Self-crosslinked recombinant humanized collagen polymer biomaterial and preparation method thereof

Abstract

The present invention relates to a self-crosslinked recombinant humanized collagen polymer biomaterial and a preparation method thereof. The present invention provides a collagen self-assembly element, which comprises at least 21 consecutive amino acids at the C-terminus of the amino acid sequence shown in SEQ ID NO.1; or an amino acid sequence in which one or more amino acids are replaced, deleted or added in the amino acid sequence as described in (i), and the function of promoting the self-crosslinking of recombinant type III humanized collagen to form a triple helix structure is retained; or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence as described in (i), and the function of promoting the self-crosslinking of recombinant type III humanized collagen to form a triple helix structure is retained. The element can effectively promote the self-crosslinking of recombinant type III humanized collagen to form a triple helix structure.

Description

Technical Field

[0001] The present invention belongs to the field of synthetic biotechnology, and specifically relates to a self-crosslinked recombinant humanized collagen polymer biomaterial and a preparation method thereof; more specifically, it 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 corresponding recombinant type III humanized collagen with a triple helix structure. Background Art

[0002] Collagen is a type of protein widely distributed in human connective tissue. It is also the most abundant protein in the human body, accounting for 25% to 35% of the total protein. The main functions of collagen are to maintain the extracellular environment, maintain the normal physiological functions of tissues and organs, and repair body damage. Collagen is a natural biological resource with bio-tissue compatibility, cell support elasticity and degradability that other polymer materials cannot match. Therefore, collagen can be widely used in industries such as medicine and cosmetics.

[0003] Natural collagen molecules can form a special superhelical structure, which is a left-handed helix with three amino acid residues as the basic repeating body. These three amino acid residues are usually Gly-X-Pro. Gly is necessary for the formation of hydrogen bonds in collagen. It has no side chain itself, which makes collagen densely packed. At a higher structural level, collagen superhelices will further associate into collagen fibrils. In organisms, the synthesis and modification of collagen starts from procollagen, undergoes many chemical changes such as hydroxylation, glycosylation, and mutual cross-linking, and is subject to complex regulation by a variety of biological enzymes. In addition to collagen chains, procollagen also contains globular heads and tails. Without these heads and tails, the collagen chains will not fold into the correct triple helix, thus lacking the biological activity of collagen. Therefore, it is difficult for collagen prepared according to the original gene sequence to spontaneously organize into the correct spatial structure in vitro. Such difficulties have seriously hindered the research and development and production of human collagen.

[0004] People have conducted appropriate research on this. For example, Reference 1 discloses that the hinge region amino acid sequence GPPGPCCGGG (SEQ ID NO. 15) can be added to the C-terminus of the expressed polypeptide to help gel formation. However, Reference 2 discloses that if the hinge region amino acid sequence shown in SEQ ID NO. 15 is added to the C-terminus of the polypeptide sequence provided, the polypeptide will form a gelatinous precipitate during fermentation and cannot be dissolved and purified, and the yield is greatly reduced.

[0005] Therefore, there is an urgent need for a method that can promote the 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 through a biosynthetic method and can have a triple helix structure and exert its specific functions.

[0006] In addition, circular dichroism spectroscopy is a method used to detect the secondary structure of proteins. When it detects natural collagen, an absorption peak near 221nm will appear under appropriate detection conditions. This absorption peak has a certain relationship with its triple helix structure, but it is also affected by the content of proline and hydroxyproline (polyproline itself has a strong 221nm peak, but does not form a triple helix). Therefore, circular dichroism spectroscopy cannot be used as direct evidence of the triple helix structure. However, since the circular dichroism spectroscopy experiment is simple to detect, it can be used to assist in the characterization of the higher-level structure of collagen. For different collagens, the conditions and results of circular dichroism spectroscopy experiments vary greatly. The main reason is that the sequence length and amino acid composition of recombinant collagen (especially the content of proline and hydroxyproline) are different from those of natural full-length collagen. Therefore, the circular dichroism spectroscopy detection conditions (temperature, concentration, solution formula, etc.) of different collagens cannot be kept consistent. In order to characterize whether collagen can form a triple helix (for example, collagen denatured during production or collagen with defects in design cannot form a triple helix structure at all), collagen can be characterized under different conditions by circular dichroism spectroscopy.

[0007] References:

[0008] Cited literature 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. 2004Nov; 136(5):643-9.

[0009] Reference 2: CN 109593126 B. Summary of the invention

[0010] Problem that the invention aims to solve

[0011] At present, the prior art has conducted appropriate research on the problem that, for example, collagen prepared according to the original gene sequence is difficult to spontaneously organize into the correct spatial structure in vitro, resulting in its inability to effectively perform physiological functions, but it still cannot be said to be sufficient. In this regard, the present invention provides a collagen self-assembly element, which can promote the self-crosslinking of recombinant type III humanized collagen to form a triple helix structure, and helps the recombinant type III humanized collagen to self-crosslink to form a triple helix structure by making the end of the amino acid sequence of the recombinant type III humanized collagen contain the amino acid sequence of the hinge region of the human type III collagen, and the recombinant type III humanized collagen biomaterial with a triple helix structure can be successfully prepared by a biosynthetic method.

[0012] Solutions for solving problems

[0013] The first aspect of the present invention provides a collagen self-assembly element, wherein the collagen self-assembly element comprises a sequence as shown in any one of the following (i) to (iii):

[0014] (i) at least 21 consecutive amino acids at the C-terminus of the amino acid sequence shown in SEQ ID NO.1;

[0015] (ii) an amino acid sequence in which one or more amino acids are substituted, deleted or added in the amino acid sequence described in (i), and the amino acid sequence retains the function of promoting the self-crosslinking of recombinant humanized type III collagen to form a triple helical structure;

[0016] (iii) an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence described in (i), and retains the function of promoting the self-crosslinking of recombinant humanized type III collagen to form a triple helical structure.

[0017] In some embodiments, 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.

[0018] The second aspect of the present invention provides use of the collagen self-assembly element according to the first aspect of the present invention in promoting self-crosslinking of recombinant type III humanized collagen to form a triple helical structure.

[0019] In some embodiments, the collagen self-assembly element functions by being present at the end of the amino acid sequence of the recombinant type III humanized collagen;

[0020] Preferably, the end of the amino acid sequence of the recombinant humanized type III collagen is the C-terminus.

[0021] The third aspect of the present invention provides a recombinant type III humanized collagen, wherein the end of the recombinant type III humanized collagen comprises the collagen self-assembly element according to claim 1 or 2;

[0022] Preferably, the terminus of the recombinant humanized type III collagen is the C-terminus.

[0023] In some embodiments, the recombinant humanized type III collagen comprises a sequence as shown in any one of the following (a) to (c):

[0024] (a) the amino acid sequence shown in SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 or SEQ ID NO.9;

[0025] (b) an amino acid sequence in which one or more amino acids are substituted, deleted or added in the amino acid sequence as described in (i), and the amino acid sequence retains the ability to self-crosslink to form a triple helical structure;

[0026] (c) an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence described in (i) and retains the ability to self-crosslink to form a triple helical structure.

[0027] The fourth aspect of the present invention provides a polynucleotide encoding the collagen self-assembly element according to the first aspect of the present invention, or encoding the recombinant type III humanized collagen according to the third aspect of the present invention.

[0028] In some embodiments, the polynucleotide comprises the sequence shown in (z1) or (z2):

[0029] (z1) the nucleotide sequence shown in SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13 or SEQ ID NO.14;

[0030] (z2) A nucleotide sequence that hybridizes with the nucleotide sequence shown in (z1) under stringent conditions and encodes a protein having the ability to self-crosslink to form a triple helical structure, wherein the stringent conditions are medium stringent conditions, medium-high stringent conditions, high stringent conditions or very high stringent conditions.

[0031] The fifth aspect of the present invention provides a recombinant expression vector, which comprises the polynucleotide according to the fourth aspect of the present invention.

[0032] The sixth aspect of the present invention provides a recombinant host cell, which comprises the recombinant expression vector according to the fifth aspect of the present invention.

[0033] The seventh aspect of the present invention provides a method for preparing the recombinant humanized type III collagen according to the third aspect of the present invention, the preparation method comprising the following steps:

[0034] S1: constructing a recombinant expression vector comprising the polynucleotide encoding the recombinant humanized type III collagen according to the third aspect according to the fourth aspect, and constructing a recombinant host cell by transformation;

[0035] S2: culturing the recombinant host cell obtained in S1 in a culture medium and producing a protein;

[0036] S3: harvesting and purifying the protein, preferably purifying the protein using a Ni column and / or anion exchange chromatography; and

[0037] S4: Optionally, the protein is cleaved by enzymes, preferably by using TEV protease.

[0038] Effects of the Invention

[0039] Through the implementation of the above technical solution, the present invention achieves the following technical effects:

[0040] First, the present invention provides a collagen self-assembly element. By making the end of the amino acid sequence of recombinant type III humanized collagen contain the collagen self-assembly element, that is, containing the amino acid sequence of the hinge region of human type III collagen, the recombinant type III humanized collagen can be effectively assisted to self-cross-link to form a triple helix structure, thereby greatly improving the availability of recombinant type III humanized collagen prepared by a biosynthetic method.

[0041] At the same time, the recombinant type III humanized collagen provided by the present invention, which contains the collagen self-assembly element at the end, that is, contains the amino acid sequence of the hinge region of human type III collagen, can form a good triple helical structure, and this result is supported by the circular dichroism spectrum detection result. And the recombinant type III humanized collagen can be prepared in large quantities by a biosynthetic method. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is the electrophoresis detection result during the preparation of recombinant type III humanized collagen TE16c.

[0043] Figure 2 This is the electrophoresis detection result during the preparation of recombinant type III humanized collagen C3T16H1.

[0044] Figure 3 This is the electrophoresis detection result during the preparation of recombinant type III humanized collagen C3T16H2.

[0045] Figure 4This is the electrophoresis detection result during the preparation of recombinant type III humanized collagen C3T16H3.

[0046] Figure 5 This is the electrophoresis detection result during the preparation of recombinant type III humanized collagen C3T16H4.

[0047] Figure 6 This is the circular dichroism UV scanning analysis result of recombinant type III humanized collagen C3T16H1, C3T16H2, C3T16H3, C3T16H4 and TE16c, and a positive peak near 221nm was detected. DETAILED DESCRIPTION

[0048] The following describes the embodiments of the present invention, but the present invention is not limited thereto. The present invention is not limited to the various structures described below, and various changes can be made within the scope of the invention claim, and the embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments and examples are also included in the technical scope of the present invention.

[0049] In the present invention, a numerical range expressed using “a numerical value A to a numerical value B” or “a numerical value A-a numerical value B” means a range including the endpoints A and B.

[0050] In the present invention, a numerical range expressed using "above" or "below" means a numerical range including the number.

[0051] In the present invention, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes instances where the event occurs and instances where it does not occur.

[0052] In the present invention, the term "a" or "an" or "the" may mean "one", and may also mean "one or more", "at least one" and "one or more than one".

[0053] In the present invention, the terms "comprising", "having", "including" or "containing" may mean inclusive or open-ended, and do not exclude additional, uncited elements or method steps. At the same time, "comprising", "having", "including" or "containing" may also mean closed, excluding additional, uncited elements or method steps.

[0054] In the present invention, the term "about" may mean that a value includes the standard deviation of the error of the device or method used to determine the value. The numerical ranges and parameters used to define the present invention are all approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value essentially inevitably contains standard deviations caused by the aforementioned test devices or methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in the present invention are modified by "about". Here, "about" generally means that the actual value is within ±10%, ±5%, ±3%, ±1% or ±0.5% of a particular value or range.

[0055] In the present invention, the terms "polypeptide", "protein" interchangeably refer to a string of at least two amino acid residues linked to each other by covalent bonds (e.g., peptide bonds), which may be recombinant polypeptides, natural polypeptides, or synthetic polypeptides. A polypeptide may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. The term also includes amino acid polymers that have been modified (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component).

[0056] In the present invention, the term "amino acid" may include natural amino acids, unnatural amino acids, amino acid analogs, and all their D and L stereoisomers.

[0057] In the present invention, amino acid modification may include modification of the natural sequence, such as modification of functional groups, intramolecular covalent bonding (e.g., cyclization between side chains), methylation, acylation, ubiquitination, phosphorylation, aminohexanylation, biotinylation, etc.

[0058] In the present invention, amino acid deletion may refer to deleting 1, 2 or 3 or more amino acids from an amino acid sequence, as long as the altered sequence fully or partially retains the activity of the original amino acid sequence.

[0059] In the present invention, amino acid addition may refer to adding 1, 2 or 3 or more amino acids at the C-terminus, N-terminus or any position between the C-terminus and the N-terminus of the amino acid sequence, as long as the altered sequence fully or partially retains the activity of the original amino acid sequence.

[0060] In the present invention, amino acid substitution may refer to the replacement of an amino acid at a certain position of an amino acid sequence by another amino acid, as long as the altered sequence retains the activity of the original amino acid sequence in whole or in part. Amino acid substitution may be a conservative amino acid substitution, which means that compared with the original amino acid sequence, several amino acids are replaced by amino acids with similar or similar properties to form a peptide (conservative variant peptide). Exemplarily, these conservative variant peptides can be generated according to the following amino acid substitutions: substitution of Ala by Val, Leu or Ile, substitution of Arg by Lys, Gln, Asn or His, substitution of Asn by Gln, His, Lys or Arg, substitution of Asn by Glu or Asn, substitution of Cys by Ser or Ala, substitution of Gln by Asn or Glu, substitution of Glu by Asp or Gln, substitution of Gly by Ala, substitution of His by Asn, Lys, Gln or Arg, substitution of Leu, Met, Ala, Val, Phe or norleucine by The amino acid substitutions may also be non-conservative amino acid substitutions.

[0061] In the present invention, the terms "sequence identity" and "percentage of identity" refer to the percentage of nucleotides or amino acids that are identical (i.e., identical) between two or more polynucleotides or polypeptides. The sequence identity between two or more polynucleotides or polypeptides can be determined by the following method: the nucleotide or amino acid sequences of the polynucleotides or polypeptides are aligned and the number of positions containing the same nucleotide or amino acid residue in the aligned polynucleotides or polypeptides is scored, and compared with the number of positions containing different nucleotides or amino acid residues in the aligned polynucleotides or polypeptides. The polynucleotides may differ at one position, for example, by containing different nucleotides (i.e., substitutions or mutations) or missing nucleotides (i.e., nucleotide insertions or nucleotide deletions in one or two polynucleotides). The polypeptides may differ at one position, for example, by containing different amino acids (i.e., substitutions or mutations) or missing amino acids (i.e., amino acid insertions or amino acid deletions in one or two polypeptides). The 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 polynucleotide or polypeptide. For example, percent identity can be calculated by dividing the number of positions containing the identical nucleotide or amino acid residue by the total number of nucleotides or amino acid residues in the polynucleotide or polypeptide and multiplying by 100.

[0062] In the present invention, the terms "moderate stringency conditions", "medium-high stringency conditions", "high stringency conditions" or "very high stringency conditions" describe conditions for nucleic acid hybridization and washing. Guidance for conducting hybridization reactions can be found 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 document, and either can be used. For example, specific hybridization conditions are as follows: (1) low stringency hybridization conditions are in 6× sodium chloride / sodium citrate (SSC) at about 45°C, followed by two washes in 0.2×SSC, 0.1% SDS at at least 50°C (for low stringency conditions, the washing temperature can be increased to 55°C); (2) moderate stringency hybridization conditions are in 6×SSC at about 45°C, followed by one or more washes in 0.2×SSC, 0.1% SDS at 60°C; (3) high stringency hybridization conditions are in 6×SSC at about 45°C, followed by one or more washes in 0.2×SSC, 0.1% SDS at 65°C and preferably; (4) very high stringency hybridization conditions are 0.5 M sodium phosphate, 7% SDS at 65°C, followed by one or more washes in 0.2×SSC, 1% SDS at 65°C.

[0063] In the present invention, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides, or their analogs. A polynucleotide may have any three-dimensional structure and may perform any known or unknown function.

[0064] In the present invention, the term "recombinant expression vector" refers to a DNA structure used to express, for example, a polynucleotide encoding a desired polypeptide. The recombinant expression vector may include, for example, a collection of genetic elements that regulate gene expression, such as promoters and enhancers; ii) a structural or coding sequence that is transcribed into mRNA and translated into a protein; and iii) a transcription subunit that is suitable for transcription and translation initiation and termination sequences. The recombinant expression vector is constructed in any suitable manner. The nature of the vector is not important, and any vector may be used, including plasmids, viruses, phages, and transposons. Possible vectors for use in the present invention include, but are not limited to, chromosomal, non-chromosomal, and synthetic DNA sequences, such as viral plasmids, bacterial plasmids, phage DNA, yeast plasmids, and vectors derived from a combination of plasmids and phage DNA, DNA from viruses such as lentivirus, retrovirus, vaccinia, adenovirus, fowlpox, baculovirus, SV40, and pseudorabies.

[0065] In the present invention, 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 or Salmonella; Bacillaceae such as Bacillus subtilis; Pneumococcus; Streptococcus 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.

[0066] In the present invention, the term "codon optimization" means that the nucleotide sequence encoding the polypeptide has been configured to contain codons preferred by the host cell or organism to improve gene expression in the host cell or organism and increase translation efficiency.

[0067] Unless otherwise defined, other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0068] The technical solution provided by the present invention is described in more detail below:

[0069] Collagen self-assembly elements

[0070] The present invention provides a collagen self-assembly element, which can effectively promote the self-crosslinking of recombinant type III humanized collagen to form a triple helix structure.

[0071] In some embodiments, the collagen self-assembly element comprises a sequence as shown in any one of the following (i) to (iii):

[0072] (i) at least 21 consecutive amino acids at the C-terminus of the amino acid sequence shown in SEQ ID NO.1;

[0073] (ii) an amino acid sequence in which one or more amino acids are substituted, deleted or added in the amino acid sequence described in (i), and the amino acid sequence retains the function of promoting the self-crosslinking of recombinant humanized type III collagen to form a triple helical structure;

[0074] (iii) an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence described in (i), and retains the function of promoting the self-crosslinking of recombinant humanized type III collagen to form a triple helical structure.

[0075] SEQ ID NO.1: GERGSEGSPGHPGQPGPPGPPGAPGPCCGG, which is a peptide segment of human type III collagen.

[0076] The present invention unexpectedly discovered that collagen self-assembly elements of different lengths have different abilities to promote the self-crosslinking of recombinant type III humanized collagen to form a triple helical structure.

[0077] In some specific embodiments, the amino acid sequence of the collagen self-assembly element comprises a sequence as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4.

[0078] In some more specific embodiments, the amino acid sequence of the collagen self-assembly element is a sequence as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4.

[0079] SEQ ID NO. 2: GSEGSPGHPGQPGPPGPPGAPGPCCGG.

[0080] SEQ ID NO. 3: GSPGHPGQPGPPGPPGAPGPCCGG.

[0081] SEQ ID NO. 4: GHPGQPGPPGPPGAPGPCCGG.

[0082] In order to effectively exert the function of the collagen self-assembly element in promoting the 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 end of the recombinant type III humanized collagen, preferably the C-terminus.

[0083] The recombinant humanized type III collagen described in the present invention refers to the full-length or partial amino acid sequence fragment encoded by the type III human collagen gene prepared by DNA recombination technology, or a combination containing functional fragments of type III human collagen.

[0084] In some specific embodiments, the amino acid sequence of the recombinant type III humanized collagen contains a collagen self-assembly element at the end, which can be achieved by the following method: designing the amino acid sequence of the recombinant type III humanized collagen containing a collagen self-assembly element at the end and its gene sequence, and constructing an expression vector containing the gene sequence, which is transformed into a host cell, and then the host cell can be fermented and cultured, and the recombinant type III humanized collagen can be induced to express.

[0085] Recombinant humanized type III collagen

[0086] The present invention provides a recombinant type III humanized collagen, the end of which contains a collagen self-assembly element. The collagen self-assembly element is as described above. Since the recombinant type III humanized collagen provided by the present invention contains the collagen self-assembly element, it can self-cross-link to form a triple helix structure after expression, and the protein can be prepared in large quantities by a biosynthetic method.

[0087] In order to better form a triple helix structure, the collagen self-assembly element is located at the C-terminus of the recombinant type III humanized collagen.

[0088] In some specific embodiments, the recombinant humanized type III collagen comprises a sequence as shown in any one of the following (a) to (c):

[0089] (a) the amino acid sequence shown in SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 or SEQ ID NO.9;

[0090] (b) an amino acid sequence in which one or more amino acids are substituted, deleted or added in the amino acid sequence as described in (i), and the amino acid sequence retains the ability to self-crosslink to form a triple helical structure;

[0091] (c) an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence described in (i) and retains the ability to self-crosslink to form a triple helical structure.

[0092] In some preferred embodiments, the amino acid sequence of the recombinant humanized type III collagen is a sequence as shown in SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 or SEQ ID NO.9.

[0093] Polynucleotide, recombinant expression vector, recombinant host cell

[0094] The present invention provides polynucleotides encoding the collagen self-assembly element and the recombinant type III humanized collagen.

[0095] In some embodiments, the polynucleotide encoding the recombinant humanized type III collagen comprises the sequence shown in (z1) or (z2):

[0096] (z1) the nucleotide sequence shown in SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13 or SEQ ID NO.14;

[0097] (z2) A nucleotide sequence that hybridizes with the nucleotide sequence shown in (z1) under stringent conditions and encodes a protein having the ability to self-crosslink to form a triple helical structure, wherein the stringent conditions are medium stringent conditions, medium-high stringent conditions, high stringent conditions or very high stringent conditions.

[0098] The present invention provides a recombinant expression vector comprising the polynucleotide.

[0099] The expression vector selected in the present invention can stably exist in various hosts of prokaryotic or eukaryotic cells and can replicate autonomously, such as conventional plasmids (pET series), shuttle vector PNV 18.1, phage or virus vectors in the art, etc. The sequence of the polynucleotide of the present invention is cloned into the vector through molecular biological operations such as enzyme digestion and ligation on the vector to construct a recombinant expression vector.

[0100] The present invention provides a recombinant host cell comprising the above recombinant expression vector.

[0101] The host cell of the present invention is selected from prokaryotic cells, yeast or eukaryotic cells, and can be further selected from Escherichia coli, Rhodococcus erythrorhizium, Bacillus subtilis and yeast. The above-mentioned recombinant expression vector of the present invention is transformed into the host cell to obtain the corresponding genetically engineered bacteria.

[0102] Preparation method of recombinant humanized type III collagen

[0103] The present invention provides a method for preparing the above-mentioned recombinant humanized type III collagen, the preparation method comprising the following steps:

[0104] S1: constructing a recombinant expression vector comprising a polynucleotide encoding the recombinant humanized type III collagen, and constructing a recombinant host cell by transformation;

[0105] S2: culturing the recombinant host cell in a culture medium and producing a protein;

[0106] S3: harvesting and purifying the protein, preferably purifying the protein using a Ni column and / or anion exchange chromatography; and

[0107] S4: Optionally, the protein is cleaved by enzymes, preferably by using collagenase.

[0108] Example

[0109] The embodiments of the present invention will be described in detail below in conjunction with examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out under normal conditions or conditions recommended by the manufacturer. Unless otherwise specified, the materials or instruments used are all commercially available conventional products.

[0110] Example 1: Construction and expression of recombinant humanized type III collagen

[0111] 1. The amino acid sequences of different lengths of the hinge region of human type III collagen are combined with the amino acid sequence of recombinant type III humanized collagen (TE16c, recorded in the prior patent ZL201811438582.6 of the applicant of the present application) to obtain recombinant type III humanized collagen containing hinge regions of different lengths.

[0112] (1) Amino acid sequence

[0113] Amino acid sequence of recombinant humanized collagen type III TE16c: GERGAPGFRGPAGPNGIPGEKGPAGERGAPGE RGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAP(SEQ ID NO.5).

[0114] Amino acid sequence of recombinant type III humanized collagen C3T16H1: GERGAPGFRGPAGPNGIPGEKGPAGERGAPGE RGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAP GER GSEGSPGHPGQPGPPGPPGAPGPCCGG (SEQ ID NO.6).

[0115] Amino acid sequence of recombinant type III humanized collagen C3T16H2: GERGAPGFRGPAGPNGIPGEKGPAGERGAP GERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAP GSEGSPGHPGQPGPPGPPGAPGPCCGG (SEQ ID NO.7).

[0116] Amino acid sequence of recombinant type III humanized collagen C3T16H3: GERGAPGFRGPAGPNGIPGEKGPAGERGAP GERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAP GSPGHPGQPGPPGPPGAPGPCCGG (SEQ ID NO.8).

[0117] The amino acid sequence of recombinant humanized type III collagen C3T16H4: GERGAPGFRGPAGPNGIPGEKGPAGERGAP GERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPN GIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAP GHPGQPGPPGPPGAPGPCCGG (SEQ ID NO.9).

[0118] In the above sequence, the underlined part is the amino acid sequence of different lengths in the hinge region of human type III collagen, that is, the collagen self-assembly elements of different lengths.

[0119] (2) Nucleotide sequence

[0120]

[0121]

[0122]

[0123]

[0124]

[0125] 2. The gene sequences of recombinant type III humanized collagen C3T16H1, C3T16H2, C3T16H3, and C3T16H4 were synthesized, and each of the above encoding nucleotide sequences (a collagenase cleavage site was added at the 5' end, the amino acid sequence of the collagenase cleavage site was ENLYFQ (SEQ ID NO.16), and the nucleotide sequence was gaaaacctgtatttccag (SEQ ID NO.17)) was inserted between the KpnI and XhoI cleavage sites of the pET-28a-Trx-His expression vector. The TE16c recombinant expression plasmid is an existing plasmid, which is recorded in the prior patent ZL201811438582.6 of the applicant of this application.

[0126] 3. Transform the successfully constructed expression plasmid into E. coli competent cells BL21 (DE3). The specific process is as follows: (1) Take out the E. coli competent cells BL21 (DE3) from the ultra-low temperature refrigerator and place them on ice. When they are half melted, take 2 μl of the plasmid to be transformed and add it to the E. coli competent cells BL21 (DE3), and mix it slightly 2-3 times. (2) Place the mixture on ice for 30 minutes, then heat shock it in a 42°C water bath for 45-90 seconds, take it out and place it on ice for 2 minutes. (3) Transfer it to a biological safety cabinet, add 700 μl of liquid LB culture medium, and then culture it at 37°C and 220 rpm for 60 minutes. (4) Take 200 μl of the bacterial solution and evenly spread it on an LB plate containing ampicillin sodium. (5) Culture the plate in a 37°C incubator for 15-17 hours until colonies of uniform size grow.

[0127] 4. Pick 5-6 single colonies from the transformed LB plate and place them in a shake flask containing antibiotic stock solution (the single colonies transformed with C3T16H1, C3T16H2, C3T16H3, and C3T16H4 recombinant expression plasmids are added to the antibiotic stock solution containing kanamycin sulfate, and the single colonies transformed with TE16c recombinant expression plasmids are added to the antibiotic stock solution containing ampicillin sodium) and incubate in a constant temperature shaker at 220rpm and 37℃ for 7h. Then cool the shake flask after culture to 16℃, add IPTG to induce expression for a period of time, divide the bacterial solution into centrifuge bottles, centrifuge at 8000rpm and 4℃ for 10min, collect the bacteria, record the weight of the bacteria, and take samples for electrophoresis detection.

[0128] 5. Resuspend the collected bacteria with balanced working solution (200mM sodium chloride, 25mM Tris, 20mM imidazole, pH8.0), cool the bacterial solution to ≤15°C, homogenize, and homogenize twice under high pressure (mark the homogenized sample: homogenize), and collect the bacterial solution after completion. The homogenized bacterial solution is divided into centrifuge bottles, centrifuged at 17000rpm and 4°C for 30min, and the supernatant is collected. The supernatant (marked: supernatant) and the precipitate (marked: precipitate) are taken for electrophoresis detection.

[0129] 6. The collected supernatant was purified and digested by enzymes. The specific process is as follows: (1) Crude purification: a. Wash the column material (Ni6FF, Cytiva) with water for 5 CVs. b. Equilibrate the column material with equilibration solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole, pH 8.0) for 5 CVs. c. Loading: Add the collected supernatant to the column material until the liquid flows out, and take the flow-through for electrophoresis inspection (marked: flow-through). d. Wash impurities: Add 25 mL of washing solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole, pH 8.0) until the liquid flows out, and take the wash impurities flow-through for electrophoresis inspection (marked: wash impurities). e. Collect the target protein: add 20 mL of eluent (200 mM sodium chloride, 25 mM Tris, 250 mM imidazole, pH 8.0), collect the flow-through (marked: elution), detect the protein concentration by UV-visible spectrophotometry, and perform electrophoresis. f. Wash the column with 1 M imidazole working solution (marked: 1 M wash). g. Wash the column with purified water. (2) Enzyme digestion: add collagen enzyme at a protein mass to collagen enzyme mass ratio of 20:1, digest at 16°C for 4 h, and take a sample (marked: enzyme digestion) for electrophoresis. Put the protein solution after digestion into a dialysis bag with dialysis solution A (20 mM Tris, 20 mM sodium chloride, pH 8.0), dialyze at 4°C for 2 h, then transfer to new dialysis solution for overnight dialysis at 4°C, take a sample (marked: change solution) for electrophoresis. (3) Purification: a. Equilibrate the column (Capto Q, Cytiva): Use solution A (20 mM Tris, 20 mM sodium chloride, pH 8.0) to equilibrate the column at a flow rate of 10 ml / min. b. Load the sample: Load the sample at a flow rate of 5 ml / min and collect the flow-through (labeled as Q flow-through) for electrophoresis detection. c. Binary mobile phase system gradient elution: set 0% (V / V)-15% (V / V) B solution (20mMTris, 1M sodium chloride, pH8.0) and 100% (V / V)-85% (V / V) A solution for 2 min and then keep for 3 CV, 15% (V / V)-30% (V / V) B solution and 85% (V / V)-70% (V / V) A solution for 2 min and then keep for 3 CV, 30% (V / V)-50% (V / V) B solution and 70% (V / V)-50% (V / V) A solution for 2 min and then keep for 3 CV, 50% (V / V)-100% (V / V) B solution and 50% (V / V)-0% (V / V) A solution for 2 min and then keep for 3 CV, collect the peaks (marked: B wash) and perform electrophoresis detection. d. Clean the column material. e. Detect the target protein content and calculate the protein yield, and store the protein at 4°C.

[0130] 7. Concentration detection

[0131] (1) Detection of crude pure protein concentration

[0132] Accurately measure an appropriate amount of sample, dilute it 10-50 times with the eluent, and stir it thoroughly with a glass rod. Use a UV-visible spectrophotometer to measure the absorbance at 280nm, and calculate the protein concentration according to the formula C (mg / ml) = A280 × absorbance coefficient × dilution factor (Note: the absorbance value must be between 0.1 and 1).

[0133] The concentration test results are as follows:

[0134] Plasmids TE16c C3T16H1 C3T16H2 C3T16H3 C3T16H4 Absorption coefficient 3.48 4.18 4.15 4.13 4.13 Dilution multiple 1 1 1 1 1 A280 0.624 0.720 0.612 0.932 0.766 concentration 2.17mg / ml 3.01mg / ml 2.54mg / ml 3.85mg / ml 3.16mg / ml Elution protein volume 20ml 20ml 20ml 20ml 20ml Protein Amount 43.40mg 60.20mg 50.80mg 76.98mg 63.27mg

[0135] The protein expression levels were C3T16H3>C3T16H4>C3T16H1>C3T16H2>TE16c.

[0136] (2) Detection of purified protein concentration

[0137] Accurately measure an appropriate amount of sample, dilute it 10-50 times with the eluent, and stir it thoroughly with a glass rod. Use a UV-visible spectrophotometer to measure the absorbance at 215 and 225 nm, and calculate the protein concentration according to the formula C (mg / ml) = (A215-A225) × 144 × dilution factor ÷ 1000 (Note: the absorbance value must be between 0.1 and 1).

[0138] The concentration test results are as follows:

[0139] Plasmids TE16c C3T16H1 C3T16H2 C3T16H3 C3T16H4 Dilution multiple 10 10 10 10 10 A215 0.301 0.505 0.513 0.584 0.717 A225 0.121 0.167 0.156 0.252 0.322 concentration 0.26mg / ml 0.49mg / ml 0.51mg / ml 0.48mg / ml 0.57mg / ml Collected protein volume 51ml 53ml 48ml 51ml 60ml Protein Amount 13.26mg 25.80mg 24.68mg 24.38mg 34.13mg

[0140] The protein amount after purification is C3T16H4>C3T16H1>C3T16H2>C3T16H3>TE16c.

[0141] 8. Electrophoresis detection

[0142] The specific process is as follows: take 40 μl of sample solution, add 10 μl 5× protein loading buffer (250 mM Tris-HCl (pH 6.8), 10% (V / V) SDS, 0.5% (V / V) bromophenol blue, 50% (V / V) glycerol, 5% (V / V) β-mercaptoethanol), place in 100°C boiling water and boil for 10 min, then add 10 μl per well to SDS-PAGE protein gel, after electrophoresis at 80 V for 2 h, use Coomassie Brilliant Blue staining solution (0.1% (V / V) Coomassie Brilliant Blue R-250, 25% (V / V) isopropanol, 10% (V / V) glacial acetic acid) for protein staining for 20 min, and then use protein decolorizing solution (10% (V / V) acetic acid, 5% (V / V) ethanol) for decolorization.

[0143] The electrophoresis results are as follows Figures 1 to 5As shown, the expression levels of C3T16H1, C3T16H2, C3T16H3, C3T16H4, and TE16c proteins were all good, and the purity of the purified collagen was good without obvious mixed bands.

[0144] Example 2: UV scanning analysis of recombinant humanized type III collagen by circular dichroism

[0145] Experimental methods

[0146] (1) Instrument parameter setting

[0147] Band width: 1.0nm;

[0148] Step: 1.0nm;

[0149] Measurement range: 190-260nm (far-UV regionscan) / 250-340nm (near-UV region scan);

[0150] Time-per-point: 0.5s;

[0151] Repeats: 3 times;

[0152] Cell Length: 10mm|0.5mm;

[0153] Temperature: Room Temperature.

[0154] (2) Standard product near and far UV scanning

[0155] The scanning wavelength was set to 180-340 nm for background test and blank buffer test, and then the near and far UV absorption of circular dichroism of 1 mg / mL CSA standard solution in the range of 180-340 nm was collected.

[0156] (3) Sample processing

[0157] Purified C3T16H1, C3T16H2, C3T16H3, C3T16H4, and TE16c protein samples were taken and concentrated to a protein concentration of 1 mg / ml using 10 kDa ultrafiltration concentrators (Millipore).

[0158] (4) Sample far-UV scanning

[0159] Soak the cuvette in 2M HNO3 overnight, rinse with deionized water and dry, collect the background first, then collect the blank buffer, then add an appropriate amount of the test sample to the cuvette, perform far-UV scanning at 190-260nm according to the above parameters and collect data.

[0160] (5) Sample near-ultraviolet scanning

[0161] Soak the cuvette in 2M HNO3 overnight, rinse with deionized water and dry, collect the background first, then collect the blank buffer, and then add an appropriate amount of the test sample to the cuvette to perform near-UV scanning at 250-340nm according to the above parameters and collect data.

[0162] (6) Scanning spectrum processing

[0163] All scanned images were processed by subtract baseline and smoothing using the software Pro-Data Viewer.

[0164] Experimental results and analysis

[0165] The experimental results are as follows Figure 6 As shown, C3T16H1, C3T16H2, C3T16H3, C3T16H4, and TE16c all have positive peaks at 221nm, and the positive peak values ​​are C3T16H2>C3T16H4≥C3T16H3>C3T16H1≥TE16c. This indicates that C3T16H1, C3T16H2, C3T16H3, C3T16H4, and TE16c all have triple helical structures under the same protein concentration conditions, and the amino acid sequence of the hinge region of human type III collagen contributes to the formation of the triple helical structure of recombinant type III humanized collagen. The amino acid sequences of hinge regions of different lengths have different abilities to promote the formation of the triple helical structure of recombinant type III humanized collagen.

Claims

1. A collagen self-assembly element, characterized in that: The amino acid sequence of the collagen self-assembly element is the amino acid sequence shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.

4.

2. Use of the collagen self-assembly element according to claim 1 in promoting the self-crosslinking of recombinant type III humanized collagen to form a triple helix structure; the collagen self-assembly element exerts its effect by being located at the C-terminus of the amino acid sequence of the recombinant type III humanized collagen, and the amino acid sequence of the recombinant type III humanized collagen is an amino acid sequence as shown in SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 or SEQ ID NO.

9.

3. A recombinant humanized type III collagen, characterized in that: The C-terminus of the recombinant humanized type III collagen is the collagen self-assembly element according to claim 1, and the amino acid sequence of the recombinant humanized type III collagen is the amino acid sequence shown in SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 or SEQ ID NO.

9.

4. A polynucleotide, characterized in that The polynucleotide encodes the recombinant humanized type III collagen according to claim 3.

5. The polynucleotide according to claim 4, characterized in that The sequence of the polynucleotide is a nucleotide sequence as shown in SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13 or SEQ ID NO.

14.

6. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the polynucleotide according to claim 4 or 5.

7. A recombinant host cell, characterized in that The recombinant host cell comprises the recombinant expression vector according to claim 6.

8. The method for preparing recombinant humanized type III collagen according to claim 3, characterized in that: The preparation method comprises the following steps: S1: constructing a recombinant expression vector comprising the polynucleotide encoding the recombinant type III humanized collagen according to claim 3 according to claim 4 or 5, and constructing a recombinant host cell by transformation; S2: culturing the recombinant host cell obtained in S1 in a culture medium and producing a protein; S3: harvesting and purifying the protein; and S4: performing enzymatic digestion on the protein.

9. The method according to claim 8, characterized in that In the S3 step, the protein is purified using a Ni column and / or anion exchange chromatography.

10. The method according to claim 8, characterized in that In the step S4, the protein is cleaved by TEV protease.

Images