CN113683678B - Recombinant I-type humanized collagen C1L2T and preparation method and application thereof - Google Patents

Recombinant I-type humanized collagen C1L2T and preparation method and application thereof Download PDF

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CN113683678B
CN113683678B CN202111080699.3A CN202111080699A CN113683678B CN 113683678 B CN113683678 B CN 113683678B CN 202111080699 A CN202111080699 A CN 202111080699A CN 113683678 B CN113683678 B CN 113683678B
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pro
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c1l2t
collagen
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CN113683678A (en
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杨霞
武庚风
张永健
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Shanxi Jinbo Bio Pharmaceutical Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/18—Peptides; Protein hydrolysates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A61K8/65—Collagen; Gelatin; Keratin; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/10—General cosmetic use
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host

Abstract

The invention discloses a recombinant I-type humanized collagen C1L2T and a preparation method and application thereof. The recombinant I-type humanized collagen C1L2T provided by the invention comprises a sequence shown as SEQ ID No.3, and the recombinant I-type humanized collagen C1L2T optionally comprises a sequence shown as SEQ ID No.2, preferably the sequence shown as SEQ ID No.2 and the sequence shown as SEQ ID No.3 are directly connected. Compared with the type I human collagen, the recombinant type I humanized collagen C1L2T prepared by the invention has better cell adhesion effect, the amino acid sequence is selected from the amino acid sequence of the natural type I human collagen, the possibility of being applied to human body to generate immune response is low, and the preparation method is simple.

Description

Recombinant I-type humanized collagen C1L2T and preparation method and application thereof
Technical Field
The invention belongs to the technical field of genetic engineering, and relates to recombinant I-type humanized collagen C1L2T and a preparation method and application thereof.
Background
Collagen (collagen) is the most abundant class of proteins in mammals and is widely distributed in the skin, bones, tendons, ligaments and blood vessels of animals. The collagen content in human body is 25% -30% of total protein, which is an important structural protein and plays an important role in protecting organism and supporting organ.
There are 28 types of collagen, and different types of collagen have been found to have different functions. Two general categories are known: one type is fibroblast collagen; another class is non-fibroblastic collagen; the fibroblast collagen comprises I, II, III, V, VI and XXVI type collagen, and the rest is non-fibrous collagen. The most abundant type I collagen in humans is approximately 85% or more, and is present in bone, skin, tendons and cornea, type II in cartilage, intervertebral discs and vitreous, and type III in blood vessels, neoskin and scar tissue.
At present, the collagen is mainly derived from animal tissues, is a mixture of collagen with different molecular weights, is insoluble in water and has poor biocompatibility; in addition, since it is derived from animal tissue, there is a risk of cross infection for animal-derived diseases or human infectious diseases, and it is not compatible with the human body, resulting in the occurrence of immune rejection and allergic symptoms.
The traditional method for producing collagen is to treat animal-derived tissues by acid, alkali and enzymolysis methods to extract collagen derivatives. The collagen extracted by the methods has lost the original biological activity and cannot be applied to the biomedical field to play a real function. Therefore, collagen can only be used in cosmetics and health care products at present, and the original biological functions of collagen cannot be exerted at all.
Disclosure of Invention
Problems to be solved by the invention
Aiming at the problems that the heterologous collagen in the field has poor bioactivity, is difficult to exert the original function and is easy to cause immune response, the invention provides a recombinant I-type humanized collagen C1L2T, and simultaneously provides a preparation method and application thereof.
Solution for solving the problem
In a first aspect, the present invention provides a recombinant type I humanized collagen C1L2T, wherein the recombinant type I humanized collagen C1L2T comprises the sequence shown in SEQ ID No. 3.
Further, the recombinant type I humanized collagen C1L2T described above optionally comprises the sequence shown in SEQ ID No. 2; preferably, the sequence shown in SEQ ID No.2 and the sequence shown in SEQ ID No.3 are directly linked.
Further, the recombinant type I humanized collagen C1L2T comprises at least one of the following sequences: an amino acid sequence shown in SEQ ID No. 4; an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID No.4, and which retains the cell adhesion effect of the amino acid sequence shown in SEQ ID No. 4; an amino acid sequence of 1 or more amino acid residues added, substituted, deleted or inserted in the amino acid sequence shown in SEQ ID No.4, and which retains the cell adhesion effect of the amino acid sequence shown in SEQ ID No. 4;
an amino acid sequence encoded by a nucleotide sequence that hybridizes with a polynucleotide sequence encoding the amino acid sequence set forth in SEQ ID No.4 under stringent conditions, which are medium-high stringent conditions, high stringent conditions or very high stringent conditions, and which retains the cell adhesion effect of the amino acid sequence set forth in SEQ ID No.4.
In a second aspect, the invention provides a polynucleotide encoding the recombinant type I humanized collagen C1L2T described above.
In a third aspect, the present invention provides an expression vector comprising a polynucleotide as described above.
In a fourth aspect, the invention provides a host cell comprising an expression vector as described above.
In a fifth aspect, the present invention provides a method for producing the recombinant type I humanized collagen C1L2T, comprising the steps of: (1) culturing the host cell provided in the fourth aspect of the present invention described above in a medium and producing the protein; (2) harvesting and purifying the protein, preferably purifying the protein with a Ni column and/or ion exchange chromatography; (3) optionally, the protein is cleaved, preferably with a PPase protease.
In a sixth aspect, the present invention provides the use of the recombinant type I humanized collagen C1L2T described above for the preparation of a product, wherein the product is preferably a tissue engineering product, a cosmetic, a health product or a pharmaceutical.
In a seventh aspect, the present invention provides a product comprising the recombinant type I humanized collagen C1L2T described above, wherein the product is preferably a tissue engineering product, a cosmetic, a health product or a pharmaceutical.
In an eighth aspect, the present invention provides the use of the recombinant type I humanized collagen C1L2T described above for the preparation of a product having a cell adhesion promoting effect.
ADVANTAGEOUS EFFECTS OF INVENTION
Through implementation of the technical scheme, the recombinant type I humanized collagen C1L2T prepared by the invention has better cell adhesion effect compared with the type I human collagen, the amino acid sequence is selected from natural type I human collagen amino acid sequences, the possibility of generating immune response when being applied to human bodies is low, and the preparation method is simple.
Drawings
FIG. 1 is a diagram of recombinant expression plasmid pET32a-C1L2T, wherein the corresponding amino acid sequence of C1L2T is SEQ ID No.4.
FIG. 2 shows a gel electrophoresis diagram of the protein C1L2T after induction expression and purification, wherein the lane 1 is a molecular weight Marker, the lane 2 is a C1L2T protein purified by a Ni column, the lane 3 is a C1L2T protein after PPase cleavage, and the lane 4 is a C1L2T protein purified by a Capto Q column.
FIG. 3 shows the results of cell adhesion activity assays for commercially available human collagen and protein C1L2T.
Detailed Description
The following describes embodiments of the present invention, but the present invention is not limited thereto. Unless otherwise indicated, the instrumentation, reagents, materials, etc., used in the present invention are all available through conventional commercial means.
In the present invention, the meaning of "can" includes both the meaning of performing a certain process and the meaning of not performing a certain process. In this specification, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event occurs and instances where it does not.
In the present invention, the "tissue engineering product" refers to a product for tissue engineering. Tissue engineering is an emerging discipline for constructing tissues or organs in vitro or in vivo by combining cell biology and material science.
In the present invention, "medical device" refers to instruments, devices, appliances, in vitro diagnostic reagents and calibrators, materials and other similar or related items that are used directly or indirectly for the human body.
As used herein, "medium stringency conditions", "medium-high stringency conditions", "high stringency conditions" or "very high stringency conditions" describe conditions for hybridization and washing of nucleic acids. Guidance for performing hybridization reactions is provided in Current Protocols in Molecular Biology, john Wiley & Sons, n.y. (1989), 6.3.1-6.3.6, incorporated herein by reference. Aqueous and non-aqueous processes are described in this document, and either may be used. For example, specific hybridization conditions are as follows: (1) Low stringency hybridization conditions are washed 2 times in 6 x sodium chloride/sodium citrate (SSC), at about 45 ℃, then at least 50 ℃, in 0.2 x SSC,0.1% sds (for low stringency conditions the wash temperature can be raised to 55 ℃); (2) Medium stringency hybridization conditions are washed 1 or more times in 6 XSSC, at about 45℃followed by 0.2 XSSC, 0.1% SDS at 60 ℃; (3) High stringency hybridization conditions are washed 1 or more times in 6 XSSC, at about 45℃followed by 65℃in 0.2 XSSC, 0.1% SDS and preferably; (4) Very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS, washed 1 or more times in 0.2 XSSC, 1% SDS at 65℃followed by 65 ℃.
In the present invention, the host cell may be a prokaryotic cell, such as a bacterium of the enterobacteriaceae family, or a eukaryotic cell, such as fungi and yeasts. One skilled in the art can replace E.coli strains as host cells by other expression strains.
In the present invention, the nucleic acid molecule comprises a nucleic acid sequence encoding the recombinant type I humanized collagen C1L2T of the present invention. The nucleic acid may be DNA or cDNA. The nucleic acid molecule may consist essentially of a nucleic acid sequence encoding a protein of the invention, or may consist of only a nucleic acid sequence encoding a protein of the invention. Such nucleic acid molecules can be synthesized using methods known in the art. Because of the degeneracy of the genetic code, nucleic acid molecules of different nucleic acid sequences can encode the same amino acid sequence.
In the present invention, the nucleic acid sequence of the present invention is included in the vector. Suitable vectors are known in the art of vector construction and include selection of promoters and other regulatory elements, such as enhancer elements. The vectors of the present invention include sequences suitable for introduction into cells. For example, the vector may be an expression vector in which the coding sequence of the protein is under the control of its own cis-acting regulatory element, the vector being designed to facilitate gene integration or gene replacement in a host cell, etc.
In the present invention, the term "vector" includes DNA molecules, such as plasmids, phages, viruses or other vectors, which contain one or more heterologous or recombinant nucleic acid sequences. Suitable phage and viral vectors include, but are not limited to: lambda phage, EMBL phage, simian virus, bovine wart virus, epstein-Barr virus, adenovirus, herpes virus, mouse sarcoma virus, murine breast cancer virus, lentivirus, etc.
In the present invention, recombinant type I humanized collagen C1L2T can be obtained byThe preparation is carried out by conventional methods in the art. For example, it can be produced by the steps of: (1) construction of escherichia coli genetic engineering bacteria: a. obtaining a target gene fragment; b. inserting the obtained target gene fragment into a pET-32a expression vector to obtain a recombinant expression plasmid; c. transferring the recombinant expression plasmid into escherichia coli competent cells BL21 (DE 3), and screening to obtain positive escherichia coli genetic engineering bacteria; (2) Fermentation culture of escherichia coli genetic engineering bacteria and induction and expression of protein: a. selecting a single colony of the optimized escherichia coli genetic engineering bacteria from an LB plate, placing the single colony in a liquid culture medium containing ampicillin antibiotics, culturing at 37 ℃ and 220rpm for 5 hours, and cooling to 16 ℃; b. induction was performed by adding IPTG at a final concentration of 0.25mM, and culturing was performed for 18 hours. Centrifuging at 3000rpm and 4 ℃ for 20min to collect thalli; (3) purification of the protein and optional cleavage: a. bacteria were resuspended in Tris buffer (25mM Tris,200mM NaCl,pH8.0), homogenized and broken, centrifuged at 17000rpm for 20min at 4℃and the supernatant collected; b. by Ni 6 FF affinity column binding protein, rinsing the hybrid protein with wash buffer containing 20mM imidazole (20 mM imidazole, 25mM Tris,200mM NaCl,pH 8.0), eluting the protein of interest with a solution containing 250mM imidazole (250 mM imidazole, 25mM Tris,200mM NaCl,pH 8.0); c. adding a proper amount of Prescission Protease (PPase) protease with His tag into the eluted protein sample, and performing enzyme digestion for 2h at the temperature of 16 ℃; d. and (3) transferring the protein dialyzing solution after the enzyme digestion into a solution A (20mM Tris,10mM NaCl,pH 8.0), flowing through a Capto Q ion exchange column, and collecting the flow-through solution, namely the target protein for removing the carrier protein.
In practical use, the protein polypeptide of the present invention or a pharmaceutically acceptable salt thereof, a derivative thereof or a pharmaceutically acceptable salt thereof, the above conjugate, the above multimer and the above composition may be administered to a patient as a drug directly or after being mixed with a suitable carrier or excipient. The carrier materials herein include, but are not limited to, water soluble carrier materials (e.g., polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (e.g., ethylcellulose, cholesterol stearate, etc.), enteric carrier materials (e.g., cellulose acetate phthalate, carboxymethyl ethyl cellulose, etc.). Among them, preferred is a water-soluble carrier material. The materials can be prepared into various dosage forms, including but not limited to tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories, freeze-dried powder injection and the like. The suppository can be pessary, vaginal ring, ointment, cream or gel suitable for vaginal application. The protein polypeptide dosage form can be a common preparation, a slow release preparation, a controlled release preparation and various microparticle administration systems. For the purpose of shaping the unit dosage form into a tablet, various carriers known in the art can be widely used. Examples of carriers are, for example, diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.; humectants and binders such as water, glycerin, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, dextrose solution, acacia slurry, gelatin slurry, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone, and the like; disintegrants such as dry starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid ester, sodium dodecyl sulfonate, methylcellulose, ethylcellulose, etc.; disintegration inhibitors such as sucrose, glyceryl tristearate, cocoa butter, hydrogenated oils and the like; absorption promoters such as quaternary ammonium salts, sodium lauryl sulfate, and the like; lubricants such as talc, silica, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, and the like. The tablets may be further formulated into coated tablets, such as sugar coated tablets, film coated tablets, enteric coated tablets, or bilayer and multilayer tablets. For the purpose of formulating the unit dosage form into a pill, various carriers well known in the art can be widely used. Examples of carriers are, for example, diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oils, polyvinylpyrrolidone, gelucire, kaolin, talc, etc.; binders such as acacia, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or batter, and the like; disintegrants such as agar powder, dry starch, alginate, sodium dodecyl sulfate, methylcellulose, ethylcellulose, etc. For preparing a unit dosage form into a suppository, various carriers well known in the art can be widely used. Examples of carriers include polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semisynthetic glycerides, and the like. For preparing unit dosage forms into injectable preparations such as solutions, emulsions, lyophilized powders and suspensions, all diluents commonly used in the art, for example, water, ethanol, polyethylene glycol, 1, 3-propanediol, ethoxylated isostearyl alcohol, polyoxyisostearyl alcohol, polyoxyethylene sorbitol fatty acid esters, etc. may be used. In addition, in order to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose or glycerin may be added to the preparation for injection, and further, a conventional cosolvent, a buffer, a pH adjuster, and the like may be added. In addition, colorants, preservatives, flavors, flavoring agents, sweeteners, or other materials may also be added to the pharmaceutical formulation, if desired.
The preparation can be administrated by injection, including subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, intracisternal injection or infusion, etc.; administration via the luminal tract, such as rectally, vaginally, and sublingually; respiratory tract administration, such as via the nasal cavity; mucosal administration. The above route of administration is preferably injection, and the preferred route of injection is subcutaneous injection.
The dosage of the protein polypeptide of the present invention or a pharmaceutically acceptable salt thereof, a derivative thereof or a pharmaceutically acceptable salt thereof, the conjugate, the polymer and the composition will depend on many factors such as the nature and severity of the disease to be prevented or treated, the sex, age, weight and individual response of the patient or animal, the specific active ingredient used, the route of administration and the number of times of administration, etc. The above-mentioned doses may be administered in a single dosage form or divided into several, for example two, three or four dosage forms. For any particular patient, the particular therapeutically effective dose level will depend on a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific active ingredient employed; the specific composition employed; age, weight, general health, sex and diet of the patient; the time of administration, route of administration and rate of excretion of the particular active ingredient employed; duration of treatment; a medicament for use in combination or simultaneously with the particular active ingredient employed; and similar factors well known in the medical arts. For example, it is common in the art to start doses of the active ingredient below the level required to obtain the desired therapeutic effect and to gradually increase the dose until the desired effect is obtained.
In the present invention, the type I human collagen sequence is as follows:
MFSFVDLRLLLLLAATALLTHGQEEGQVEGQDEDIPPITCVQNGLRYHDRDVWKPEPCRICVCDNGKVLCDDVICDETKNCPGAEVPEGECCPVCPDGSESPTDQETTGVEGPKGDTGPRGPRGPAGPPGRDGIPGQPGLPGPPGPPGPPGPPGLGGNFAPQLSYGYDEKSTGGISVPGPMGPSGPRGLPGPPGAPGPQGFQGPPGEPGEPGASGPMGPRGPPGPPGKNGDDGEAGKPGRPGERGPPGPQGARGLPGTAGLPGMKGHRGFSGLDGAKGDAGPAGPKGEPGSPGENGAPGQMGPRGLPGERGRPGAPGPAGARGNDGATGAAGPPGPTGPAGPPGFPGAVGAKGEAGPQGPRGSEGPQGVRGEPGPPGPAGAAGPAGNPGADGQPGAKGANGAPGIAGAPGFPGARGPSGPQGPGGPPGPKGNSGEPGAPGSKGDTGAKGEPGPVGVQGPPGPAGEEGKRGARGEPGPTGLPGPPGERGGPGSRGFPGADGVAGPKGPAGERGSPGPAGPKGSPGEAGRPGEAGLPGAKGLTGSPGSPGPDGKTGPPGPAGQDGRPGPPGPPGARGQAGVMGFPGPKGAAGEPGKAGERGVPGPPGAVGPAGKDGEAGAQGPPGPAGPAGERGEQGPAGSPGFQGLPGPAGPPGEAGKPGEQGVPGDLGAPGPSGARGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGAAGLPGPKGDRGDAGPKGADGSPGKDGVRGLTGPIGPPGPAGAPGDKGESGPSGPAGPTGARGAPGDRGEPGPPGPAGFAGPPGADGQPGAKGEPGDAGAKGDAGPPGPAGPAGPPGPIGNVGAPGAKGARGSAGPPGATGFPGAAGRVGPPGPSGNAGPPGPPGPAGKEGGKGPRGETGPAGRPGEVGPPGPPGPAGEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGASGERGPPGPMGPPGLAGPPGESGREGAPGAEGSPGRDGSPGAKGDRGETGPAGPPGAPGAPGAPGPVGPAGKSGDRGETGPAGPAGPVGPVGARGPAGPQGPRGDKGETGEQGDRGIKGHRGFSGLQGPPGPPGSPGEQGPSGASGPAGPRGPPGSAGAPGKDGLNGLPGPIGPPGPRGRTGDAGPVGPPGPPGPPGPPGPPSAGFDFSFLPQPPQEKAHDGGRYYRADDANVVRDRDLEVDTTLKSLSQQIENIRSPEGSRKNPARTCRDLKMCHSDWKSGEYWIDPNQGCNLDAIKVFCNMETGETCVYPTQPSVAQKNWYISKNPKDKRHVWFGESMTDGFQFEYGGQGSDPADVAIQLTFLRLMSTEASQNITYHCKNSVAYMDQQTGNLKKALLLQGSNEIEIRAEGNSRFTYSVTVDGCTSHTGAWGKTVIEYKTTKTSRLPIIDVAPLDVGAPDQEFGFDVGPVCFL(SEQ ID No.1)。
in the present invention, the sequence of SEQ ID No.2 used is: GAPGPCCGG (SEQ ID No. 2), which is a terminal sequence peptide that enhances collagen activity.
In some specific embodiments, the recombinant type I humanized collagen C1L2T described herein comprises the sequence shown in SEQ ID No.3, wherein the sequence shown in SEQ ID No.3 is a type I human collagen peptide fragment.
GQPGAKGANGAPGIAGAPGFPGARGPSGPQGPGGPPGPKGNSGEPGAPGSKGDTGAKGEPGPVGVQGPPGPAGEEGKRGARGEPGPTGLPGPPGERGGPGSRGFPGADGVAGPKGPAGERGSPGPAGPKGSPGEAGRPGEAGLPGAKGLTGSPGSPGPDGKTGPPGPAGQDGRPGPPGPPGARGQAGVMGFPGPKGAAGEPGKAGERGVPGPP (SEQ ID No. 3) in other specific embodiments, the recombinant humanized collagen type I C1L2T according to the present invention comprises the sequence shown as SEQ ID No.3 and the sequence shown as SEQ ID No.2, wherein the sequence shown as SEQ ID No.3 and the sequence shown as SEQ ID No.2 are directly linked.
GQPGAKGANGAPGIAGAPGFPGARGPSGPQGPGGPPGPKGNSGEPGAPGSKGDTGAKGEPGPVGVQGPPGPAGEEGKRGARGEPGPTGLPGPPGERGGPGSRGFPGADGVAGPKGPAGERGSPGPAGPKGSPGEAGRPGEAGLPGAKGLTGSPGSPGPDGKTGPPGPAGQDGRPGPPGPPGARGQAGVMGFPGPKGAAGEPGKAGERGVPGPPGAPGPCCGG(SEQ ID No.4)
In other preferred embodiments, the recombinant type I humanized collagen C1L2T of the present invention comprises a sequence of substitution, deletion, insertion and/or addition of one or more amino acids in the amino acid sequence set forth in SEQ ID No.4 or in the sequence set forth in SEQ ID No.4, provided that the recombinant type I humanized collagen C1L2T of the present invention retains the cell adhesive effect of the amino acid sequence of SEQ ID No.4. The "plurality" may be 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11.
Amino acid addition refers to the addition of an amino acid at the C-or N-terminus of an amino acid sequence, e.g., the amino acid sequence shown in SEQ ID No.4, provided that the recombinant type I humanized collagen C1L2T of the present invention retains the cell adhesion effect of the amino acid sequence shown in SEQ ID No.4.
Amino acid substitution refers to the replacement of a certain amino acid residue at a certain position in an amino acid sequence, for example, as shown in SEQ ID No.4, with another amino acid residue, as long as the recombinant humanized collagen type I C1L2T of the present invention retains the cell adhesion effect of the amino acid sequence shown in SEQ ID No.4.
Amino acid insertion refers to the insertion of amino acid residues at appropriate positions in an amino acid sequence, e.g., as shown in SEQ ID No.4, which may also be all or partially adjacent to each other, or none of the inserted amino acids, as long as the recombinant humanized collagen type I C1L2T of the present invention retains the cell adhesion effect of the amino acid sequence shown in SEQ ID No.4.
Amino acid deletion means that 1, 2 or 3 or more amino acids can be deleted from the amino acid sequence, for example, from the amino acid sequence shown in SEQ ID No.4, as long as the recombinant type I humanized collagen C1L2T of the present invention retains the cell adhesion effect of the amino acid sequence shown in SEQ ID No.4.
In the present invention, the substitution may be a conservative amino acid substitution, meaning that 3, more preferably 2 or 1 amino acids are replaced with amino acids having similar or similar properties to the amino acid sequence shown in SEQ ID No.4 to form a peptide. These conservatively mutated peptides may be generated by amino acid substitutions according to the table below.
Initial residues Representative substitution Preferred substitution
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
In order to more clearly describe the technical solution of the present invention, the following description is further given by way of specific examples, but not by way of limitation, only some examples of the present invention.
Example 1: preparation of recombinant type I humanized collagen C1L2T
1. Construction of C1L2T Gene expression vector
The total length of the amino acid sequence of the recombinant I-type humanized collagen C1L2T used in the embodiment is shown as SEQ ID No.4, 222aa is added, the total length of the corresponding gene is 666bp, the codon optimization is carried out on the codon of the escherichia coli, and the optimized sequence is as follows: GGACAACCCGGAGCAAAAGGTGCTAATGGGGCGCCTGGCATCGCCGGTGCTCCGGGATTCCCGGGCGCGAGAGGCCCGAGCGGTCCGCAGGGTCCGGGCGGTCCGCCGGGGCCGAAGGGAAACAGCGGTGAGCCGGGTGCGCCAGGCTCCAAAGGTGATACGGGTGCTAAAGGCGAACCGGGCCCGGTTGGTGTCCAGGGTCCACCGGGACCGGCTGGTGAGGAAGGTAAACGTGGTGCTCGCGGTGAGCCGGGTCCGACCGGTCTGCCGGGTCCGCCGGGCGAGCGCGGCGGTCCTGGTAGCCGTGGCTTTCCGGGCGCGGACGGCGTTGCAGGTCCGAAGGGCCCGGCCGGTGAGCGTGGTTCCCCGGGCCCAGCGGGCCCCAAGGGCAGCCCGGGCGAAGCGGGTCGTCCGGGTGAAGCAGGCCTGCCGGGTGCCAAAGGTTTGACCGGCTCTCCGGGCTCGCCGGGCCCAGATGGTAAAACCGGTCCGCCAGGTCCGGCGGGCCAAGACGGCCGTCCGGGTCCGCCGGGTCCTCCGGGCGCGCGTGGTCAAGCAGGCGTGATGGGTTTCCCGGGCCCGAAGGGTGCGGCAGGCGAGCCGGGTAAGGCGGGCGAACGCGGGGTGCCGGGCCCGCCGGGTGCACCGGGTCCGTGTTGTGGTGGT (SEQ ID No. 5).
The synthesis of the gene fragment is carried out by Beijing Cheng Yuanke lovely gene biotechnology limited company, and the synthesized gene fragment is inserted between BamHI and XhoI restriction sites of the pET-32a expression vector to obtain a corresponding recombinant expression plasmid pET32a-C1L2T, and the detailed diagram is shown in figure 1.
2. Transformation of recombinant expression plasmids
Transferring the recombinant expression plasmid into an escherichia coli competent cell BL21 (DE 3), and screening to obtain the positive escherichia coli genetic engineering bacteria.
The specific process is as follows: (1) taking 1 mu L of the plasmid into 100 mu L of escherichia coli competent cells BL21 (DE 3), and standing on ice for 30min; (2) heating the mixture in a water bath at a temperature of 42 ℃ for 90s, and then rapidly standing on ice for 2min; (3) to this mixture, 700. Mu.L of a non-resistant LB liquid medium (10 g/L peptone, 5g/L yeast extract, 10g/L sodium chloride) was added, and the mixture was cultured at 37℃for 1 hour at 220 rpm; (4) 200. Mu.L of the bacterial liquid was uniformly spread on LB solid medium plates (10 g/L peptone, 5g/L yeast extract, 10g/L sodium chloride, 15g/L agar, 100. Mu.g/mL ampicillin) containing ampicillin; (5) the plates were incubated upside down in a 37℃incubator for about 16 hours to allow for the growth of clearly visible colonies.
3. Inducible expression of a protein of interest
And selecting a single colony of the optimized escherichia coli genetic engineering bacteria from the LB plate, placing the single colony into an LB liquid culture medium containing ampicillin antibiotics, culturing at 37 ℃ and 220rpm for 5 hours, cooling to 16 ℃, and adding IPTG with the final concentration of 0.25mM for induction and culturing for 18 hours. The cells were collected by centrifugation at 3000rpm and 4℃for 20 min.
Purification of C1L2T
Bacteria were resuspended in Tris buffer (25mM Tris,200mM NaCl,pH8.0), lysed, homogenized and broken, centrifuged at 17000rpm for 20min at 4℃and the supernatant was collected; washing the Ni affinity column with clear water, balancing the column with buffer 1 (25mM Tris,200mM NaCl,pH8.0), loading, rinsing the hybrid protein with a wash buffer containing 20mM imidazole (20 mM imidazole, 25mM Tris,200mM NaCl,pH 8.0), eluting the protein of interest with a solution containing 250mM imidazole (250 mM imidazole, 25mM Tris,200mM NaCl,pH 8.0); the column was washed with a solution containing 1M imidazole, then with water, and finally with 20% ethanol.
An appropriate amount of His-tagged Prescission Protease (PPase) protease was added to the eluted protein sample, and the mixture was digested at 16℃for 2 hours.
Dialyzing the digested protein to obtain solution A (20mM Tris,10mM NaCl,pH 8.0); and (3) balancing a Capto Q (Cytiva company, product number: 17531610) ion exchange column by using a solution A with the volume of 5 times of the column volume, allowing the protein after enzyme switching liquid to flow through the Capto Q column, and collecting the flowing-through liquid to obtain the target protein for removing the carrier protein.
Electrophoresis detection of C1L2T
The purity of the obtained C1L2T was checked by SDS-PAGE. The specific process is as follows: 20. Mu.L of purified protein solution was added with 5. Mu.L of 5 Xprotein loading buffer (250 mM Tris-HCl (pH 6.8), 10% SDS,0.5% bromophenol blue, 50% glycerol, 5% beta-mercaptoethanol), and the mixture was placed in boiling water at 100℃for 5 minutes, then 10. Mu.L of each well was added to SDS-PAGE protein gel, and after electrophoresis at a voltage of 150V for 1 hour, protein staining was performed for 3 minutes with Coomassie brilliant blue staining solution (0.1% Coomassie brilliant blue R-250, 25% ethanol, 10% glacial acetic acid), and further protein staining was performed with protein staining solution (10% acetic acid, 5% ethanol).
The detection result is shown in FIG. 2, the apparent molecular weight of C1L2T obtained by electrophoresis is 33kDa, and the molecular weight corresponds to that of recombinant type I humanized collagen C1L2T, which indicates that the protein C1L2T is correctly expressed.
Example 2: biological activity detection of recombinant type I humanized collagen C1L2T
Methods for detecting collagen activity can be found in references 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:
(1) The concentration of the protein sample to be detected is detected by an ultraviolet absorption method, and the protein sample to be detected comprises commercial human collagen (Sigma, C7774) and recombinant I-type humanized collagen C1L2T provided by the invention.
Specifically, the ultraviolet absorbance of the samples at 215nm and 225nm, respectively, was measured, and the protein concentration was calculated using the empirical formula C (μg/mL) =144× (a 215-a 225), taking care of detection at a215< 1.5. After the detection of the protein concentration, the concentration of all the proteins to be tested was adjusted to 0.5mg/ml with PBS.
(2) 100. Mu.L of each protein solution was added to the 96-well plate and allowed to stand at room temperature for 60min in comparison with a blank PBS solution.
(3) 10 is added into each hole 5 3T3 cells with good culture state are incubated for 60min at 37 ℃.
(4) Each well was washed 4 times with PBS.
(5) Using LDH detection kitRoche, 04744926001) detection of absorbance OD at 492nm 492nm . The cell attachment rate can be calculated from the values of the blank. The calculation formula is as follows: cell attachment rate = (test well-blank well) ×100%/(positive well-blank well).
As shown in FIG. 3, it is understood from the comparison that the recombinant type I humanized collagen C1L2T of the present invention was added to the hole OD compared with the commercial human collagen 492nm Larger means that the cell attachment rate of the well is greater. The protein C1L2T has higher bioactivity than commercial human collagen, can provide a high-quality external environment for cells in a shorter time, and helps the cells to adhere to the wall.
Sequence listing
<110> Shanxi brocade biological medicine Co., ltd
<120> a recombinant I-type humanized collagen C1L2T, and preparation method and use thereof
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Glu Asp Ile Pro Pro Ile Thr Cys Val Gln Asn Gly Leu Arg Tyr His
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Asp Arg Asp Val Trp Lys Pro Glu Pro Cys Arg Ile Cys Val Cys Asp
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Asn Gly Lys Val Leu Cys Asp Asp Val Ile Cys Asp Glu Thr Lys Asn
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Cys Pro Gly Ala Glu Val Pro Glu Gly Glu Cys Cys Pro Val Cys Pro
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Asp Gly Ser Glu Ser Pro Thr Asp Gln Glu Thr Thr Gly Val Glu Gly
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Pro Lys Gly Asp Thr Gly Pro Arg Gly Pro Arg Gly Pro Ala Gly Pro
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Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Leu Gly Gly Asn Phe Ala
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Pro Gln Leu Ser Tyr Gly Tyr Asp Glu Lys Ser Thr Gly Gly Ile Ser
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Val Pro Gly Pro Met Gly Pro Ser Gly Pro Arg Gly Leu Pro Gly Pro
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Pro Gly Ala Pro Gly Pro Gln Gly Phe Gln Gly Pro Pro Gly Glu Pro
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Gly Glu Pro Gly Ala Ser Gly Pro Met Gly Pro Arg Gly Pro Pro Gly
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Gly Leu Pro Gly Glu Arg Gly Arg Pro Gly Ala Pro Gly Pro Ala Gly
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Ala Arg Gly Asn Asp Gly Ala Thr Gly Ala Ala Gly Pro Pro Gly Pro
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Thr Gly Pro Ala Gly Pro Pro Gly Phe Pro Gly Ala Val Gly Ala Lys
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Gly Glu Ala Gly Pro Gln Gly Pro Arg Gly Ser Glu Gly Pro Gln Gly
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Val Arg Gly Glu Pro Gly Pro Pro Gly Pro Ala Gly Ala Ala Gly Pro
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Ala Gly Asn Pro Gly Ala Asp Gly Gln Pro Gly Ala Lys Gly Ala Asn
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Gly Ala Pro Gly Ile Ala Gly Ala Pro Gly Phe Pro Gly Ala Arg Gly
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Pro Ser Gly Pro Gln Gly Pro Gly Gly Pro Pro Gly Pro Lys Gly Asn
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Ser Gly Glu Pro Gly Ala Pro Gly Ser Lys Gly Asp Thr Gly Ala Lys
435 440 445
Gly Glu Pro Gly Pro Val Gly Val Gln Gly Pro Pro Gly Pro Ala Gly
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Glu Glu Gly Lys Arg Gly Ala Arg Gly Glu Pro Gly Pro Thr Gly Leu
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Pro Gly Pro Pro Gly Glu Arg Gly Gly Pro Gly Ser Arg Gly Phe Pro
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Gly Ala Asp Gly Val Ala Gly Pro Lys Gly Pro Ala Gly Glu Arg Gly
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Ser Pro Gly Pro Ala Gly Pro Lys Gly Ser Pro Gly Glu Ala Gly Arg
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Gln Asp Gly Arg Pro Gly Pro Pro Gly Pro Pro Gly Ala Arg Gly Gln
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Gly Phe Gln Gly Leu Pro Gly Pro Ala Gly Pro Pro Gly Glu Ala Gly
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Lys Pro Gly Glu Gln Gly Val Pro Gly Asp Leu Gly Ala Pro Gly Pro
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Ser Gly Ala Arg Gly Glu Arg Gly Phe Pro Gly Glu Arg Gly Val Gln
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Gly Pro Pro Gly Pro Ala Gly Pro Arg Gly Ala Asn Gly Ala Pro Gly
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Ala Asp Gly Ser Pro Gly Lys Asp Gly Val Arg Gly Leu Thr Gly Pro
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Gly Pro Ser Gly Pro Ala Gly Pro Thr Gly Ala Arg Gly Ala Pro Gly
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Gly Ala Lys Gly Asp Ala Gly Pro Pro Gly Pro Ala Gly Pro Ala Gly
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Pro Pro Gly Pro Ile Gly Asn Val Gly Ala Pro Gly Ala Lys Gly Ala
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Arg Gly Ser Ala Gly Pro Pro Gly Ala Thr Gly Phe Pro Gly Ala Ala
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Gly Arg Val Gly Pro Pro Gly Pro Ser Gly Asn Ala Gly Pro Pro Gly
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Thr Gly Pro Ala Gly Arg Pro Gly Glu Val Gly Pro Pro Gly Pro Pro
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Gly Pro Ala Gly Glu Lys Gly Ser Pro Gly Ala Asp Gly Pro Ala Gly
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Ala Pro Gly Thr Pro Gly Pro Gln Gly Ile Ala Gly Gln Arg Gly Val
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Val Gly Leu Pro Gly Gln Arg Gly Glu Arg Gly Phe Pro Gly Leu Pro
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Gly Pro Ser Gly Glu Pro Gly Lys Gln Gly Pro Ser Gly Ala Ser Gly
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Gly Arg Asp Gly Ser Pro Gly Ala Lys Gly Asp Arg Gly Glu Thr Gly
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Pro Ala Gly Pro Pro Gly Ala Pro Gly Ala Pro Gly Ala Pro Gly Pro
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Val Gly Pro Ala Gly Lys Ser Gly Asp Arg Gly Glu Thr Gly Pro Ala
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Gly Pro Ala Gly Pro Val Gly Pro Val Gly Ala Arg Gly Pro Ala Gly
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Arg Gly Ile Lys Gly His Arg Gly Phe Ser Gly Leu Gln Gly Pro Pro
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Gly Pro Pro Gly Ser Pro Gly Glu Gln Gly Pro Ser Gly Ala Ser Gly
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Pro Ala Gly Pro Arg Gly Pro Pro Gly Ser Ala Gly Ala Pro Gly Lys
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Asp Gly Leu Asn Gly Leu Pro Gly Pro Ile Gly Pro Pro Gly Pro Arg
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Gly Arg Thr Gly Asp Ala Gly Pro Val Gly Pro Pro Gly Pro Pro Gly
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1235 1240 1245
Glu Gly Ser Arg Lys Asn Pro Ala Arg Thr Cys Arg Asp Leu Lys Met
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Cys His Ser Asp Trp Lys Ser Gly Glu Tyr Trp Ile Asp Pro Asn Gln
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Gly Cys Asn Leu Asp Ala Ile Lys Val Phe Cys Asn Met Glu Thr Gly
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Glu Thr Cys Val Tyr Pro Thr Gln Pro Ser Val Ala Gln Lys Asn Trp
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Tyr Ile Ser Lys Asn Pro Lys Asp Lys Arg His Val Trp Phe Gly Glu
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Ser Met Thr Asp Gly Phe Gln Phe Glu Tyr Gly Gly Gln Gly Ser Asp
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Pro Ala Asp Val Ala Ile Gln Leu Thr Phe Leu Arg Leu Met Ser Thr
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Glu Ala Ser Gln Asn Ile Thr Tyr His Cys Lys Asn Ser Val Ala Tyr
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Ser Asn Glu Ile Glu Ile Arg Ala Glu Gly Asn Ser Arg Phe Thr Tyr
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Ser Val Thr Val Asp Gly Cys Thr Ser His Thr Gly Ala Trp Gly Lys
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ggacaacccg gagcaaaagg tgctaatggg gcgcctggca tcgccggtgc tccgggattc 60
ccgggcgcga gaggcccgag cggtccgcag ggtccgggcg gtccgccggg gccgaaggga 120
aacagcggtg agccgggtgc gccaggctcc aaaggtgata cgggtgctaa aggcgaaccg 180
ggcccggttg gtgtccaggg tccaccggga ccggctggtg aggaaggtaa acgtggtgct 240
cgcggtgagc cgggtccgac cggtctgccg ggtccgccgg gcgagcgcgg cggtcctggt 300
agccgtggct ttccgggcgc ggacggcgtt gcaggtccga agggcccggc cggtgagcgt 360
ggttccccgg gcccagcggg ccccaagggc agcccgggcg aagcgggtcg tccgggtgaa 420
gcaggcctgc cgggtgccaa aggtttgacc ggctctccgg gctcgccggg cccagatggt 480
aaaaccggtc cgccaggtcc ggcgggccaa gacggccgtc cgggtccgcc gggtcctccg 540
ggcgcgcgtg gtcaagcagg cgtgatgggt ttcccgggcc cgaagggtgc ggcaggcgag 600
ccgggtaagg cgggcgaacg cggggtgccg ggcccgccgg gtgcaccggg tccgtgttgt 660
ggtggt 666

Claims (10)

1. The amino acid sequence of the recombinant type I humanized collagen C1L2T is shown as SEQ ID No.4.
2. A polynucleotide encoding the recombinant type I humanized collagen C1L2T according to claim 1.
3. An expression vector comprising the polynucleotide of claim 2.
4. A host cell comprising the expression vector of claim 3.
5. The method for producing recombinant type I humanized collagen C1L2T according to claim 1, comprising the steps of:
(1) culturing the host cell of claim 4 in a medium and producing the protein;
(2) harvesting and purifying the protein;
(3) optionally, cleaving the protein.
6. The method according to claim 5, wherein in step (2), the protein is purified using a Ni column and/or ion exchange chromatography.
7. The method according to claim 5, wherein in step (3), the protein is cleaved with PPase protease.
8. Use of recombinant type I humanized collagen C1L2T according to claim 1 in the manufacture of a product, wherein the product is a tissue engineering product, a cosmetic or a pharmaceutical.
9. A product comprising the recombinant type I humanized collagen C1L2T according to claim 1, wherein the product is a tissue engineering product, a cosmetic or a pharmaceutical.
10. Use of recombinant type I humanized collagen C1L2T according to claim 1 for the preparation of a product having a cell adhesion promoting effect.
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