thickness is generally 1000-3000μm), while Pro.STP-6 could only carry large molecular protein BSA to the epidermis of pig skin.

[0083] Example 8: Transdermal experiment of nonapeptide-1 carried by superconductor transport protein Pro.STP

[0084] The superconductor carrier protein Pro.STP was dissolved in pure water, and the superconductor carrier protein Pro.STP and FITC-labeled nonapeptide-1 (molecular weight 1.2 kD) were mixed and placed at 25° C. for 30 min. The experimental group and the blank control group were set according to Table 7.

[0085] Table 7: Experimental design

[0086] <![CDATA[Superconductive carrier protein a > <![CDATA[FITC-labeled nonapeptide-1 b > Pure water total Experimental Group +17.5μL +12.5μL - 30μL Blank control group - +12.5μL +17.5μL 30μL

[0087] a The superconductor carrier proteins are Pro.STP-1-12 and Pro.STP-14, and the final concentration of the added proteins is 50 μM.

[0088] b Nonapeptide-1 was added to a final concentration of 694 μM.

[0089] The mixed reagents were evenly dripped onto the prepared pigskin to ensure that the smear reagent formed a uniform film on the surface of the pigskin. After standing for 15 minutes in the dark, the experimental pigskin was cut and the experimental results of the superconductor carrier protein carrying nonapeptide-1 through the skin were observed under a fluorescence microscope. The results are shown in Table 8. All Pro.STPs showed the biological function of carrying nonapeptide-1 through the skin relative to the blank control group, mainly using Pro.STP-6 and Pro.STP-7 as examples.

[0090] Table 8: Depth of nonapeptide-1 carried by different Pro.STPs penetrating into pig skin

[0091]

[0092]

[0093] The transdermal results of Pro.STP-6 and Pro.STP-7 are as follows Figure 6 As shown, Figure 6 A is a fluorescence image of Pro.STP-6 carrying nonapeptide-1 penetrating pig skin; Figure 6 B is a fluorescence image of Pro.STP-7 carrying nonapeptide-1 penetrating pig skin; Figure 6 C is a fluorescence image of the nonapeptide-1 penetrating pig skin using pure water as a control. The fluorescence detection depth of the blank control group was 436μm, while that of Pro.STP-6 was 944μm and that of Pro.STP-7 was 915μm. Both Pro.STP-6 and Pro.STP-7 showed the biological function of the transport protein relative to the blank control. In the experimental results of the small molecule protein nonapeptide-1 transport protein, Pro.STP-6 and Pro.STP-7 showed comparable transport protein capabilities, both of which could carry the small molecule protein nonapeptide-1 to penetrate the epidermis of pig skin, reach the dermis of pig skin or even deeper (data not shown), while the fluorescence of the small molecule polypeptide in the blank control group only stayed in the shallow dermis.

[0094] Example 9: Transdermal experiment of superconductor transport protein Pro.STP carrying type III collagen

[0095] The superconductor carrier protein Pro.STP was dissolved in pure water, and the superconductor carrier protein Pro.STP and FITC-labeled type III collagen (molecular weight 13 kD) were mixed and placed at 25°C for 30 minutes. The experimental group and the blank control group were set according to Table 6.

[0096] Table 9: Experimental design

[0097]

[0098] aThe superconductor carrier proteins were Pro.STP1-14, and the final concentration of the added proteins was 50 μM. When Pro.STP-13 was added, FITC-labeled type III collagen was not added, and the type III collagen fused and expressed by Pro.STP-13 was labeled with FITC.

[0099] b The final concentration of type III collagen added was 64 μM.

[0100] c Glycyrrhizic acid and ethyl acetate were added to a final concentration of 0.5% (v / v)

[0101] The mixed reagents were evenly dripped onto the prepared pigskin to ensure that the smear reagent formed a uniform film on the surface of the pigskin. After standing for 15 minutes in the dark, the experimental pigskin was cut and the experimental results of the superconductor carrier protein carrying type III collagen transdermal were observed under a fluorescence microscope. The results are shown in Table 10. All Pro.STPs showed the biological function of carrying type III collagen transdermal relative to the blank control group, mainly using Pro.STP-6 and Pro.STP-7 as examples.

[0102] Table 10: Depth of type III collagen carried by different Pro.STPs penetrating into pig skin

[0103] Serial number Pro.STP Transdermal depth / μm 1 Pro.STP-1 890 2 Pro.STP-2 952 3 Pro.STP-3 971 4 Pro.STP-4 968 5 Pro.STP-5 949 6 Pro.STP-6 961 7 Pro.STP-7 451 8 Pro.STP-8 460 9 Pro.STP-9 458 10 Pro.STP-10 448 11 Pro.STP-11 432 12 Pro.STP-12 450 13 Pro.STP-13 967 14 Pro.STP-14 962 15 Blank control 165 16 0.5% Glycyrrhizic acid 248 17 0.5% ethyl acetate 201 18 CA glue curing 88

[0104] The transdermal results of Pro.STP-6, Pro.STP-7, Pro.STP-13, and Pro.STP-14 are as follows Figure 7 As shown, Figure 7 The ability of Pro.STP of the present invention to deliver type III collagen observed by fluorescence microscopy was demonstrated. Figure 7 A is a fluorescence image of Pro.STP-6 of the present invention carrying type III collagen penetrating pig skin; Figure 7 B is a fluorescence image of Pro.STP-13 of the present invention carrying type III collagen penetrating pig skin; Figure 7 C is a fluorescence image of Pro.STP-14 of the present invention carrying type III collagen penetrating pig skin; Figure 7 D is a fluorescence image of Pro.STP-7 of the present invention carrying type III collagen penetrating pig skin; Figure 7 E is the fluorescence image of 0.5% glycyrrhizic acid-loaded type III collagen penetrating pig skin; Figure 7 F is the fluorescence image of type III collagen carried by 0.5% ethyl acetate penetrating pig skin; Figure 7 G is a fluorescence image of type III collagen penetrating pig skin with pure water as control; Figure 7H is the fluorescence image of type III collagen solidified by CA glue. The fluorescence detection depth of the blank control group was 165μm, while that of Pro.STP-7 was 451μm, that of Pro.STP-6 was 961μm, that of Pro.STP-13 was 967μm, that of Pro.STP-14 was 962μm, that of 0.5% glycyrrhizic acid was 248μm, that of 0.5% ethyl acetate was 201μm, and that of the CA glue control group was 88μm. Pro.STP-6, Pro.STP-7, Pro.STP-13, and Pro.STP-14 all showed the biological function of the transport protein relative to the blank control. Pro.STP-6, which is composed of two pep-1s, was obtained by column-free purification, Pro.STP-14 was obtained by nickel column purification, and Pro.STP-13 was obtained by fusion expression of type III collagen on the basis of Pro.STP-6. In the experimental results of carrying type III collagen, Pro.STP-6, Pro.STP-13 and Pro.STP-14 showed comparable abilities, and all of them could carry type III collagen to penetrate the epidermis of pig skin, and could reach the dermis of pig skin or even deeper (data not shown). The data showed that the superconductor carrier proteins obtained by different purification methods all had the biological function of carrier proteins, and the abilities of fusion expression and covalent bond formation to transport proteins were comparable; Pro.STP-7, which is composed of pep-1 and TAT, could also carry type III collagen to penetrate the epidermis of pig skin, but its ability was relatively poor compared with Pro.STP-6, Pro.STP-13 and Pro.STP-14. Compared with glycyrrhizic acid and ethyl acetate, which are commonly used reagents on the market to increase the transdermal penetration of cosmetic raw materials, Pro.STP-1~14 has a better ability to carry type III collagen through pig skin.

Claims

1. A superconductor carrier protein Pro.STP, characterized in that Its amino acid sequence is shown in any one of SEQ ID NOs. 1-14.

2. The coding gene of the superconductor carrier protein Pro.STP according to claim 1.

3. The coding gene according to claim 2, characterized in that The nucleotide sequence thereof is shown in any one of SEQ ID NOs.15-28.

4. The expression vector of the superconductor carrier protein Pro.STP according to claim 1.

5. A host bacteria of the superconductor carrier protein Pro.STP as claimed in claim 1.

6. The biosynthesis method of the superconductor carrier protein Pro.STP according to claim 1, characterized in that The steps include: (1) constructing the expression vector according to claim 4 and transforming it into a host bacterium; (2) Cultivating host bacteria and inducing protein expression; (3) Purify the protein to obtain the superconductor carrier protein Pro.STP as claimed in claim 1.

7. Use of the superconductor carrier protein Pro.STP according to claim 1 in transporting target proteins across membranes for non-medical purposes.

8. The use according to claim 7, characterized in that: The superconductor carrier protein Pro.STP is fused with the target protein for expression.

9. The use according to claim 7, characterized in that: The superconductor carrier protein Pro.STP is mixed with the target protein to form a complex, and then carries the target protein across the membrane.

10. The use according to any one of claims 7 to 9, characterized in that: The target protein is BAS, type III collagen or nonapeptide-1.

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