Recombinant II-type collagen and application thereof in blood coagulation and tissue regeneration

Abstract

The invention provides recombinant II-type collagen and application thereof in blood coagulation and tissue regeneration, and relates to the technical field of genetic engineering. The recombinant II type collagen realizes recombinant expression in pichia pastoris, and verifies that the recombinant expression protein can be self-assembled to form a triple helix structure and has no biotoxicity. The invention also provides an application of the prepared recombinant II-type collagen in promoting blood coagulation and fibroblast differentiation, shows the blood coagulation capability superior to that of a commercial blood coagulation agent rat tail collagen I, and has huge value and potential in the fields of cosmetics, medicine and tissue engineering.

Description

Technical Field

[0001] The present application relates to the field of genetic engineering technology, and in particular to a recombinant type II collagen and its application in blood coagulation and tissue regeneration. Background Art

[0002] Collagen is an extracellular matrix protein present throughout the human body and has multiple important biological functions in normal physiology. Collagen can self-assemble into striated fibrils, which not only provide support for cell growth but are also responsible for the mechanical elasticity of connective tissue. Twenty-eight different collagens (I–XXVIII) have been identified based on differences in amino acid sequence, function, and protein structure. Despite the expanding market demand for collagen in the fields of biomedicine, cosmetics, and health foods, the traditional method of extracting collagen from animals is expensive, inefficient, and unsuitable to meet the growing industrial demand. Therefore, various expression systems (including insects, animal cells, plants, bacteria, and yeast) have been developed to synthesize recombinant collagen. With increasing usage, the collagen market is expected to reach US$6.63 billion by 2025. Although the synthesis of recombinant collagen has achieved good commercialization, further research and expansion are needed to improve biosafety, yield, and functionality. In addition, full-length collagen is poorly soluble and has low yields, so it is necessary to identify new functional collagen fragments that can be commercially produced for use as biomaterials.

[0003] Various systems, including insects, animal cells, plants, bacteria, and yeast, have been developed for the biosynthesis of recombinant collagen, and each system has unique advantages and disadvantages. The main advantage of insect cell expression systems for the production of recombinant proteins is that they are safe and efficient, while animal cells can be used to produce recombinant collagen with improved physiological activity. However, large-scale production using existing systems is limited by cost and procedural complexity. Plant expression systems are also suitable for large-scale production of recombinant proteins and can achieve much higher yields than insect and mammalian cells. However, plants require longer culture periods than microorganisms and increase the risk of environmental contamination.

[0004] Escherichia coli cells are commonly used as low-cost and efficient hosts for the production of recombinant proteins. However, it is difficult to secrete recombinant proteins into the extracellular medium, resulting in high separation and purification costs. As an efficient platform commonly used for the production of recombinant proteins, Pichia pastoris is not only suitable for high-density and large-scale production, but also has a mature secretion system for secreting proteins into the external environment. It is currently the best choice for the synthesis of recombinant collagen fragments.

[0005] Type II collagen (COL2) is a rod-shaped structure composed of three mutually coded peptide chains, which is mainly synthesized by human and animal chondrocytes and is the main component of animal cartilage. COL2 has good biological activity, not only affecting cell growth potential, but also stimulating cell growth and redifferentiation, and is used in medicine and cartilage tissue engineering. It is necessary to further improve the efficiency of microbial production of recombinant COL2 to reduce the production cost for commercial use. Summary of the invention

[0006] In view of the above problems, the object of the present invention is to provide a new functional recombinant COL2 with high production efficiency and a preparation method thereof.

[0007] In one aspect, the present application provides a recombinant type II collagen, wherein the amino acid sequence of the recombinant type II collagen includes any one or more of the following:

[0008] A1) the amino acid sequence of the recombinant type II collagen comprises one or more repeating units of amino acid residues, the repeating units comprise the sequence shown in SEQ ID NO.1, the number of repetitions is n, 20≤n≤30; A2) the amino acid sequence of the recombinant type II collagen comprises one or more repeating units of amino acid residues, the repeating units comprise the sequence shown in SEQ ID NO.2, the number of repetitions is n, 1≤n≤10;

[0009] A3) The amino acid sequence of the recombinant type II collagen comprises one or more repeating units of amino acid residues, wherein the repeating unit comprises the sequence shown in SEQ ID NO.3, and the number of repeats is n, 1≤n≤15.

[0010] in,

[0011] A1) The amino acid sequence of the recombinant type II collagen comprises one or more repeating units of amino acid residues, wherein the repeating unit comprises the sequence shown in SEQ ID NO.1, the number of repetitions is n, and n is a natural number. Optionally, the value range of the number of repetitions n can be: 20≤n≤30, 21≤n≤30, 22≤n≤30, 23≤n≤30, 24≤n≤30, 25≤n≤30, 26≤n≤30, 27≤n≤30, 28≤n≤30, 29≤n≤30, 20≤n≤29, 21≤n≤29, 22≤n≤29, 23≤n≤29, 24≤n≤29, 25≤n≤29, 26≤n≤29, 27≤n≤29, 28≤n≤29, 20≤n≤28, 21≤n≤28, 22≤n≤28, 23≤n≤ 28, 24≤n≤28, 25≤n≤28, 26≤n≤28, 27≤n≤28, 20≤n≤27, 21≤n≤27, 22≤n≤27, 23≤n≤27, 24≤n≤27, 25≤n≤27, 26≤n≤27, 20≤n≤26, 21≤n≤26, 22≤n≤26, 23≤n≤26, 24≤n≤26, 25≤n≤26, 20≤n≤25, 21≤n≤25, 22≤n≤25, 23≤n≤25, 24≤n≤25, 20≤n≤24, 21≤n≤24, 22≤n≤24, 23≤n≤24 and any range therebetween;

[0012] For example, the value of n, or the upper limit value of n, or the lower limit value of n, n can be selected from any value of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.

[0013] A2) The amino acid sequence of the recombinant type II collagen comprises one or more repeating units of amino acid residues, wherein the repeating unit comprises For the sequence shown in NO.2, the number of repetitions is n, where n is a natural number. Optionally, the value range of the number of repetitions n can be: 1≤n≤10, 2≤n≤10, 3≤n≤10, 4≤n≤10, 5≤n≤10, 6≤n≤10, 7≤n≤10, 8≤n≤10, 9≤n≤10, 1≤n≤9, 2≤n≤9, 3≤n≤9, 4≤n≤9, 5≤n≤9, 6≤n≤9, 7≤n≤9, 8≤n≤9, 1≤n≤8, 2≤n≤8, 3≤n≤8, 4≤n≤8, 5≤n≤8, 6≤n≤8, 7≤n≤8, 1≤n≤7, 2≤n≤7, 3≤n≤7, 4≤n≤7, 5≤n≤7, 6≤n≤7 and any range therebetween;

[0014] For example, the value of n, or the upper limit value of n, or the lower limit value of n, n can be selected from any value of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0015] A3) The amino acid sequence of the recombinant type II collagen comprises one or more repeating units of amino acid residues, wherein the repeating unit comprises the sequence shown in SEQ ID NO.3, the number of repetitions is n, and n is a natural number. Optionally, the value range of the number of repetitions n can be: 1≤n≤15, 2≤n≤15, 3≤n≤15, 4≤n≤15, 5≤n≤15, 6≤n≤15, 7≤n≤15, 8≤n≤15, 9≤n≤15, 10≤n≤15, 11≤n≤15, 12≤n≤15, 13≤n≤15, 14≤n≤15, 15≤n≤15, 16≤n≤15, 17≤n≤15, 18≤n≤15, 19≤n≤15 , 2≤n≤14, 3≤n≤14, 4≤n≤14, 5≤n≤14, 6≤n≤14, 7≤n≤14, 8≤n≤14, 9≤n≤14, 10≤n≤14, 11≤n≤14, 12≤n≤14, 13≤n≤14, 1≤n≤13, 2≤n≤13, 3≤n≤13, 4≤n≤13, 5≤n≤13, 6≤n≤13, 7≤n≤13, 8≤n≤13, 9≤n≤ 13, 10≤n≤13, 11≤n≤13, 12≤n≤13, 1≤n≤12, 2≤n≤12, 3≤n≤12, 4≤n≤12, 5≤n≤12, 6≤n≤12, 7≤n≤12, 8≤n≤12, 9≤n≤12, 10≤n≤12, 11≤n≤12, 1≤n≤11, 2≤n≤11, 3≤n≤11, 4≤n≤11, 5≤n≤11, 6≤n≤11, 7 ≤n≤11, 8≤n≤11, 9≤n≤11, 10≤n≤11, 1≤n≤10, 2≤n≤10, 3≤n≤10, 4≤n≤10, 5≤n≤10, 6≤n≤10, 7≤n≤10, 8≤n≤10, 9≤n≤10, 1≤n≤9, 2≤n≤9, 3≤n≤9, 4≤n≤9, 5≤n≤9, 6≤n≤9, 7≤n≤9, 8≤n≤9, and any range therebetween;

[0016] For example, the value of n, or the upper limit value of n, or the lower limit value of n, n can be selected from any value of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15.

[0017] Furthermore, the amino acids of the collagen include an amino acid sequence as shown in SEQ ID NO.4 or an amino acid sequence that is at least 95% identical to SEQID NO.4, and / or an amino acid sequence as shown in SEQ ID NO.6 or an amino acid sequence that is at least 95% identical to SEQID NO.6, and / or an amino acid sequence as shown in SEQ ID NO.8 or an amino acid sequence that is at least 95% identical to SEQID NO.8.

[0018] in,

[0019] The amino acids of the collagen protein include the amino acid sequence shown in SEQ ID NO.4 or an amino acid sequence having 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identity with SEQ ID NO.4,

[0020] and / or, the amino acid sequence as set forth in SEQ ID NO.6, or an amino acid sequence having 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identity to SEQ ID NO.6,

[0021] and / or, the amino acid sequence as shown in SEQ ID NO.8 or an amino acid sequence having 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identity with SEQ ID NO.8.

[0022] It is understandable that those skilled in the art can select a suitable gene editing system and gene editing method according to actual conditions to complete the construction of the above-mentioned recombinant type II collagen.

[0023] On the other hand, the present application also provides a biomaterial, which comprises any one of the following B1)-B5):

[0024] B1) a nucleic acid molecule, wherein the nucleic acid molecule contains the nucleic acid molecule of the recombinant type II collagen;

[0025] B2) an expression cassette, the expression cassette containing the nucleic acid molecule described in B1);

[0026] B3) a recombinant vector, the recombinant vector comprising the nucleic acid molecule described in B1) and / or the expression cassette described in B2);

[0027] B4) a recombinant microorganism, which contains the nucleic acid molecule described in B1), the expression cassette described in B2) and / or the recombinant vector described in B3);

[0028] B5) A recombinant cell, wherein the recombinant cell contains the nucleic acid molecule described in B1), the expression cassette described in B2) and / or the recombinant vector described in B3).

[0029] Further, the nucleic acid molecule includes: a nucleotide sequence as shown in SEQ ID NO.5 or a nucleotide sequence having at least 90% identity with SEQ ID NO.5, and / or a nucleotide sequence as shown in SEQ ID NO.7 or a nucleotide sequence having at least 90% identity with SEQ ID NO.7, and / or a nucleotide sequence as shown in SEQ ID NO.9 or a nucleotide sequence having at least 90% identity with SEQ ID NO.9.

[0030] Wherein, the nucleic acid molecule comprises: a nucleotide sequence as shown in SEQ ID NO.5 or a nucleotide sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identity with SEQ ID NO.5,

[0031] and / or, the nucleotide sequence as set forth in SEQ ID NO.7, or a nucleotide sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identity to SEQ ID NO.7,

[0032] and / or, the nucleotide sequence as shown in SEQ ID NO.9 or a nucleotide sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identity to SEQ ID NO.9.

[0033] In a preferred embodiment, the amino acids of the collagen protein comprise the amino acid sequence shown in SEQ ID NO.4, and the nucleotide sequence of the nucleic acid molecule encoding the collagen protein is shown in SEQ ID NO.5.

[0034] In a preferred embodiment, the amino acids of the collagen protein comprise the amino acid sequence shown in SEQ ID NO.6, and the nucleotide sequence of the nucleic acid molecule encoding the collagen protein is shown in SEQ ID NO.7.

[0035] In a preferred embodiment, the amino acids of the collagen protein comprise the amino acid sequence shown in SEQ ID NO.8, and the nucleotide sequence of the nucleic acid molecule encoding the collagen protein is shown in SEQ ID NO.9.

[0036] Optionally, the expression cassette refers to a DNA capable of expressing the above-mentioned protein in a host cell. The expression cassette may also include a single-stranded or double-stranded nucleic acid molecule of all regulatory sequences necessary for expressing the nucleic acid molecule of any of the above-mentioned proteins. The regulatory sequence can guide the coding sequence to express any of the above-mentioned proteins in a suitable host cell under its compatible conditions. The regulatory sequence includes, but is not limited to, a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal sequence and a transcription terminator. At a minimum, the regulatory sequence should include a promoter and termination signals for transcription and translation. In order to introduce specific restriction enzyme sites of the vector so as to connect the regulatory sequence to the coding region of the nucleic acid sequence encoding the protein, a regulatory sequence with a joint can be provided. The regulatory sequence can be a suitable promoter sequence, that is, a nucleic acid sequence that can be recognized by the host cell expressing the nucleic acid sequence. The promoter sequence contains a transcriptional regulatory sequence that mediates protein expression. The promoter can be any nucleic acid sequence that is transcriptionally active in the selected host cell, including mutant, truncated and hybrid promoters, and can be obtained from a gene encoding an extracellular or intracellular protein that is homologous or heterologous to the host cell. The regulatory sequence may also be a suitable transcription termination sequence, i.e., a sequence that can be recognized by the host cell to terminate transcription. The termination sequence may be operably linked to the 3' end of the nucleic acid sequence encoding the protein. Any terminator that can function in the selected host cell may be used in the present invention. The regulatory sequence may also be a suitable leader sequence, i.e., an untranslated region of mRNA that is important for translation of the host cell. The leader sequence may be operably linked to the 5' end of the nucleic acid sequence encoding the protein. Any leader sequence that can function in the selected host cell may be used in the present invention. The regulatory sequence may also be a signal peptide coding region that encodes an amino acid sequence attached to the amino terminus of the protein that can guide the encoded protein into the cell secretory pathway. Signal peptide coding regions that can guide the expressed protein into the secretory pathway of the host cell used may be used in the present invention. It may also be desirable to add regulatory sequences that can regulate protein expression according to the growth of the host cell. Examples of regulatory systems are those that can respond to chemical or physical stimuli (including in the presence of regulatory compounds) to open or close gene expression. Other examples of regulatory sequences are those that can amplify genes. In these cases, the nucleic acid sequence encoding the protein should be operably linked to the regulatory sequences.

[0037] Optionally, the vector may include a nucleic acid molecule encoding the above-mentioned protein, a promoter, and transcription and translation termination signals. When preparing a recombinant vector, the nucleic acid molecule encoding the above-mentioned protein may be located in the vector so as to be operably linked to an appropriate expression control sequence. The recombinant vector may be any vector (e.g., a plasmid or virus) that is convenient for recombinant DNA manipulation and expression of the nucleic acid sequence. The choice of the vector generally depends on the compatibility of the vector with the host cell into which it is to be introduced. The vector may be a linear or closed-loop plasmid. The vector may be an autonomously replicating vector (i.e., a complete structure present outside the chromosome that can be replicated independently of the chromosome), such as a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any mechanism that ensures self-replication. Alternatively, the vector is a vector that, when introduced into a host cell, will be integrated into the genome and replicated together with the chromosome into which it is integrated. In addition, a single vector or plasmid may be used, or two or more vectors or plasmids or transposons that generally contain all the DNA that will be introduced into the host cell genome may be included. The vector may contain one or more selection markers that are convenient for selecting transformed cells. A selectable marker is a gene whose product confers resistance to biocides or viruses, resistance to heavy metals, or confers prototrophy to an auxotroph, etc. Examples of bacterial selectable markers are the dal genes of Bacillus subtilis or Bacillus licheniformis, or resistance markers to antibiotics such as ampicillin, kanamycin, chloramphenicol, or tetracycline. The vector contains elements that enable the vector to be stably integrated into the host cell genome, or that ensure that the vector replicates autonomously in the cell independently of the cell genome. In the case of autonomous replication, the vector may also contain an origin of replication that enables the vector to replicate autonomously in the target host cell. The origin of replication may carry a mutation that makes it temperature-sensitive in the host cell (see, e.g., fEhrlich, 1978, Proceedings of the National Academy of Sciences of the United States of America 75:1433). One or more copies of a nucleic acid molecule encoding any of the above proteins of the present invention may be inserted into the host cell to increase the yield of the gene product. The number of copies of the nucleic acid molecule can be increased by inserting at least one additional copy of the nucleic acid molecule into the host cell genome, or inserting an amplifiable selection marker together with the nucleic acid molecule, and selecting cells containing amplified copies of the selectable marker gene and thus the additional copies of the nucleic acid molecule by culturing the cells in the presence of a suitable selection agent. The operations for connecting the above-mentioned elements to construct the recombinant expression vector of the present invention are well known to those skilled in the art (see, for example, Sambrook et al., Molecular Cloning Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989).

[0038] The term "operably linked" is defined herein as a configuration in which a regulatory sequence is appropriately positioned relative to the coding sequence of a DNA sequence such that the regulatory sequence directs the expression of a protein.

[0039] Optionally, the microorganism comprises a polynucleotide of the present invention operably linked to one or more control sequences that instruct the production of the collagen of the present invention. The construct comprising the polynucleotide is introduced into the microorganism so that the construct is maintained as a chromosomal integrant or as an autonomously replicating extrachromosomal vector. The term "microorganism" encompasses any parent cell progeny that is not identical to the parent cell due to mutations that occur during replication. The selection of microorganisms will depend to a large extent on the genes encoding collagen or polypeptides and their sources.

[0040] Optionally, the microorganism is selected from any one of Corynebacterium glutamicum, Bacillus subtilis, Escherichia coli and yeast. In an optional embodiment, the expression cassette of the nucleic acid molecule is located on a recombinant vector or is imported into a recombinant microorganism using a recombinant vector. It should be noted that, as known to those skilled in the art, the nucleic acid molecule can be selected to insert the genome of the starting strain, or can be present in a plasmid in a free state, as long as the expression of the nucleic acid molecule or the synthesis of collagen can be achieved.

[0041] Furthermore, the recombinant microorganism is selected from one or more of Streptococcus, Bacillus, Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris; preferably, Pichia pastoris.

[0042] In a preferred embodiment, the Pichia pastoris is GS115. Those skilled in the art will appreciate that conventional fermentation strains or any known industrial strains can be used as starting strains, as long as they can achieve the expression of the recombinant type II collagen described in the present application, and the specific strains are not limited in detail herein.

[0043] On the other hand, the present application also provides a method for preparing recombinant type II collagen, the method comprising: constructing a recombinant microorganism that expresses the recombinant type II collagen, and culturing the recombinant microorganism.

[0044] Preferably, the amino acids of the collagen contain the amino acid sequence shown in SEQ ID NO.4 or an amino acid sequence that is at least 95% identical to SEQID NO.4, and / or the amino acid sequence shown in SEQ ID NO.6 or an amino acid sequence that is at least 95% identical to SEQID NO.6, and / or the amino acid sequence shown in SEQ ID NO.8 or an amino acid sequence that is at least 95% identical to SEQID NO.8.

[0045] More preferably, the nucleic acid molecule encoding the collagen includes: a nucleotide sequence as shown in SEQ ID NO.5 or a nucleotide sequence having at least 90% identity with SEQ ID NO.5, and / or a nucleotide sequence as shown in SEQ ID NO.7 or a nucleotide sequence having at least 90% identity with SEQ ID NO.7, and / or a nucleotide sequence as shown in SEQ ID NO.9 or a nucleotide sequence having at least 90% identity with SEQ ID NO.9.

[0046] Preferably, the recombinant microorganism is selected from one or more of Streptococcus, Bacillus, Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris; more preferably, Pichia pastoris.

[0047] In one embodiment, a method for preparing recombinant type II collagen comprises: step 1, constructing a recombinant microorganism that expresses the recombinant type II collagen;

[0048] Step 2: Cultivate the recombinant microbial cells at 25°C-37°C for 20-24h to obtain a seed solution, then inoculate the cells into 10-50mL of a culture medium at an inoculum size of 1-5% (v / v), add 1-5% methanol for induction, terminate the fermentation after induction at 25°C-37°C for 24-48h, and purify the supernatant after centrifugation of the fermentation solution to obtain collagen.

[0049] The above purification process can be carried out using general methods.

[0050] On the other hand, the present application also provides a composition, which contains the recombinant type II collagen or the recombinant type II collagen prepared by the preparation method.

[0051] In the present application, it has been verified through experiments that the recombinant type II collagen can self-assemble to form a triple helical structure, has biological activity, and is non-cytotoxic.

[0052] On the other hand, the present application also provides the use of the recombinant type II collagen or the biomaterial or the recombinant type II collagen prepared by the preparation method or the composition in promoting coagulation or preparing a coagulation-promoting product.

[0053] Compared with the commercial rat tail collagen I currently sold on the market, the recombinant type II collagen described in the present application has a better coagulation promoting function, that is, it promotes the aggregation of blood cells. When treated with a concentration of 1-2 mg / mL for 30 minutes, the blood coagulation rate can reach up to 37.9%.

[0054] On the other hand, the present application also provides the use of the recombinant type II collagen or the biomaterial or the recombinant type II collagen prepared by the preparation method or the composition in promoting tissue regeneration or preparing products promoting tissue regeneration.

[0055] Preferably, the promotion of tissue regeneration is achieved by promoting cell proliferation and / or differentiation.

[0056] On the other hand, the present application also provides the use of the recombinant type II collagen or the biomaterial or the recombinant type II collagen prepared by the preparation method or the composition in promoting cell proliferation and / or differentiation or preparing products that promote cell proliferation and / or differentiation.

[0057] On the other hand, the present application also provides the use of the recombinant type II collagen or the biomaterial or the recombinant type II collagen prepared by the preparation method or the composition in increasing the mRNA level of VEGF or preparing a product for increasing the mRNA level of VEGF.

[0058] The present invention has the following beneficial effects:

[0059] The present invention intercepts and screens type II collagen fragments, obtains polypeptide sequences with self-assembly potential based on the screened fragments, and provides new functional fragments with biological activity for collagen.

[0060] The present invention realizes recombinant expression of the designed polypeptide (recombinant type II collagen) in Pichia pastoris, and verifies that the recombinantly expressed protein can self-assemble to form a triple helical structure and has no biological toxicity.

[0061] The present invention also provides the use of the prepared recombinant type II collagen in promoting coagulation and fibroblast differentiation, showing coagulation ability superior to that of the commercial coagulant rat tail collagen I, and has great value and potential in the fields of cosmetics, medicine and tissue engineering. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0063] Figure 1 This is the SDS-PAGE analysis of the recombinant expression of type II collagen;

[0064] Figure 2 220-222 nm CD spectrum analysis diagram of type II collagen samples 205, 208, and 209;

[0065] Figure 3 Figure 2 is a graph of cytotoxicity and in vitro blood coagulation test, where A represents the addition of 2 mg / mL Ca 2+ , COL1 and samples 205, 208 and 209 were used to culture L929 cells, and the cell proliferation rate statistics and in vitro coagulation rate diagrams obtained by cell counting using the CCK-8 kit, and B is a schematic diagram of an in vitro coagulation test based on mouse blood cells;

[0066] Figure 4 Statistical graph of VEGF mRNA expression level. DETAILED DESCRIPTION

[0067] Identity: refers to the degree of similarity between the nucleotide sequences of two nucleic acid molecules or between the amino acid sequences of two protein molecules in molecular evolution research.

[0068] Recombination: Broadly speaking, any gene exchange process that causes genotype changes is called recombination.

[0069] Expression cassette: An expression cassette is a group of DNA sequences consisting of a promoter, a target gene, and a reporter gene, which can be expressed in specific tissues and easily detected.

[0070] Recombinant vector: A recombinant vector is a vector that transfers the target gene into the basic skeleton of a cloning vector, thereby enabling the target gene to be expressed.

[0071] Recombinant microorganism: A fungal cell strain that uses genetic engineering methods to efficiently express exogenous genes.

[0072] Recombinant cell: The term "recombinant cell" means any cell type that is susceptible to transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "recombinant cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

[0073] In order to more clearly explain the overall concept of the present application, the following is described in detail in conjunction with the accompanying drawings of the specification by way of embodiment. In the following description, a large number of specific details are provided to provide a more thorough understanding of the present invention. However, it is obvious to those skilled in the art that the present invention can be implemented without one or more of these details. In other examples, in order to avoid confusion with the present invention, some technical features well known in the art are not described.

[0074] Unless otherwise specified, in the following embodiments, reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased from the market. If no specific conditions are indicated in the examples, conventional conditions or conditions recommended by the manufacturer are followed.

[0075] The plasmids, endonucleases, PCR enzymes, column-based DNA extraction kits, and DNA gel recovery kits used in the following examples are commercial products, and the specific operations are performed in accordance with the kit instructions.

[0076] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in the field of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields, and can be specifically carried out according to Molecular Cloning: A Laboratory Manual (Fourth Edition).

[0077] Example 1: Extraction of Type II Collagen Sequence

[0078] In this example, three COL2 variants with different characteristics (sample names are 205, 208, and 209) were selected from full-length human type II collagen (COL2) (Gene Bank No. NG_008072.1) to explore the factors that may affect the assembly and function of collagen (COL). The amino acid sequences and characteristic data of the variants are shown in Table 1.

[0079] Table 1 Type II collagen sequence

[0080]

[0081]

[0082] Example 2 Recombinant expression of type II collagen in Pichia pastoris

[0083] In this example, the three COL2 obtained in Example 1 were transferred into Pichia pastoris for recombinant expression, as follows:

[0084] (1) Recombinant expression of type II collagen in Pichia pastoris

[0085] The COL2 gene in Example 1 was optimized and synthesized by Genscript (Nanjing, China), and the specific sequence is shown in Table 2.

[0086] The gene was cloned into pPICk9k, and the plasmid was linearized through the SalI site and integrated into the Pichia pastoris GS115 genome for transformation. The single colony with the correct colony PCR (the verification primer is shown in Table 3 below) was inoculated into YPD medium as a seed culture, cultured in a shaker at 30°C and 220rpm for 24h, and then inoculated into BMMY medium, 1% methanol was added to induce protein expression, and cultured in a shaker at 30°C and 220rpm for 48h, of which 1% methanol was supplemented once every 24h, and the fermentation supernatant was collected.

[0087] Table 2 COL2 gene sequence

[0088]

[0089]

[0090]

[0091]

[0092]

[0093] Table 3

[0094]

[0095] (2) Extraction and purification of type II collagen from Pichia pastoris

[0096] The fermentation broth obtained in step (1) was centrifuged to collect the supernatant, and the protein was purified by molecular chromatography using a G-25 desalting column (GE Healthcare, New York, USA), and eluted using PBS (pH 7.5) to obtain a high-purity type II collagen variant sample.

[0097] Example 3 Identification and structural characterization of type II collagen

[0098] The high-purity type II collagen sample obtained by Pichia yeast fermentation in Example 2 was subjected to SDS-PAGE analysis. Figure 1 As shown, the recombinant type II collagen samples 205, 208, and 209 expressed in Pichia pastoris as a host were secreted into the extracellular space in a soluble form, and were verified by SDS-PAGE to show obvious bands. The protein was confirmed to be the target protein by Maldi-Tof.

[0099] The high-purity type II collagen samples prepared in Example 2 were subjected to circular dichroism (CD) spectroscopy detection:

[0100] 0.2 mg / mL sample solutions were prepared in PBS (pH 7.5) and incubated at 4°C for 12 h before CD measurement. CD spectra were recorded at 4°C using a Photophysics Chirascan instrument equipped with a Peltier temperature controller (Model 110-OS; Hellma, Shanghai, China) and a quartz cuvette with an optical path length of 1 mm (Model 110-OS; Hellma). The scanning wavelength range was 190-250 nm.

[0101] CD was performed on the expressed proteins to confirm triple helix formation. Samples 205, 208, and 209 purified from Pichia pastoris showed positive peaks at 220-222 nm (e.g. Figure 2 It is shown in Figure 3), demonstrating that it can self-assemble to form a triple helical structure.

[0102] Example 4 Cytotoxicity Detection of Type II Collagen

[0103] In this example, the high-purity type II collagen sample obtained by fermentation of Pichia pastoris in Example 2 and commercial rat collagen I (Merck, Shanghai, China) were prepared as sponges for culturing L929 fibroblasts (ATCC, China) to evaluate their toxicity.

[0104] Specifically, L929 cells were cultured for 48 h and 10 4 The cell density of L929 cells was transferred to a new culture plate. The cells were allowed to adhere for 12 h, and then 10 mL of culture medium containing 2 mg / mL sample sponge was added (the culture medium contained 10% (v / v) fetal bovine serum (Ausbian, Australia) and 1% (v / v) penicillin-streptomycin). The cells were cultured for 48 h, centrifuged, the solution was discarded, and the cell number was measured using a CCK-8 kit (Sangon). The proliferation rate of L929 cells was evaluated by using the following cell counting formula: Proliferation rate (%) = (total number of cells after culture) / (total cells before culture).

[0105] The results are as follows Figure 3 As shown, when supplemented with 2 mg / mL of the selected samples, the cell proliferation rates ranged from 177% to 192%, indicating that all tested samples were non-toxic.

[0106] Example 5 Application of Type II Collagen in Promoting Blood Coagulation

[0107] In this example, the high-purity type II collagen samples 205, 208, and 209 prepared in Example 2 were used for in vitro coagulation tests.

[0108] Specifically, samples 205, 208, 209 and commercial rat tail collagen I (Merck, Shanghai, China) were dissolved in PBS solution at a concentration of 1 mg / mL and supplemented to 2 mg / mL with platelet-poor plasma during the culture period. The platelet-poor plasma was centrifuged at 2000×g for 10 min to extract the supernatant. After culture at 25°C for 30 min and centrifugation at 2000×g for 20 min, the supernatant was collected and the cells were counted using a CCK-8 kit. The coagulation rate was calculated according to the following formula: blood coagulation rate (%) = 1-(cells from the supernatant after culture) / (total cells before culture).

[0109] The results are as follows Figure 3As shown, after treatment at 25°C for 30 minutes, samples 205 and 209 induced 29.9% and 37.9% blood cell coagulation, respectively. In contrast, commercial rat tail collagen I only induced 20.7% cell coagulation, demonstrating that samples 205 and 209 have better procoagulant function.

[0110] Example 6 Application of Type II Collagen in Promoting Cell Regeneration

[0111] In this example, the ability of type II collagen prepared in Example 2 to promote fibroblast differentiation was tested:

[0112] VEGF mRNA levels can be used to assess fibroblast differentiation.

[0113] The cell experiment was carried out as follows. 1×10 4 Mouse embryonic fibroblasts (L929 cells) were cultured in a 37°C incubator with Gibco MEM (Invitrogen, Shanghai, China), 10% (v / v) fetal bovine serum and 1% (v / v) penicillin-streptomycin (supplemented with 2 mg / mL liquid Example 2 collagen samples 205, 208, 209 or collagen I) for 72 h.

[0114] The ability of type II collagen samples to promote fibroblast differentiation was tested as follows, with commercial rat tail collagen I (Merck, Shanghai, China) as a positive control. Gene expression levels were measured by real-time quantitative PCR (RT-qPCR). Total RNA was extracted from L929 cells using TRIzol reagent (Invitrogen). RNA was reverse transcribed into cDNA using the One Step RT-qPCR kit (Sangon) and the QuantStudio 3RT-qPCR instrument (Thermo Fisher, Shanghai, China). The primers are shown in Table 4, and the gene expression levels were referenced to GAPDH. The results are shown in Table 4. Figure 4 As shown, sample 205 exhibited the ability to upregulate VEGF relative to collagen I, given that the mRNA levels of the three collagen types II were all higher than those using BSA. These results indicate that the designed type II collagen can promote cell differentiation.

[0115] Table 4 RT-qPCR primers

[0116]

[0117] The above is only an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Claims

1. Recombinant type II collagen, characterized in that: The amino acid sequence of the recombinant type II collagen includes any one or more of the following: A1) the amino acid sequence of the recombinant type II collagen comprises one or more repeating units of amino acid residues, wherein the repeating unit comprises the sequence shown in SEQ ID NO.2, and the number of repeats is n, 1≤n≤10; A2) The amino acid sequence of the recombinant type II collagen comprises one or more repeating units of amino acid residues, wherein the repeating unit comprises the sequence shown in SEQ ID NO.3, and the number of repeats is n, 1≤n≤15.

2. The recombinant type II collagen according to claim 1, characterized in that: The amino acids of the collagen include the amino acid sequence shown in SEQ ID NO.6 or an amino acid sequence having at least 95% identity with SEQ ID NO.6, and / or the amino acid sequence shown in SEQ ID NO.8 or an amino acid sequence having at least 95% identity with SEQ ID NO.

8.

3. Biomaterial, characterized in that The biological material includes any one of the following B1)-B5): B1) a nucleic acid molecule comprising a nucleic acid molecule encoding the recombinant type II collagen according to claim 1 or 2; B2) an expression cassette, the expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector, the recombinant vector comprising the nucleic acid molecule described in B1) and / or the expression cassette described in B2); B4) a recombinant microorganism, which contains the nucleic acid molecule described in B1), the expression cassette described in B2) and / or the recombinant vector described in B3); B5) A recombinant cell, wherein the recombinant cell contains the nucleic acid molecule described in B1), the expression cassette described in B2) and / or the recombinant vector described in B3).

4. The biomaterial according to claim 3, characterized in that The nucleic acid molecule comprises: a nucleotide sequence as shown in SEQ ID NO.7 or a nucleotide sequence having at least 90% identity with SEQ ID NO.7, and / or a nucleotide sequence as shown in SEQ ID NO.9 or a nucleotide sequence having at least 90% identity with SEQ ID NO.

9.

5. The biomaterial according to claim 3 or 4, characterized in that: The recombinant microorganism is selected from one or more of Streptococcus, Bacillus, Escherichia coli, Saccharomyces cerevisiae and Pichia pastoris.

6. A method for preparing recombinant type II collagen, characterized in that: The method comprises: constructing a recombinant microorganism expressing the recombinant type II collagen as claimed in claim 1 or 2, and culturing the recombinant microorganism.

7. A composition, characterized in that It contains the recombinant type II collagen according to claim 1 or 2.

8. Use of the recombinant type II collagen as claimed in claim 1 or 2, or the biomaterial as claimed in any one of claims 3 to 5, or the composition as claimed in claim 7 in the preparation of products promoting blood coagulation and / or promoting tissue regeneration.

9. Use of the recombinant type II collagen as claimed in claim 1 or 2, or the biomaterial as claimed in any one of claims 3 to 5, or the composition as claimed in claim 7 in the preparation of products promoting cell proliferation and / or differentiation.

10. Use of the recombinant type II collagen as claimed in claim 1 or 2, or the biomaterial as claimed in any one of claims 3 to 5, or the composition as claimed in claim 7 in daily chemicals and / or medical devices and / or engineered tissue materials.

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