CN113683681B - Recombinant I-type humanized collagen C1L3T and preparation method and application thereof - Google Patents

Recombinant I-type humanized collagen C1L3T and preparation method and application thereof Download PDF

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CN113683681B
CN113683681B CN202111082095.2A CN202111082095A CN113683681B CN 113683681 B CN113683681 B CN 113683681B CN 202111082095 A CN202111082095 A CN 202111082095A CN 113683681 B CN113683681 B CN 113683681B
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gly
pro
ala
collagen
c1l3t
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CN113683681A (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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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 C1L3T and a preparation method and application thereof. The recombinant I-type humanized collagen C1L3T provided by the invention comprises a sequence shown as SEQ ID No.3, and the recombinant I-type humanized collagen C1L3T 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. The amino acid sequence of the recombinant type I humanized collagen C1L3T prepared by the invention is derived from the amino acid sequence of natural collagen, not only has the molecular weight smaller than that of the natural type I collagen, but also has better cell adhesion effect compared with the type I collagen, and meanwhile, the preparation method is simple, and the collagen with higher yield can be obtained.

Description

Recombinant I-type humanized collagen C1L3T 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 C1L3T and a preparation method and application thereof.
Background
Collagen (collagen) is a biopolymer, the main component in animal connective tissue, and is also the most abundant and most widely distributed functional protein in mammals, which may account for 25% -30% of the total protein, and in some organisms the collagen content is even up to more than 80%. Collagen is mainly distributed on the skin, blood vessels, bones, tendons, teeth, cartilage and the like of a human body, is a main matrix and a bracket of the tissues, protects and connects various tissues, and plays an important physiological function in the body.
The most common structural feature of collagen is the triple helix structure formed by 3 peptide chains, i.e. the formation of proteins from 3 a peptide chains in a right-hand supercoiled manner, such triple helix regions being termed collagen regions. Each A peptide chain is formed into a left-handed helix by repeatedly appearing Gly-X-Y (X, Y represents any amino acid residue except Gly), X is Pro, Y is Hyp) peptide fragments, and 3 chains form a stable triple-handed helix structure in a right-handed supercoiled mode by taking the same axis as the center under the interaction of amino acid residues. In organisms, collagen synthesis and modification begin from procollagen, undergo various chemical changes such as hydroxylation, glycosylation, cross-linking and the like, and are subjected to complex regulation and control of various biological enzymes. Procollagen contains globular heads and tails in addition to collagen chains. Without these heads and tails, the collagen chains do not fold into the correct triple helix, thus lacking the biological activity of collagen. Thus, collagen prepared according to the original gene sequence cannot spontaneously form a correct spatial structure in vitro. Such difficulties have severely hampered the development and production of human collagen.
Collagen products are currently marketed as being derived from animal tissues such as swine, bovine, fish, etc. In terms of the amino acid composition of collagen: the similarity of mammal pigs and cows to human is 95% and the similarity of fish to human is 65%. Although mammalian pigs, cattle, etc. have a high degree of similarity to human collagen, it is difficult to avoid the risk of viral infection and sensitization. However, the utilization rate of collagen extracted from edible fish is lower than 65% and cannot be completely absorbed and utilized by human body.
In summary, the collagen can only be used in cosmetics and health care products, and the original biological functions of the collagen cannot be exerted at all.
Disclosure of Invention
Problems to be solved by the invention
Aiming at the situation that the collagen prepared according to the original gene sequence in the field is difficult to form a correct spatial structure in vitro and spontaneous tissues, and the heterologous collagen is easy to induce immune reaction and has low bioavailability, the invention provides a recombinant I-type humanized collagen C1L3T, 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 C1L3T, wherein the recombinant type I humanized collagen C1L3T comprises the sequence shown in SEQ ID No. 3.
Further, the recombinant type I humanized collagen C1L3T 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 C1L3T comprises one or more of the following sequences: an amino acid sequence shown in SEQ ID No. 4; an amino acid sequence having 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% 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 C1L3T described above.
In a third aspect, the present invention provides an expression vector comprising a polynucleotide as provided in the second aspect above.
In a fourth aspect, the present invention provides a host cell comprising the expression vector provided in the third aspect above.
In a fifth aspect, the present invention provides a method for producing the recombinant type I humanized collagen C1L3T, 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 C1L3T 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 C1L3T 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 C1L3T 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 amino acid sequence of the recombinant type I humanized collagen C1L3T prepared by the invention is derived from a natural collagen amino acid sequence, so that the molecular weight is smaller than that of the natural type I collagen, the recombinant type I humanized collagen has better cell adhesion effect compared with the type I collagen, and meanwhile, the preparation method is simple, and the collagen with higher yield can be obtained.
Drawings
FIG. 1 is a plasmid map of recombinant expression vector pET32a-C1L3T, wherein the corresponding amino acid sequence of C1L3T is SEQ ID No.4.
FIG. 2 shows gel electrophoresis of the protein C1L3T after induction expression and purification, wherein the molecular weight Marker is shown in lane 1, the C1L3T protein after PPase cleavage is shown in lane 2, the C1L3T protein after purification by a Ni column is shown in lane 3, and the C1L3T protein after purification by a Capto Q ion exchange column is shown in lane 4.
FIG. 3 shows the results of cell adhesion activity assays for commercially available human collagen and protein C1L3T.
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 or a eukaryotic cell, such as enterobacteriaceae, fungi, yeast, and the like. 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 C1L3T 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, the recombinant type I humanized collagen C1L3T may be prepared by a conventional method 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 target gene fragment into a pET-32a expression vector to obtain a corresponding 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, re-suspending the single colony in an LB liquid culture medium containing ampicillin antibiotics, culturing at 37 ℃ and 220rpm for 5 hours, and cooling to 16 ℃; b. IPTG was added to the mixture to a final concentration of 0.25mM for induction and the mixture was cultured for 18 hours. The cells were collected by centrifugation at 3000rpm and 4℃for 20 min. (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 a solution 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 into the eluted protein sample, and performing enzyme digestion for 2 hours at the temperature of 16 ℃; d. purifying target protein by Capto Q ion exchange column, and dialyzing the digested protein to obtain solution A(20mM Tris,10mM NaCl,pH 8.0) and passing through Capto Q column, collecting the flow-through liquid, and removing the target protein of carrier protein.
In the present invention, the recombinant type I humanized collagen C1L3T amino acid sequence portion is derived from type I human collagen. The type I human collagen has the following sequence:
MFSFVDLRLLLLLAATALLTHGQEEGQVEGQDEDIPPITCVQNGLRYHDRDVWKPEPCRICVCDNGKVLCDDVICDETKNCPGAEVPEGECCPVCPDGSESPTDQETTGVEGPKGDTGPRGPRGPAGPPGRDGIPGQPGLPGPPGPPGPPGPPGLGGNFAPQLSYGYDEKSTGGISVPGPMGPSGPRGLPGPPGAPGPQGFQGPPGEPGEPGASGPMGPRGPPGPPGKNGDDGEAGKPGRPGERGPPGPQGARGLPGTAGLPGMKGHRGFSGLDGAKGDAGPAGPKGEPGSPGENGAPGQMGPRGLPGERGRPGAPGPAGARGNDGATGAAGPPGPTGPAGPPGFPGAVGAKGEAGPQGPRGSEGPQGVRGEPGPPGPAGAAGPAGNPGADGQPGAKGANGAPGIAGAPGFPGARGPSGPQGPGGPPGPKGNSGEPGAPGSKGDTGAKGEPGPVGVQGPPGPAGEEGKRGARGEPGPTGLPGPPGERGGPGSRGFPGADGVAGPKGPAGERGSPGPAGPKGSPGEAGRPGEAGLPGAKGLTGSPGSPGPDGKTGPPGPAGQDGRPGPPGPPGARGQAGVMGFPGPKGAAGEPGKAGERGVPGPPGAVGPAGKDGEAGAQGPPGPAGPAGERGEQGPAGSPGFQGLPGPAGPPGEAGKPGEQGVPGDLGAPGPSGAR GERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGAAGLPGPKGDRGDAGPKG ADGSPGKDGVRGLTGPIGPPGPAGAPGDKGESGPSGPAGPTGARGAPGDRGEPGPPGPAGFAGPPGADGQPGAKGE PGDAGAKGDAGPPGPAGPAGPPGPIGNVGAPGAKGARGSAGPPGATGFPGAAGRVGPPGPSGNAGPPGPPGPAGKEGGKGPRGETGPAGRPGEVGPPGPPGPAGEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGASGERGPPGPMGPPGLAGPPGESGREGAPGAEGSPGRDGSPGAKGDRGETGPAGPPGAPGAPGAPGPVGPAGKSGDRGETGPAGPAGPVGPVGARGPAGPQGPRGDKGETGEQGDRGIKGHRGFSGLQGPPGPPGSPGEQGPSGASGPAGPRGPPGSAGAPGKDGLNGLPGPIGPPGPRGRTGDAGPVGPPGPPGPPGPPGPPSAGFDFSFLPQPPQEKAHDGGRYYRADDANVVRDRDLEVDTTLKSLSQQIENIRSPEGSRKNPARTCRDLKMCHSDWKSGEYWIDPNQGCNLDAIKVFCNMETGETCVYPTQPSVAQKNWYISKNPKDKRHVWFGESMTDGFQFEYGGQGSDPADVAIQLTFLRLMSTEASQNITYHCKNSVAYMDQQTGNLKKALLLQGSNEIEIRAEGNSRFTYSVTVDGCTSHTGAWGKTVIEYKTTKTSRLPIIDVAPLDVGAPDQEFGFDVGPVCFL(SEQ ID No.1)。
in some embodiments, the recombinant type I humanized collagen C1L3T 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, i.e., the underlined portion of SEQ ID No. 1.
GAVGPAGKDGEAGAQGPPGPAGPAGERGEQGPAGSPGFQGLPGPAGPPGEAGKPGEQGVPGDLGAPGPSGARGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGAAGLPGPKGDRGDAGPKGADGSPGKDGVRGLTGPIGPPGPAGAPGDKGESGPSGPAGPTGARGAPGDRGEPGPPGPAGFAGPPGADGQPGAKGEP(SEQ ID No.3)
In some preferred embodiments, the recombinant type I humanized collagen C1L3T of the present invention comprises the sequence shown in SEQ ID No.3 and the sequence shown in SEQ ID No.2 (GAPGPCCGG), wherein the sequence shown in SEQ ID No.2 is a peptide fragment that enhances collagen activity, preferably the sequence shown in SEQ ID No.3 and the sequence shown in SEQ ID No.2 are directly linked.
In some more preferred embodiments, the recombinant type I humanized collagen C1L3T 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 C1L3T 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.
GAVGPAGKDGEAGAQGPPGPAGPAGERGEQGPAGSPGFQGLPGPAGPPGEAGKPGEQGVPGDLGAPGPSGARGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGAAGLPGPKGDRGDAGPKGADGSPGKDGVRGLTGPIGPPGPAGAPGDKGESGPSGPAGPTGARGAPGDRGEPGPPGPAGFAGPPGADGQPGAKGEPGAPGPCCGG(SEQ ID No.4)
In the present invention, amino acid addition means addition of an amino acid at the amino acid sequence, for example, at the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID No.4, as long as the recombinant type I humanized collagen C1L3T of the present invention retains the cell adhesion effect of the amino acid sequence shown in SEQ ID No.4.
In the present invention, amino acid substitution means that a certain amino acid residue at a certain position in an amino acid sequence, for example, an amino acid sequence shown in SEQ ID No.4 is replaced with another amino acid residue, as long as the recombinant type I humanized collagen C1L3T of the present invention retains the cell adhesion effect of the amino acid sequence shown in SEQ ID No.4.
In the present invention, amino acid insertion means insertion of an amino acid residue at an appropriate position in an amino acid sequence, for example, in the amino acid sequence shown in SEQ ID No.4, and the inserted amino acid residues may be all or partially adjacent to each other or none of the inserted amino acids adjacent to each other, as long as the recombinant type I humanized collagen C1L3T of the present invention retains the cell adhesion effect of the amino acid sequence shown in SEQ ID No.4.
In the present invention, 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 C1L3T 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 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 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 C1L3T
1. Construction of C1L3T Gene expression vector
The total length of the amino acid sequence of the recombinant I-type humanized collagen C1L3T used in the embodiment is shown as SEQ ID No.4, 234aa is added, the total length of the corresponding gene is 702bp, the codon optimization is carried out on the codon of the escherichia coli, and the optimized sequence is as follows: GGAGCAGTAGGACCCGCTGGGAAAGATGGTGAGGCAGGCGCGCAGGGTCCACCGGGCCCGGCTGGCCCGGCTGGCGAACGTGGCGAGCAAGGTCCGGCGGGTTCTCCGGGATTCCAGGGTCTGCCTGGCCCGGCGGGTCCGCCTGGCGAAGCTGGCAAACCGGGCGAGCAAGGCGTTCCGGGGGACCTGGGTGCACCGGGCCCGAGCGGTGCCCGTGGTGAAAGAGGTTTTCCGGGCGAGCGCGGTGTCCAGGGCCCACCGGGGCCGGCGGGTCCGCGTGGTGCGAACGGTGCCCCTGGTAATGATGGTGCGAAAGGTGATGCAGGTGCGCCAGGCGCGCCAGGCTCGCAAGGTGCTCCGGGCTTACAGGGTATGCCGGGCGAGCGCGGTGCGGCAGGCCTGCCGGGGCCCAAGGGCGACCGTGGTGACGCAGGTCCGAAGGGCGCCGATGGTAGCCCGGGTAAAGATGGCGTGCGTGGTTTGACCGGTCCCATCGGTCCGCCGGGCCCGGCGGGCGCGCCGGGCGACAAAGGCGAAAGCGGTCCGTCCGGCCCGGCGGGCCCGACGGGTGCTCGTGGTGCGCCAGGTGACCGCGGTGAACCGGGCCCACCGGGTCCGGCGGGCTTCGCCGGCCCGCCGGGCGCGGATGGTCAGCCGGGTGCCAAGGGTGAGCCGGGTGCACCGGGTCCGTGTTGTGGTGGT (SEQ ID No. 5).
The synthesis of the gene fragment is carried out by Beijing Cheng Yuanke lovely gene biotechnology Co., ltd, the synthesized gene fragment is inserted between multiple cloning sites of pET-32a expression vector by utilizing restriction enzymes BamHI and XhoI, and the corresponding recombinant expression plasmid pET32a-C1L3T is obtained, and the detailed plasmid map 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) 1. Mu.L of the recombinant expression plasmid was added to 100. Mu.L of E.coli competent cell BL21 (DE 3), and left on ice for 30min; (2) placing the mixture in a water bath kettle at 42 ℃ for heat shock for 90s, and then rapidly placing the mixture on ice for standing for 2min; (3) to this mixture, 700. Mu.L of LB (10 g/L peptone, 5g/L yeast extract, 10g/L sodium chloride) containing no antibiotic was added, and the mixture was cultured at 37℃and 220rpm for 1 hour; (4) 200. Mu.L of the bacterial liquid was uniformly spread on LB plates containing ampicillin (10 g/L peptone, 5g/L yeast extract, 10g/L sodium chloride, 15g/L agar, 100. Mu.g/mL 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
Selecting a single colony of the optimized escherichia coli genetic engineering bacteria, placing the single colony in an LB liquid culture medium containing ampicillin antibiotics, culturing for 5 hours at 37 ℃ and 220rpm, cooling to 16 ℃, and then 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 C1L3T
(1)Ni 6 FF affinity column purified protein
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; 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 solution containing 20mM imidazole (20 mM imidazole, 25mM Tris,200mM NaCl,pH 8.0), eluting the target protein 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.
(2) Enzyme cutting purified protein
And adding a proper amount of Prescission Protease (PPase) protease with His tag into the eluted protein sample, and performing enzyme digestion for 2 hours at the temperature of 16 ℃.
(3) Capto Q ion exchange column purified protein
Dialyzing the digested protein to change the liquid into a liquid 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 5 times of column volume, passing the protein after enzyme switching solution through the Capto Q column, and collecting the flow-through solution to obtain the target protein for removing the carrier protein.
Electrophoresis detection of C1L3T
Purity of the C1L3T protein obtained above was checked by SDS-PAGE. The specific process is as follows: 20. Mu.L of purified protein solution was taken, 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) was added, the mixture was put 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 the C1L3T obtained by electrophoresis is 35kDa, and the molecular weight corresponds to the protein of the C1L3T, which indicates that the recombinant type I humanized collagen C1L3T is correctly expressed.
Example 2: biological activity detection of recombinant type I humanized collagen C1L3T
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 utilizing an ultraviolet absorption method, and the protein sample to be detected comprises commercial human collagen (Sigma, C7774) and recombinant I-type humanized collagen C1L3T 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. The principle of the method is as follows: the characteristic absorption of peptide bond under far ultraviolet light is measured, the influence of chromophore content is avoided, the interference substances are few, the operation is simple and convenient, and the method is suitable for detecting the human collagen and analogues thereof which are not developed by coomassie brilliant blue. 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) Detection of OD per well with LDH detection kit (Roche, 04744926001) 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). The cell attachment rate can be used for reflecting the activity of the collagen. The higher the activity of the protein, the better the environment can be provided for the cells in a short time, and the cell attachment is assisted.
As shown in fig. 3, it is understood from the comparison that the recombinant type I humanized collagen C1L3T of the present invention has more excellent bioadhesive activity than the commercial human collagen.
Sequence listing
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ggagcagtag gacccgctgg gaaagatggt gaggcaggcg cgcagggtcc accgggcccg 60
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ctgcctggcc cggcgggtcc gcctggcgaa gctggcaaac cgggcgagca aggcgttccg 180
ggggacctgg gtgcaccggg cccgagcggt gcccgtggtg aaagaggttt tccgggcgag 240
cgcggtgtcc agggcccacc ggggccggcg ggtccgcgtg gtgcgaacgg tgcccctggt 300
aatgatggtg cgaaaggtga tgcaggtgcg ccaggcgcgc caggctcgca aggtgctccg 360
ggcttacagg gtatgccggg cgagcgcggt gcggcaggcc tgccggggcc caagggcgac 420
cgtggtgacg caggtccgaa gggcgccgat ggtagcccgg gtaaagatgg cgtgcgtggt 480
ttgaccggtc ccatcggtcc gccgggcccg gcgggcgcgc cgggcgacaa aggcgaaagc 540
ggtccgtccg gcccggcggg cccgacgggt gctcgtggtg cgccaggtga ccgcggtgaa 600
ccgggcccac cgggtccggc gggcttcgcc ggcccgccgg gcgcggatgg tcagccgggt 660
gccaagggtg agccgggtgc accgggtccg tgttgtggtg gt 702

Claims (10)

1. The amino acid sequence of the recombinant type I humanized collagen C1L3T is shown as SEQ ID No.4.
2. A polynucleotide encoding the recombinant type I humanized collagen C1L3T of 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 C1L3T of 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 by 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 the recombinant type I humanized collagen C1L3T of 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 C1L3T of claim 1, wherein the product is a tissue engineering product, cosmetic or pharmaceutical.
10. Use of the recombinant type I humanized collagen C1L3T of claim 1 for the preparation of a product having cell adhesion promoting effect.
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