Human-derived XVII type collagen-conotoxin recombinant fusion protein, expression method thereof, expression recombinant plasmid and construction method of human-derived XVII type collagen-conotoxin recombinant fusion protein

Abstract

The invention discloses a human XVII type collagen-conotoxin recombinant fusion protein as well as an expression method, an expression recombinant plasmid and a construction method thereof, and belongs to the technical field of gene engineering. The preparation method comprises the following steps: acquiring a human-derived XVII type collagen gene segment 17A, a mu type conotoxin gene segment and orthologous promoter segments from different methylotrophic yeast by virtue of PCR (Polymerase Chain Reaction) cloning, and connecting the human-derived XVII type collagen gene segment 17A, the mu type conotoxin gene segment and the orthologous promoter segments by virtue of a Gibson Assessment seamless connection technology, so as to construct an expression recombinant plasmid pPIC9K-FMD-mu-CnIIIC-17A; the expression recombinant plasmid can express a human-derived X VII type collagen-conotoxin recombinant fusion protein with excellent cell adhesion activity, cell migration promotion activity and biological activity of promoting tissue regeneration and hair follicle repair and regeneration in a pichia pastoris GS115 expression system. And the phenomena of difficult exocytosis and easy degradation of the current human X VII type collagen are overcome.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a human type XVII collagen-conotoxin recombinant fusion protein and an expression method thereof, an expression recombinant plasmid and a construction method thereof. Background Art

[0002] Conotoxin (CTX) is a class of bioactive peptides composed of 10 to 50 amino acid residues secreted by the venom glands of the venom tube and venom sac of the marine mollusk cone snail. Conotoxins can specifically act on a variety of ion channels and neural receptors, selectively targeting different types of neurotransmitter receptors or voltage-gated ion channels, thereby affecting signal transduction in cells or nerves. Therefore, they are often used as molecular tools for neuroscience research and drug development. According to the highly conserved signal peptide sequence of conotoxins, conotoxins can be divided into 14 superfamilies, including A, M, O, P, S, T, I, V, Y, and J. According to their pharmacological targets, they are further divided into subfamilies such as α, μ, の, K, 8, V, σ, p, γ, vasopressin, convulsant, and sleep peptide.

[0003] Among them, μ-conotoxin (μ-CnIIIC) in the O superfamily is a sequence of 22 amino acids, in which three pairs of disulfide bonds are formed between the amino acids at positions 3 and 15, 4 and 21, and 10 and 22. μ-conotoxin (μ-CnIIIC) has a stable structure and efficient biological activity. It can be used as a blocker of NaV1.4 channels and exhibits potent and long-lasting properties. In addition, it also exhibits biological functions of analgesia, anesthesia and muscle relaxation. This highly specific mechanism of action gives μ-conotoxin (μ-CnIIIC) a unique advantage in promoting cell repair. For example, NaV1.4 channels are widely present in nerve cells and muscle cells, and are involved in the generation and propagation of action potentials. μ-conotoxin (μ-CnIIIC) can specifically act on Nav1.4 channels. It has certain transdermal properties and blocks Nav1.4 channels and inhibits their activity after entering the skin, thereby regulating the microenvironment of hair follicle stem cells, thereby promoting the migration and differentiation of hair follicle stem cells and accelerating the regeneration process of hair follicles.

[0004] Collagen is one of the most important and abundant proteins in mammals. It is a structural protein found in human skin, connective tissue, bones and other tissues. Collagen is generally white, transparent, unbranched fibrils. It is the basic support for skin and bones, accounting for 25% to 35% of the total protein. It is mainly distributed in human skin, blood vessels, bones, tendons, teeth and cartilage. It is the main matrix and scaffold of these tissues, protecting and connecting various tissues, and plays an important physiological function in the body. Therefore, collagen is widely used in industries such as medicine and cosmetics. The human body contains 28 different types of collagen, which are divided into common fibrous collagen and uncommon non-fibrous collagen. Type I and type II in human skin are all fibrous collagen.

[0005] Among non-fibrous collagens, there is a very important collagen subtype, which is type XVII collagen. Natural type XVII collagen is a transmembrane non-fibrogenic collagen. It is a homogeneous trimer composed of three identical α1 (XVII) chains, with a single-chain molecular weight of 180kDa. The N-terminus is located in the cytoplasm and the C-terminus is located in the extracellular matrix. It is divided into three major structural domains: intracellular, transmembrane, and extracellular. It is mainly expressed in epidermal basal keratinocytes. Human type XVII collagen is an important component of hemidesmosomes in cells. It plays an important role in maintaining the tight connection between the basement membrane and the extracellular matrix. It can regulate the adhesion, separation and developmental differentiation of epithelial cells, and plays an important role in the differentiation and regeneration of keratinocytes. Studies have shown that the expression of type XVII collagen can help inhibit the aging of hair follicle stem cells, thereby delaying hair loss. Not only that, in terms of anti-aging, studies have found that type XVII collagen can promote the removal of senescent cells, thereby delaying the aging of the body.

[0006] However, the content of type XVII collagen in the human body is extremely low, and the content of type XVII collagen in animals is also extremely rare. It is very difficult to extract and cannot be mass-produced. Animal-derived collagen products inevitably have problems such as animal-derived disease infection, immune rejection or allergic reactions, and production capacity limitations. Therefore, type XVII collagen, especially human type XVII collagen, can only be used in scientific research and cannot be widely used, further limiting the functional research and market application of human type XVII collagen.

[0007] Recombinant collagen produced by genetic engineering technology generally has the same properties as human collagen, and recombinant collagen has the advantages of strong water solubility, high biological activity, low non-immunogenicity and high biocompatibility. Therefore, the production of human type XVII collagen and its recombinant fusion protein using biotechnology such as genetic engineering is feasible, effective and relatively superior. However, transmembrane proteins are generally not secreted outside the cell when expressed, but are fixed on the cell membrane. Type XVII collagen is a transmembrane non-fibroblastic collagen with a total length of 1497 amino acids, including intracellular domains, transmembrane domains and extracellular domains, which are further divided into 16 non-triple helical regions and 15 triple helical regions, which are staggered. Therefore, in theory, it is difficult to effectively secrete it outside the cell and it is easy to degrade. When using biotechnology to produce human type XVII collagen and its recombinant fusion protein, it is also necessary to focus on the selection of related sequences.

[0008] In existing biotechnology, the E. coli system is generally used for secretory expression of human type XVII collagen. For example, the Chinese patent with application number CN118324899A and application date June 13, 2024 discloses a recombinant type XVII humanized collagen, a preparation method and its application. A humanized type XVII collagen gene is designed based on the characteristic sequence of collagen Gly-XY, and amplified and cloned into the pPIC9K vector after codon optimization. After enzyme linearization, it is transformed into E. coli cells for expression to obtain the target protein. The prepared type XVII humanized collagen has good hydrophilicity, high protein expression level and stability, and the obtained protein has better cell adhesion and proliferation performance and other biological activities for commercially available type XVII collagen. However, since E. coli is a prokaryotic organism and cannot perform post-translational modification on proteins, it is difficult for the E. coli system to express triple-helical collagen with a natural higher-order structure. The protein obtained by expression in E. coli also has problems such as endotoxin contamination and low biological activity.

[0009] Pichia pastoris protein overcomes the defects of collagen expressed by animal sources and Escherichia coli. The yeast expression system has molecular chaperones and enzymes for post-translational modification of proteins (such as glycosylation, hydroxylation, acetylase, etc.), which are very suitable for the formation of triple-helical collagen with a higher structure. At the same time, since little is known about the amino acid sequence, structure, function, etc. of type XVII collagen in existing research, how to select an amino acid sequence so that it can achieve more biological functions while maintaining the advantages of the Pichia pastoris expression system (especially secretory expression) is an outstanding difficulty. For example, a Chinese patent with application number CN116640205A and application date May 26, 2023 discloses recombinant type XVII collagen and its expression strain. It uses a Pichia pastoris expression system, selects truncated sequences of the non-helical region NC16, the C15 helical region and the C-terminus of type XVII collagen for splicing and combination to obtain a high-copy Pichia pastoris genetically engineered bacterium expressing recombinant type XVII collagen. The expressed recombinant type XVII collagen has excellent cell proliferation, migration and adhesion properties that promote human hair follicle stem cells and human fibroblasts, and can be used to repair skin and hair follicle tissues and enhance the resistance of skin and hair follicles.

[0010] Combining the rational transformation strategy of protein engineering, rationally designing recombinant human type XVII collagen with high biological performance and achieving its efficient expression in heterologous hosts will greatly contribute to the application of type XVII collagen in industrial production. However, the current research on recombinant human type XVII collagen mainly focuses on the collagen itself, and there is still a large gap in the research on functional fusion proteins of human type XVII collagen. Therefore, the use of genetic engineering technology to construct genetically engineered strains and further explore and study high-quality functional fusion proteins of human type XVII collagen is of great significance to promoting the application of rare human-like collagen in daily chemicals, skin care, health care and medical fields. Summary of the invention

[0011] In order to solve the problems existing in the prior art, the present invention provides a human type XVII collagen-conotoxin recombinant fusion protein and its expression method and recombinant plasmid. The recombinant plasmid can efficiently secrete and express the human type XVII collagen-conotoxin recombinant fusion protein with excellent cell adhesion activity, cell migration promotion activity, and biological activity of promoting tissue regeneration and hair follicle repair and regeneration through Pichia pastoris cells GS115.

[0012] The technical solution of the present invention is as follows: One of the purposes of the present invention is to provide a human type XVII collagen-conotoxin recombinant fusion protein and an expression method thereof, an expression recombinant plasmid and a construction method, comprising the following steps: optimizing the amino acid sequence of the α1 chain of human type XVII collagen according to the codon preference of Pichia pastoris, analyzing and selecting stable fragments in the extracellular domain 489-1497aa for splicing, obtaining a human type XVII collagen gene fragment 17A with an amino acid sequence as shown in SEQ ID NO.1, and selecting a μ-type conotoxin gene fragment derived from conus and an orthologous promoter fragment derived from different methylotrophic yeasts, obtaining the above three connection fragments by PCR cloning and mixing, connecting the human type XVII collagen gene fragment 17A, the μ-type conotoxin gene fragment and the orthologous promoter fragment by Gibson Assembly seamless connection technology, and constructing a recombinant plasmid pPIC9K-FMD-μ-CnIIIC-17A. Furthermore, the nucleotide sequence of the human type XVII collagen gene fragment 17A is shown in SEQ ID NO.2.

[0013] Furthermore, the amino acid sequence of the μ-type conotoxin gene fragment derived from cone snail is shown as SEQ ID NO.3, and the nucleotide sequence thereof is shown as SEQ ID NO.4.

[0014] Further, the orthologous promoter fragment is derived from Pichia pastoris AOX1 promoter or Hansenula polymorpha FMD promoter. Furthermore, the Hansenula polymorpha The nucleotide sequence of the FMD promoter is shown in SEQ ID NO.5.

[0015] Furthermore, primers pPIC9K-F / R, μ-CnIIIC -F / R, 17A-F / R and FMD-F / R were used to perform PCR cloning on the human type XVII collagen gene fragment 17A, the μ-type conotoxin gene fragment and the orthologous promoter fragment, respectively, to obtain three connecting fragments.

[0016] Furthermore, the nucleotide sequence of pPIC9K-F is shown in SEQ ID NO.6; The nucleotide sequence of the primer pPIC9K-R is shown in SEQ ID NO.7; The nucleotide sequence of the primer μ-CnIIIC-F is shown in SEQ ID NO.8; The nucleotide sequence of the primer μ-CnIIIC-R is shown in SEQ ID NO.9; The nucleotide sequence of primers 17A-F is shown in SEQ ID NO.10; The nucleotide sequence of the primer 17A-R is shown in SEQ ID NO.11; The nucleotide sequence of the primer FMD-F is shown in SEQ ID NO.12; The nucleotide sequence of the primer FMD-R is shown in SEQ ID NO.13.

[0017] Furthermore, the human type XVII collagen fragment 17A, the μ-type conotoxin fragment, the orthologous promoter fragment and the expression vector pPIC9K fragment in the connection fragment are mixed at a concentration ratio of 3:4:2:1.

[0018] Furthermore, the Gibson Assembly seamless connection and expression recombinant plasmid construction process is as follows: The ligated fragment mixture including human type XVII collagen fragment 17A, μ-conotoxin fragment, promoter fragment and pPIC9K expression vector backbone fragment was incubated in a metal bath at 45-55°C for 0.5-1.5 h, and then the ligated fragment mixture was transformed into Escherichia coli DH5α competent cells by heat shock method. After overnight incubation, the positive single clones grown on plates containing sodium ampicillin and kanamycin were picked for preliminary verification by colony PCR. Finally, the successfully verified single colony was inoculated in LB test tubes, and the plasmid was extracted and sequenced for confirmation to construct the expression recombinant plasmid pPIC9K-FMD-μ-CnIIIC-17A.

[0019] Furthermore, the successfully verified single colony was inoculated in a 5 mL LB test tube and cultured at 37° C. for 16 h.

[0020] The second object of the present invention is to provide a recombinant plasmid pPIC9K-FMD-μ-CnIIIC-17A for expressing human type XVII collagen-conotoxin recombinant fusion protein.

[0021] The third object of the present invention is to provide a method for expressing a human type XVII collagen-conotoxin recombinant fusion protein, comprising the following steps: (1) Prepare competent cells according to the Pichia pastoris sorbitol method, and linearize the expression recombinant plasmid pPIC9K-FMD-μ-CnIIIC-17A fragment and yeast competent cells. P. pastoris After mixing, GS115 was quickly transferred to the electroporation cup, and the recombinant plasmid fragment was transformed by electroporation to enter the competent cells. P. pastoris GS115; (2) Transform the competent cells P. pastorisGS115 bacterial solution was spread on MD plates and cultured until a single colony appeared on the plate. A single colony was selected for culture and genome extraction for PCR detection and sequencing verification. (3) Screening for high-copy recombinant strains of the recombinants that were verified to be correct by sequencing, and then transfer them to YPD liquid medium for overnight culture and activation; (4) Transfer the overnight activated bacterial solution to BMGY liquid medium at a 1% inoculum volume and culture for 24 h until the OD 600 When the concentration reaches 1.0 to 1.2, the bacteria are collected, and then suspended and centrifuged in BMMY liquid culture medium to collect the bacteria and ferment and culture them. Methanol is added to the culture medium every 24 hours to induce the expression of the human type XVII collagen-conotoxin recombinant fusion protein.

[0022] Furthermore, 1 μg of the linearized expression recombinant plasmid pPIC9K-FMD-μ-CnIIIC-17A fragment in step (1) was mixed with 100 μL of yeast competent cell GS115, and then quickly transferred to an electroporation cup for electroporation transformation to allow the recombinant plasmid fragment to enter the competent cell GS115.

[0023] Furthermore, in step (2), the transformed bacterial solution is spread on the MD plate and then inverted in a 30° C. constant temperature incubator for 3-4 days.

[0024] Furthermore, the screening step in step (3) is to transfect the recombinant bacteria grown on the MD plate onto a YPD plate with a G418 concentration of 500 μg / mL-4 mg / mL for screening, and activate the recombinant bacteria that can grow on the high-concentration G418 plate and then preserve the bacteria.

[0025] Furthermore, the YPD liquid culture medium culture conditions in step (3) are 30°C, 220 rpm overnight culture for 16 h.

[0026] Furthermore, in step (4), the overnight activated bacterial solution is transferred to a total system of 5 mL of BMGY liquid culture medium at a 1% inoculation rate, cultured at 30°C and 220 rpm for 24 h, and then centrifuged at 5000 rpm for 10 min at 4°C to collect the bacteria, and the centrifuged bacteria are suspended in 50 mL of BMMY liquid culture medium and fermented at 30°C and 220 rpm, and methanol with a final concentration of 0.5-1% is added to the culture solution every 24 h to induce expression.

[0027] The fourth object of the present invention is to provide a human type XVII collagen-conotoxin recombinant fusion protein.

[0028] Compared with the prior art, the beneficial effects of the present invention are: 1. The present invention innovates an expression recombinant plasmid that can be used to express human type XVII collagen-conotoxin recombinant fusion protein. By analyzing and selecting the stable human type XVII collagen gene fragment 17A, the μ-type conotoxin gene fragment derived from conus, and the orthologous promoter fragments derived from different methylotrophic yeasts, a new expression recombinant plasmid pPIC9K-FMD-μ-CnIIIC-17A was constructed. The expression recombinant plasmid overcomes the difficulty of extracellular secretion and easy degradation of human type XVII collagen as a transmembrane protein, and effectively improves the expression amount of human type XVII collagen-conotoxin recombinant fusion protein.

[0029] 2. The present invention discloses for the first time a human type XVII collagen-conotoxin recombinant fusion protein, which has both the unique advantages of μ-conotoxin (μ-CnIIIC) in promoting cell repair and the important role of human type XVII collagen in regulating the adhesion, separation and developmental differentiation of epithelial cells. On the basis of retaining the same properties of the original human collagen, it is endowed with the advantages of strong water solubility, high biological activity, low non-immunogenicity and high biocompatibility through genetic engineering technology. It has excellent cell adhesion activity, cell migration promotion activity, and biological activity of promoting tissue regeneration and hair follicle repair and regeneration, which is helpful for the repair of skin and hair follicle tissues, improves the resistance of skin and hair follicles, inhibits the aging of hair follicle stem cells and thus delays hair loss. The human type XVII collagen-conotoxin recombinant fusion protein fills the gap in the current research reports on human type XVII collagen functionalized fusion protein.

[0030] 3. The present invention provides a method for expressing a human type XVII collagen-conotoxin recombinant fusion protein, combining a protein engineering rational transformation strategy to rationally design a recombinant human type XVII collagen with high biological performance, and achieve its efficient expression in a heterologous host. The expression method selects Pichia pastoris GS115, which is conducive to the formation of high-order triple-helical collagen, as an expression system, and transforms the constructed expression recombinant plasmid pPIC9K-FMD-μ-CnIIIC-17A into competent cells through electroporation. P. pastoris GS115. The recombinant fusion protein expressed in this way not only has higher yield and better solubility, but also significantly reduces the degradation of the recombinant fusion protein in the later stage of fermentation, and also has the inherent biocompatibility of natural collagen and biosafety with low immune rejection reaction. The expression method has simple operation steps, can realize the industrial production of functional recombinant fusion proteins, and is suitable for wide application in the fields of biomedical materials, tissue engineering, cosmetics and food. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1This is a schematic diagram of the structure of the expression recombinant plasmid pPIC9K-FMD-μ-CnIIIC-17A of the present invention; Figure 2 The electrophoresis result of the human type XVII collagen-conotoxin recombinant fusion protein described in the performance test of the present invention, wherein M is a marker, and lanes 1-4 are respectively schematic diagrams of the expression of the supernatant of the human type XVII collagen-conotoxin recombinant fusion protein fermented for 24h, 48h, 72h and 96h; Figure 3 This is a comparison diagram of the effects of human type XVII collagen-conotoxin recombinant fusion protein and fetal bovine serum (BSA) on the relative adhesion of human hair follicle stem cells in the performance test of the present invention; Figure 4 This is a comparison diagram of the effects of human type XVII collagen-conotoxin recombinant fusion protein and fetal bovine serum (BSA) on the relative adhesion of human fibroblasts in the performance test of the present invention; Figure 5 This is a comparison diagram of the effects of human type XVII collagen-conotoxin recombinant fusion protein and fetal bovine serum (BSA) on the migration rate of human hair follicle stem cells in the performance test of the present invention; Figure 6 This is a comparison diagram of the effects of human type XVII collagen-conotoxin recombinant fusion protein and fetal bovine serum (BSA) on the migration rate of human fibroblasts in the performance test of the present invention; Figure 7 This is a comparison chart of the effects of human type XVII collagen-conotoxin recombinant fusion protein and fetal bovine serum (BSA) on the proliferation rate of human hair follicle stem cells in the performance test of the present invention; Figure 8 This is a comparison chart of the effects of human type XVII collagen-conotoxin recombinant fusion protein and fetal bovine serum (BSA) on the proliferation rate of human fibroblasts in the performance test of the present invention. DETAILED DESCRIPTION

[0032] The present invention is further described below in conjunction with preferred embodiments. The endpoints of the ranges disclosed in the present invention and any values ​​are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be deemed to be specifically disclosed herein. The experimental methods in the following examples, unless otherwise specified, are conventional methods and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0033] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources. In the following examples, the gene fragments human type XVII collagen fragment 17A, μ-conotoxin fragment, and FMD promoter fragment were sent to Nanjing GenScript Biotech Co., Ltd. for full sequence synthesis.

[0034] Example 1 This embodiment provides a method for constructing a recombinant plasmid expressing a human type XVII collagen-conotoxin recombinant fusion protein, comprising the following steps: S1. The nucleotide sequence of the α1 chain of human type XVII collagen was optimized and synthesized according to the codon preference of Pichia pastoris, and the human type XVII collagen gene fragment 17A was obtained by analyzing and selecting stable fragments for splicing. The amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2; S2, select the μ-type conotoxin gene fragment derived from cone snail, the amino acid sequence is shown in SEQ ID NO.3, the nucleotide sequence is shown in SEQ ID NO.4, and the Hansenula polymorpha FMD promoter fragment; S3, using primers pPIC9K-F / R, μ-CnIIIC -F / R, 17A-F / R and FMD-F / R to perform PCR cloning, respectively, to obtain human type XVII collagen fragment 17A, μ-conotoxin fragment and FMD promoter fragment as connecting fragments; S4, the cloned connection fragments were mixed with the expression vector pPIC9K fragment at a concentration ratio of 3:4:2:1, and the human type XVII collagen fragment 17A, μ-type conotoxin fragment and FMD promoter fragment were connected to the expression vector pPIC9K fragment using the Gibson Assembly seamless connection technology. The connection mixture was incubated in a 50°C metal bath for 1 h, and then the connection fragment mixture was transformed into Escherichia coli DH5α competent cells by heat shock method, wherein the metal bath incubation conditions can be adjusted to incubate at 45-55°C for 0.5-1.5 h according to actual operation; S5. After overnight culture at 37°C, the positive single clones grown on the plates containing sodium ampicillin and kanamycin were picked for preliminary verification by colony PCR. The verification primers used were TES-F / R. The successfully verified single colonies were inoculated in 5 mL LB test tubes and cultured at 37°C for 16 h. After the plasmid was extracted, sequencing was performed to confirm whether the recombinant gene was consistent with the design. After confirmation, the expression recombinant plasmid pPIC9K-FMD-μ-CnIIIC-17A was obtained.

[0035] Example 2 This embodiment provides a method for expressing a human type XVII collagen-conotoxin recombinant fusion protein, comprising the following steps: S1. Activate the bacterial liquid of Pichia pastoris GS115 by streaking on a YPD plate, culture in a 30°C incubator until a single colony grows, pick a single colony and inoculate it into 5 mL YPD liquid medium, and culture it at 30°C and 220 rpm for 18 h; inoculate the seed liquid into 50 mL YPD at a 1% inoculum, culture it until OD600 is 1.4, collect the bacterial liquid and centrifuge it at 4°C and 5000 rpm for 5 min, discard the supernatant, repeat twice, resuspend the bacterial cells with 1 M / L D-sorbitol solution and dispense into small portions at 100 μL / tube; S2, take 1 μg of the linearized expression recombinant plasmid pPIC9K-FMD-μ-CnIIIC-17A fragment prepared in Example 1, mix it with 100 μL of Pichia pastoris competent GS115 in S1, and quickly transfer it to a 0.2 cm pre-cooled electroporation cup after mixing, place it on ice for 5 minutes, and then perform electroporation transformation under the conditions of 2 kV electric shock voltage, 25 μF capacitance, 200 Ω resistance, and 5 msec electric shock time; S3. Immediately after the electroporation, add 650 μL of pre-cooled 1M sorbitol solution to the electroporation cup, gently blow and mix, and quickly transfer to a 1.5mL centrifuge tube. Place it in a 30℃ constant temperature incubator and culture it for 1.5 hours to allow the recombinant plasmid fragment to enter the competent cells. P. pastoris GS115; S4. Take 100 μL of the recombinant strain and spread it on the MD plate, then invert it and culture it in a 30°C constant temperature incubator for 3 days until a single colony appears on the plate. Pick a single colony for culture, extract the genome, perform PCR verification, and then send it for sequencing to verify whether the transformation is successful. S5. The recombinant bacteria grown on the MD plate were respectively transfected on the YPD plate with a G418 concentration of 500 μg / mL-4 mg / mL for selection, and the recombinant bacteria that can grow on the high-concentration G418 plate were selected for activation and preservation; S6. Pick a verified recombinant single colony and inoculate it into a new 5 mL YPD liquid medium. Incubate it at 30°C, 220 rpm overnight for 16 h. Then transfer the overnight activated bacterial solution to 5 mL BMGY liquid medium with a total system of 1% inoculation volume. Incubate it at 30°C, 220 rpm for 24 h. Centrifuge it at 4°C, 5000 rpm for 10 min to collect the bacteria. S7. Use 50 mL of BMMY liquid culture medium to suspend the collected bacteria by centrifugation, and ferment and culture them at 30°C and 220 rpm. Add methanol at a final concentration of 0.7% to the culture medium every 24 hours to induce the expression of human type XVII collagen-conotoxin recombinant fusion protein.

[0036] The final methanol concentration can be adjusted to 0.5% or 1% according to actual operation.

[0037] Performance Testing 1. Expression test According to the method described in Example 2, the expression of human type XVII collagen-conotoxin recombinant fusion protein was induced, and 4 test groups were set up, and the expression was induced for 24h, 48h, 72h and 96h respectively. Then the culture medium was centrifuged at 4°C, 12000rpm for 10min, and the supernatant was collected for SDS-PAGE protein electrophoresis to verify the protein expression.

[0038] The experimental results are shown in Figure 2 : M is Marker; Lane 1 is the supernatant of 24h fermentation of human type XVII collagen-conotoxin recombinant fusion protein, Lane 2 is the supernatant of 48h fermentation of human type XVII collagen-conotoxin recombinant fusion protein, Lane 3 is the supernatant of 72h fermentation of human type XVII collagen-conotoxin recombinant fusion protein, Lane 4 is the supernatant of 96h fermentation of human type XVII collagen-conotoxin recombinant fusion protein.

[0039] The protein molecular weight of the human type XVII collagen-conotoxin recombinant fusion protein is slightly above the 20 kDa position, which is consistent with the protein theoretical molecular weight of the human type XVII collagen-conotoxin recombinant fusion protein of 21 kDa.

[0040] 2. Cell adhesion test The centrifugation method was used, and human hair follicle stem cells and human fibroblasts were used as experimental cells to detect the ability of the human type XVII collagen-conotoxin recombinant fusion protein induced and expressed in Example 2 and BSA to promote cell adhesion.

[0041] The test results are as follows Figure 3 and 4 As shown. Figure 3-4 From the comparison chart of the effects on relative adhesion, it can be seen that the human type XVII collagen-conotoxin recombinant fusion protein can exhibit a better ability to promote cell adhesion than BSA.

[0042] 3. Cell migration rate test The cell migration movement was determined by cell scratch method. Human hair follicle stem cells and human fibroblasts were used as experimental cells to detect the ability of human type XVII collagen-conotoxin recombinant fusion protein and BSA induced and expressed in Example 2 to promote cell migration. The test results are as follows Figure 5 and 6 shown.

[0043] Depend on Figure 5-6 From the comparison chart of the effects on cell migration rate, it can be seen that the human type XVII collagen-conotoxin recombinant fusion protein can show a better ability to promote cell migration than BSA.

[0044] 4. Cell proliferation rate test The MTT method was used to detect the ability of the human type XVII collagen-conotoxin recombinant fusion protein and BSA induced and expressed in Example 2 to promote cell proliferation using human hair follicle stem cells and human fibroblasts as experimental cells. Figure 7 and 8 shown.

[0045] Depend on Figure 7-8 From the comparison chart of the effects on cell proliferation rate, it can be seen that the human type XVII collagen-conotoxin recombinant fusion protein exhibits a better ability to promote cell proliferation than BSA.

[0046] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for constructing a recombinant plasmid expressing a human type XVII collagen-conotoxin recombinant fusion protein, characterized in that: The method comprises the following steps: optimizing the amino acid sequence of the α1 chain of human type XVII collagen according to the codon preference of Pichia pastoris, analyzing and selecting stable fragments in the extracellular domain 489-1497aa for splicing, obtaining a human type XVII collagen gene fragment 17A with an amino acid sequence as shown in SEQ ID NO.1, selecting a μ-type conotoxin gene fragment derived from cone snails and orthologous promoter fragments derived from different methylotrophic yeasts, obtaining the above three connection fragments by PCR cloning and mixing them, connecting the human type XVII collagen gene fragment 17A, the μ-type conotoxin gene fragment and the orthologous promoter fragment by Gibson Assembly seamless connection technology, and constructing a recombinant plasmid pPIC9K-FMD-μ-CnIIIC-17A.

2. The method for constructing a recombinant plasmid expressing a human type XVII collagen-conotoxin recombinant fusion protein according to claim 1, characterized in that: The nucleotide sequence of the human type XVII collagen gene fragment 17A is shown as SEQ ID NO.

2.

3. The method for constructing a recombinant plasmid expressing a human type XVII collagen-conotoxin recombinant fusion protein according to claim 1, characterized in that: The amino acid sequence of the μ-type conotoxin gene fragment derived from cone snail is shown in SEQ ID NO.3, and the nucleotide sequence thereof is shown in SEQ ID NO.

4.

4. The method for constructing a recombinant plasmid expressing a human type XVII collagen-conotoxin recombinant fusion protein according to claim 1, characterized in that: The orthologous promoter fragment is derived from Pichia pastoris AOX1 promoter or Hansenula polymorpha FMD promoter.

5. The method for constructing a recombinant plasmid expressing a human type XVII collagen-conotoxin recombinant fusion protein according to claim 1, characterized in that: Primers pPIC9K-F / R, μ-CnIIIC -F / R, 17A-F / R and FMD-F / R were used to perform PCR cloning on human type XVII collagen gene fragment 17A, μ-conotoxin gene fragment and orthologous promoter fragment to obtain three connecting fragments.

6. The method for constructing a recombinant plasmid expressing a human type XVII collagen-conotoxin recombinant fusion protein according to claim 1, characterized in that: The human type XVII collagen fragment 17A, the μ-type conotoxin fragment, the orthologous promoter fragment and the expression vector pPIC9K fragment in the connection fragment are mixed at a concentration ratio of 3:4:2:

1.

7. The method for constructing a recombinant plasmid expressing a human type XVII collagen-conotoxin recombinant fusion protein according to claim 1, characterized in that: The Gibson Assembly seamless connection and plasmid construction process is as follows: A ligated fragment mixture including human type XVII collagen fragment 17A, μ-conotoxin fragment, promoter fragment and pPIC9K expression vector backbone fragment was incubated in a metal bath at 45-55°C for 0.5-1.5 h, and then the ligated fragment mixture was transformed into Escherichia coli DH5α competent cells by heat shock method. After overnight incubation, positive single clones grown on plates containing sodium ampicillin and kanamycin were picked for preliminary verification by colony PCR. Finally, the successfully verified single colony was inoculated in an LB test tube, and the plasmid was extracted and sequenced for confirmation to construct the recombinant plasmid pPIC9K-FMD-μ-CnIIIC-17A.

8. A recombinant plasmid pPIC9K-FMD-μ-CnIIIC-17A for expressing human type XVII collagen-conotoxin recombinant fusion protein constructed by the method according to any one of claims 1 to 7.

9. A method for expressing human type XVII collagen-conotoxin recombinant fusion protein using the expression recombinant plasmid pPIC9K-FMD-μ-CnIIIC-17A as claimed in claim 8, characterized in that: The steps include: (1) Prepare competent cells according to the Pichia pastoris sorbitol method, and linearize the expression recombinant plasmid pPIC9K-FMD-μ-CnIIIC-17A fragment and yeast competent cells. P. pastoris After mixing, GS115 was quickly transferred to the electroporation cup, and the recombinant plasmid fragment was transformed by electroporation to enter the competent cells. P. pastoris GS115; (2) Transform the competent cells P. pastoris GS115 bacterial solution was spread on MD plates and cultured until a single colony appeared on the plate. A single colony was selected for culture and genome extraction for PCR detection and sequencing verification. (3) Screening for high-copy recombinant strains of the recombinants that were verified to be correct by sequencing, and then transfer them to YPD liquid medium for overnight culture and activation; (4) Transfer the overnight activated bacterial solution to BMGY liquid medium at a 1% inoculum volume and culture for 24 h until the OD 600 When the concentration reaches 1.0 to 1.2, the bacteria are collected, and then suspended and centrifuged in BMMY liquid culture medium to collect the bacteria and ferment and culture them. Methanol is added to the culture medium every 24 hours to induce the expression of the human type XVII collagen-conotoxin recombinant fusion protein.

10. A human type XVII collagen-conotoxin recombinant fusion protein expressed by the method of claim 9.

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