阅读说明：本技术 一种重组i型人源化胶原蛋白多肽及其制备方法和用途 (Recombinant I-type humanized collagen polypeptide and preparation method and application thereof ) 是由 杨霞 陆晨阳 兰小宾 何振瑞 王建 王玲玲 于 2021-08-23 设计创作，主要内容包括：本发明公开了一种重组I型人源化胶原蛋白多肽及其制备方法和用途。本发明提供的重组I型人源化胶原蛋白多肽包含以SEQ ID No.1所示的序列的n个重复,n为大于等于1的整数,其中当n为大于等于2的整数时,各重复序列之间是直接连接的；任选地,在所述重组I型人源化胶原蛋白多肽的N末端包含能够通过TEV蛋白酶切除的氨基酸序列。本发明提供的重组I型人源化胶原蛋白多肽具有促进细胞黏附的活性,所述重组蛋白的氨基酸序列选自天然胶原蛋白氨基酸序列,应用于人体不会产生免疫反应,并且其制备方法简单,在低成本下即可获得较高产量的胶原蛋白。(The invention discloses a recombinant I-type humanized collagen polypeptide and a preparation method and application thereof. The recombinant humanized collagen I polypeptide provided by the invention comprises n repeats of a sequence shown as SEQ ID No.1, wherein n is an integer which is more than or equal to 1, and when n is an integer which is more than or equal to 2, the repeated sequences are directly connected; optionally, the recombinant type I humanized collagen polypeptide comprises an amino acid sequence at the N-terminus that is cleavable by TEV protease. The recombinant I-type humanized collagen polypeptide provided by the invention has the activity of promoting cell adhesion, the amino acid sequence of the recombinant protein is selected from natural collagen amino acid sequences, the recombinant protein can not generate immune reaction when being applied to a human body, the preparation method is simple, and the collagen with higher yield can be obtained at low cost.)

1. A recombinant type I humanized collagen polypeptide, wherein,

the recombinant humanized collagen I polypeptide comprises n repeats of a sequence shown as SEQ ID No.1, wherein n is an integer which is more than or equal to 1, and when n is an integer which is more than or equal to 2, the repeated sequences are directly connected; optionally, the recombinant type I humanized collagen polypeptide comprises an amino acid sequence at the N-terminus that is cleavable by TEV protease.

2. The recombinant humanized collagen type I polypeptide according to claim 1, characterized in that said amino acid sequence cleavable by TEV protease comprises the sequence shown in SEQ ID No. 4; preferably, the sequence shown as SEQ ID No.4 is directly linked to the sequence shown as SEQ ID No. 1.

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

a) an amino acid sequence shown as SEQ ID No. 3;

b) an amino acid sequence which has 90%, 92%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID No.3 and which retains the cell adhesion effect of the amino acid sequence of SEQ ID No. 3;

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.3, and which retains the cell adhesion effect of the amino acid sequence of SEQ ID No. 3; or

d) An amino acid sequence encoded by a nucleotide sequence that hybridizes with a polynucleotide sequence encoding the amino acid sequence of SEQ ID No.3 under stringent conditions, which are medium stringency conditions, medium-high stringency conditions, high stringency conditions or very high stringency conditions, and which retains the cell adhesion effect of the amino acid sequence of SEQ ID No. 3.

4. A polynucleotide encoding the recombinant type I humanized collagen polypeptide 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. A method of producing a recombinant type I humanized collagen polypeptide according to any one of claims 1 to 3, comprising the steps of:

cultivating the host cell according to claim 6 in a medium and producing the polypeptide;

② harvesting and purifying the polypeptide, preferably purifying the polypeptide using Ni column and/or anion exchange chromatography; and

③ optionally cleaving said polypeptide, preferably with TEV protease.

8. Use of a recombinant type I humanized collagen polypeptide according to any of claims 1 to 3 for the preparation of a product, preferably a tissue engineering product, a cosmetic product, a nutraceutical product or a pharmaceutical product.

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

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

Technical Field

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

Background

Collagen (collagen) is a family of proteins, generally white, transparent and unbranched fibrils, is a basic support of skin and bones, can account for 25% -35% of the total amount of protein, is mainly distributed in skin, blood vessels, bones, tendons, teeth, cartilage and the like of a human body, is a main matrix and a support of the tissues, protects and binds various tissues, and plays an important physiological function in vivo. Therefore, the collagen can be widely applied to industries such as medicine, cosmetics and the like. Collagen can be classified into interstitial collagen, basement membrane collagen and extracellular and peripheral collagen according to its distribution and functional characteristics in vivo. The interstitial collagen molecules account for the vast majority of collagen in the whole body, and comprise collagen molecules I, II and III, wherein the collagen I is mainly distributed in tissues such as skin, tendon and the like, and is most widely applied in medicine.

However, natural collagen is insoluble in water, has inhomogeneous properties, is difficult to be directly utilized by human body, and is often used after being treated by chemical means. Moreover, the collagen products sold in the current market are all taken from animal tissues such as pigs, cows, fish and the like, so that the risk of virus infection is difficult to avoid, and immune rejection and allergic symptoms can be caused because the collagen products cannot be compatible with the human body. If collagen is extracted from human placenta raw materials, the source is limited, and the collagen is subjected to strict punishment of law. Therefore, at present, the collagen can only be used in cosmetics and health care products, and the original biological function of the collagen cannot be exerted at all.

The conventional method for producing collagen is to treat animal-derived tissues by acid, alkali, and enzymatic hydrolysis 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. Most prepared collagen products have weak tensile strength, the pure collagen is degraded quickly in vivo, potential antigenicity possibly exists, the nutritional ingredients and the feeding value of the products are different due to the difference of the collagen source, the processing technology and the raw material proportion, and the leather not only needs to contact with a plurality of chemical substances in the processing process, but also is easily polluted by bacteria, so the application of the collagen is severely limited by the problems. Although foreign research institutions have obtained human collagen-containing milk by breeding mice containing human collagen genes, the production cost is too high, the production period is too long, and large-scale production cannot be achieved.

TEV Protease is a cysteine Protease of His-tagged (6 × His tag) Tobacco Etch Virus (TEV) which is expressed recombinantly in Escherichia coli, can specifically recognize heptapeptide sequence Glu-Asn-Leu-Tyr-Phe-Gln-Gly/Ser, and is cleaved between Gln and Gly/Ser amino acid residues, and is commonly used for removing Glutathione S-transferase (GST), His or other tags of fusion proteins.

Therefore, it is urgent to find a product which can be rapidly absorbed by human body, has simple and easily available preparation method and low cost, and can play the role and characteristics of human collagen.

Disclosure of Invention

Problems to be solved by the invention

Aiming at the conditions that the heterologous collagen in the field has low biological activity and is easy to induce immune response, and the collagen has low utilization rate caused by a series of problems of high production cost, long period, incapability of large-scale production and the like of human collagen, the invention provides a recombinant I-type humanized collagen polypeptide and a preparation method and application thereof.

Means for solving the problems

In a first aspect, the invention provides a recombinant humanized collagen type I polypeptide, wherein the recombinant humanized collagen type I polypeptide comprises n repeats of a sequence shown as SEQ ID No.1, n is an integer greater than or equal to 1, wherein when n is an integer greater than or equal to 2, the repeats are directly linked; optionally, the recombinant type I humanized collagen polypeptide comprises an amino acid sequence at the N-terminus that is cleavable by TEV protease.

Further, in the above recombinant type I humanized collagen polypeptide, the amino acid sequence capable of being cleaved by TEV protease comprises a sequence represented by SEQ ID No. 4; preferably, the sequence shown as SEQ ID No.4 is directly linked to the sequence shown as SEQ ID No. 1.

Further, in the above recombinant type I humanized collagen polypeptide, the recombinant type I humanized collagen polypeptide comprises: a. an amino acid sequence shown as SEQ ID No. 3; b. an amino acid sequence which has 90%, 92%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID No.3 and which retains the cell adhesion effect of the amino acid sequence of SEQ ID No. 3; 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.3 and which retains the cell adhesion effect of the amino acid sequence of SEQ ID No. 3; an amino acid sequence encoded by a nucleotide sequence that hybridizes under stringent conditions to a polynucleotide sequence encoding the amino acid sequence of SEQ ID No.3 and which retains the cell adhesion effect of the amino acid sequence of SEQ ID No.3, said stringent conditions being 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 above recombinant type I humanized collagen polypeptide.

In a third aspect, the present invention provides an expression vector comprising the polynucleotide described above.

In a fourth aspect, the present invention provides a host cell comprising the above-described expression vector.

In a fifth aspect, the present invention provides a method for producing the above recombinant type I humanized collagen polypeptide, comprising the steps of: culturing a host cell according to the fourth aspect of the present invention in a medium and producing a polypeptide; ② harvesting and purifying the polypeptide, preferably purifying the polypeptide using Ni column and/or anion exchange chromatography; and (iii) optionally cleaving said polypeptide, preferably with TEV protease.

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

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

ADVANTAGEOUS EFFECTS OF INVENTION

Through the implementation of the technical scheme, compared with other fragments of the type I collagen, the recombinant type I humanized collagen prepared by the invention has better cell adhesion effect, and because the amino acid sequence of the recombinant type I humanized collagen is selected from the amino acid sequence of natural collagen, the recombinant type I humanized collagen can not generate immune reaction when being applied to a human body, and meanwhile, the preparation method is simple, and the collagen with higher yield can be obtained at low cost.

Drawings

FIG. 1 is an electrophoresis chart of the protein after the recombinant type I humanized collagen TC1R4 is induced to express; the first lane is a molecular weight Marker, and the second lane is purified TC1R4 protein; the TC1R4 protein has an electrophoretically detectable molecular weight of about 38kDa and corresponds to a protein comprising the amino acid sequence of SEQ ID No. 3.

FIG. 2 shows the results of the detection of the biological adhesion activity of human collagen, C1S4T, C1S5T, and TC1R4 protein polypeptides.

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 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 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.

The present invention is based in part on the following findings of the inventors in previous studies: when a polypeptide comprising a plurality of repeats such as GEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGAS (SEQ ID No.1) is expressed in E.coli, the hinge region structure of the full-length protein is lacking because the polypeptide is often a truncated protein. Therefore, in order to improve the gelling property Of a recombinantly expressed polypeptide, it is often necessary to add a sequence such as amino acid GPPGPCCGGG (SEQ ID No.2) Of the hinge region to the C-terminus Of the polypeptide to aid in gelling (refer to Journal Of biochemistry.2004; 136:643 649). Therefore, in the invention application with application number 201811438582.6, a polypeptide sequence containing the polypeptide sequence of SEQ ID No.2 at the C-terminal end, such as T16a, was designed, but it was found that most of the target polypeptide forms a colloidal precipitate and cannot be purified by dissolution when the polypeptide with the sequence of T16a is cultured in a shake flask or fermentation, resulting in a low yield. To solve this problem, polypeptides comprising multiple repeats are obtained by adding non-collagenous amino acid linkers between the repeats (SEQ ID No.1), and the engineering of such polypeptides has also focused mainly on linker amino acid residues and C-terminal hinge region amino acids, also known as C-terminal stabilizing sequences.

Surprisingly, the inventors have found, after resolving the crystal structure of SEQ ID No.1, that the region of SEQ ID No.1 can form a very stable collagen trimer structure without the involvement of a hinge region. In the invention application with application number 201811438582.6, the inventors continued to engineer polypeptide sequences and found that the polypeptides claimed in the above application do not require the involvement of a C-terminal stabilizing sequence. And meanwhile, the polypeptide does not form a colloidal structure prematurely to influence later protein purification while being expressed recombinantly.

In the present invention, the SEQ ID No.1 repeat sequence used is: GEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGAS (SEQ ID No. 1). The polypeptide of the invention may comprise a plurality of repeats as shown in SEQ ID No.1, provided that there is no linker between the repeats. Herein, the linker may be 1 or more amino acid residues. The present invention has no limitation on the number of repetitive sequences as long as it can achieve the characteristics verified in the adhesion examples. Preferably, the number of repeated sequences is 4, i.e. a polypeptide comprising the sequence shown in SEQ ID No. 3:

GEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGASGEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGASGEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGASGEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGAS(SEQ ID No.3)。

the polypeptide sequence of the invention should be added with ENLYFQ (SEQ ID No.4) sequence at the N terminal when in expression, and the polypeptide with the sequence shown as SEQ ID No.3 can be directly obtained by TEV protease excision. Preferably, the sequence of ENLYFQ (SEQ ID No.4) is directly linked to the N-terminus of the first repeat sequence, i.e.

ENLYFQGEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGASGEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGASGEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGASGEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGAS(SEQ ID No.5)。

In the research, the inventors found that the polypeptide having the sequence shown in SEQ ID No.3 has higher activity than the recombinant humanized collagen type I described in the invention application with application number 201811254050.7.

In the present invention, the polypeptides are recombinant type I humanized collagens, which are used interchangeably herein with recombinant humanized collagens.

In the present invention, the recombinant humanized collagen may 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) inducing and expressing the recombinant humanized collagen; (4) purifying the recombinant humanized collagen and optionally carrying out enzyme digestion.

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 genetic engineering bacteria and the induction and expression of the recombinant humanized collagen can be carried out by the following steps: a. selecting the preferred single colony of the Escherichia coli genetic engineering bacteria from the transformed LB plate, placing the single colony in 1000ml LB culture medium containing ampicillin at 37 ℃, and culturing at 220rpm until the OD of the bacterial liquid₆₀₀In the range of 0.9-1.1; b. the cells were induced by adding IPTG to a final concentration of 0.5mM, cultured overnight at 18 ℃ and centrifuged to collect the cells.

In the step (4), the purification and the enzyme digestion of the recombinant humanized collagen polypeptide can be performed by the following steps: a. resuspending the cells in Tris buffer (25mM Tris, 200mM NaCl, pH 8.0), disrupting the cells, and centrifuging to collect the supernatant; b. by using Ni₆And combining the FF affinity column with recombinant humanized collagen, rinsing the hybrid protein by using a washing buffer solution containing 20mM imidazole, eluting the target protein by using a solution containing 250mM imidazole, adding TEV protease at 16 ℃, and carrying out enzyme digestion for 2h to finally obtain the target collagen polypeptide.

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

The invention also provides nucleic acid molecules comprising a nucleic acid sequence encoding a polypeptide 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 peptide according to the invention, or may consist of only a nucleic acid sequence encoding a peptide 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 polypeptide is under the control of its own cis-acting regulatory elements, which are designed to facilitate gene integration or gene replacement in a host cell, and 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.

The polypeptide of the present invention comprises a sequence in which one or more amino acids are substituted, deleted, inserted and/or added in the sequence shown as SEQ ID No.3 or the sequence shown as SEQ ID No.3, as long as the polypeptide of the present invention retains the cell adhesion effect of the amino acid sequence of SEQ ID No. 3. 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 amino acid sequence, e.g., the C-terminus or N-terminus of SEQ ID No.3, as long as the polypeptide of the present invention retains the cell adhesion effect of the amino acid sequence of SEQ ID No. 3.

Amino acid substitution refers to the substitution of an amino acid residue at a certain position of an amino acid sequence, such as the sequence of SEQ ID No.3, with another amino acid residue, as long as the polypeptide of the present invention retains the cell adhesion effect of the amino acid sequence of SEQ ID No. 3.

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.3, and the inserted amino acid residues may be adjacent to each other in whole or in part, or none of the inserted amino acids may be adjacent to each other, as long as the polypeptide of the present invention retains the cell adhesion effect of the amino acid sequence of SEQ ID No. 3. The position of insertion of amino acids is not in between the repeats in this context.

Amino acid deletion means that 1, 2 or 3 or more amino acids can be deleted from an amino acid sequence, such as the sequence of SEQ ID No.3, as long as the polypeptide of the present invention retains the cell adhesion effect of the amino acid sequence of SEQ ID No. 3.

In the present invention, the substitution may be a conservative amino acid substitution, which means that 3, 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.3 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 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 polypeptide dosage form can be common preparation, sustained release preparation, controlled release preparation and various particle 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 dosage of the 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 to be administered depends on many factors, such as the nature and severity of the disease to be prevented or treated, sex, age, body weight and individual response of the patient or animal, the specific active ingredient used, the administration route and the administration frequency, etc. 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.

The human collagen C1S4T and the human collagen C1S5T of the present invention are described in the patent application having the application number of 201811254050.7 and the title of "polypeptide, method for producing the same, and use thereof".

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 TC1R4

Construction of TC1R4 Gene expression vector

The sequence of the full-length recombinant humanized collagen type I TC1R4 used in the present example is shown inThe amino acid sequence shown in SEQ ID No.3, the full length is 240 aa. In order to purify the TC1R4 protein after expression, the N end of the amino acid sequence shown in SEQ ID No.3 is connected with the amino acid sequence shown in SEQ ID No.4 to form the amino acid sequence shown in SEQ ID No.5, and the total length of the amino acid sequence is 246 aa. The coding sequence corresponding to the amino acid sequence is codon optimized aiming at the codon of the escherichia coli, and the whole length of the corresponding gene is 738bp (see the sequence marked by underline in SEQ ID No. 6). In order to facilitate the construction of a subsequent expression vector, a restriction enzyme site sequence is added at the 5 'end of the coding sequence, and a stop codon sequence and a restriction enzyme site sequence are added at the 3' end. The optimized gene sequence is as follows: GGTACCGAAAACCTGTATTTCCAGGGTGAAAA AGGTAGTCCGGGTGCAGATGGTCCGGCCGGTGCACCTGGTACACCGGGTCCTCAGGGCATTGCAGGCCAGCGTGGT GTTGTGGGCCTGCCGGGTCAGCGCGGTGAACGTGGTTTTCCGGGTCTGCCGGGTCCGAGCGGCGAACCTGGTAAAC AGGGCCCGAGTGGTGCAAGCGGTGAAAAAGGCAGTCCGGGCGCAGATGGTCCTGCAGGTGCCCCTGGTACACCTGG TCCGCAGGGTATTGCAGGTCAGCGCGGCGTGGTGGGCCTGCCTGGTCAACGTGGCGAACGTGGTTTCCCGGGCCTG CCGGGACCGTCAGGTGAACCTGGCAAACAGGGCCCTAGCGGTGCAAGTGGCGAAAAAGGTAGCCCGGGCGCAGACG GCCCGGCAGGTGCACCTGGCACACCTGGTCCTCAGGGTATTGCGGGCCAGCGCGGCGTTGTTGGTCTGCCTGGCCA GCGTGGCGAACGCGGTTTTCCGGGCCTGCCTGGCCCGTCAGGCGAACCTGGAAAACAGGGCCCAAGTGGTGCAAGT GGTGAAAAAGGAAGTCCGGGCGCCGATGGCCCGGCCGGTGCGCCTGGTACGCCTGGTCCTCAAGGCATTGCCGGTC AGCGCGGAGTTGTTGGCCTGCCGGGCCAGCGTGGAGAACGCGGTTTCCCGGGTCTGCCTGGTCCGAGTGGTGAACC GGGTAAACAGGGTCCGAGCGGTGCATCATAACTCGAG(SEQ ID No.6)。

The synthesis of gene fragment was entrusted to Beijing Shengyue Gegen Biotech Co., Ltd, and the synthesized TC1R4 gene fragment was inserted into pET-32a expression vector (EMD Biosciences (Novagen)) via the restriction enzyme sites of KpnI (NEB Co., Ltd.: R0136L) and XhoI (NEB Co., Ltd.: R0146L) to obtain a recombinant expression plasmid.

2. Transformation of recombinant expression plasmids

Coli competent cells BL21(DE3) (Merck) were transformed with the successfully constructed expression plasmid. 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 (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 containing ampicillin (10g/L peptone, 5g/L yeast extract, 10g/L sodium chloride, 15g/L agar, 100 mu.g/mL ampicillin); fifthly, the plate is inversely cultured in a 37 ℃ incubator for about 16 hours until a clear and visible colony grows out.

3. Induced expression of recombinant type I humanized collagen TC1R4

Monoclonal colonies were picked from transformed LB plates and cultured in 1000mL LB medium (containing 100. mu.g/mL ampicillin) at 37 ℃ and 220rpm to OD₆₀₀At 0.9-1.1, IPTG (Sigma; cat # I5502-1G) was added to a final concentration of 0.5mM for induction expression, and the culture was carried out overnight at 220rpm at 18 ℃. Finally, the thalli are collected by centrifugation and stored at the temperature of minus 20 ℃ or immediately purified in the next step.

4. Purification of recombinant type I humanized collagen TC1R4

The cell pellet was resuspended (1L) in about 100ml of Tris buffer (25mM Tris, 200mM NaCl, pH 8.0), disrupted by a homogenizer (GEA), and centrifuged at 17000rpm for 30min to separate the soluble protein from the inclusion bodies.

Ni was equilibrated with 5 column volumes of Binding buffer (25mM Tris, 200mM NaCl, pH 8.0)₆FF (Cytiva, cat # 30210) affinity column. Then adding the protein supernatant to make the target recombinant protein fully combined on the column material. The hetero-protein was washed with a washing buffer (washing buffer) containing 20mM imidazole (Sigma) (20mM imidazole, 25mM Tris, 200mM NaCl, pH 8.0), and the target protein was eluted with a solution containing 250mM imidazole (250mM imidazole, 25mM Tris, 200mM NaCl, pH 8.0). Then adding a proper amount of TEV protease (Sigma, T4455), incubating for 2h at 16 ℃, and collecting the transudate, namely the target collagen from which the carrier protein is removed. Dialyzing the obtained product overnight, and freeze-drying the product to obtain dry powder for later use。

5. Purity detection of recombinant I-type humanized collagen TC1R4

The purity of the obtained TC1R4 protein 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.

As shown in FIG. 1, the apparent molecular weight of TC1R4 obtained by electrophoresis is 38kDa, and the molecular weight corresponds to that of TC1R4, which indicates that the polypeptide TC1R4 is correctly expressed.

Example 2: mass spectrometry detection of recombinant I-type humanized collagen TC1R4

Mass spectrometric detection of the TC1R4 protein was performed according to the conditions shown in Table 1.

TABLE 1 conditions for Mass spectrometric detection

After DTT reduction and iodoacetamide alkylation treatment of TC1R4 protein samples, trypsin is added for enzymolysis overnight. And desalting the peptide segment obtained after enzymolysis by C18ZipTip, and mixing the peptide segment with a matrix alpha-cyano-4-hydroxybinamic acid (CHCA) to form a dot plate. Finally, matrix-assisted laser desorption ionization-time-of-flight mass spectrometry MALDI-TOF/TOF FlextreemeTM, Brucker, Germany is used for analysis (the technology of peptide fingerprinting can be referred to: Protein J.201635: 212-7).

Database retrieval is handled by a Peptide Mass Fingerprint page from the local mascot website. The protein identification result is obtained according to the primary mass spectrum of the peptide fragment generated after enzymolysis. And (3) retrieving parameters: trypsin is subjected to enzymolysis, and two missed cutting sites are arranged. The alkylation of cysteine is set as a fixed modification and the oxidation of methionine as a variable modification. The database used for identification was Swissprot.

The mass spectrometric detection of the molecular weights and the corresponding polypeptides are shown in table 2.

TABLE 2 Mass spectrometric determination of molecular weights and corresponding polypeptides

Sequence compared to polypeptide:

GEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGASGEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGASGEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGASGEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGAS

the coverage rate of the peptide fragment is 98.75 percent through calculation, and the detection result is very credible.

Example 3: biological activity detection of recombinant I-type humanized collagen TC1R4

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 to be detected comprises commercial human collagen (Sigma, C7774), recombinant I type humanized collagen TC1R4 provided by the invention, recombinant type III collagen C1S4T and recombinant type III collagen C1S5T (wherein the sequences of the proteins C1S4T and C1S5T are referred to the patent application with the application number of 201811254050.7).

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 hole⁵Well-cultured 3T3 cells (from the Physician of the university of Qinghua) 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 the cells in a short time, and the cells are attached to the wall.

As shown in fig. 2, it is understood from comparison that the recombinant type I humanized collagen TC1R4 of the present invention has more excellent bioadhesive activity than the commercial human collagen, the recombinant type III collagen C1S4T, and the recombinant type III collagen C1S 5T.

Sequence listing

<110> Shanxi brocade biomedical products Ltd

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

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Val Val Gly Leu Pro Gly Gln Arg Gly Glu Arg Gly Phe Pro Gly Leu

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ggtaccgaaa acctgtattt ccagggtgaa aaaggtagtc cgggtgcaga tggtccggcc 60

ggtgcacctg gtacaccggg tcctcagggc attgcaggcc agcgtggtgt tgtgggcctg 120

ccgggtcagc gcggtgaacg tggttttccg ggtctgccgg gtccgagcgg cgaacctggt 180

aaacagggcc cgagtggtgc aagcggtgaa aaaggcagtc cgggcgcaga tggtcctgca 240

ggtgcccctg gtacacctgg tccgcagggt attgcaggtc agcgcggcgt ggtgggcctg 300

cctggtcaac gtggcgaacg tggtttcccg ggcctgccgg gaccgtcagg tgaacctggc 360

aaacagggcc ctagcggtgc aagtggcgaa aaaggtagcc cgggcgcaga cggcccggca 420

ggtgcacctg gcacacctgg tcctcagggt attgcgggcc agcgcggcgt tgttggtctg 480

cctggccagc gtggcgaacg cggttttccg ggcctgcctg gcccgtcagg cgaacctgga 540

aaacagggcc caagtggtgc aagtggtgaa aaaggaagtc cgggcgccga tggcccggcc 600

ggtgcgcctg gtacgcctgg tcctcaaggc attgccggtc agcgcggagt tgttggcctg 660

ccgggccagc gtggagaacg cggtttcccg ggtctgcctg gtccgagtgg tgaaccgggt 720

aaacagggtc cgagcggtgc atcataactc gag 753