Self-crosslinking recombinant humanized collagen polymeric biomaterial and its preparation method

Abstract

The present invention provides a self-crosslinking recombinant humanized collagen polymer biomaterial and a method for preparing the same. The present invention also provides a collagen self-assembly element comprising: (i) at least 21 consecutive amino acids from the C-terminus of the amino acid sequence set forth in SEQ ID NO. 1; or an amino acid sequence in which one or more amino acid substitutions, deletions, or additions have occurred in the amino acid sequence of (i) and which retains the function of promoting self-crosslinking of recombinant type III humanized collagen to form a triple helix structure; or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of (i) and which retains the function of promoting self-crosslinking of recombinant type III humanized collagen to form a triple helix structure. This element can effectively promote self-crosslinking of recombinant type III humanized collagen to form a triple helix structure.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This disclosure claims priority to an invention patent application filed with the China Patent Office on August 4, 2023, bearing application number 202310983181.3 and entitled "Self-crosslinking recombinant humanized collagen polymer biomaterial and preparation method thereof," the entire contents of which are incorporated herein by reference.

[0002] The present disclosure belongs to the technical field of synthetic biology, and specifically relates to a self-crosslinking recombinant humanized collagen polymer biomaterial and a method for preparing the same, and more specifically relates to a collagen self-assembly element that promotes the self-crosslinking of recombinant type III humanized collagen to form a triple helix structure, and a recombinant type III humanized collagen having the corresponding triple helix structure. [Background technology]

[0003] Collagen is a type of protein widely distributed in the connective tissues of the human body and is the most abundant protein in the human body, accounting for 25-35% of the total protein. Its main functions include maintaining the extracellular environment, maintaining the normal physiological functions of tissues and organs, and repairing physical injuries. Collagen is a natural biological resource, possessing unparalleled biocompatibility, cell-supporting elasticity, and biodegradability compared to other polymeric materials. Therefore, collagen can be widely used in industries such as pharmaceuticals and cosmetics.

[0004] Native collagen molecules can form a unique supercoiled structure, a left-handed helix with three amino acid residues as the basic repeat. These three amino acid residues are typically Gly-X-Pro. Gly is necessary for the formation of collagen hydrogen bonds, and its lack of side chains allows collagen to pack tightly together. At higher structural levels, collagen supercoils are further combined to form collagen fibrils. In vivo, collagen synthesis and modification begins with tropocollagen and undergoes numerous chemical transformations, including hydroxylation, glycosylation, and cross-linking, under the complex control of multiple biological enzymes. Tropocollagen contains globular heads and tails in addition to collagen chains. Without these heads and tails, collagen chains cannot fold correctly into triple helices, resulting in the loss of collagen's biological activity. Therefore, collagen prepared according to its original genetic sequence has difficulty spontaneously assembling into the correct spatial structure in vitro. This difficulty significantly hinders the research, development, and production of human collagen.

[0005] Appropriate research has already been conducted on this issue. For example, Reference 1 shows that gelation can be promoted by adding a sequence such as the amino acids GPPGPCCGGG (SEQ ID NO. 15) of the hinge region to the C-terminus of an expressed polypeptide. However, Reference 2 discloses that when the amino acids of the hinge region shown in SEQ ID NO. 15 are added to the C-terminus of a provided polypeptide sequence, the polypeptide forms colloidal precipitates during fermentation, making it impossible to dissolve and purify, and significantly reducing the yield.

[0006] Therefore, there is an urgent need for a method to promote cross-linking of recombinant humanized type III collagen to form a triple helix structure, so that recombinant humanized type III collagen can be mass-produced by biosynthetic methods and have a triple helix structure to exert its specific functions.

[0007] Furthermore, circular dichroism spectroscopy is a method used to detect the secondary structure of proteins. When detecting native collagen, an absorption peak appears around 221 nm under appropriate detection conditions. This absorption peak is somewhat related to its triple helical structure, but it is also affected by factors such as proline and hydroxyproline content (polyproline itself has a strong peak at 221 nm but does not form a triple helical structure). Therefore, circular dichroism spectra cannot be used as direct evidence of a triple helical structure. However, experimental detection of circular dichroism spectra is simple and can aid in the characterization of the higher-order structure of collagen. The experimental conditions and results of circular dichroism spectra vary significantly depending on the type of collagen. This is mainly because the sequence length and amino acid composition (especially the proline and hydroxyproline content) of recombinant collagen differ from those of native full-length collagen. Therefore, it is difficult to maintain constant detection conditions (e.g., temperature, concentration, solution composition) for circular dichroism spectra of different collagens. Circular dichroism spectroscopy can be used to characterize collagen under various conditions to characterize whether it can form triple helices (e.g., collagen that has been denatured during preparation or has design defects cannot form triple helical structures at all).

[0008] References: Citation 1: Yao J, Yanagisawa S, Asakura T. Design, expression and characterization of collagen-like proteins based on the cell adhesive and crosslinking sequences derived from native collagens. J Biochem. 2004 Nov;136(5):643-9. Cited Document 2: Chinese Patent No. 109593126 Summary of the Invention [Problem to be solved by the invention]

[0009] For example, collagen prepared according to its original gene sequence has difficulty spontaneously assembling into the correct spatial structure in vitro, resulting in its inability to effectively perform physiological functions. While appropriate research has been conducted using existing technologies to address this issue, however, these studies remain insufficient. In this regard, the present disclosure provides a collagen self-assembly element capable of promoting the self-crosslinking of recombinant humanized type III collagen to form a triple helix structure. By incorporating the amino acid sequence of the hinge region of human type III collagen at the end of the amino acid sequence of recombinant humanized type III collagen, the self-crosslinking of recombinant humanized type III collagen to form a triple helix structure is promoted, and the recombinant humanized type III collagen biomaterial having a triple helix structure can be conveniently prepared by biosynthesis. [Means for solving the problem]

[0010] A first aspect of the present disclosure provides a collagen self-assembly element, the collagen self-assembly element comprising a sequence shown in any one of (i) to (iii) below: (i) at least 21 contiguous amino acids at the C-terminus of the amino acid sequence set forth in SEQ ID NO. 1 (ii) An amino acid sequence in which one or more amino acid substitutions, deletions, or additions occur in the amino acid sequence of (i), and which retains the function of promoting self-crosslinking of recombinant humanized type III collagen to form a triple helix structure. (iii) an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of (i) and retaining the function of promoting self-crosslinking of recombinant humanized type III collagen to form a triple helix structure. In some embodiments, the sequence set forth in (i) above is the amino acid sequence set forth in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, or SEQ ID NO. 4.

[0011] A second aspect of the present disclosure provides the use of a collagen self-assembling element according to the first aspect of the present disclosure in promoting the formation of a triple helix structure by self-crosslinking of recombinant humanized type III collagen.

[0012] In some embodiments, the collagen self-assembly element exerts its effect by being present at the terminus of the amino acid sequence of the recombinant humanized type III collagen; Preferably, the end of the amino acid sequence of the recombinant humanized type III collagen is the C-terminus.

[0013] A third aspect of the present disclosure provides a recombinant type III humanized collagen, comprising the collagen self-assembly element according to claim 1 or 2 at an end of the recombinant type III humanized collagen; Preferably, the terminus of the recombinant humanized type III collagen is the C-terminus.

[0014] In some embodiments, the recombinant humanized type III collagen comprises a sequence set forth in any of (a) to (c) below: (a) the amino acid sequence shown in SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8 or SEQ ID NO. 9 (b) an amino acid sequence in which one or more amino acid substitutions, deletions, or additions occur in the amino acid sequence of (i) and which retains the ability to self-crosslink and form a triple helix structure; (c) an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of (i) and retaining the ability to self-crosslink to form a triple helix structure;

[0015] A fourth aspect of the present disclosure provides a polynucleotide, wherein the polynucleotide encodes a collagen self-assembly element according to the first aspect of the present disclosure, or a recombinant humanized type III collagen according to the third aspect of the present disclosure.

[0016] In some embodiments, the polynucleotide comprises the sequence shown in (z1) or (z2) below. (z1) the nucleotide sequence set forth in SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13 or SEQ ID NO. 14 (z2) A nucleotide sequence encoding a protein capable of hybridizing to the nucleotide sequence shown in (z1) under stringent conditions and self-crosslinking to form a triple helix structure, wherein the stringent conditions are moderately stringent, moderately-highly stringent, highly stringent, or very highly stringent.

[0017] A fifth aspect of the present disclosure provides a recombinant expression vector, said recombinant expression vector comprising a polynucleotide according to the fourth aspect of the present disclosure.

[0018] A sixth aspect of the present disclosure provides a recombinant host cell, said recombinant host cell comprising the recombinant expression vector according to the fifth aspect of the present disclosure.

[0019] A seventh aspect of the present disclosure provides a method for preparing the recombinant type III humanized collagen according to the third aspect of the present disclosure, said method comprising: Step S1 of constructing a recombinant expression vector comprising the polynucleotide according to the fourth aspect encoding the recombinant type III humanized collagen according to the third aspect, and constructing a recombinant host cell by transformation; Step S2 of culturing the recombinant host cell obtained in step S1 in a medium to produce a protein; Step S3 of obtaining and purifying the protein, preferably by Ni column and / or anion exchange chromatography; Optionally, a step S4 of enzymatically digesting the protein, preferably with TEV protease. [Effects of the Invention]

[0020] By implementing the above technical solutions, the present disclosure achieves the following technical effects:

[0021] First, the present disclosure provides a collagen self-assembly element, which contains the amino acid sequence of the hinge region of human type III collagen at the end of the amino acid sequence of recombinant humanized type III collagen, effectively promoting the self-crosslinking of recombinant humanized type III collagen to form a triple helix structure, thereby significantly improving the utility of recombinant humanized type III collagen prepared by biosynthesis.

[0022] Furthermore, the recombinant humanized type III collagen according to the present disclosure, which contains a collagen self-assembly element at its terminal, i.e., the amino acid sequence of the hinge region of human type III collagen, can form a good triple helix structure, as confirmed by the results of circular dichroism spectroscopy. Furthermore, the recombinant humanized type III collagen can be mass-produced by biosynthesis. [Brief explanation of the drawings]

[0023] [Figure 1] Electrophoresis detection results during the preparation of recombinant type III humanized collagen TE16c. [Figure 2] Electrophoresis detection results during the preparation of recombinant type III humanized collagen C3T16H1. [Figure 3] Electrophoresis detection results during the preparation of recombinant type III humanized collagen C3T16H2. [Figure 4] Electrophoresis detection results during the preparation of recombinant type III humanized collagen C3T16H3. [Figure 5] Electrophoresis detection results during the preparation of recombinant type III humanized collagen C3T16H4. [Figure 6] Ultraviolet scanning analysis results of circular dichroism spectra of recombinant type III humanized collagens C3T16H1, C3T16H2, C3T16H3, C3T16H4, and TE16c, with a positive peak detected around 221 nm. DETAILED DESCRIPTION OF THE INVENTION

[0024] Hereinafter, embodiments of the present disclosure will be described, but the present disclosure is not limited thereto. The present disclosure is not limited to the configurations described below, and various modifications are possible within the scope of the disclosure claims. Embodiments and examples that appropriately combine the technical means disclosed in different embodiments and examples are also included in the technical scope of the present disclosure.

[0025] In the present disclosure, a numerical range expressed as "numeric value A to numerical value B" or "numeric value A minus numerical value B" means a range including the endpoint numerical values ​​A and B.

[0026] In this disclosure, a numerical range expressed as "greater than or equal to" or "less than or equal to" means an inclusive numerical range.

[0027] In this disclosure, "any" or "optionally" means that the event or circumstance described below may or may not occur, and the description includes the occurrence of the event and the absence of the event.

[0028] In this disclosure, "a" or "an" or "the" can mean "one," "one or more," "at least one," or "one or more."

[0029] In this disclosure, the terms "comprises," "has," "comprises," or "including" may mean an inclusive or open sense and do not exclude additional, unrecited elements or method steps, whereas "comprises," "has," "comprises," or "comprising" may mean a closed sense and exclude additional, unrecited elements or method steps.

[0030] In this disclosure, the term "about" may mean that a value includes the standard deviation of error for the device or method used to measure the value. All numerical ranges and parameters used to define this disclosure are approximate, and the relevant numerical values ​​in specific embodiments are presented herein as precisely as possible. However, any numerical value necessarily includes the standard deviation resulting from the testing device or method described above. Therefore, unless expressly stated otherwise, all ranges, quantities, values, and percentages used in this disclosure should be understood to be modified by "about." Here, "about" typically means that the actual numerical value is within ±10%, ±5%, ±3%, ±1%, or ±0.5% of the specified value or range.

[0031] In this disclosure, the terms "polypeptide" and "protein" refer interchangeably to a chain of at least two amino acid residues joined together by a covalent bond (e.g., a peptide bond), and may be a recombinant, natural, or synthetic polypeptide. Polypeptides may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The term also includes amino acid polymers that have been modified (e.g., by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or other manipulation such as conjugation with a labeling component).

[0032] In this disclosure, the term "amino acid" includes natural amino acids, unnatural amino acids, amino acid analogs, and all D and L stereoisomers thereof.

[0033] In the present disclosure, modifications of amino acids may include modifications to the native sequence such as functional group modifications, intramolecular covalent bonds (e.g., cyclization between side chains), methylation, acylation, ubiquitination, phosphorylation, aminohexanylation, biotinylation, etc.

[0034] In the present disclosure, amino acid deletion may refer to the removal of one, two, three or more amino acids from an amino acid sequence, so long as the modified sequence fully or partially retains the activity of the original amino acid sequence.

[0035] In the present disclosure, the addition of amino acids may refer to the addition of one, two, three or more amino acids to the C-terminus, N-terminus, or any position between the C-terminus and N-terminus of an amino acid sequence, as long as the modified sequence fully or partially retains the activity of the original amino acid sequence.

[0036] In the present disclosure, amino acid substitution may refer to the replacement of an amino acid at a position in an amino acid sequence with another amino acid, as long as the modified sequence fully or partially retains the activity of the original amino acid sequence. Amino acid substitution may be conservative, which refers to a peptide (conservative variant peptide) formed by replacing some amino acids with amino acids having similar or close properties compared to the original amino acid sequence. Illustratively, these conservative variant peptides can be generated by the following amino acid substitutions: replacement of Ala with Val, Leu, or Ile; replacement of Arg with Lys, Gln, Asn, or His; replacement of Asn with Gln, His, Lys, or Arg; replacement of Asp with Glu or Asn; replacement of Cys with Ser or Ala; replacement of Gln with Asn or Glu; replacement of Glu with Asp or Gln; replacement of Gly with Ala; replacement of His with Asn, Lys, Gln, or Arg; Leu, Met, Ala, Val, Phe, or N-methyl. substitution of Ile with Ile, substitution of Leu with Ile, Met, Ala, Val, Phe or norleucine, substitution of Lys with Asn, Gln or Arg, substitution of Met with Ile, Leu or Phe, substitution of Phe with Leu, Val, Ile, Ala or Tyr, substitution of Pro with Ala, substitution of Ser with Thr, substitution of Thr with Ser or Val, substitution of Trp with Phe or Tyr, substitution of Tyr with Trp, Phe, Thr or Ser, and substitution of Val with Phe, Ala, Met, Ile, Leu or norleucine. The amino acid substitution may be a non-conservative amino acid substitution.

[0037] In this disclosure, the terms "sequence identity" and "percent identity" refer to the percentage of nucleotides or amino acids that are the same (i.e., identical) between two or more polynucleotides or polypeptides. Sequence identity between two or more polynucleotides or polypeptides can be determined by aligning the nucleotide or amino acid sequences of the polynucleotides or polypeptides, scoring the number of positions in the aligned polynucleotides or polypeptides that contain the same nucleotide or amino acid residue, and comparing this to the number of positions in the aligned polynucleotides or polypeptides that contain a different nucleotide or amino acid residue. Polynucleotides can differ at a single position, for example, by containing a different nucleotide (i.e., substitution or mutation) or by deleting a nucleotide (i.e., inserting or deleting a nucleotide in one or both polynucleotides). Polypeptides can differ at a single position, for example, by containing a different amino acid (i.e., substitution or mutation) or by deleting an amino acid (i.e., inserting or deleting an amino acid in one or both polypeptides). Sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of amino acid residues in the polynucleotides or polypeptides. For example, the percentage identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of nucleotides or amino acid residues in the polynucleotide or polypeptide and multiplying by 100.

[0038] In this disclosure, the terms "moderately stringent conditions," "moderately to highly stringent conditions," "highly stringent conditions," or "very highly stringent conditions" describe conditions for nucleic acid hybridization and washing. Guidelines for conducting hybridization reactions are described in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6, which is incorporated herein by reference. Aqueous and non-aqueous methods are described in this publication, and either method can be used. For example, specific hybridization conditions are as follows: (1) For low stringency hybridization conditions, 6× sodium chloride / sodium citrate (SSC) at approximately 45°C, followed by two washes in 0.2× SSC, 0.1% SDS at at least 50°C (for low stringency conditions, the wash temperature can be increased to 55°C). (2) For moderate stringency hybridization conditions, 6×SSC at about 45° C. is followed by one or more washes in 0.2×SSC, 0.1% SDS at 60° C. (3) For high stringency hybridization conditions, 6×SSC at about 45° C. is followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C. Preferably, (4) for very high stringency hybridization conditions, 0.5 M sodium phosphate, 7% SDS at 65° C. is followed by one or more washes in 0.2×SSC, 1% SDS at 65° C.

[0039] In this disclosure, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, such as deoxyribonucleotides or ribonucleotides, or their analogs. Polynucleotides may have any three-dimensional structure and may perform any function, known or unknown.

[0040] In this disclosure, the term "recombinant expression vector" refers to a DNA construct used to express, for example, a polynucleotide encoding a desired polypeptide. A recombinant expression vector may include, for example, i) a collection of genetic elements that act to control gene expression, such as a promoter and enhancer, ii) a structural or coding sequence that is transcribed into mRNA and translated into protein, and iii) a transcription subunit with appropriate transcription and translation initiation and termination sequences. Recombinant expression vectors are constructed in any suitable manner. The nature of the vector is not critical, and any vector can be used, including plasmids, viruses, phages, and transposons. Vectors that can be used in the present disclosure include, but are not limited to, chromosomal, non-chromosomal, and synthetic DNA sequences, such as vectors derived from viral plasmids, bacterial plasmids, phage DNA, yeast plasmids, and combinations of plasmids and phage DNA, as well as DNA from viruses such as lentiviruses, retroviruses, vaccinia viruses, adenoviruses, fowlpox viruses, baculoviruses, SV40 viruses, and pseudorabies viruses.

[0041] In this disclosure, the term "recombinant host cell" refers to a cell into which an expression vector has been introduced. Host cells may include bacterial, microbial, plant, or animal cells. Easily transformed bacteria include members of the Enterobacteriaceae family, such as strains of Escherichia coli and Salmonella, Bacillaceae bacteria, such as Bacillus subtilis, Streptococcus pneumoniae, Streptococcus pyogenes, and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese hamster ovary cell line) and NS0 cells.

[0042] In the present disclosure, the term "codon optimization" means that a nucleotide sequence encoding a polypeptide is configured to contain codons preferred by a host cell or organism in order to improve gene expression and increase translation efficiency in the host cell or organism.

[0043] Unless defined otherwise, other technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art.

[0044] The technical solutions provided by the present disclosure are described in more detail below.

[0045] Collagen self-assembly elements The present disclosure provides collagen self-assembly elements that can effectively promote the self-crosslinking of recombinant humanized type III collagen to form triple helix structures.

[0046] In some embodiments, the collagen self-assembly element comprises a sequence set forth in any of (i) to (iii) below: (i) at least 21 contiguous amino acids at the C-terminus of the amino acid sequence set forth in SEQ ID NO. 1 (ii) An amino acid sequence in which one or more amino acid substitutions, deletions, or additions occur in the amino acid sequence of (i), and which retains the function of promoting self-crosslinking of recombinant humanized type III collagen to form a triple helix structure. (iii) an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of (i) and retaining the function of promoting self-crosslinking of recombinant humanized type III collagen to form a triple helix structure.

[0047] SEQ ID NO. 1: GERGSEGSPGHPGQPGPPGPPGAPGPCCGG is a human type III collagen peptide segment.

[0048] The present disclosure has unexpectedly discovered that collagen self-assembling elements of different lengths have different abilities to promote self-crosslinking of recombinant humanized type III collagen to form triple helical structures.

[0049] In some specific embodiments, the amino acid sequence of the collagen self-assembly element comprises the sequence set forth in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, or SEQ ID NO.4.

[0050] In some more specific embodiments, the amino acid sequence of the collagen self-assembly element is the sequence set forth in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, or SEQ ID NO.4. SEQ ID NO.2:GSEGSPGHPGQPGPPGPPGAPGPCCGG SEQ ID NO.3: GSPGHPGQPGPPGPPGAPGPCCGG SEQ ID NO.4:GHPGQPGPPGPPGAPGPCCGG

[0051] In order for the collagen self-assembly element to effectively exert its function of promoting self-crosslinking of recombinant type III humanized collagen to form a triple helix structure, in some embodiments, the collagen self-assembly element is located at the terminus, preferably the C-terminus, of the recombinant type III humanized collagen.

[0052] The recombinant type III humanized collagen of the present disclosure refers to the full-length or partial amino acid sequence fragment encoded by the type III human collagen gene prepared by DNA recombinant technology, or a combination containing a functional fragment of type III human collagen.

[0053] In some specific embodiments, the recombinant humanized type III collagen contains a collagen self-assembly element at its terminal amino acid sequence, which can be achieved by the following method: The amino acid sequence of recombinant humanized type III collagen containing a collagen self-assembly element at its terminal and its gene sequence are designed, an expression vector containing the gene sequence is constructed, and host cells are transformed with the vector. The host cells are then fermented and cultured to induce expression of the recombinant humanized type III collagen.

[0054] Recombinant Type III Humanized Collagen The present disclosure provides a recombinant type III humanized collagen containing collagen self-assembly elements at its termini, the collagen self-assembly elements being as described above. Because the recombinant type III humanized collagen of the present disclosure contains collagen self-assembly elements, it can self-crosslink to form a triple helix structure upon expression, and the protein can be mass-produced by biosynthesis.

[0055] To better form a triple helix structure, the collagen self-assembly element is located at the C-terminus of the recombinant humanized type III collagen.

[0056] In some specific embodiments, the recombinant humanized type III collagen comprises a sequence shown in any of (a) to (c) below: (a) the amino acid sequence shown in SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8 or SEQ ID NO. 9 (b) an amino acid sequence in which one or more amino acid substitutions, deletions, or additions occur in the amino acid sequence of (i) and which retains the ability to self-crosslink and form a triple helix structure; (c) an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of (i) and retaining the ability to self-crosslink to form a triple helix structure;

[0057] In some preferred embodiments, the amino acid sequence of the recombinant humanized type III collagen is the sequence set forth in SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 or SEQ ID NO.9.

[0058] Polynucleotides, recombinant expression vectors, recombinant host cells The present disclosure provides polynucleotides encoding the above collagen self-assembly elements and the above recombinant type III humanized collagen.

[0059] In some embodiments, the polynucleotide encoding the recombinant type III humanized collagen comprises the sequence shown in (z1) or (z2) below. (z1) the nucleotide sequence set forth in SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13 or SEQ ID NO. 14 (z2) A nucleotide sequence encoding a protein capable of hybridizing to the nucleotide sequence shown in (z1) under stringent conditions and self-crosslinking to form a triple helix structure, wherein the stringent conditions are moderately stringent, moderately-highly stringent, highly stringent, or very highly stringent.

[0060] The present disclosure provides a recombinant expression vector comprising the above-described polynucleotide.

[0061] The expression vectors selected in the present disclosure can exist stably in various prokaryotic or eukaryotic hosts and can replicate autonomously, such as conventional plasmids (pET series) in the art, shuttle vector PNV 18.1, phage vectors, or viral vectors. The sequences of the above polynucleotides of the present disclosure are cloned into vectors through molecular biological manipulations such as enzyme digestion and ligation to construct recombinant expression vectors.

[0062] The present disclosure provides a recombinant host cell comprising the above-described recombinant expression vector.

[0063] The host cells of the present disclosure can be selected from prokaryotic cells, yeast, or eukaryotic cells, and can also be selected from Escherichia coli, Rhodococcus ruber, Bacillus subtilis, and yeast. The recombinant expression vectors of the present disclosure can be transformed into host cells to obtain corresponding recombinant bacteria. Method for preparing recombinant humanized type III collagen

[0064] The present disclosure provides a method for preparing the above recombinant type III humanized collagen, the method comprising: Step S1: constructing a recombinant expression vector containing a polynucleotide encoding the recombinant type III humanized collagen and constructing a recombinant host cell by transformation; Step S2 of culturing the recombinant host cell in a medium to produce a protein; Step S3 of obtaining and purifying the protein, preferably by Ni column and / or anion exchange chromatography; Optionally, a step S4 of enzymatically digesting the protein, preferably with a collagen tool enzyme.

[0065] Example Hereinafter, the embodiments of the present disclosure will be described in detail with reference to examples. However, those skilled in the art should understand that the following examples are for the purpose of illustrating the present disclosure and do not limit the scope of the present disclosure. Unless otherwise specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the materials and instruments used were commercially available.

[0066] Example 1: Construction and expression of recombinant humanized collagen type III 1. The amino acid sequences of different lengths of the hinge region of human type III collagen were combined with the amino acid sequence of recombinant type III humanized collagen (TE16c, described in the applicant's prior patent ZL201811438582.6) to obtain recombinant type III humanized collagen containing hinge regions of different lengths. (1) Amino acid sequence Amino acid sequence of recombinant type III humanized collagen TE16c: GERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIP GEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGE RGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAP(SEQ ID NO.5). Recombinant type III humanized collagen C3T16H1 amino acid sequence: GERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAP RGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIP GEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGSEGSPGHPGQPGPPGPPGAPGPCCGG(SEQ ID NO.6). Amino acid sequence of recombinant type III humanized collagen C3T16H2: GERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAP GERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPN GIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGSEGSPGHPGQPGPPGPPGAPGPCCGG(SEQ ID NO.7). Amino acid sequence of recombinant type III humanized collagen C3T16H3: GERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGA PGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAG PNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGSPGHPGQPGPPGPPGAPGPCCGG(SEQ ID NO.8). Amino acid sequence of recombinant type III humanized collagen C3T16H4:GERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGER GAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGP AGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGHPGQPGPPGPPGAPGPCCGG(SEQ ID NO.9). In the above sequence, the underlined parts are amino acid sequences of different lengths in the hinge region of human type III collagen, i.e., collagen self-assembly elements of different lengths. (2) Nucleotide sequence

[0067] 2. The gene sequences of recombinant type III humanized collagen C3T16H1, C3T16H2, C3T16H3, and C3T16H4 were synthesized, and the above-mentioned various coding nucleotide sequences (with a collagen tool enzyme added to the 5' end. The amino acid sequence of the collagen tool enzyme enzyme digestion site is ENLYFQ (SEQ ID NO. 16), and the nucleotide sequence is gaaaacctgtatttccag (SEQ ID NO. 17)) were inserted between the KpnI and XhoI enzyme digestion sites of the pET-28a-Trx-His expression vector. The TE16c recombinant expression plasmid was an existing plasmid described in the applicant's prior patent ZL201811438582.6.

[0068] 3. The successfully constructed expression plasmid was transformed into E. coli competent cells BL21(DE3). The specific process was as follows: (1) E. coli competent cells BL21(DE3) were removed from the ultra-low temperature refrigerator and placed on ice. When half-melted, 2 μl of the plasmid to be transformed was added to the E. coli competent cells BL21(DE3) and gently mixed 2-3 times to homogenize. (2) The mixture was placed on ice and treated with ice bath for 30 minutes, then heat-shocked in a 42°C water bath for 45-90 seconds, removed, and placed on ice and treated with ice bath for 2 minutes. (3) The mixture was transferred to a biosafety cabinet, and 700 μl of liquid LB medium was added. The mixture was then cultured at 37°C and 220 rpm for 60 minutes. (4) 200 μl of the bacterial suspension was evenly spread on an LB plate containing sodium ampicillin. (5) The plate was cultured in a 37°C incubator for 15-17 hours until uniformly sized colonies grew.

[0069] 4. Five to six single colonies from the transformed LB plates were transferred to shake flasks containing antibiotic stock solution (single colonies transformed with the C3T16H1, C3T16H2, C3T16H3, and C3T16H4 recombinant expression plasmids were added to antibiotic stock solution containing kanamycin sulfate, while single colonies transformed with the TE16c recombinant expression plasmid were added to antibiotic stock solution containing ampicillin sodium) and cultured in a shaker at 220 rpm for 7 hours at 37°C. The cultured shake flasks were then cooled to 16°C, and IPTG was added to induce expression for a certain period. After that, the culture was dispensed into centrifuge bottles and centrifuged at 8000 rpm for 10 minutes at 4°C. The bacterial cells were collected, their weights recorded, and samples were taken for electrophoresis.

[0070] 5. The collected bacterial cells were resuspended in an equilibrated working solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole, pH 8.0). The bacterial solution was cooled to below 15°C and then homogenized twice under high pressure (the homogenized sample is labeled "homogenized"). After completion, the bacterial solution was collected. The homogenized bacterial solution was dispensed into centrifuge bottles and centrifuged at 17,000 rpm at 4°C for 30 minutes. The supernatant was collected, and the supernatant (labeled "supernatant") and precipitate (labeled "precipitate") were detected by electrophoresis.

[0071] 6. The collected supernatant was purified and subjected to enzymatic digestion. The specific process was as follows: (1) Crude purification: a. The column (Ni6FF, Cytiva) was washed with water for 5 CV. b. The column was equilibrated with an equilibration solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole, pH 8.0) for 5 CV. c. Sample loading: The collected supernatant was added to the column. After the liquid had completely flowed through, the flow-through fraction was collected and subjected to electrophoresis (labeled "Flow-through"). d. Washing of non-target proteins: 25 mL of washing solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole, pH 8.0) was added until the liquid had completely flowed through. The flow-through fraction from washing of non-target proteins was collected and subjected to electrophoretic detection (labeled "Washing of non-target proteins"). e. Collection of target protein: 20 mL of eluent (200 mM sodium chloride, 25 mM Tris, 250 mM imidazole, pH 8.0) was added, and the flow-through liquid (labeled "Elution") was collected. Protein concentration was detected by UV-visible spectroscopy, followed by electrophoretic detection. f. The column was washed with 1 M imidazole working solution (labeled "1 M Wash"). g. The column was washed with purified water. (2) Enzymatic digestion: Collagen tool enzyme was added to a protein to collagen tool enzyme mass ratio of 20:1, and digestion was performed at 16°C for 4 hours. A sample (labeled "Enzymatic digestion") was collected and subjected to electrophoretic detection. The protein solution after enzymatic digestion was placed in a dialysis bag and dialyzed against dialysate A (20 mM Tris, 20 mM sodium chloride, pH 8.0) for 2 hours at 4°C. It was then transferred to fresh dialysate and dialyzed overnight at 4°C. A sample was collected, the liquid was replaced (labeled "Liquid exchange"), and electrophoretic detection was performed. (3) Purification: a. Column (CaptoQ, Cytiva) equilibration: The column was equilibrated with solution A (20 mM Tris, 20 mM sodium chloride, pH 8.0) at a flow rate of 10 ml / min. b. Sample loading: The sample was loaded at a flow rate of 5 ml / min, and the flow-through (labeled "Q flow-through") was collected and subjected to electrophoretic detection. Gradient elution with a binary mobile phase system: 0% (V / V) to 15% (V / V) solution B (20 mM Tris, 1 M sodium chloride, pH 8.0) and 100% (V / V) to 85% (V / V) solution A, respectively, for 2 minutes, followed by a 3 CV hold.The following was done: 15% (V / V) to 30% (V / V) solution B and 85% (V / V) to 70% (V / V) solution A, 2 minutes later, held for 3 CV; 30% (V / V) to 50% (V / V) solution B and 70% (V / V) to 50% (V / V) solution A, 2 minutes later, held for 3 CV; 50% (V / V) to 100% (V / V) solution B and 50% (V / V) to 0% (V / V) solution A, 2 minutes later, held for 3 CV. The peak (labeled "B Wash") was collected and subjected to electrophoretic detection. d. The column was washed. e. The target protein content was detected, the protein yield was calculated, and the protein was stored at 4°C.

[0072] 7. Concentration detection (1) Determination of crude protein concentration An appropriate amount of sample was accurately weighed, diluted 10-50 times with the eluent, and stirred thoroughly with a glass rod. The absorbance at 280 nm was measured using a UV-Vis spectrophotometer. The protein concentration was calculated using the formula: C (mg / ml) = A280 × absorbance coefficient × dilution factor (Note: The absorbance value should be in the range of 0.1-1). The concentration detection results are as follows: [Table 1] The protein expression levels were C3T16H3>C3T16H4>C3T16H1>C3T16H2>TE16c.

[0073] (2) Detection of purified protein concentration An appropriate amount of sample was accurately weighed, diluted 10-50 times with the eluent, and stirred thoroughly with a glass rod. The absorbance at 215 nm and 225 nm was measured using a UV-Vis spectrophotometer, and the protein concentration was calculated using the formula C (mg / ml) = (A215 - A225) × 144 × dilution factor ÷ 1000 (Note: The absorbance should be in the range of 0.1-1). The results of the concentration detection are as follows: [Table 2] The protein content after purification was C3T16H4>C3T16H1>C3T16H2>C3T16H3>TE16c.

[0074] 8. Electrophoretic Detection The specific process is as follows: 40 μl of sample solution was taken, 10 μl of 5x protein loading buffer (250 m M Tris-HCl (pH 6.8), 10% (V / V) SDS, 0.5% (V / V) bromophenol blue, 50% (V / V) glycerol, 5% (V / V) β-mercaptoethanol) was added, and the mixture was placed in boiling water at 100°C for 10 minutes. Then, 10 μl of SDS-PAGE protein gel was added per well and electrophoresed at 80 V for 2 hours. After that, the proteins were stained for 20 minutes using Coomassie Brilliant Blue staining solution (0.1% (V / V) Coomassie Brilliant Blue R-250, 25% (V / V) isopropanol, 10% (V / V) glacial acetic acid) and then destained using protein destaining solution (10% (V / V) acetic acid, 5% (V / V) ethanol). The results of electrophoresis detection are shown in Figures 1 to 5. The expression levels of C3T16H1, C3T16H2, C3T16H3, C3T16H4, and TE16c proteins were all good, and the purity of the purified collagen was also good, with no obvious impurity bands observed.

[0075] Example 2: Ultraviolet scanning analysis of circular dichroism spectra of recombinant type III humanized collagen Experimental Method (1) Setting the fixture parameters Band width: 1.0 nm Step: 1.0 nm Measurement range: 190-260nm (far-UV region scan) / 250-340nm (near-UV region scan) Time-per-point: 0.5 seconds Repeats: 3 times Cell length: 10mm|0.5mm Temperature: Room Temperature (2) Near-UV and far-UV scans of the standard A background test and a blank buffer test were performed with the scanning wavelength set at 180-340 nm, and then circular dichroism near-ultraviolet absorption of a 1 mg / mL CSA standard solution was collected in the range of 180-340 nm. (3) Sample processing Purified C3T16H1, C3T16H2, C3T16H3, C3T16H4, and TE16c protein samples were collected and concentrated to a protein concentration of 1 mg / ml using 10 kDa ultrafiltration concentration tubes (Millipore). (4) Far-UV scanning of the sample The cell was soaked in 2M HNO3 overnight, rinsed with deionized water, and air-dried. The background was collected first, followed by a blank buffer solution. An appropriate amount of test sample was then added to the cell, and a far-UV scan was performed from 190 to 260 nm according to the parameters described above. Data was then collected. (5) Near-UV scanning of the sample The cell was soaked in 2M HNO3 overnight, rinsed with deionized water, and air-dried. A background sample was collected first, followed by a blank buffer solution. An appropriate amount of sample was then added to the cell, and a near-UV scan was performed from 250 to 340 nm according to the parameters described above. (6) Scanning spectrum processing All scanned spectra were subjected to baseline subtraction and smoothing using Pro-Data Viewer software.

[0076] Experimental results and analysis The experimental results are shown in Figure 6. C3T16H1, C3T16H2, C3T16H3, C3T16H4, and TE16c all have positive peaks at 221 nm, with the positive peak values ​​being C3T16H2 > C3T16H4 ≥ C3T16H3 > C3T16H1 ≥ TE16c. The results showed that C3T16H1, C3T16H2, C3T16H3, C3T16H4, and TE16c all formed triple helical structures under identical protein concentrations. The amino acid sequence of the hinge region of human type III collagen contributed to the formation of the triple helical structure of recombinant humanized type III collagen. Different lengths of amino acid sequences in the hinge region had different abilities to contribute to the formation of the triple helical structure of recombinant humanized type III collagen.

Claims

1. A collagen self-assembly element, characterized in that the collagen self-assembly element comprises any of the sequences shown in (i) to (iii) below: (i) at least 21 contiguous amino acids at the C-terminus of the amino acid sequence set forth in SEQ ID NO. 1 (ii) An amino acid sequence in which one or more amino acid substitutions, deletions, or additions occur in the amino acid sequence of (i), and which retains the function of promoting self-crosslinking of recombinant humanized type III collagen to form a triple helix structure. (iii) an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of (i) and retaining the function of promoting self-crosslinking of recombinant humanized type III collagen to form a triple helix structure.

2. The collagen self-assembly element of claim 1, wherein the sequence shown in (i) is an amino acid sequence shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, or SEQ ID NO.

4.

3. 3. Use of the collagen self-assembly element of claim 1 or 2 in promoting the formation of a triple helix structure by self-crosslinking of recombinant type III humanized collagen.

4. The collagen self-assembly element exerts its effect by being present at the end of the amino acid sequence of the recombinant humanized type III collagen, The use according to claim 3, characterized in that the end of the amino acid sequence of the recombinant humanized type III collagen is preferably the C-terminus.

5. 1. A recombinant type III humanized collagen comprising: The end of the recombinant type III humanized collagen comprises the collagen self-assembly element of claim 1 or 2, Preferably, the recombinant type III humanized collagen is characterized in that the terminus of the recombinant type III humanized collagen is the C-terminus.

6. The recombinant type III humanized collagen according to claim 5, characterized in that the recombinant type III humanized collagen comprises any of the sequences shown in (a) to (c) below: (a) the amino acid sequence set forth in SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, or SEQ ID NO. 9; (b) an amino acid sequence in which one or more amino acid substitutions, deletions, or additions occur in the amino acid sequence of (i), and which retains the ability to self-crosslink and form a triple helix structure; (c) an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of (i) and retaining the ability to self-crosslink to form a triple helix structure.

7. A polynucleotide comprising: The polynucleotide encodes the collagen self-assembly element of claim 1 or 2, or the recombinant humanized type III collagen of claim 5 or 6, Preferably, the polynucleotide has the following structure: １ ) or (z ２ ) A polynucleotide comprising the sequence shown in (z １ ) The nucleotide sequence shown in SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13 or SEQ ID NO. 14 (z ２ ) under stringent conditions (z １ ) and encoding a protein capable of self-crosslinking to form a triple helix structure, wherein the stringent conditions are medium stringent conditions, medium-high stringent conditions, high stringent conditions, or very high stringent conditions.

8. 1. A recombinant expression vector comprising: A recombinant expression vector, characterized in that the recombinant expression vector comprises the polynucleotide of claim 7.

9. 1. A recombinant host cell comprising: A recombinant host cell, characterized in that the recombinant host cell comprises the recombinant expression vector of claim 8.

10. 7. A method for preparing recombinant type III humanized collagen according to claim 5 or 6, comprising: The preparation method comprises: Step S1: constructing a recombinant expression vector containing the polynucleotide according to claim 7 encoding the recombinant type III humanized collagen according to claim 5 or 6, and constructing a recombinant host cell by transformation; Step S2 of culturing the recombinant host cell obtained in step S1 in a medium to produce a protein; Step S3 of obtaining and purifying the protein, preferably by Ni column and / or anion exchange chromatography; Optionally, a step S4 of enzymatically digesting the protein, preferably with TEV protease.

Images