CN113683679A

CN113683679A - Recombinant I-type humanized collagen C1L6T and preparation method and application thereof

Info

Publication number: CN113683679A
Application number: CN202111082075.5A
Authority: CN
Inventors: 杨霞; 武庚风; 张永健
Original assignee: Shanxi Jinbo Bio Pharmaceutical Co Ltd
Current assignee: Shanxi Jinbo Bio Pharmaceutical Co Ltd
Priority date: 2021-09-15
Filing date: 2021-09-15
Publication date: 2021-11-23
Anticipated expiration: 2041-09-15
Also published as: CN113683679B

Abstract

The invention discloses a recombinant I-type humanized collagen C1L6T and a preparation method and application thereof. The recombinant type I humanized collagen C1L6T provided by the present invention comprises the sequence shown in SEQ ID No.3; optionally, the recombinant type I humanized collagen C1L6T comprises the sequence shown in SEQ ID No.2 sequence; preferably, the sequence shown in SEQ ID No.3 is directly linked to the N-terminus of the sequence shown in SEQ ID No.2. The amino acid sequence of the recombinant type I humanized collagen C1L6T prepared by the present invention is partially derived from the amino acid sequence of natural type I collagen, and it will not produce immune response when applied to the human body. In addition, the protein has a correct folded structure and strong stability. , the advantages of high expression, and experiments show that this protein has better cell adhesion effect than type I collagen itself.

Description

Recombinant I-type humanized collagen C1L6T and preparation method and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and relates to a recombinant I-type humanized collagen C1L6T, and a preparation method and application thereof.

Background

Collagen (collagen) accounts for 25% to 30% of the total protein in humans, with collagen type I being the most abundant, above about 85%, being high in bone, skin, tendon and cornea, type II being present in cartilage, intervertebral discs and vitreous, and type III being present in blood vessels, neogenetic skin and scar tissue.

The collagen has good biocompatibility, biodegradability and bioactivity, and can be widely applied to the industries of pharmaceutical chemicals, foods, cosmetics and the like. The clinical application of the medicine comprises: collagen membrane for treating burn and wound, medical collagen injection for beautifying and orthopedic treatment, collagen hemostatic sponge for wound hemostasis and the like. In the field of cosmetics, collagen is an important component of skin extracellular matrix, and has the effects of moisturizing, supplementing skin collagen, resisting aging and the like.

At present, most of commercially available collagen products are taken from animal tissues such as pigs, cows, fish and the like, the risk of virus infection is difficult to avoid, and simultaneously, the collagen products are not compatible with human bodies, so that the collagen products are used for the human bodies and are easy to cause immunological rejection and allergic symptoms. Meanwhile, the conventional method for producing collagen is to treat animal-derived tissues by acid, alkali, and enzymatic hydrolysis methods to extract collagen derivatives. The collagen extracted by the methods loses the original biological activity and cannot be applied to the biomedical field to play the real function. Therefore, people now shift the research idea of collagen production to genetic engineering means.

The basic structure of collagen is procollagen, each procollagen molecule consists of three a peptide chains, each a peptide chain forms a left-hand helix, and the three left-hand helices are twisted together to form a large right-hand triple helix structure. Collagen consists of a repeating amino acid sequence of a specific Gly-Xaa-Yaa, where Xaa is usually proline and Yaa is usually hydroxyproline or hydroxylysine. Because of the specific amino acid composition of collagen, how to improve the stability and expression quantity of collagen and whether a correct folding structure can be formed by the collagen prepared by a genetic engineering mode is a key aspect for restricting the application of the collagen. Therefore, in the process of preparing collagen by a genetic engineering mode, a proper collagen structural domain needs to be selected, and the amino acid sequence of the selected collagen domain needs to be reasonably optimized, so that the preparation efficiency of the collagen can be improved, and the stability and the activity of the prepared collagen can be improved.

Disclosure of Invention

Problems to be solved by the invention

Aiming at the problems of low bioactivity, low bioavailability, easy induction of immune response and the like of the collagen extracted by the traditional method in the field and the requirement of stabilizing the protein and forming a correct folding structure when the collagen is prepared by a genetic engineering method, the invention provides the recombinant I-type humanized collagen C1L6T, and also provides a preparation method and application thereof.

Means for solving the problems

In a first aspect, the invention provides a recombinant humanized collagen type I C1L6T, wherein the recombinant humanized collagen type I C1L6T comprises a sequence shown as SEQ ID No. 3.

Further, in the above recombinant type I humanized collagen C1L6T, optionally, the recombinant type I humanized collagen C1L6T comprises a sequence represented by SEQ ID No. 2; preferably, the sequence shown as SEQ ID No.3 is directly linked to the N-terminus of the sequence shown as SEQ ID No. 2.

Further, the recombinant type I humanized collagen C1L6T described above comprises: a) an amino acid sequence shown as SEQ ID No. 4; b) an amino acid sequence having homology of not less than 90% with the amino acid sequence of SEQ ID No.4 retaining the cell adhesion effect of the amino acid sequence of SEQ ID No. 4; c) an amino acid sequence in which 1 or more amino acid residues are added, substituted, deleted or inserted in the amino acid sequence of SEQ ID No.4, which retains the cell adhesion effect of the amino acid sequence of SEQ ID No. 4; or d) an amino acid sequence encoded by a nucleotide sequence that retains the cell adhesion effect of the amino acid sequence of SEQ ID No.4, which nucleotide sequence hybridizes with the polynucleotide sequence encoding the amino acid sequence of SEQ ID No.4 under stringent conditions, which are medium stringency conditions, medium-high stringency conditions, high stringency conditions or very high stringency conditions.

In a second aspect, the present invention provides a polynucleotide encoding the recombinant type I humanized collagen C1L6T described above.

In a third aspect, the present invention provides an expression vector comprising the polynucleotide 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 above recombinant type I humanized collagen C1L6T, comprising the steps of: culturing the host cell provided by the fourth aspect in a culture medium and producing a protein; ② harvesting and purifying the protein, preferably purifying the protein with Ni column and/or ion exchange chromatography; and (iii) optionally cleaving said protein, preferably with a PPase protease.

In a sixth aspect, the invention provides the use of the above-mentioned recombinant type I humanized collagen C1L6T in the preparation of a product, wherein the product is preferably a tissue engineering product, a cosmetic, a health product or a medicament.

In a seventh aspect, the present invention provides a product comprising the above-described recombinant type I humanized collagen C1L6T, wherein the product is preferably a tissue engineering product, a cosmetic, a nutraceutical or a pharmaceutical.

In an eighth aspect, the invention provides a use of the recombinant humanized collagen type I C1L6T in preparation of products with a cell adhesion promoting effect.

ADVANTAGEOUS EFFECTS OF INVENTION

Through the implementation of the technical scheme, the amino acid sequence part of the recombinant I-type humanized collagen C1L6T prepared by the invention is derived from the natural I-type collagen amino acid sequence, and the protein can not generate immune reaction when being applied to a human body.

Drawings

FIG. 1 is a map of recombinant expression plasmid pET32a-C1L6T, wherein the corresponding amino acid sequence of C1L6T is SEQ ID No. 4.

FIG. 2 is a gel electrophoresis diagram of protein C1L6T after induction expression and purification, lane 1 is molecular weight Marker, lane 2 is C1L6T protein purified by Ni column, lane 3 is C1L6T protein digested by PPase, lane 4 is C1L6T protein purified by Capto Q column.

FIG. 3 shows the results of cell adhesion activity assays for commercial human collagen and protein C1L 6T.

Detailed Description

The following describes embodiments of the present invention, but the present invention is not limited to these embodiments. Unless otherwise indicated, the instrumentation, reagents, materials, etc. used in the present invention are commercially available in a conventional manner.

In the present specification, the meaning of "may" 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 specification, "tissue engineering product" refers to a product used for tissue engineering. Tissue engineering is an emerging discipline for the construction of tissues or organs in vitro or in vivo, combining cell biology and material science.

In this specification, "medical device" refers to instruments, devices, instruments, in-vitro diagnostic reagents and calibrators, materials, and other similar or related items that are used directly or indirectly on the human body.

In the present invention, the type I collagen sequence is selected as the sequence to be optimized for screening. The sequence of human collagen type I COL1a1 is the NCBI reference sequence: NP-000079 (SEQ ID No.1), seehttps://www.ncbi.nlm.nih.gov/ protein/NP_000079.2。

The amino acid sequences selected in the present invention are underlined in bold in the above sequences. The applicant has found through extensive studies that the selected above sequence achieves better adhesion effect than commercial human collagen. In the present invention, the recombinant type I humanized collagen C1L6T is not the full-length sequence of 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 peptide segment that enhances collagen activity.

The protein in the present invention may be C1L6T, and is a single chain structure comprising 222 amino acid residues. Wherein the N-terminal amino acid sequence is GERGGPGSRGFPGADGVAGPKGPAGERGSPGPAGPKGSPGEAGRPGEAGLPGAKGLTGSPGSPGPDGKTGPPGPAGQDGRPGPPGPPGARGQAGVMGFPGPKGAAGEPGKAGERGVPGPPGAVGPAGKDGEAGAQGPPGPAGPAGERGEQGPAGSPGFQGLPGPAGPPGEAGKPGEQGVPGDLGAPGPSGARGERGFPGERGVQGPPGPAGPR (SEQ ID No.3), which is a human collagen I-type peptide segment; the C-terminal amino acid sequence is GAPGPCCGG (SEQ ID No. 2). Namely, the amino acid sequence of the recombinant humanized collagen I C1L6T is as follows: GERGGPGSRGFPGADGVAGPKGPAGERGSPGPAGPKGSPGEAGRPGEAGLPGAKGLTGSPGSPGPDGKTGPPGPAGQDGRPGPPGPPGARGQAGVMGFPGPKGAAGEPGKAGERGVPGPPGAVGPAGKDGEAGAQGPPGPAGPAGERGEQGPAGSPGFQGLPGPAGPPGEAGKPGEQGVPGDLGAPGPSGARGERGFPGERGVQGPPGPAGPRGAPGPCCGG (SEQ ID No. 4).

In the present invention, the recombinant type I humanized collagen C1L6T can be prepared by a method conventional in the art. For example, it can be produced by the following steps: (1) constructing escherichia coli genetic engineering bacteria; (2) fermenting and culturing the escherichia coli genetic engineering bacteria; (3) induction and expression of proteins; (4) purification of the protein and optional enzymatic cleavage.

In the step (1), the construction of the engineered Escherichia coli can be carried out by the following steps: a. obtaining a target gene segment; 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 an escherichia coli competent cell BL21(DE3), and screening to obtain the positive escherichia coli genetic engineering bacteria.

In the steps (2) and (3), the fermentation culture of the escherichia coli genetically engineered bacteria and the induction and expression of the protein can be carried out by the following steps: a. selecting a single colony of the optimized escherichia coli genetic engineering bacteria, placing the single colony in a culture medium containing ampicillin, culturing at 37 ℃ and 220rpm for 5 hours, and cooling to 16 ℃; b. induction was carried out by adding IPTG at a final concentration of 0.25mM, and culturing was carried out for 18 hours. The cells were collected by centrifugation at 3000rpm at 4 ℃ for 20 min.

In the step (4) above, the purification and cleavage of the protein may be performed by: a. resuspending the bacteria in Tris buffer (25mM Tris, 200mM NaCl, pH8.0), homogenizing and disrupting, centrifuging at 17000rpm at 4 ℃ for 20 minutes, and collecting the supernatant; b. by using Ni₆FF affinity column binding protein, rinsing the hybrid protein with a wash buffer solution containing 20mM imidazole (20mM imidazole, 25mM Tris, 200mM NaCl, pH8.0), eluting the protein of interest with a solution containing 250mM imidazole (250mM imidazole, 25mM Tris, 200mM NaCl, pH 8.0); c. in the eluted eggAdding a proper amount of Prescission Protease (PPase) Protease with His labels into the white sample, and performing enzyme digestion for 2h at 16 ℃ to obtain target protein; d. purifying target protein by using a Capto Q ion exchange column, dialyzing the enzyme-cut protein, changing the solution into A solution (20mM Tris, 10mM NaCl, pH8.0), passing through the Capto Q column, and collecting flow-through solution, namely the target protein without carrier protein.

The invention also provides nucleic acid molecules comprising a nucleic acid sequence encoding a protein of the invention. The nucleic acid may be DNA or cDNA. The nucleic acid molecule may consist essentially of a nucleic acid sequence encoding a protein according to the invention or may consist of only a nucleic acid sequence encoding a protein according to the invention. Such nucleic acid molecules can be synthesized using methods known in the art. Due to the degeneracy of the genetic code, it will be understood by those skilled in the art that nucleic acid molecules of different nucleic acid sequences may encode the same amino acid sequence.

The invention also provides a vector comprising a nucleic acid sequence according to the invention. Suitable vectors are known in the art of vector construction and include promoter selection and other regulatory elements, such as enhancer elements. The vectors of the invention include sequences suitable for introduction into a cell. For example, the vector may be an expression vector in which the coding sequence for the protein is under the control of its own cis-acting regulatory elements, a vector designed to facilitate gene integration or gene replacement in a host cell, or the like.

It will be understood by those of ordinary skill in the art that, in the present invention, the term "vector" includes DNA molecules, e.g., 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, verruca bovis, Epstein-Barr virus, adenovirus, herpes virus, murine sarcoma virus, murine mammary carcinoma virus, lentivirus, and the like.

In the present invention, the host cell may be a eukaryotic cell, such as fungi and yeast, a prokaryotic cell, such as a bacterium of the Enterobacteriaceae family. It is understood that one skilled in the art may substitute the above E.coli strain for other expression strains as host cells.

In the present invention, "homology" refers to the degree of similarity between the nucleotide sequences of two nucleic acid molecules or between the amino acid sequences of two protein molecules.

The recombinant humanized collagen type I C1L6T of the present invention comprises a sequence represented by SEQ ID No.4 or a sequence represented by SEQ ID No.4 in which one or more amino acids are substituted, deleted, inserted and/or added, as long as the recombinant humanized collagen type I C1L6T of the present invention retains the cell adhesion 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 amino acids to the amino acid sequence, such as the C-terminus or N-terminus of SEQ ID No.4, as long as the recombinant type I humanized collagen C1L6T of the present invention retains the cell adhesion effect of the amino acid sequence of SEQ ID No. 4.

Amino acid substitution refers to the substitution of an amino acid residue at a position in the amino acid sequence, e.g., the sequence of SEQ ID No.4, with another amino acid residue, so long as the recombinant type I humanized collagen C1L6T of the present invention retains the cell adhesion effect of the amino acid sequence of SEQ ID No. 4.

The amino acid insertion refers to the insertion of amino acid residues at appropriate positions of an amino acid sequence, such as the sequence of SEQ ID No.4, and the inserted amino acid residues may be adjacent to each other in whole or in part, or none of the inserted amino acids is adjacent to each other, as long as the recombinant humanized collagen type I C1L6T of the present invention retains the cell adhesion effect of the amino acid sequence of SEQ ID No. 4. The position of insertion of amino acids is not in between the repeats in this context.

The amino acid deletion means that 1, 2 or 3 or more amino acids can be deleted from the amino acid sequence, for example, the sequence of SEQ ID No.4, as long as the recombinant type I humanized collagen C1L6T of the present invention retains the cell adhesion effect of the amino acid sequence of SEQ ID No. 4.

In the present invention, the substitution may be a conservative amino acid substitution, which means that 3, more preferably 2 or 1 amino acids are substituted with amino acids having similar or similar properties to the amino acid sequence of SEQ ID No.4 to form a peptide. These conservative variant peptides can be generated by amino acid substitutions according to the following table.

Initial residue(s)	Representative substitutions	Preferred substitutions
			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

As used herein, the terms "medium stringency conditions", "medium-high stringency conditions", "high stringency conditions" or "very high stringency conditions" describe conditions for nucleic acid hybridization and washing. For guidance in performing hybridization reactions see Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which is incorporated herein by reference. Aqueous and non-aqueous methods 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) moderate stringency hybridization conditions are 1 or more washes in 6 XSSC, at about 45 ℃, then 60 ℃ in 0.2 XSSC, 0.1% SDS; (3) high stringency hybridization conditions are 1 or more washes in 6 XSSC, at about 45 ℃, then 65 ℃ in 0.2 XSSC, 0.1% SDS and preferably; (4) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS, 1 or more washes in 0.2 XSSC, 1% SDS at 65 ℃.

In practical applications, the protein polypeptide of the present invention or its pharmaceutically acceptable salt, its derivative or its pharmaceutically acceptable salt, the conjugate, the polymer and the composition can be administered directly to a patient as a medicament or can be administered to a patient after being mixed with a suitable carrier or excipient. The carrier material herein includes, but is not limited to, water-soluble carrier materials (e.g., polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (e.g., ethyl cellulose, cholesterol stearate, etc.), enteric carrier materials (e.g., cellulose acetate phthalate, carboxymethyl cellulose, etc.). Among these, water-soluble carrier materials are preferred. 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 injections and the like. Wherein the suppository can be vaginal suppository, vaginal ring, ointment, cream or gel suitable for vaginal application. The protein polypeptide dosage form can be common preparation, sustained release preparation, controlled release preparation and various particle drug delivery systems. In order to prepare the unit dosage form into tablets, various carriers well known in the art can be widely used. Examples of the carrier 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 and the like; wetting agents and binders such as water, glycerin, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethylcellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone and the like; disintegrating agents such as dried starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid ester, sodium dodecylsulfate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors such as sucrose, glyceryl tristearate, cacao butter, hydrogenated oil and the like; absorption accelerators such as quaternary ammonium salts, sodium lauryl sulfate and the like; lubricants, for example, 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 double-layer and multi-layer tablets. In order to prepare the dosage form for unit administration into a pill, various carriers well known in the art can be widely used. Examples of the carrier are, for example, diluents and absorbents such as glucose, lactose, starch, cacao butter, hydrogenated vegetable oil, polyvinylpyrrolidone, Gelucire, kaolin, talc and the like; binders such as acacia, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or batter, etc.; disintegrating agents, such as agar powder, dried starch, alginate, sodium dodecylsulfate, methylcellulose, ethylcellulose, etc. In order to prepare the unit dosage form into suppositories, various carriers known in the art can be widely used. As examples of the carrier, there may be mentioned, for example, polyethylene glycol, lecithin, cacao butter, higher alcohols, esters of higher alcohols, gelatin, semisynthetic glycerides and the like. In order to prepare the unit dosage form into preparations for injection, 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, polyoxylated isostearyl alcohol, polyoxyethylene sorbitol fatty acid esters, etc., can be used. In addition, for the preparation of isotonic injection, sodium chloride, glucose or glycerol may be added in an appropriate amount to the preparation for injection, and conventional cosolvents, buffers, pH adjusters and the like may also be added. In addition, colorants, preservatives, flavors, flavorings, sweeteners or other materials may also be added to the pharmaceutical preparation, if desired.

The preparation can be used for injection administration, including subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, intracisternal injection or infusion, and the like; for buccal administration, e.g., rectally, vaginally, and sublingually; administration to the respiratory tract, e.g., nasally; administration to the mucosa. The above route of administration is preferably by injection, and the preferred route of injection is subcutaneous injection.

The administration dose of the protein polypeptide of the present invention or its pharmaceutically acceptable salt, its derivative or its pharmaceutically acceptable salt, the above conjugate, the above multimer and the above composition depends on many factors, such as the nature and severity of the disease to be prevented or treated, sex, age, body weight and individual reaction of the patient or animal, the specific active ingredient used, the administration route and administration frequency, and the like. The above-mentioned dosage may be administered in a single dosage form or divided into several, e.g. two, three or four dosage forms. For any particular patient, the specific therapeutically effective dose level will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the particular active ingredient employed; the specific composition employed; the 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; the duration of treatment; drugs used in combination or concomitantly with the specific active ingredient employed; and similar factors known in the medical arts. For example, it is common in the art to start doses of the active ingredient at levels below those required to achieve the desired therapeutic effect and to gradually increase the dose until the desired effect is achieved.

To more clearly illustrate the technical solutions of the present invention, the following embodiments are further described, but the present invention is not limited thereto, and these embodiments are only some examples of the present invention.

Example 1: preparation of recombinant type I humanized collagen C1L6T

Construction of C1L6T Gene expression vector

The full-length protein sequence of the human collagen C1L6T used in the embodiment is the sequence shown in SEQ ID No.4, the full length is 222aa, and the corresponding gene has the full length of 666 bp. Codon optimization is carried out aiming at codons of escherichia coli, and the optimized sequence is as follows: GGAGAAAGAGGCGGTCCCGGATCAAGGGGGTTCCCGGGCGCGGATGGTGTTGCAGGTCCGAAGGGTCCGGCGGGCGAACGTGGTTCGCCGGGCCCGGCTGGTCCGAAAGGTTCTCCGGGTGAGGCTGGACGCCCAGGCGAAGCGGGCCTGCCGGGAGCGAAAGGCTTGACCGGTAGCCCGGGCAGCCCGGGTCCGGACGGTAAGACGGGTCCGCCAGGCCCAGCTGGCCAAGATGGTCGTCCGGGCCCTCCGGGCCCACCGGGTGCCCGTGGTCAGGCAGGCGTTATGGGTTTTCCGGGTCCGAAGGGTGCGGCAGGCGAACCGGGCAAAGCAGGTGAAAGAGGCGTGCCGGGTCCGCCTGGAGCCGTTGGTCCGGCGGGCAAGGACGGTGAGGCAGGTGCGCAGGGTCCGCCGGGTCCGGCGGGCCCGGCTGGCGAGCGCGGTGAGCAAGGTCCGGCGGGCTCCCCGGGCTTTCAGGGTCTGCCGGGTCCGGCGGGCCCACCGGGTGAGGCCGGCAAACCGGGCGAACAAGGTGTGCCGGGCGACCTGGGTGCCCCTGGCCCAAGCGGTGCGCGTGGTGAACGTGGCTTCCCGGGCGAGCGCGGTGTCCAGGGTCCGCCGGGACCGGCGGGCCCGCGTGGTGCACCGGGTCCGTGTTGTGGTGGT (SEQ ID No. 5).

The synthesis of the gene fragment was entrusted to beijing shengyuan gemeng biotechnology limited, and the synthesized gene fragment was inserted between the BamHI and XhoI restriction sites of the expression vector pET-32a to obtain the corresponding recombinant expression plasmid pET32a-C1L6T, and the vector map of the recombinant expression plasmid is shown in fig. 1.

2. Transformation of recombinant expression plasmids

The recombinant expression plasmid is transferred into an escherichia coli competent cell BL21(DE3), and positive escherichia coli genetic engineering bacteria are obtained through screening.

The specific process is as follows: putting 1 μ L of the plasmid into 100 μ L of Escherichia coli competent cell BL21(DE3), and standing on ice for 30 min; secondly, thermally shocking the mixture for 90s in a water bath kettle at 42 ℃, and then rapidly placing the mixture on ice for standing for 2 min; ③ adding 700. mu.L of nonresistant LB liquid medium (10g/L peptone, 5g/L yeast extract, 10g/L sodium chloride) to the mixture, and culturing at 37 ℃ and 220rpm for 1 h; mu.L of the bacterial liquid is evenly coated on an LB plate (10g/L peptone, 5g/L yeast extract, 10g/L sodium chloride, 15g/L agar, 100 mu.g/mL ampicillin) containing ampicillin (Amp); fifthly, the plate is inversely cultured in a 37 ℃ incubator for about 16 hours until a clear and visible colony grows out.

3. Inducible expression of a protein of interest

A single colony of the preferred Escherichia coli genetic engineering bacteria is picked up, placed in a liquid LB culture medium containing ampicillin, cultured at 37 ℃ and 220rpm for 5 hours, cooled to 16 ℃, induced by adding IPTG (final concentration of 0.25mM) and cultured for 18 hours. The cells were collected by centrifugation at 3000rpm at 4 ℃ for 20 min.

Purification of C1L6T

Resuspending the bacteria in Tris buffer (25mM Tris, 200mM NaCl, pH8.0), lysing, homogenizing, disrupting, centrifuging at 17000rpm at 4 deg.C for 20min, and collecting the supernatant; washing the Ni affinity column with clean water, equilibrating the column with buffer 1(25mM Tris, 200mM NaCl, pH8.0), loading, rinsing the hybrid protein with a solution containing 20mM imidazole (20mM imidazole, 25mM Tris, 200mM NaCl, pH8.0), eluting the protein of interest with a solution containing 250mM imidazole (250mM imidazole, 25mM Tris, 200mM NaCl, pH 8.0); washing the column with a solution containing 1M imidazole, washing the column with water, and finally filling the column with 20% ethanol; adding a proper amount of Prescission Protease (PPase) Protease with His labels into the eluted protein sample, and performing enzyme digestion for 2h at 16 ℃; exchanging the digested protein dialysis solution into solution A (20mM Tris, 10mM NaCl, pH 8.0); the method comprises the steps of balancing a Capto Q (Cytiva company, a cargo number: 17531610) ion exchange column with 5 times of column volume of solution A, enabling protein after enzyme digestion and liquid exchange to flow through the Capto Q column, collecting flow-through liquid, namely target protein without carrier protein, and then carrying out electrophoresis detection.

Electrophoretic detection of C1L6T

The purity of the recombinant humanized collagen type I C1L6T was checked by SDS-PAGE. The specific process is as follows: 20 mu L of purified protein solution is taken, 5 mu L of 5 multiplied protein loading buffer solution (250mM Tris-HCl (pH 6.8), 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5% beta-mercaptoethanol) is added, the mixture is placed in boiling water at 100 ℃ for boiling for 5min, 10 mu L of each hole is added into SDS-PAGE protein gel, after electrophoresis is carried out for 1h at the voltage of 150V, protein staining is carried out for 3min by Coomassie brilliant blue staining solution (0.1% Coomassie brilliant blue R-250, 25% ethanol, 10% glacial acetic acid), and then protein destaining solution (10% acetic acid, 5% ethanol) is utilized for destaining.

The detection result is shown in fig. 2, and the apparent molecular weight of the protein obtained by electrophoresis of C1L6T is 33kDa, and the molecular weight corresponds to C1L6T, which indicates that the recombinant type I humanized collagen C1L6T is correctly expressed.

Example 2: biological activity detection of recombinant I-type humanized collagen C1L6T

Methods for detecting Collagen activity may be found in Juming Yao, Satoshi Yanagiwa, Tetsuo Asakura, Design, Expression and Characterization of Collagen-Like Proteins Based on the Cell additive and Crosslinking Sequences Derived from Native Collagen, J biochem.136,643-649 (2004). The specific implementation method comprises the following steps:

(1) the concentration of a protein sample to be detected is detected by using an ultraviolet absorption method, and the protein sample comprises commercial human collagen (Sigma, C7774) and the recombinant I-type humanized collagen C1L6T provided by the invention.

Specifically, the protein concentration was calculated by using the empirical formula C (μ g/mL) 144 × (a215-a225) to measure the ultraviolet absorption at 215nm and 225nm, respectively, and it was noted that the detection was performed in the case of a215< 1.5. The principle of the method is as follows: the characteristic absorption of peptide bond under far ultraviolet light is measured, the detection is not influenced by chromophore content, interference substances are few, the operation is simple and convenient, and the method is suitable for detecting human collagen and analogues thereof which do not develop color in Coomassie brilliant blue. (reference is made to Walker JM. the Protein Protocols Handbook, second edition. HumanaPress.43-45.). After the protein concentration was determined, the concentration of all proteins to be tested was adjusted to 0.5mg/mL with PBS.

(2) 100 μ L of each protein solution and a blank PBS solution control were added to a 96-well plate and allowed to stand at room temperature for 60 min.

(3) Adding 10 into each hole⁵3T3 cells in good culture state were incubated at 37 ℃ for 60 min.

(4) Each well was washed 4 times with PBS.

(5) OD detection with LDH detection kit (Roche, 04744926001)_492nmAbsorbance of (b). According to the value of the blank control, the adherence rate of the cells can be calculated. The calculation formula is as follows: cell adherence rate ═ 100%/(positive well-blank well). The anchorage rate of the cells can reflect the activity of the collagen. The higher the activity of the protein, the better the external environment can be provided for cells in a short time, and the cells can be attached to the wall.

As shown in fig. 3, it is understood from the comparison that the recombinant type I humanized collagen C1L6T of the present invention has more excellent bioadhesive activity than the commercialized human collagen.

Sequence listing

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<120> recombinant I-type humanized collagen C1L6T, and preparation method and application thereof

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Claims

1 . A recombinant type I humanized collagen C1L6T, wherein the recombinant type I humanized collagen C1L6T comprises the sequence shown in SEQ ID No.3.

2. The recombinant type I humanized collagen C1L6T according to claim 1, wherein, optionally, the recombinant type I humanized collagen C1L6T comprises the sequence shown in SEQ ID No.2; Preferably, the sequence shown in SEQ ID No.3 is directly linked to the N-terminus of the sequence shown in SEQ ID No.2.

3. The recombinant type I humanized collagen C1L6T according to claim 1 or 2, wherein the recombinant type I humanized collagen C1L6T comprises:

a) the amino acid sequence shown in SEQ ID No.4;

b) an amino acid sequence having no less than 90% homology with the amino acid sequence of SEQ ID No.4 that retains the cell adhesion effect of the amino acid sequence of SEQ ID No.4;

c) an amino acid sequence with the addition, substitution, deletion or insertion of 1 or more amino acid residues in the amino acid sequence of SEQ ID No. 4 that retains the cell adhesion effect of the amino acid sequence of SEQ ID No. 4; or

d) an amino acid sequence encoded by a nucleotide sequence that retains the cell adhesion effect of the amino acid sequence of SEQ ID No. 4 under stringent conditions with the polynucleotide sequence encoding the amino acid sequence of SEQ ID No. 4 hybridize under conditions of moderate stringency, medium-high stringency, high stringency or very high stringency.

4. A polynucleotide encoding the recombinant type I humanized collagen C1L6T of any one of claims 1 to 3.

5. An expression vector comprising the polynucleotide of claim 4.

6. A host cell comprising the expression vector of claim 5.

7. the production method of the recombinant I type humanized collagen C1L6T described in any one of claim 1 to 3, it comprises the following steps:

1. Cultivate the host cell according to claim 6 in a culture medium and produce protein;

② Harvest and purify the protein, preferably by Ni column and/or ion exchange chromatography; and

③ Optionally, the protein is digested with enzyme, preferably, the protein is digested with PPase protease.

8. Use of the recombinant type I humanized collagen C1L6T according to any one of claims 1 to 3 in the preparation of a product, wherein the product is preferably a tissue engineering product, a cosmetic, a health care product or a medicine.

9. A product comprising the recombinant type I humanized collagen C1L6T according to any one of claims 1 to 3, wherein the product is preferably a tissue engineering product, a cosmetic, a health product or a drug.

10. Use of the recombinant type I humanized collagen C1L6T according to any one of claims 1 to 3 in the preparation of a product with the effect of promoting cell adhesion.