CN113683679B

CN113683679B - A kind of recombinant type I humanized collagen C1L6T and its preparation method and use

Info

Publication number: CN113683679B
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: 2023-10-31
Anticipated expiration: 2041-09-15
Also published as: CN113683679A

Abstract

The invention discloses a recombinant I-type humanized collagen C1L6T and a preparation method and application thereof. The recombinant I-type humanized collagen C1L6T provided by the invention comprises a sequence shown in SEQ ID No. 3; optionally, the recombinant type I humanized collagen C1L6T comprises the sequence shown in 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. The amino acid sequence part of the recombinant I-type humanized collagen C1L6T prepared by the invention is derived from the amino acid sequence of the natural I-type collagen, and the recombinant I-type humanized collagen is applied to a human body without generating immune reaction.

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 recombinant I-type humanized collagen C1L6T and a preparation method and application thereof.

Background

Collagen (collagen) accounts for 25% -30% of total protein in human body, wherein the most content is type I collagen, which accounts for more than about 85%, the type I collagen has high content in bone, skin, tendon and cornea, type II exists in cartilage, intervertebral disc and vitreous body, and type III exists in blood vessel, neogenesis 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, collagen medical injection for caring skin and correcting shape, collagen hemostatic sponge for stopping wound, etc. In the cosmetic field, collagen is an important component of skin extracellular matrix, and has the effects of moisturizing, supplementing skin collagen, resisting aging and the like.

Most of the collagen products sold in the market at present are taken from animal tissues such as pigs, cows, fishes and the like, so that the risk of virus infection is difficult to avoid, and meanwhile, the collagen products are not compatible with human bodies, so that immune rejection and allergic symptoms are easy to occur when the collagen products are used for the human bodies. Meanwhile, the conventional method for producing collagen is to treat animal-derived tissues by acid, alkali or enzymolysis 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 the real functions. Therefore, the research thought of collagen production is now transferred 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-handed helix, and the three left-handed helices are twisted together to form a large right-handed triple helix structure. Collagen consists of a repetitive amino acid sequence of a specific Gly-Xaa-Yaa, where Xaa is typically proline and Yaa is typically hydroxyproline or hydroxylysine. Because of the specific amino acid composition of the collagen, how to improve the stability and the expression level of the collagen and whether the collagen can form a correct folding structure prepared by a genetic engineering mode is a key aspect for restricting the application of the collagen. Therefore, in the preparation process of the collagen through the genetic engineering manner, a proper collagen domain needs to be selected, and the amino acid sequence of the selected collagen domain is reasonably optimized, so that the preparation efficiency of the collagen can be improved, and the stability and activity of the prepared collagen can be improved.

Disclosure of Invention

Problems to be solved by the invention

Aiming at a series of problems of low bioactivity, low bioavailability, easy induction of immune response and the like of collagen extracted by the traditional method in the field and the requirement that the collagen needs to be stabilized and a correct folding structure is formed by using a genetic engineering method for preparing the collagen, the invention provides a recombinant I-type humanized collagen C1L6T and also 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 C1L6T, wherein the recombinant type I humanized collagen C1L6T comprises the sequence shown in SEQ ID No. 3.

Further, in the above recombinant type I humanized collagen C1L6T, optionally, the recombinant type I humanized collagen C1L6T comprises the sequence shown in 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 in SEQ ID No. 4; b) An amino acid sequence having a homology of not less than 90% with 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; c) An amino acid sequence of 1 or more amino acid residues 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 hybridizes with a polynucleotide sequence encoding the amino acid sequence of SEQ ID No.4 under stringent conditions, which are medium-high, medium-high or very high stringent 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 a 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 recombinant type I humanized collagen C1L6T, comprising the steps of: (1) culturing the host cell provided in the fourth aspect 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; and (3) optionally cleaving the protein, preferably with a PPase protease.

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

In an eighth aspect, the present invention provides the use of the recombinant type I humanized collagen C1L6T 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 part of the recombinant I-type humanized collagen C1L6T prepared by the invention is derived from the natural I-type collagen amino acid sequence, so that immune response can not be generated when the recombinant I-type humanized collagen is applied to a human body, and the recombinant I-type humanized collagen has the advantages of being high in stability and high in expression quantity while being correct in folding structure, and experiments prove that the recombinant I-type humanized collagen has better cell adhesion effect compared with the I-type collagen.

Drawings

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

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

FIG. 3 shows the results of cell adhesion activity assays for commercially available human collagen and protein C1L6T.

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 specification, 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 specification, "tissue engineering product" means 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 this specification, "medical device" refers to instruments, devices, appliances, in vitro diagnostic reagents and calibrators, materials and other similar or related items for direct or indirect use on the human body.

In the present invention, the type I collagen sequence is selected as the screening optimized sequence. The sequence of human collagen type I COL1A1 is NCBI reference sequence: NP-000079 (SEQ ID No. 1), seehttps://www.ncbi.nlm.nih.gov/ protein/NP_000079.2。

The bold underlined parts of the above sequences are the amino acid sequences selected in the present invention. The applicant has found through extensive research that the above sequences are selected to achieve better adhesion 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 sequence peptide that enhances collagen activity.

The protein may be C1L6T in the present invention, in 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 fragment; the C-terminal amino acid sequence was GAPGPCCGG (SEQ ID No. 2). Namely, the amino acid sequence of the recombinant type I humanized collagen C1L6T is as follows: GERGGPGSRGFPGADGVAGPKGPAGERGSPGPAGPKGSPGEAGRPGEAGLPGAKGLTGSPGSPGPDGKTGPPGPAGQDGRPGPPGPPGARGQAGVMGFPGPKGAAGEPGKAGERGVPGPPGAVGPAGKDGEAGAQGPPGPAGPAGERGEQGPAGSPGFQGLPGPAGPPGEAGKPGEQGVPGDLGAPGPSGARGERGFPGERGVQGPPGPAGPRGAPGPCCGG (SEQ ID No. 4).

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

In the step (1), the construction of the escherichia coli genetically engineered bacterium can be performed by the following steps: 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.

In the above steps (2) and (3), the fermentation culture of the E.coli genetically engineered bacterium and the induction and expression of the protein may be performed 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 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. The cells were collected by centrifugation at 3000rpm and 4℃for 20 min.

In the above step (4), the purification and cleavage of the protein may be performed by: 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 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 2 hours at the temperature of 16 ℃ to obtain target protein; d. purifying target protein by using a Capto Q ion exchange column, dialyzing and changing the digested protein into A solution (20mM Tris,10mM NaCl,pH8.0), and collecting the flow-through solution by using the Capto Q column to obtain the target protein for removing 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 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, 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 the nucleic acid sequence of the invention. 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.

As understood by those of ordinary skill in the art, 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, bovine wart virus, epstein-Barr virus, adenovirus, herpes virus, mouse sarcoma virus, murine breast cancer virus, lentivirus, etc.

In the present invention, the host cell may be a eukaryotic cell, such as fungi and yeasts, a prokaryotic cell, such as a bacterium of the enterobacteriaceae family. It will be appreciated that the person skilled in the art may replace the E.coli strain described above as host cell by another expression strain.

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

The recombinant type I humanized collagen C1L6T of the present invention comprises a sequence represented by SEQ ID No.4 or a sequence in which one or more amino acids are substituted, deleted, inserted and/or added in the sequence represented by 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. 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., SEQ ID No.4, provided that 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 replacement of a certain amino acid residue at a certain position of an amino acid sequence, e.g., the sequence of SEQ ID No.4, with another amino acid residue, 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 insertion refers to the insertion of amino acid residues at appropriate positions in an amino acid sequence, such as the sequence of SEQ ID No.4, which may also 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 C1L6T of the present invention retains the cell adhesion effect of the amino acid sequence of SEQ ID No.4. The insertion position of an amino acid is not between the repeated sequences in this context.

Amino acid deletion means that 1, 2 or 3 or more amino acids can be deleted from an amino acid sequence, e.g., 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, a 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 of 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

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. 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 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 C1L6T

Construction of C1L6T Gene expression vector

The full-length protein sequence of the human collagen C1L6T used in the embodiment is shown in SEQ ID No.4, the full length is 222aa, and the full length of the corresponding gene is 666bp. Codon optimization is carried out on the codons of the escherichia coli, and the optimized sequence is as follows: GGAGAAAGAGGCGGTCCCGGATCAAGGGGGTTCCCGGGCGCGGATGGTGTTGCAGGTCCGAAGGGTCCGGCGGGCGAACGTGGTTCGCCGGGCCCGGCTGGTCCGAAAGGTTCTCCGGGTGAGGCTGGACGCCCAGGCGAAGCGGGCCTGCCGGGAGCGAAAGGCTTGACCGGTAGCCCGGGCAGCCCGGGTCCGGACGGTAAGACGGGTCCGCCAGGCCCAGCTGGCCAAGATGGTCGTCCGGGCCCTCCGGGCCCACCGGGTGCCCGTGGTCAGGCAGGCGTTATGGGTTTTCCGGGTCCGAAGGGTGCGGCAGGCGAACCGGGCAAAGCAGGTGAAAGAGGCGTGCCGGGTCCGCCTGGAGCCGTTGGTCCGGCGGGCAAGGACGGTGAGGCAGGTGCGCAGGGTCCGCCGGGTCCGGCGGGCCCGGCTGGCGAGCGCGGTGAGCAAGGTCCGGCGGGCTCCCCGGGCTTTCAGGGTCTGCCGGGTCCGGCGGGCCCACCGGGTGAGGCCGGCAAACCGGGCGAACAAGGTGTGCCGGGCGACCTGGGTGCCCCTGGCCCAAGCGGTGCGCGTGGTGAACGTGGCTTCCCGGGCGAGCGCGGTGTCCAGGGTCCGCCGGGACCGGCGGGCCCGCGTGGTGCACCGGGTCCGTGTTGTGGTGGT (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 an expression vector pET-32a to obtain a corresponding recombinant expression plasmid pET32a-C1L6T, and the vector diagram of the recombinant expression plasmid 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 plates containing ampicillin (Amp) (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 single colony of optimized escherichia coli genetic engineering bacteria, placing the single colony in a liquid LB culture medium containing ampicillin antibiotics, culturing for 5 hours at 37 ℃ and 220rpm, cooling to 16 ℃, adding IPTG (final concentration is 0.25 mM), and culturing for 18 hours. The cells were collected by centrifugation at 3000rpm and 4℃for 20 min.

Purification of C1L6T

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 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); 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 tag into the eluted protein sample, and performing enzyme digestion for 2h at the temperature of 16 ℃; 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 5 times of column volume, allowing the protein after enzyme switching liquid to flow through the Capto Q column, collecting the flowing-through liquid, namely removing target protein of carrier protein, and then performing electrophoresis detection.

Electrophoresis detection of C1L6T

The purity of the obtained recombinant type I humanized collagen C1L6T is detected 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 the C1L6T obtained by electrophoresis is 33kDa, and the molecular weight corresponds to the protein of the C1L6T, which indicates that the recombinant type I humanized collagen C1L6T is correctly expressed.

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

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 C1L6T 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. (reference is Walker JM. The Protein Protocols Handbook, second edition. HumanaPress. 43-45.). After the protein concentration was detected, all the protein concentrations to be tested were 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 with LDH detection kit (Roche, 04744926001) _492nm Is a solid phase, and is a liquid phase. 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 C1L6T of the present invention has more excellent bioadhesive activity than the commercial human collagen.

Sequence listing

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Claims

1. A recombinant type I humanized collagen C1L6T, wherein the amino acid sequence of the recombinant type I humanized collagen is shown as SEQ ID No.4.

2. A polynucleotide encoding the recombinant type I humanized collagen C1L6T 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 C1L6T 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; and

the presence or absence (3) of cleavage of 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 of claim 5, wherein the protein is cleaved with PPase protease when step (3) is present.

8. Use of the recombinant type I humanized collagen C1L6T of claim 1 in the manufacture of a product, wherein the product is a tissue engineering product or a cosmetic.

9. A product comprising the recombinant type I humanized collagen C1L6T of claim 1, wherein the product is a tissue engineering product, cosmetic or pharmaceutical.

10. Use of the recombinant type I humanized collagen C1L6T of claim 1 for the preparation of a product having cell adhesion promoting effect.