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WO2024229988A1 - 生物合成重组人源化纤连蛋白以及制备方法 - Google Patents

生物合成重组人源化纤连蛋白以及制备方法 Download PDF

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WO2024229988A1
WO2024229988A1 PCT/CN2023/110757 CN2023110757W WO2024229988A1 WO 2024229988 A1 WO2024229988 A1 WO 2024229988A1 CN 2023110757 W CN2023110757 W CN 2023110757W WO 2024229988 A1 WO2024229988 A1 WO 2024229988A1
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materials
polypeptide
composition
amino acid
cell
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PCT/CN2023/110757
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English (en)
French (fr)
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张永健
张国梁
赵雪
何振瑞
杨霞
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山西锦波生物医药股份有限公司
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Publication of WO2024229988A1 publication Critical patent/WO2024229988A1/zh

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  • the present invention relates to the field of synthetic biotechnology, and in particular to a biosynthetic method of recombinant humanized fibronectin.
  • Reactive oxygen species also participate in cell signal regulation, including inducing cell apoptosis after activation of the p38MAPK signaling pathway, leading to cell growth stagnation.
  • the aging of the immune system is parallel to the aging of the body. Studies have shown that the number and function of immune cells in the elderly are damaged to varying degrees. The immune system can not only protect the body from the invasion of pathogens, but also remove aging cells in time to maintain the stability of the body's internal environment.
  • integrins also called integrins
  • FN fibronectin
  • mice have shown that the reduction of FN levels in aged skeletal muscle stem cells (MuSCs) leads to a lack of quantity (only a significant number is included in the young group), function and muscle maintenance.
  • the restoration of FN in aged mice also restores the phenotype, level, muscle maintenance and function of skeletal muscle stem cells to the young group.
  • FN is the preferred adhesion substrate of MuSCs, regulating the aging pathway of p38MAPK through integrins and focal adhesion kinase (FAK). Restoring attachment to FN in the aged niche reactivates FAK signaling in MuSCs, thereby restoring the regenerative capacity of aged skeletal muscle.
  • FN also exhibits a conditioning effect, aimed at promoting the absorption of tissue debris by phagocytes and providing a good internal and external environment for the metabolism of new cells.
  • Fibronectin is a high molecular weight glycoprotein with a molecular weight of about 450 Daltons, which is widely present in blood, body fluids and various tissues. It is a dimer composed of two subunits with a molecular weight of 220 000 Daltons connected by disulfide bonds. The entire molecule is composed of two similar A chains and B chains, which are V-shaped.
  • fibronectin is a multi-domain glycoprotein composed of a series of multiple repeating module structures, 12 FNI type repeat sequences (FN I), 2 FNII type repeat sequences (FN II), 15 constitutively expressed and 2 alternatively spliced FN III type (FN III) repeat sequences, and a non-homologous variable (V) or type III connecting segment (IIICS) region.
  • the multi-module structure and inter-module region make the fibronectin (FN) molecule flexible and participate in regulating its function.
  • fibronectin FN
  • FN fibronectin
  • fibronectin Due to its unique value, some domestic biotechnology companies have also launched some beauty products containing fibronectin (FN). Therefore, fibronectin is widely used in beauty, medicine and other fields. In recent years, with the development of functional skin care products, China has discovered that fibronectin has unique effects in medical beauty during the process of exploring the medical application of fibronectin. Dermatologists have studied the application of fibronectin in functional skin care, but there are currently no medical device products.
  • a fibronectin that can be used as a medical device, and it has broad application prospects in beauty aspects such as wrinkle removal, freckle lightening and whitening, and in medical fields such as tumors, anti-infection, and skin wound repair.
  • the inventors prepared different human fibronectin The inventors have found that different human fibronectin fragments encounter technical problems such as inability to perform enzyme cleavage, low expression levels, and poor fragment stability during recombinant expression. After a series of screening, the inventors obtained a human fibronectin fragment that is easy to recombinantly express, has a high expression level, and has high stability. Surprisingly, the inventors also found that the human fibronectin fragment has excellent cell adhesion activity.
  • the inventors provide a polypeptide: SSSGPVEVFITETPSQPNSHPIQWNAPQPSHISKYILRWRPKNSVGRWKEATIPGHLNSYTIKGLKPGVVYEGQLISIQQYGHQEVTRFDFTTTSTSTP.
  • the polypeptide is derived from human fibronectin, is a natural polypeptide, and does not cause an immune response in the human body.
  • the polypeptide can be a biosynthesized recombinant humanized fibronectin.
  • the inventors generated the polypeptide described herein by genetic engineering methods. Specifically, the inventors achieved the generation of the polypeptide by functional region screening, strain construction; large-scale biological fermentation culture and protein induced expression; purification of humanized fibronectin and optional enzyme cleavage. The inventors found that compared with other peptide segments of human fibronectin, the polypeptide of the present invention is easy to purify, stable, and has the effect of promoting cell adhesion.
  • polypeptide comprising the following amino acid sequence:
  • SEQ ID NO: 1 SSSGPVEVFITETPSQPNSHPIQWNAPQPSHISKYILRWRPKNSVGRWKEATIPGHLNSYTIKGLKPGVVYEGQLISIQQYGHQEVTRFDFTTTSTSTP;
  • the polypeptide is derived from human fibronectin.
  • amino acid substitutions are conservative amino acid substitutions.
  • the amino acid additions are referenced to the amino acid additions of human fibronectin, such that the polypeptide is a native fragment of human fibronectin.
  • the polypeptide has the function of fibronectin.
  • the present invention provides a fusion protein comprising one or more polypeptides described herein as repeating units.
  • the repeating units can be directly or indirectly connected through a linker.
  • the fusion protein also comprises another proteinaceous part, such as a polypeptide having a beauty/make-up function.
  • nucleic acids are provided herein that encode a polypeptide or fusion protein as described herein.
  • the nucleic acid comprises the nucleotide sequence shown in SEQ ID NO:2.
  • vectors comprising a nucleic acid as described herein.
  • the vector comprises nucleotides encoding a purification tag, nucleotides encoding a leader, and/or a regulatory element.
  • the purification tag is selected from a His tag, a GST tag, an MBP tag, a SUMO tag or a NusA tag.
  • the regulatory element is selected from a promoter, a terminator and/or an enhancer.
  • a host cell comprising a nucleic acid as described herein or a vector as described herein.
  • the host cell is a eukaryotic cell or a prokaryotic cell.
  • the eukaryotic cell is a yeast cell, an animal cell and/or an insect cell, and/or the prokaryotic cell is an Escherichia coli cell, such as Escherichia coli BL21.
  • methods for producing a polypeptide as described herein comprising:
  • Purifying the fusion protein for example, including (1) purifying the polypeptide on a Ni affinity chromatography column; (2) adding TEV enzyme for cleavage; and/or (3) purifying the polypeptide on an ion exchange column.
  • composition comprising a polypeptide described herein, a fusion protein described herein, a nucleic acid described herein, a vector described herein, and/or a host cell described herein.
  • the composition is a pharmaceutical composition, a food composition or a cosmetic composition.
  • the cosmetic composition is a skin anti-wrinkle, spot lightening and/or skin whitening cosmetic composition.
  • the composition is one or more of biological dressings, human bionic materials, plastic surgery materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue injection filling materials, ophthalmic materials, obstetrics and gynecology biomaterials, nerve repair and regeneration materials, liver tissue materials and vascular repair and regeneration materials, 3D printed artificial organ biomaterials, cosmetic raw materials, pharmaceutical excipients and food additives.
  • the composition comprises a pharmaceutically, cosmetically and/or food acceptable carrier.
  • the composition is a solid, liquid or gel composition.
  • the composition is a kit.
  • the composition is a liquid formulation comprising a polypeptide described herein or a fusion protein described herein and a pharmaceutically, cosmetically and/or food acceptable carrier.
  • the liquid formulation comprises sodium chloride, Tris and imidazole.
  • the sodium chloride concentration is 100-300 mM, such as 120, 150, 170, 200, 220, 250 or 270 mM.
  • the Tris concentration is 10-50 mM, such as 20, 30 or 40 nM.
  • the imidazole concentration is 100-300 nM, such as 120, 150, 170, 200, 220, 250 or 270 mM.
  • the pH of the liquid formulation is 6-10, such as 6.5, 7.5, 8, 8.5, 9, or 9.5.
  • the liquid formulation comprises 200 mM sodium chloride, 25 mM Tris, 250 mM imidazole, pH 8.0
  • cosmetic or beauty methods comprising applying to the skin a polypeptide, fusion protein and/or composition described herein, and optionally applying to the skin other cosmetic and/or beauty products.
  • the cosmetic or beauty method is a skin anti-wrinkle, lightening and/or whitening method.
  • administration is topical or dermal administration.
  • a polypeptide, fusion protein, nucleic acid, vector and/or host cell described herein in a pharmaceutical, cosmetic or beauty product.
  • the product is a topical product.
  • the cosmetic or beauty product is a skin anti-wrinkle, oil control, repair and/or soothing product.
  • the product is selected from biological dressings, human bionic materials, plastic surgery materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue injection filling materials, ophthalmic materials, obstetrics and gynecology biomaterials, nerve repair and regeneration materials, liver tissue materials and vascular repair and regeneration materials, and 3D printed artificial organ biomaterials.
  • the present invention provides the use of the polypeptides, fusion proteins, nucleic acids, vectors and/or host cells described herein in preparing a kit for treating or assisting in the treatment of tumors, anti-infection and/or skin wound repair.
  • the present invention provides the core functional region and amino acid sequence of recombinant humanized fibronectin.
  • the present invention successfully biosynthesized recombinant humanized fibronectin for the first time, which is easy to purify and stable during storage.
  • the polypeptide prepared by the present invention eg, recombinant humanized fibronectin
  • has a good cell adhesion effect is derived from natural human protein, and will not produce an immune response when applied to the human body.
  • the present invention adopts a biosynthetic method to achieve large-scale preparation of recombinant humanized fibronectin.
  • the recombinant humanized fibronectin prepared by the present invention has the effects of wrinkle removal, spot lightening and whitening, etc. It is expected to be widely used in the medical fields such as tumor, anti-infection, and skin wound repair.
  • the FST5 protein of the present invention is expressed in E. coli at a relatively high protein expression level and has relatively high stability when stored at room temperature, indicating that the FST5 protein has relatively high storage stability.
  • FIG 1 shows the electrophoresis detection of TEV enzyme digestion of the purified proteins of FST1, FST2, FST3, and FST4. After the expression of the FST1, FST2, FST3, and FST4 polypeptides, the protein structure is abnormal and cannot be digested by enzymes.
  • FIG. 2 shows the electrophoresis results during the expression of FST8.
  • the target protein was precipitated after homogenization.
  • FIG3 shows the results of electrophoresis detection of FST5.
  • FIG4 shows the electrophoresis detection results of FST6.
  • FIG5 shows the results of electrophoresis detection of FST7.
  • FIG6 shows the stability results of FST5 and FST7 after being placed at room temperature for 2 h.
  • FIG. 7 shows the results of a cell adhesion experiment of the recombinant humanized fibronectin FST5 of the present invention.
  • fibronectin is a high molecular weight glycoprotein with a molecular weight of about 450 Daltons, which is widely present in blood, body fluids and various tissues. It is a dimer formed by two subunits with a molecular mass of 220,000 Daltons connected by disulfide bonds, and the entire molecular shape is composed of two similar A chains and B chains, which are V-shaped.
  • fibronectin is a multi-domain glycoprotein composed of a series of multiple repeating module structures, 12 FNI type repeat sequences (FN I), 2 FNII type repeat sequences (FN II), 15 constitutively expressed and 2 alternately spliced FN III type (FN III) repeat sequences, and a non-homologous variable (V) or III type connecting fragment (IIICS) region.
  • the multi-module structure and the inter-module region make the fibronectin (FN) molecule flexible and participate in regulating its function.
  • a peptide or polypeptide refers to a plurality of amino acid residues connected by peptide bonds.
  • the polypeptide described herein may be a natural polypeptide derived from human fibronectin, or a fragment of human fibronectin.
  • the polypeptide or fragment described herein retains various functions of human fibronectin, such as adhesion effect, wrinkle removal effect, spot lightening and whitening and other beauty effects.
  • nucleic acid refers to a plurality of nucleotides connected by internucleotides. Internucleotide connections can be, for example, phosphodiester bonds.
  • the nucleic acid herein can include a polynucleotide encoding a polypeptide of the present invention.
  • the nucleic acid of the present invention can also include nucleotides encoding a purification tag, such as a His tag, a GST tag, an MBP tag, a SUMO tag, or a NusA tag, and a nucleotide sequence encoding a leader sequence when necessary.
  • the nucleic acid described herein can be a nucleic acid encoding a polypeptide described herein.
  • the nucleic acid can be codon-optimized for an expression host.
  • the encoding nucleic acid is codon-optimized for Escherichia coli. Codon-optimized methods are known to those skilled in the art.
  • vector is a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • a vector can express the protein encoded by the inserted polynucleotide
  • the vector is called an expression vector.
  • the vector can be introduced into a host cell by transformation, transduction or transfection so that the genetic material elements it carries are expressed in the host cell.
  • Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or artificial chromosomes (PAC) derived from P1; bacteriophages such as lambda phage or M13 phage and animal viruses, etc.
  • the vector may contain a variety of elements for controlling expression, including but not limited to promoter sequences, transcription start sequences, enhancer sequences, selection elements and reporter genes.
  • the vector may also contain a replication initiation site.
  • the vector may contain a nucleic acid of the present invention to facilitate introduction into cells for expression.
  • the vector may contain expression control elements such as promoters, terminators and/or enhancers that are operably linked to the nucleic acid.
  • the term "host cell” is a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. These techniques include transfection of viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration.
  • the host cell can be a eukaryotic cell or a prokaryotic cell.
  • a eukaryotic cell is a yeast cell, an animal cell, and/or an insect cell.
  • a prokaryotic cell can be an E. coli cell.
  • sequence identity As used herein, the degree of relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".
  • sequence identity is described using the EMBOSS software package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. [Genetics Trends] 16: 276-277) (preferred 5.0.0 version or updated version) Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol. [Molecular Biology] 48: 443-453) is used to determine the sequence identity between two amino acid sequences.
  • the parameters used are gap opening penalty 10, gap extension penalty 0.5 and EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.
  • the output (obtained using the non-simplified option) of Needleman, which is labeled as "longest identity”, is used as identity percentage and is calculated as follows:
  • the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS software package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, supra) (preferably version 5.0.0 or later) is used to determine the sequence identity between two deoxynucleotide sequences.
  • the parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix.
  • the output of Needle labeled "longest identity" (obtained using the non-simplified option) is used as the identity percentage and is calculated as follows:
  • the polypeptide of the present invention may have an amino acid sequence of SSSGPVEVFITETPSQPNSHPIQWNAPQPSHISKYILRWRPKNSVGRWKEATIPGHLNSYTIKGLKPGVVYEGQLISIQQYGHQEVTRFDFTTTSTSTP, but may also have certain mutations.
  • the amino acid sequence may have substitutions, deletions, additions or insertions of amino acid residues.
  • the present invention may use variants as long as the variants retain the activity of fibronectin.
  • the variants may have a certain percentage identity with the specified sequence SEQ ID NO:1, such as 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 95%, 96%, 97%, 98%, 99% identity.
  • polypeptide of the present invention can be prepared by any suitable means, for example, by synthesis.
  • the polypeptide of the present invention can be prepared by recombinant means or genetic engineering.
  • the polypeptide of the present invention can be prepared as a fusion protein.
  • the fusion protein can include one or more polypeptides described herein as repeating units.
  • the repeating units can be directly or indirectly connected through a linker.
  • the fusion protein can include 2-10, such as 3, 4, 5, 6, 7, 8 or 9 connected polypeptides described herein.
  • the polypeptide or fusion protein of the present invention can be prepared as a composition.
  • the composition can include the polypeptide, fusion protein, nucleic acid, vector and/or host cell described herein.
  • the composition can also include a pharmaceutical, cosmetic and/or food acceptable carrier or solvent.
  • the composition can be a pharmaceutical composition, a food composition or a cosmetic composition, used for the purpose of medicine, food and/or cosmetics.
  • the composition is one or more of a biological dressing, a human bionic material, a plastic surgery material, an organoid culture material, a cardiovascular stent material, a coating material, a tissue injection filling material, an ophthalmic material, a gynecological biomaterial, a nerve repair and regeneration material, a liver tissue material and a vascular repair and regeneration material, a 3D printed artificial organ biomaterial, a cosmetic raw material, a pharmaceutical excipient and a food additive.
  • the cosmetic composition may be a skin anti-wrinkle, spot lightening and/or skin whitening cosmetic composition.
  • the part to which the cosmetic composition is applied is not particularly limited, and may be the face, hands, legs, trunk, etc.
  • composition is not particularly limited as long as the intended function can be achieved.
  • the composition is a solid, liquid or gel composition.
  • a cosmetic or beauty method which comprises applying the polypeptide, fusion protein and/or composition described herein to the skin.
  • Application to the skin may be topical application, for example, application to a skin part that requires skin anti-wrinkle, spot lightening and/or whitening.
  • the method may also comprise applying other beauty and/or cosmetic products to the skin.
  • the types of other beauty and/or cosmetic products are not particularly limited, and may be other cosmetics with skin anti-wrinkle, spot lightening and/or whitening effects, such as collagen peptides.
  • the present invention also provides the use of polypeptides, fusion proteins, nucleic acids, vectors and/or host cells in medicine, cosmetics or beauty products.
  • These products can be external products, such as external cosmetics or beauty products.
  • These cosmetics or beauty products can be skin anti-wrinkle, oil control, repair and/or soothing products.
  • the product can be selected from biological dressings, human bionic materials, plastic surgery materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue injection filling materials, ophthalmic materials, obstetrics and gynecology biomaterials, nerve repair and regeneration materials, liver tissue materials and vascular repair and regeneration materials, 3D printing artificial organ biomaterials.
  • the polypeptides, nucleic acids, vectors and/or host cells described herein in preparing a kit for treating or assisting in the treatment of tumors, anti-infection and/or skin wound repair.
  • the present invention provides a method for biosynthesizing recombinant humanized fibronectin, the specific process of which includes: (1) screening of functional regions and construction of bacterial strains; (2) large-scale biological fermentation culture and induced expression of proteins; (3) purification of humanized fibronectin and optional enzymatic cleavage.
  • the functional region screening and strain construction can be carried out as follows: (1) large-scale functional region screening to obtain the functional region of the target gene; (2) inserting the obtained functional region of the target gene into the pET-32a expression vector to obtain a recombinant expression plasmid; (3) transferring the recombinant expression plasmid into the Escherichia coli competent cell BL21 (DE3) to screen and obtain positive Escherichia coli genetically engineered bacteria.
  • large-scale biological fermentation can be carried out as follows: add the positive Escherichia coli genetically engineered bacteria obtained by screening into a shake flask containing antibiotic stock solution and culture it in a constant temperature shaker at 220 rpm and 37°C for 7 hours.
  • the induced expression of the protein can be carried out as follows: (1) cooling the shake flask after culture to 16°C; (2) adding IPTG mother solution to induce expression; (3) placing the bacterial solution after induced expression in a centrifuge bottle, centrifuging at 8000 rpm and 4°C for 10 min, and then collecting the bacteria.
  • the purification and optional enzymatic cleavage of recombinant humanized fibronectin can be carried out as follows: (1) crudely purifying recombinant humanized fibronectin on a Ni affinity chromatography column; (2) adding TEV enzyme for enzymatic cleavage at a certain ratio; (3) purifying recombinant humanized fibronectin on an ion exchange column.
  • the functional regions screened out are as follows: FST5 amino acid sequence: SSSGPVEVFITETPSQPNSHPIQWNAPQPSHISKYILRWRPKNSVGRWKEATIPGHLNSYTIKGLKPGVVYEGQLISIQQYGHQEVTRFDFTTTSTSTP.
  • the amino acid sequence of the recombinant humanized fibronectin prepared by the present invention is derived from the functional region of natural human fibronectin, including this functional region and similar functional regions, and proteins whose amino acid sequences are mutated and modified.
  • the recombinant humanized fibronectin prepared by the present invention has the effects of wrinkle removal, spot lightening and whitening, etc. It is expected to be widely used in the medical fields such as tumor, anti-infection, and skin wound repair.
  • Example 1 Construction, expression and screening of fibronectin fragments
  • FST5 nucleotide sequence (SEQ ID NO: 2):
  • Each of the above encoding nucleotide sequences was inserted into the pET-28a-Trx-His expression vector to obtain a recombinant expression plasmid.
  • wash the impurities Add 25 mL of washing solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole) until the liquid flows out, and take the wash flow-through for electrophoresis inspection (marked as wash impurities).
  • Wash the column with 1 M imidazole working solution marked as 1 M wash).
  • wash the column with purified water are examples of purified water.
  • Enzyme digestion add TEV enzyme at a ratio of 50:1 between the total amount of protein and the total amount of TEV enzyme, digest at 16°C for 4 h, and take samples for electrophoresis detection. Put the protein solution after enzyme digestion into a dialysis bag, dialyze at 4°C for 2 h, and then transfer to new dialysis solution for overnight dialysis at 4°C.
  • Purification a. Equilibrate the column: use solution A (20 mM Tris, 20 mM sodium chloride, pH 8.0) to equilibrate the column at a flow rate of 10 ml/min.
  • Loading Flow rate is 5 ml/min, load and collect the flow-through, and perform electrophoresis detection.
  • the protein expression levels were FST5>FST7>FST2>FST1>FST6>FST4>FST3>FST8.
  • the specific process is: take 40 ⁇ l of sample solution, add 10 ⁇ l 5 ⁇ protein loading buffer (250mM Tris-HCl (pH: 6.8), 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5% ⁇ -mercaptoethanol), place it in 100°C boiling water for 10min, then add 10 ⁇ l to each well of SDS-PAGE protein gel, run at 80V for 2h, use Coomassie Brilliant Blue staining solution (0.1% Coomassie Brilliant Blue R-250, 25% isopropanol, 10% glacial acetic acid) for protein staining for 20min, and then use protein decolorization solution (10% acetic acid, 5% ethanol) for decolorization.
  • 5 ⁇ protein loading buffer 250mM Tris-HCl (pH: 6.8), 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5% ⁇ -mercaptoethanol
  • the proteins purified from FST1, FST2, FST3, and FST4 could not be cleaved by enzymes (see Figure 1).
  • the expression of these four proteins was abnormal and their performance was poor.
  • the target protein expressed by FST8 was expressed as inclusion bodies in the precipitate after homogenization separation and was not suitable for subsequent purification (see Figure 2).
  • the target protein was expressed by FST6 (see Figure 4), but the protein expression level was low.
  • the protein purification effects of FST5 (see Figure 3) and FST7 (see Figure 5) were better.
  • FST5 and FST7 protein 5 mg/ml, 200 mM sodium chloride, 25 mM Tris, 250 mM imidazole, pH 8.0
  • FST5 and FST7 protein 5 mg/ml, 200 mM sodium chloride, 25 mM Tris, 250 mM imidazole, pH 8.0
  • the protein sample was digested overnight with trypsin.
  • the peptides obtained after digestion were desalted by C18ZipTip and mixed with the matrix ⁇ -cyano-4-hydroxycinnamic acid (CHCA) and spotted on the plate.
  • CHCA matrix ⁇ -cyano-4-hydroxycinnamic acid
  • Protein identification results were obtained based on the primary mass spectra of the peptides produced after enzymatic digestion. Detection parameters: Trypsin digestion, two missed cleavage sites. Alkylation of cysteine was set as a fixed modification. Oxidation of methionine was set as a variable modification.
  • the database used for identification was NCBprot.
  • the coverage of the detected polypeptide fragments compared with the theoretical sequence is 94%, and the detection result is very reliable.
  • the method for detecting protein adhesion activity can refer to the literature Juming Yao, Satoshi Yanagisawa, Tetsuo Asakura, Design, Expression and Characterization of Collagen-Like Proteins Based on the Cell Adhesive and Crosslinking Sequences Derived from Native Collagens, J Biochem. 136, 643-649 (2004).
  • the specific implementation method is as follows:
  • the concentration of the protein sample to be tested is detected by ultraviolet absorption method, including bovine type I collagen (China Food and Drug Inspection Institute, No.: 380002) and the recombinant humanized fibronectin FST5 provided by the present invention.
  • the principle of this method is: to measure the characteristic absorption of peptide bonds under far-ultraviolet light, which is not affected by the chromophore content, has few interfering substances, is easy to operate, and is suitable for detecting human collagen and its analogs that do not develop color with Coomassie Brilliant Blue. (The reference is Walker JM. The Protein Protocols Handbook, second edition. Humana Press. 43-45.). After the protein concentration is detected, the concentration of all proteins to be tested is adjusted to 0.5 mg/mL with PBS.
  • the cell adhesion rate can reflect the adhesion activity of each protein. The higher the activity of the protein, the better the external environment it can provide to the cells in a short time, helping the cells to adhere to the wall.
  • FIG7 describes the relative cell adhesion activity of the FST5 protein relative to bovine type I collagen, indicating that FST5 has a higher (more than 2 times) cell adhesion activity than bovine type I collagen. It is unexpected that the FST5 protein has such a high cell adhesion activity.

Abstract

提供了生物合成重组人源化纤连蛋白以及制备方法。本文所述的多肽包含SEQ ID NO:1所示的氨基酸序列。多肽是来源于人纤连蛋白的天然多肽，可用作医疗器械类的纤连蛋白，且在祛皱功效、淡斑美白等美容方面和在肿瘤、抗感染、皮肤创面修复等医疗方向，具有广阔的应用前景。

Description

生物合成重组人源化纤连蛋白以及制备方法
本申请要求申请日为2023年5月10日，申请号为202310523607.7，发明名称为“生物合成重组人源化纤连蛋白以及制备方法”的优先权权益。上述申请通过引用并入本文。
技术领域
本发明涉及合成生物技术领域，具体涉及一种重组人源化纤连蛋白的生物合成方法。
背景技术
衰老已成为全球关注的热点问题。据世界卫生组织2021年发布的信息显示，预计到2050年，全球65岁以上人口占比将达到15.6％，是5岁以下儿童(7.2％)的2倍多。我国第七次人口普查数据显示，65岁及以上人口占比13.50％。目前，关于衰老的主流学说有氧化应激、免疫衰老、DNA损伤等学说，这些学说从不同的层面揭示了衰老的发生过程。在正常情况下，机体新陈代谢会产生活性氧，维持体内氧化还原的平衡。活性氧还会参与细胞信号调节，其中包括p38MAPK信号通路活化后，诱导细胞凋亡，导致细胞生长停滞。同时，免疫系统的衰老与机体的衰老平行，研究表明，老年人免疫细胞的数量和功能都受到不同程度的损伤。免疫系统不仅可以保护机体免受病原体的侵袭，还可以及时清除衰老的细胞，维持机体内环境的稳定。
细胞通过细胞表面整合素(又叫整合蛋白)与细胞外基质的相互作用产生一系列复杂的信号事件，调节细胞生长、分化、粘附和运动的行为。研究表明，在机体生命活动中，纤连蛋白(fibronectin，FN)通过与整合素结合，特别是与细胞固定连接蛋白结合，从而参与了细胞的迁移、黏附、增殖、止血及组织修复和胚胎的发育，具有生长因子的作用，可诱导表皮细胞通过肉芽组织，并促进表皮下基底膜再建和正常角化过程。有老鼠实验研究表明，老年骨骼肌干细胞(MuSC)的FN水平降低导致数量(仅在年轻组中包含显着数量)、功能和肌肉维持的不足，老年小鼠FN的恢复则也恢复到年轻组的骨骼肌干细胞的表型、水平、肌肉维持和功能。研究发现，FN是MuSCs的首选粘附底物，通过整合素和粘附斑激酶(FAK)调节p38MAPK的衰老途径。 在老年生态位中恢复与FN的附着，使得重新激活MuSC中的FAK信号，从而恢复老年骨骼肌的再生能力。同时，FN还表现出调理作用，旨在促进吞噬细胞对组织碎片的吸收，为新生细胞的新陈代谢提供良好的内外环境。
纤连蛋白(fibronectin，FN)是一种分子量约为450道尔顿，广泛存在于血液、体液及各种组织中的高分子糖蛋白。由2个分子质量为220 000道尔顿的亚基通过二硫键连接而成的二聚体，整个分子形状是由两条相似的A链及B链组成，呈V形。根据氨基酸、基因和基因组DNA序列的分析表明，纤连蛋白(FN)是一种多结构域糖蛋白，由一系列多个重复模块结构组成，12个FNI型重复序列(FN I)，2个FNII型重复序列(FN II)，15个组成型表达和2个交替剪接FN III型(FN III)重复序列，以及一个非同源变量(V)或III型连接片段(IIICS)区域。多模块结构和模块间区域使纤连蛋白(FN)分子具有灵活性，参与调控其功能。
进入21世纪后，分子生物学进入了飞速发展的时代，这也导致了人体组织工程学的兴起，促进了纤连蛋白(FN)在微观领域的研究突破，在肿瘤与生物材料等新兴领域的研究也如雨后春笋般涌现出来。近年来，对于纤连蛋白(FN)的临床应用价值以及领域的研究也越来越多，经过众多的科研工作者及从事转化医学研究与临床应用研究的医生、学者等的努力，在肿瘤、抗感染及生物材料中的研究与应用等都有突破性的进展。在皮肤创面修复和愈合中，纤连蛋白可以缩短创面愈合时间并且减轻伤口瘢痕，在再生医学中被广泛应用，而在护肤方面，纤连蛋白(FN)具有多种用途，具体表现为：祛皱功效、淡斑美白功效。
纤连蛋白因其具有独特的价值，国内的一些生物科技公司也推出一些包含纤连蛋白(FN)的美容产品。因此，纤连蛋白被广泛应用于美容、医药等领域。近年来，随着功能性护肤品的发展，国内在探讨纤连蛋白在医学应用的过程中，发现纤连蛋白在医学美容中有独特的功效，皮肤学专家纷纷研究纤连蛋白在功能性护肤中的应用，但是，目前还没有医疗器械类产品。
亟需一种可用作医疗器械类的纤连蛋白，且在祛皱功效、淡斑美白等美容方面和在肿瘤、抗感染、皮肤创面修复等医疗方向，具有广阔的应用前景。
发明内容
针对目前的研究和应用现状，本发明人从人纤连蛋白中制备不同人纤连 蛋白片段，并且尝试以重组方式表达。发明人发现从不同人纤连蛋白片段在重组表达过程中会遇到不能进行酶切、表达量低、片段稳定性差等技术问题。在经过一系列筛选后，发明人得到了容易进行重组表达，且表达量较高，稳定性较高的人纤连蛋白片段。令人惊讶地，发明人还发现该人纤连蛋白片段具有优异的细胞粘附活性。
本发明人提供了一种多肽：SSSGPVEVFITETPSQPNSHPIQWNAPQPSHISKYILRWRPKNSVGRWKEATIPGHLNSYTIKGLKPGVVYEGQLISIQQYGHQEVTRFDFTTTSTSTP。
该多肽来源于人纤连蛋白，是天然的多肽，在人体内不会引起免疫反应。多肽可以是生物合成重组人源化纤连蛋白。发明人通过基因工程方法生成了本文所述的多肽。具体地，发明人通过功能区筛选、菌种的构建；大规模生物发酵培养和蛋白的诱导表达；人源化纤连蛋白的纯化和任选的酶切等步骤实现了多肽的生成。发明人发现与人纤连蛋白的其他肽段，本发明的多肽是易于纯化的，稳定的，并且具有促进细胞粘附的功效。
在一方面，本文提供了多肽，其包含以下氨基酸序列：
(1)SEQ ID NO:1所示的氨基酸序列：SSSGPVEVFITETPSQPNSHPIQWNAPQPSHISKYILRWRPKNSVGRWKEATIPGHLNSYTIKGLKPGVVYEGQLISIQQYGHQEVTRFDFTTTSTSTP；
(2)与SEQ ID NO:1所示的氨基酸序列具有80％、85％、90％、95％、98％或99％序列同一性的氨基酸序列；或
(3)SEQ ID NO:1所示的氨基酸序列中进行一个或多个氨基酸取代、插入、缺失或添加的氨基酸序列。
在一个实施方案中，多肽来源于人纤连蛋白。
在一个实施方案中，氨基酸取代是保守的氨基酸取代。
在一个实施方案中，氨基酸添加是参照人纤连蛋白的氨基酸添加，使得多肽为人纤连蛋白的天然片段。
在一个实施方案中，多肽具有纤连蛋白的功能。
在一方面，本文提供了融合蛋白，其包含一个或多个本文所述的多肽作为重复单元。该重复单元可以直接或通过接头间接连接。任选地，融合蛋白还包含另一个蛋白质性部分，例如具有美容/化妆功能的多肽。
在一方面，本文提供了核酸，其编码本文所述的多肽或融合蛋白。在一 个实施方案中，核酸包含SEQ ID NO:2所示的核苷酸序列。
在一方面，本文提供了载体，其包含本文所述的核酸。在一个实施方案中，载体包含编码纯化标签的核苷酸、编码前导物的核苷酸和/或调节元件。
在一个实施方案中，纯化标签选自His标签、GST标签、MBP标签、SUMO标签或NusA标签。在一个实施方案中，调节元件选自启动子、终止子和/或增强子。
在一方面，本文提供了宿主细胞，其包含本文所述的核酸或本文所述的载体。在一个实施方案中，宿主细胞是真核细胞或原核细胞。在一个实施方案中，真核细胞是酵母细胞、动物细胞和/或昆虫细胞，和/或原核细胞是大肠杆菌细胞，例如大肠杆菌BL21。
在一方面，本文提供了生产本文所述的多肽，其包括：
(1)在合适的培养条件下培养本文所述的宿主细胞；
(2)收获包含多肽的宿主细胞和/或培养基；和
(3)纯化融合蛋白，例如包括(1)在Ni亲和层析柱上粗纯多肽；(2)添加TEV酶酶切；和/或(3)离子交换柱精纯多肽。
在一方面，本文提供了组合物，其包含本文所述的多肽、本文所述的融合蛋白、本文所述的核酸、本文所述的载体和/或本文所述的宿主细胞。
在一个实施方案中，组合物是药物组合物、食品组合物或化妆品组合物。
在一个实施方案中，化妆品组合物是皮肤抗皱、淡斑和/或美白的化妆品组合物。
在一个实施方案中，组合物是生物敷料、人体仿生材料、整形美容材料、类器官培养材料、心血管支架材料、涂层材料、组织注射填充材料、眼科材料、妇产科生物材料、神经修复再生材料、肝脏组织材料及血管修复再生材料、3D打印人造器官生物材料、化妆品原料、药用辅料和食品添加剂中的一种或多种。
在一个实施方案中，组合物包含药学、化妆品和/或食品可接受的载体。
在一个实施方案中，组合物为固体、液体或凝胶组合物。
在一个实施方案中，组合物为试剂盒。
在一个实施方案中，组合物为液体配制剂，其包含本文所述的多肽或本文所述的融合蛋白和药学、化妆品和/或食品可接受的载体。
在一个实施方案中，液体配制剂包含氯化钠、Tris和咪唑。在一个实施 方案中，氯化钠浓度为100-300mM，例如120、150、170、200、220、250或270mM。在一个实施方案中，Tris浓度为10-50mM，例如20、30或40nM。在一个实施方案中，咪唑浓度为100-300nM，例如120、150、170、200、220、250或270mM。在一个实施方案中，液体配制剂的pH为6-10，例如6.5、7.5、8、8.5、9、或9.5。在一个实施方案中，液体配制剂包含200mM氯化钠、25mM Tris、250mM咪唑，pH8.0
在一方面，本文提供了化妆或美容方法，其包括对皮肤施用本文所述的多肽、融合蛋白和/或组合物，以及任选地对皮肤应用其他美容和/或化妆产品。
在一个实施方案中，化妆或美容方法是皮肤抗皱、淡斑和/或美白方法。
在一个实施方案中，施用为局部或皮肤表面施用。
在一方面，本文提供了本文所述的多肽、融合蛋白、核酸、载体和/或宿主细胞在医药、化妆或美容产品中的用途。
在一个实施方案中，产品是外用产品。
在一个实施方案中，化妆或美容产品是皮肤抗皱、控油、修复和/或舒缓产品。
在一个实施方案中，产品选自生物敷料、人体仿生材料、整形美容材料、类器官培养材料、心血管支架材料、涂层材料、组织注射填充材料、眼科材料、妇产科生物材料、神经修复再生材料、肝脏组织材料及血管修复再生材料、3D打印人造器官生物材料。
在一方面，本文提供了本文所述的多肽、融合蛋白、核酸、载体和/或宿主细胞在制备试剂盒中的用途，所述试剂盒用于治疗或辅助治疗肿瘤、抗感染和/或皮肤创面修复。
本发明的优点：
1.本发明提供了重组人源化纤连蛋白的核心功能区及氨基酸序列。
2.本发明首次成功生物合成了重组人源化纤连蛋白，其易于纯化的且在存储过程中是稳定的。
3.本发明制备得到的多肽(例如重组人源化纤连蛋白)具有良好的细胞黏附效果，来源于人天然蛋白质，应用于人体不会产生免疫反应。
4.本发明采用生物合成方法可实现大规模制备重组人源化纤连蛋白。
5.本发明制备得到的重组人源化纤连蛋白在祛皱功效、淡斑美白等美容 方向和在肿瘤、抗感染、皮肤创面修复等医疗领域有望得到广泛的应用。
6.本发明的FST5蛋白以较大的蛋白表达量在大肠杆菌中得到表达，且在常温放置时具有较大的稳定性。表明FST5蛋白具备较高的储存稳定性。
附图说明
图1显示了FST1、FST2、FST3、FST4纯化后的蛋白的TEV酶酶切的电泳检测。FST1、FST2、FST3、FST4多肽表达后蛋白结构异常导致不能被酶切。
图2显示了FST8表达过程中的电泳检测结果。目的蛋白在均质分离后沉淀。
图3显示了FST5电泳检测结果。
图4显示了FST6电泳检测结果。
图5显示了FST7电泳检测结果。
图6显示了FST5和FST7常温放置2h后稳定性结果。
图7显示了本发明的重组人源化纤连蛋白FST5的细胞粘附实验结果。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚，下面将结合本发明的实施例，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
如本文中所用，纤连蛋白(fibronectin，FN)是一种分子量约为450道尔顿，广泛存在于血液、体液及各种组织中的高分子糖蛋白。由2个分子质量为220000道尔顿的亚基通过二硫键连接而成的二聚体，整个分子形状是由两条相似的A链及B链组成，呈V形。根据氨基酸、基因和基因组DNA序列的分析表明，纤连蛋白(FN)是一种多结构域糖蛋白，由一系列多个重复模块结构组成，12个FNI型重复序列(FN I)，2个FNII型重复序列(FN II)，15个组成型表达和2个交替剪接FN III型(FN III)重复序列，以及一个非同源变量(V)或III型连接片段(IIICS)区域。多模块结构和模块间区域使纤连蛋白(FN)分子具有灵活性，参与调控其功能。
如本文中所用，肽或多肽是指通过肽键连接的多个氨基酸残基。本文所述的多肽可以是来源于人纤连蛋白的天然多肽，或者称为人纤连蛋白的片段。本文所述的多肽或片段保留了人纤连蛋白的各种功能，例如粘附效果，祛皱功效、淡斑美白等美容功效。
如本文中所用，“核酸”是指通过核苷酸间连接的多个核苷酸。核苷酸间连接可以是例如磷酸二酯键。本文中的核酸可以包含编码本发明的多肽的多核苷酸。为了便于多肽后续处理，本发明的核酸还可以包含编码纯化标签，例如His标签、GST标签、MBP标签、SUMO标签或NusA标签的核苷酸，以及当需要时编码前导序列的核苷酸序列。本文所述的核酸可以是编码本文所述的多肽的核酸。该核酸可以针对表达宿主进行密码子优化。例如，在本文中的实施例中，编码核酸针对大肠杆菌进行密码子优化。密码子优化的方法对于本领域技术人员而言是已知的。
如本文中所用，术语“载体”是可将多核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白质获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。载体可以含有多种控制表达的元件，包括但不限于启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。载体可以包含本发明的核酸以便于导入细胞进行表达。载体可以包含与所述核酸可操作连接的表达控制元件，如启动子、终止子和/或增强子。
如本文中所用，术语“宿主细胞”是已经通过分子生物学技术将核酸分子引入的细胞。这些技术包括转染病毒载体，用质粒载体转化，以及通过电穿孔、脂转染、和粒子枪加速引入裸DNA。宿主细胞可以是真核细胞或原核细胞。例如，真核细胞是酵母细胞、动物细胞和/或昆虫细胞。原核细胞可以是大肠杆菌细胞。
如本文中所用，两个氨基酸序列之间或两个核苷酸序列之间的关联度通过参数“序列同一性”来描述。出于本发明的目的，使用如在EMBOSS软件包(EMBOSS：欧洲分子生物学开放软件套件，Rice等人，2000,Trends Genet. [遗传学趋势]16:276-277)(优选5.0.0版或更新版本)的尼德尔程序所实施的尼德曼-翁施算法(Needleman和Wunsch，1970,J.Mol.Biol.[分子生物学杂志]48:443-453)来确定两个氨基酸序列之间的序列同一性。使用的参数是空位开放罚分10、空位延伸罚分0.5以及EBLOSUM62(BLOSUM62的EMBOSS版本)取代矩阵。将标记为“最长同一性”的尼德尔的输出(使用非简化选项获得)用作同一性百分比并且计算如下：
(相同的残基×100)/(比对长度-比对中的空位总数)
出于本发明的目的，使用如在EMBOSS软件包(EMBOSS：欧洲分子生物学开放软件套件，Rice等人,2000,同上)(优选5.0.0版本或更新版本)的尼德尔程序所实施的尼德曼-翁施算法(Needleman和Wunsch,1970,同上)来确定两个脱氧核苷酸序列之间的序列同一性。所使用的参数是空位开放罚分10、空位延伸罚分0.5、和EDNAFULL(NCBI NUC4.4的EMBOSS版)取代矩阵。将标记为“最长同一性”的尼德尔的输出(使用非简化选项获得)用作同一性百分比并且计算如下：
(相同的脱氧核糖核苷酸x 100)/(比对长度-比对中的空位总数)
保守取代可以通过下表中反映的氨基酸类别内的取代来定义：
表1：用于保守取代的氨基酸残基类别
多肽
在本文中，本发明的多肽可以具有SSSGPVEVFITETPSQPNSHPIQWNAPQPSHISKYILRWRPKNSVGRWKEATIPGHLNSYTIKGLKPGVVYEGQLISIQQYGHQEVTRFDFTTTSTSTP的氨基酸序列，但是也可以存在一定的突变。例如，氨基酸序列可以存在氨基酸残基的取代、缺失、添加或插入。也就是说，本发明可以使用变体，只要变体保留纤连蛋白的活性。具体而言，变体可以与指定的序列SEQ ID NO:1具备一定的百分比同一性，例如80％、85％、90％、91％、92％、93％、94％、 95％、96％、97％、98％、99％同一性。本发明的多肽可以具有以下功能中的一种或多种：皮肤抗皱、淡斑和/或美白功能。
本发明的多肽可以通过任何合适的方式进行制备，例如可以通过合成制备。优选地，本发明的多肽可以通过重组方式或基因工程制备。
本发明的多肽可以制备成融合蛋白。融合蛋白可以包含一个或多个本文所述的多肽作为重复单元。该重复单元可以直接或通过接头间接连接。例如，融合蛋白可以包含2-10，例如3、4、5、6、7、8或9个连接的本文所述的多肽。
组合物
本发明的多肽或融合蛋白可以制备为组合物。组合物可以包含本文所述的多肽、融合蛋白、核酸、载体和/或宿主细胞。组合物还可以包含药学、化妆品和/或食品可接受的载体或溶剂。组合物可以是药物组合物、食品组合物或化妆品组合物，用于药品、食品和/或化妆品的目的。例如，组合物是生物敷料、人体仿生材料、整形美容材料、类器官培养材料、心血管支架材料、涂层材料、组织注射填充材料、眼科材料、妇产科生物材料、神经修复再生材料、肝脏组织材料及血管修复再生材料、3D打印人造器官生物材料、化妆品原料、药用辅料和食品添加剂中的一种或多种。
化妆品组合物可以是皮肤抗皱、淡斑和/或美白的化妆品组合物。化妆品组合物应用的部分没有特别限制，可以是脸部、手部、腿部、躯干等。
组合物的形式没有特别限制，只要可以实现预期的功能。例如，组合物为固体、液体或凝胶组合物。
方法
本文还提供了化妆或美容方法，其包括对皮肤施用本文所述的多肽、融合蛋白和/或组合物。对皮肤施用可以是局部施用，例如对需要皮肤抗皱、淡斑和/或美白的皮肤部位进行施用。方法还可以包括对皮肤应用其他美容和/或化妆产品。其他美容和/或化妆产品的种类没有特别限制，可以是其他具有皮肤抗皱、淡斑和/或美白的化妆品，如胶原蛋白肽。
用途
本文还提供了多肽、融合蛋白、核酸、载体和/或宿主细胞在医药、化妆或美容产品中的用途。这些产品可以是外用产品，例如外用的化妆产品或美容产品。这些化妆或美容产品可以是皮肤抗皱、控油、修复和/或舒缓产品。 例如，产品可以选自生物敷料、人体仿生材料、整形美容材料、类器官培养材料、心血管支架材料、涂层材料、组织注射填充材料、眼科材料、妇产科生物材料、神经修复再生材料、肝脏组织材料及血管修复再生材料、3D打印人造器官生物材料。本文还提供了本文所述的多肽、核酸、载体和/或宿主细胞在制备试剂盒中的用途，所述试剂盒用于治疗或辅助治疗肿瘤、抗感染和/或皮肤创面修复。
示例性实施方案
1.针对目前的研究现状，本发明提供了一种生物合成重组人源化纤连蛋白的方法，具体过程包括：(1)功能区筛选、菌种的构建；(2)大规模生物发酵培养和蛋白的诱导表达；(3)人源化纤连蛋白的纯化和任选的酶切。
2.根据项1所述，功能区筛选、菌种的构建可如下进行：(1)大规模功能区筛选，得到目的基因功能区；(2)将得到的目的基因功能区插入pET-32a表达载体中得到重组表达质粒；(3)将重组表达质粒转入大肠杆菌感受态细胞BL21(DE3)中，筛选得到阳性大肠杆菌基因工程菌。
3.根据项1所述，大规模生物发酵可如下进行：将筛选得到的阳性大肠杆菌基因工程菌加入抗生素储液的摇瓶中，并在220rpm、37℃恒温摇床中培养7h。
4.根据项1所述，蛋白的诱导表达可如下进行：(1)将培养后的摇瓶降温至16℃；(2)添加IPTG母液，进行诱导表达；(3)将诱导表达后的菌液装于离心瓶中，8000rpm、4℃离心10min后收集菌体。
5.根据项1所述，重组人源化纤连蛋白的纯化和任选的酶切可如下进行：(1)在Ni亲和层析柱粗纯重组人源化纤连蛋白；(2)按一定比例添加TEV酶酶切；(3)离子交换柱精纯重组人源化纤连蛋白。
6.根据项2所述中，筛选出的功能区如下所示：FST5氨基酸序列：SSSGPVEVFITETPSQPNSHPIQWNAPQPSHISKYILRWRPKNSVGRWKEATIPGHLNSYTIKGLKPGVVYEGQLISIQQYGHQEVTRFDFTTTSTSTP。
7.本发明制备的重组人源化纤连蛋白的氨基酸序列源自于人天然的纤连蛋白的功能区，包含该功能区及类似的功能区、对氨基酸序列分别进行突变和修饰后的蛋白。
8.本发明制备得到的重组人源化纤连蛋白在祛皱功效、淡斑美白等美容 方向和在肿瘤、抗感染、皮肤创面修复等医疗领域有望得到广泛的应用。
实施例
本发明通过下述实施例进一步阐明，但任何实施例或其组合不应当理解为对本发明的范围或实施方式的限制。本发明的范围由所附权利要求书限定，结合本说明书和本领域一般常识，本领域普通技术人员可以清楚地明白权利要求书所限定的范围。在不偏离本发明的精神和范围的前提下，本领域技术人员可以对本发明的技术方案进行任何修改或改变，这种修改和改变也包含在本发明的范围内。
实施例1：纤连蛋白片段的构建、表达和筛选
1.进行大规模功能区筛选，得到以下不同的重组人源化纤连蛋白的目的基因功能区。
FST1氨基酸序列：
FST2氨基酸序列：
FST3氨基酸序列：

FST4氨基酸序列：
FST5氨基酸序列：
FST6氨基酸序列：
FST7氨基酸序列：
FST8氨基酸序列：
2.核苷酸序列
FST1核苷酸序列：

FST2核苷酸序列：
FST3核苷酸序列：

FST4核苷酸序列：

FST5核苷酸序列(SEQ ID NO:2)：
FST6核苷酸序列：

FST7核苷酸序列：
FST8核苷酸序列：
将上述每种编码核苷酸序列插入pET-28a-Trx-His表达载体中得到重组表达质粒。
3.将构建成功的表达质粒转化大肠杆菌感受态细胞BL21(DE3)。具体过程为：(1)在超低温冰箱中取出大肠杆菌感受态细胞BL21(DE3)置于冰上，待半融时取2μl待转化的质粒加入大肠杆菌感受态细胞BL21(DE3)中，稍微混匀2-3次。(2)将混合物置于冰上冰浴30min，然后于42℃水浴热激45-90s，取出后置于冰上冰浴2min。(3)转移至生物安全柜中，并加入700μl液体LB培养基，然后于37℃、220rpm条件下培养60min。(4)取200μl的菌液均匀涂布在含有氨苄西林钠LB平板上。(5)将平板在37℃的培养箱中培养15-17h，待其长出大小均匀的菌落。
4.从转化好的LB平板中挑取5-6个单菌落于含有抗生素储液的LB培养基的摇瓶中，在220rpm，37℃恒温摇床中7h。再将培养后的摇瓶降温至16℃，添加IPTG诱导表达一段时间后，将菌液分装于离心瓶中，于8000rpm、4℃离心10min，收集菌体，并记录菌体重量，取样(标注：菌液)进行电 泳检测。
5.将收集的菌体用平衡工作液(200mM氯化钠、25mM Tris、20mM咪唑，pH8.0)重悬，将菌液降温至≤15℃，进行均质，高压均质两次(分别取两次均质后的样品标注：均一、均二)，完成后收集菌液。将均质后的菌液分装至离心瓶中，于17000rpm、4℃离心30min，收集上清液，取上清液和沉淀进行电泳检测。
6.将重组人源化纤连蛋白的进行纯化和酶切，具体过程是：(1)粗纯：a.水洗柱材(Ni6FF，Cytiva)，5个CV。b.平衡液(200mM氯化钠、25mM Tris、20mM咪唑，pH8.0)平衡柱材，5个CV。c.上样：将离心后的上清液加入柱材中，直至液体流完后，取流穿进行电泳检验(标注为流穿)。d.清洗杂蛋白：添加洗杂液25mL(200mM氯化钠，25mM Tris，20mM咪唑)至液体流完，并取洗杂流穿进行电泳检验(标注为洗杂)。e.收集目的蛋白：添加20mL洗脱液(200mM氯化钠、25mM Tris、250mM咪唑，pH8.0)，并收集流穿液(标注：洗脱)，检测蛋白浓度计算蛋白量，并进行电泳检测。f.用1M咪唑工作液清洗柱材(标注为1M洗)。g.纯化水清洗柱材。(2)酶切：按蛋白总量与TEV酶总量比为50：1，添加TEV酶，16℃酶切4h，取样进行电泳检测。将酶切后的蛋白液放入透析袋，于4℃透析2h，再转移至新的透析液中4℃过夜透析。(3)精纯：a.平衡柱材：使用A液(20mM Tris，20mM氯化钠，pH8.0)将柱材平衡，流速为10ml/min。b.上样：流速为5ml/min，上样并收集流穿，并进行电泳检测。c.梯度洗脱：分别设置0-15％B液(20mM Tris，1M氯化钠，pH8.0)2min然后保持3个CV、15-30％B液2min然后保持3个CV、30-50％B液2min然后保持3个CV、50-100％B液2min然后保持3个CV，出峰收集并进行电泳检测(标注为精纯蛋白)。d.清洗柱材。将蛋白储存在4℃环境中。
7.浓度检测
精确量取适量样品，用洗脱液稀释10-50倍，用玻璃棒充分搅拌均匀。使用紫外可见分光光度计在280nm处测定吸光度，根据公式C(mg/ml)＝A280×吸光系数×稀释倍数，计算蛋白浓度(注：吸光度值需在0.1-1)。
浓度检测结果如下：

蛋白表达量FST5＞FST7＞FST2＞FST1＞FST6＞FST4＞FST3＞FST8。
8.电泳检测
具体过程为：取样品液40μl，加入10μl 5×的蛋白上样缓冲液(250mM的Tris-HCl(pH:6.8)，10％SDS,0.5％溴酚蓝，50％甘油，5％β-巯基乙醇)，置于100℃沸水中煮10min，然后每孔10μl加入SDS-PAGE蛋白胶中，电压80V跑2h后，用考马斯亮蓝染色液(0.1％考马斯亮蓝R-250，25％异丙醇，10％冰醋酸)进行蛋白染色20min，再利用蛋白脱色液(10％醋酸，5％乙醇)进行脱色。
电泳检测结果显示：
FST1、FST2、FST3、FST4纯化后的蛋白均不能被酶切(见图1)，这四个蛋白表达异常，表现较差；FST8表达的目的蛋白在均质分离后的沉淀中，为包涵体表达，不适合后续的纯化(见图2)；FST6(见图4)有目的蛋白表达，但是蛋白表达量较少；FST5(见图3)和FST7(见图5)蛋白纯化效果较好。
9.蛋白稳定性观察
将FST5和FST7纯化后蛋白溶液(蛋白5mg/ml，200mM氯化钠、25mM Tris、250mM咪唑，pH8.0)在常温放置2h后，观察蛋白性状，结果见图6。FST5澄清透亮，FST7发白、混浊稳定性较差，因此，FST5质粒表现更好。对纯化的FST5蛋白进行检测。
实施例2：重组人源化纤连蛋白FST5的质谱检测
实验方法

蛋白样品经DTT还原和碘代乙酰胺烷基化处理后，加入胰蛋白酶酶解过夜。酶解后得到的肽段再经C18ZipTip脱盐后，与基质α-cyano-4-hydroxycinnamic acid(CHCA)混合点板。最后用基质辅助激光解析电离-飞行时间质谱仪MALDI-TOF/TOF UlraflextremeTM，Brucker，Germany进行分析(肽指纹图谱的技术可以参考Protein J.2016；35:212-7)。
数据检索是通过从本地masco网站上MS/MS Ion Search页面处理的。蛋白质鉴定结果是根据酶解后所产生的肽段的一级质谱得到的。检测参数：Trypsin酶解，设两个漏切位点。设定半胱氨酸的烷基化为固定修饰。甲硫氨酸的氧化为可变修饰。鉴定所用的数据库为NCBprot。
表1：重组人源化纤连蛋白FST5质谱检出分子量及对应多肽

检出多肽片段与理论序列相比，覆盖率为94％，检测结果非常可信。
实施例3：重组人源化纤连蛋白FST5的生物活性检测
蛋白的黏附活性检测方法可以参考文献Juming Yao,Satoshi Yanagisawa,Tetsuo Asakura,Design,Expression and Characterization of Collagen-Like Proteins Based on the Cell Adhesive and Crosslinking Sequences Derived from  Native Collagens,J Biochem.136,643-649(2004)。具体实施方法如下：
(1)利用紫外吸收法检测待测蛋白样品的浓度，包括牛Ⅰ型胶原蛋白(中国食品药品检定研究院，编号：380002)、本发明提供的重组人源化纤连蛋白FST5。
具体为分别测定样品在215nm和225nm下的紫外光吸收，利用经验公式C(μg/mL)＝144×(A215-A225)计算蛋白质浓度，注意需在A215<1.5的情况下检测。该方法的原理是：测定肽键在远紫外光下的特征吸收，不受生色团含量的影响，干扰物质少，操作简便，适合检测考马斯亮蓝不显色的人胶原蛋白及其类似物。(参考文献为Walker JM.The Protein Protocols Handbook,second edition.HumanaPress.43-45.)。检测完蛋白浓度后，用PBS将所有待测蛋白浓度调整到0.5mg/mL。
(2)向96孔板中加入100μL各种蛋白溶液和空白PBS溶液对照(见图7中的NC组)，室温静置60min。
(3)每孔中加入105个培养状态良好的3T3细胞，37℃孵育60min。
(4)每孔用PBS清洗4次。
(5)用LDH检测试剂盒(Roche，04744926001)检测OD492nm的吸光度。根据空白对照的数值，可以计算出细胞的贴壁率。计算公式如下： 细胞的贴壁率即可以反映各蛋白的黏附活性。蛋白的活性越高，越能在短时间给细胞提供优质的外环境，帮助细胞贴壁。
结果如图7所示，从对比中可知，相比于牛Ⅰ型胶原蛋白(PC组，0.5mg/ml)，本发明的重组人源化纤连蛋白FST5具有更加优秀的生物黏附活性。图7描述了FST5蛋白相对于牛Ⅰ型胶原蛋白的相对细胞黏附活性，表明FST5具有比牛Ⅰ型胶原蛋白具有更高(2倍以上)的细胞黏附活性。FST5蛋白具有如此高的细胞黏附活性是出乎意料的。
尽管已参考说明性实施例描述了本发明，但所属领域的技术人员将理解，在不背离本发明的精神及范围的情况下可做出各种其它改变、省略及/或添加且可用实质等效物替代所述实施例的元件。另外，可在不背离本发明的范围的情况下做出许多修改以使特定情形或材料适应本发明的教示。因此，本文并不打算将本发明限制于用于执行本发明的所揭示特定实施例，而是打算使本发明将包含归属于所附权利要求书的范围内的所有实施例。

Claims (11)

  1. 多肽，其包含以下氨基酸序列：
    (1)SEQ ID NO:1所示的氨基酸序列；
    (2)与SEQ ID NO:1所示的氨基酸序列具有80％、85％、90％、95％、98％或99％序列同一性的氨基酸序列；或
    (3)SEQ ID NO:1所示的氨基酸序列中进行一个或多个氨基酸取代、插入、缺失或添加的氨基酸序列；
    优选地，其中多肽来源于人纤连蛋白；
    优选地，其中氨基酸取代是保守的氨基酸取代；
    优选地，其中氨基酸添加是参照人纤连蛋白的氨基酸添加，使得多肽为人纤连蛋白的片段；
    优选地，其中多肽具有纤连蛋白的功能。
  2. 融合蛋白，其包含一个或多个根据权利要求1所述的多肽，任选地还包含另一个蛋白质性部分，例如具有美容/化妆功能的多肽。
  3. 核酸，其编码根据权利要求1所述的多肽，优选包含SEQ ID NO:2所示的核苷酸序列，或者编码根据权利要求2所述的融合蛋白。
  4. 载体，其包含根据权利要求2所述的核酸，任选地，所述载体包含编码纯化标签的核苷酸、编码前导物的核苷酸和/或调节元件；
    优选地，纯化标签选自His标签、GST标签、MBP标签、SUMO标签或NusA标签；优选地，调节元件选自启动子、终止子和/或增强子。
  5. 宿主细胞，其包含根据权利要求3所述的核酸或根据权利要求4所述的载体；其中优选地，宿主细胞是真核细胞或原核细胞；其中优选地，真核细胞是酵母细胞、动物细胞和/或昆虫细胞，和/或原核细胞是大肠杆菌细胞，例如大肠杆菌BL21。
  6. 生产根据权利要求1所述的多肽，其包括：
    (1)在合适的培养条件下培养根据权利要求5所述的宿主细胞；
    (2)收获包含多肽的宿主细胞和/或培养基；和
    (3)纯化融合蛋白，例如包括(1)在Ni亲和层析柱上粗纯多肽；(2)添加TEV酶酶切；和/或(3)离子交换柱精纯多肽。
  7. 组合物，其包含根据权利要求1所述的多肽、根据权利要求2所述的融合蛋白、根据权利要求3所述的核酸、根据权利要求4所述的载体和/或根据权 利要求5所述的宿主细胞；
    优选地，组合物是药物组合物、食品组合物或化妆品组合物，
    优选地，化妆品组合物是皮肤抗皱、淡斑和/或美白的化妆品组合物；
    优选地，组合物是生物敷料、人体仿生材料、整形美容材料、类器官培养材料、心血管支架材料、涂层材料、组织注射填充材料、眼科材料、妇产科生物材料、神经修复再生材料、肝脏组织材料及血管修复再生材料、3D打印人造器官生物材料、化妆品原料、药用辅料和食品添加剂中的一种或多种；
    优选地，组合物包含药学、化妆品和/或食品可接受的载体；
    优选地，组合物为固体、液体或凝胶组合物；
    优选地，组合物为试剂盒；
    优选地，组合物为液体配制剂，其包含根据权利要求1所述的多肽和/或根据权利要求2所述的融合蛋白和药学、化妆品和/或食品可接受的载体；
    优选地，液体配制剂包含氯化钠、Tris和咪唑；
    优选地，氯化钠浓度为100-300mM；
    优选地，Tris浓度为10-50mM；
    优选地，咪唑浓度为100-300nM；
    优选地，液体配制剂的pH为6-10。
  8. 化妆或美容方法，其包括对皮肤施用根据权利要求1所述的多肽、根据权利要求2所述的融合蛋白和/或根据权利要求7的组合物，以及任选地对皮肤应用其他美容和/或化妆产品；
    优选地，所述化妆或美容方法是皮肤抗皱、淡斑和/或美白方法；
    优选地，施用为局部或皮肤表面施用。
  9. 根据权利要求1所述的多肽、根据权利要求2所述的融合蛋白、根据权利要求3所述的核酸、根据权利要求4所述的载体和/或根据权利要求5所述的宿主细胞在医药、化妆或美容产品中的用途，
    优选地，其中产品是外用产品，
    优选地，其中化妆或美容产品是皮肤抗皱、控油、修复和/或舒缓产品；
    优选地，产品选自生物敷料、人体仿生材料、整形美容材料、类器官培养材料、心血管支架材料、涂层材料、组织注射填充材料、眼科材料、妇产科生物材料、神经修复再生材料、肝脏组织材料及血管修复再生材料、3D 打印人造器官生物材料。
  10. 根据权利要求1所述的多肽、根据权利要求2所述的融合蛋白、根据权利要求3所述的核酸、根据权利要求4所述的载体、根据权利要求5所述的宿主细胞、和/或根据权利要求7的组合物在制备试剂盒中的用途，所述试剂盒用于治疗或辅助治疗肿瘤、抗感染和/或皮肤创面修复。
  11. 促进细胞粘附的方法，其包括将细胞与根据权利要求1所述的多肽和/或根据权利要求7的组合物接触的步骤。
PCT/CN2023/110757 2023-05-10 2023-08-02 生物合成重组人源化纤连蛋白以及制备方法 WO2024229988A1 (zh)

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