Recombinant collagen, preparation method and its use

Abstract

The present invention relates to recombinant collagen, preparation methods and uses thereof, in particular to recombinantly expressed full-length collagen α1 chain, preparation methods and uses thereof, which belong to the technical field of collagen expression. [Solution] The mutant of the α1 chain of human type I collagen (α1(I) chain) (designated as α1(I)M1) and the mutant of the α1 chain of human type II collagen (α1(II) chain) (designated as α1(II)M6) constructed in the present invention, when recombinantly expressed in Pichia pastoris, removes the main degradation product (main degradation band) which is generated in essentially the same proportion as the target product (target band) of the full-length α1 chain, which is generated when the natural full-length α1(I) chain and α1(II) chain are recombinantly expressed, improving the yield of the target product, and has physical and chemical properties and biological activity similar to those of the natural full-length α1(I) chain collagen and α1(II) chain collagen recombinantly expressed in Pichia pastoris, making them worthy of use in the field of biomedical materials.

Description

Technical Field

[0001] The present invention relates to recombinant collagen, a preparation method and its use, and particularly to an α1 chain of a full-length collagen recombinantly expressed, a preparation method and its use, belonging to the technical field of collagen expression.

Background Art

[0002] Types I and II collagen are typical fibroblast collagens in the human body, composed of three α-peptide chains, and each α-peptide chain contains three parts: an amino-terminal peptide region, a characteristic (G-X-Y)n triplet repeat sequence region, and a carboxyl-terminal peptide region.

[0003] Type I collagen is composed of two α1 chains and one α2 chain, and is the most abundant type of collagen in the human body, contained in muscle, skin, arterial wall, and fibrocartilage. Type II collagen is composed of three α1 chains, mainly distributed in cartilage tissue, vitreous body, and cornea, accounting for more than 90% of the total amount of adult cartilage matrix collagen, and is an essential component for the formation of cartilage and bone, bone growth, and maintenance of mature cartilage.

[0004] Collagen, an important natural biomacromolecule, has unique functional characteristics such as good biocompatibility, bioactivity, and degradability, and is widely used in many fields such as the chemical industry, pharmaceuticals, food, and cosmetics. It is particularly suitable for the preparation of various biomedical devices, is the most ideal source of biomaterials, and has broad application potential.

[0005] Commercially available collagen is mainly an extract of collagen obtained by treating animal tissues with acid, alkali, and enzymatic hydrolysis methods. It is severely degraded during processing, loses its biological activity, and the extracted collagen peptides have different lengths, heterogeneous properties, and unstable quality, posing a safety risk of virus infections such as mad cow disease and foot-and-mouth disease. At the same time, the amino acid sequences of animal-derived and human-derived collagens are significantly different, and they are heterologous proteins that may cause immune rejection and allergic symptoms.

[0006] If recombinant collagen is generated by genetic engineering techniques, these defects can be effectively avoided. In existing recombinant collagen expression methods, expression systems such as mammalian cell expression systems, insect cell (baculovirus) expression systems, and transgenic animals and plants are mainly used at the scientific research level because of their high cost, low yield, and long cycle. In large-scale industrial production, human collagen is mainly expressed by prokaryotic (E. coli) expression systems and Pichia pastoris expression systems. In E. coli, there is no post-translational modification of proteins, large-scale expression is intracellular expression, cell lysis is required, and the removal of host proteins and naturally occurring (cell wall components) endotoxins and peptidoglycans, which are impurities generated in large quantities, requires complex purification. However, for Pichia pastoris, human collagen is ultimately an exogenous protein. When expressed, it occupies more intracellular resources (the methanol metabolism pathway it depends on can express up to 30% of the cell-soluble proteins), and the cell has to make adjustments to the exogenous protein. Typically, recombinant proteins are severely degraded, but the α1 chains of human type I and II collagens are long peptide chains exceeding 1000 amino acids and are easily degraded.

[0007] The α1-chain sequences of mature human type I and type II collagen contain three parts: an amino telopeptide, a triple helix region, and a carboxyl telopeptide. The full length of the α1-chain of human type I collagen (hereinafter referred to as α1(I) in this specification) is 1057 amino acids (AA), and the full length of the α1-chain of human type II collagen (hereinafter referred to as α1(II) in this specification) is 1060 AA. There are numerous studies and patents regarding the expression of the human α1(I) chain using Pichia pastoris, but there are few studies and patents regarding the expression of the human α1(II) chain. Among the existing research results on Pichia pastoris expressing the full-length α1(I) chain and the full-length α1(II) chain, most studies expressed only a part of the α1(I) chain sequence rather than the mature full-length α1(I) chain. Some disclosed results expressed the full-length α1(I) chain, but they all generated major degradation products that basically accounted for the same proportion as the target product during expression. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), they were shown as major degradation bands (major degradation products) that basically accounted for the same proportion as the target band (target product) of the full-length α1 chain. Such degradation not only reduces the yield of the full-length α1 chain generated by expression, but also, because its characteristics are close to those related to the full-length α1 chain, to obtain a high-purity single full-length α1 chain product, two-step double affinity purification is required, but due to the increased complexity of the purification process, the purification cost increases. Therefore, keeping the full-length peptide chain intact, reducing degradation, and maintaining the biological activity of collagen without change are important issues for the production of recombinant collagen using Pichia pastoris.

Summary of the Invention

Problems to be Solved by the Invention

[0008] The object of the present invention is to overcome some technical problems existing in the prior art and provide the α1 chain of full-length collagen recombinantly expressed in Pichia pastoris, a preparation method thereof, and its use. The variant of the α1 chain of recombinant human type I collagen (denoted as α1(I)M1) and the variant of the α1 chain of recombinant human type II collagen (denoted as α1(II)M6) in the present invention, compared with the natural full-length α1(I) chain and α1(II) chain, when expressed in Pichia pastoris, the main degradation bands (main degradation products) that basically occupy the same ratio as the target band (target product) of the full-length α1 chain are removed, the yield of the target product is improved, and they have physical and chemical properties similar to those of the natural full-length α1(I) chain collagen and α1(II) chain collagen expressed in Pichia pastoris, as well as the same biological activity as commercially available human collagen, and have value for use in the field of biomedical materials.

Means for Solving the Problems

[0009] To achieve the above object of the present invention, the present invention adopts the following technical solutions. The present invention provides an α1 chain of recombinant collagen which is α1(I)M1 or α1(II)M6, wherein the above α1(I)M1 is obtained by amino acid mutation of the natural full-length amino acid sequence of the α1 chain of human type I collagen, and the above α1(II)M6 is obtained by amino acid mutation of the natural full-length amino acid sequence of the α1 chain of human type II collagen.

[0010] Preferably, the number of amino acid mutation sites of the above α1(I)M1 is 4, and the number of amino acid mutation sites of the above α1(II)M6 is 9. Preferably, the α1 chain of the above human type I collagen is shown in SEQ ID NO:1, and the amino acid mutation sites are M at position 106, R at position 109, M at position 190, and R at position 193, and specifically all are changed to P.

[0011] The α1 chain of the above-mentioned human type II collagen is shown in SEQ ID NO: 4, and the mutation sites of the above-mentioned amino acids are V at the 67th position, M at the 68th position, M at the 72nd position, M at the 75th position, R at the 78th position, M at the 108th position, R at the 111th position, M at the 162nd position, and R at the 165th position, and specifically all are changed to P.

[0012] Furthermore, the amino acid sequence of the above-mentioned α1(I)M1 is shown in SEQ ID NO: 2, and the amino acid sequence of the above-mentioned α1(II)M6 is shown in SEQ ID NO: 5.

[0013] In the present invention, when there is any change in the type of change of the corresponding amino acid at the mutation site of the amino acid sequence, that is, when the mutation sites are the same but the types of amino acids to be changed are different, the same technical effects as those of this patent can be obtained. By mutating one or several sites, the same technical effects as those of this patent can also be obtained.

[0014] The present invention further provides nucleotides encoding the α1 chain of the above-mentioned recombinant collagen, wherein the nucleotide sequence of the α1 chain of the above-mentioned coded recombinant collagen contains a nucleotide sequence encoding α1(I)M1 or α1(II)M6.

[0015] Furthermore, the nucleotide sequence encoding the above-mentioned α1(I)M1 is shown in SEQ ID NO: 3, and the nucleotide sequence encoding α1(II)M6 is shown in SEQ ID NO: 6.

[0016] The present invention further provides a recombinant expression vector containing the nucleotides encoding the α1 chain of the above-mentioned recombinant collagen.

[0017] The present invention further provides an engineering bacterium constructed with the above-mentioned recombinant expression vector, which contains the above-mentioned recombinant expression vector or expresses the α1 chain of the above-mentioned recombinant collagen.

[0018] The host strain of the above-mentioned engineered strain is preferably Pichia pastoris, and the above-mentioned engineered strain is An international deposit based on the Budapest Treaty has been made with the China Center for Type Culture Collection, The deposit date is March 11, 2021, the deposit number is CGMCC NO.21891 or CGMCC NO.21892, the taxonomic name is Pichia pastoris, the depositary institution is the General Microbiology Center of the China Microbial Culture Collection Center, and the address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Here, the engineered strain with the deposit number CGMCC NO.21891 expresses the α1 chain of recombinant α1(I)M1 collagen, and the engineered strain with the deposit number CGMCC NO.21892 expresses the α1 chain of recombinant α1(II)M6 collagen.

[0019] It should be noted that the host strain of the present invention is not limited to Pichia pastoris, and theoretically the same technical effects as those of the present invention can be obtained as long as it can be secreted and expressed in Pichia pastoris or other types of yeast according to the method of the present invention.

[0020] The present invention further provides the use of the above-mentioned recombinant expression vector or the above-mentioned engineered strain for expressing the α1 chain of the above-mentioned recombinant collagen.

[0021] The present invention further provides a method for preparing the α1 chain of the above-mentioned recombinant collagen, and the corresponding preparation method includes the following steps.

[0022] (1) Synthesizing a nucleotide sequence encoding the α1 chain of recombinant collagen By changing the corresponding amino acids at the amino acid sequence sites of the α1 chain sequence of natural collagen, the α1 chain of type I collagen is changed by 4 amino acids to obtain α1(I)M1, and the α1 chain of type II collagen is changed by 9 amino acids to obtain α1(II)M6. An affinity purification tag is added to the amino terminus and carboxy terminus of the sequence, and the DNA sequences encoding α1(I)M1 and α1(II)M6 are synthesized to include a bispecific affinity purification label, which contributes to the detection of immunological antibodies based on the two tag sequences. α1(I)M1 and α1(II)M6 have removed the main degradation bands (main degradation products) that basically occupy the same ratio as the target bands (target products) of the full-length α1 chain generated when the full-length α1(I) chain and the full-length α1(II) chain are expressed in Pichia pastoris, respectively. Compared with the original natural sequence, α1(I)M1 and α1(II)M6 have mutations in the amino acids located in the characteristic (G-X-Y)n triple repeat region. However, they are all amino acids located at X and Y, and the structural characteristics of the amino acid sequence of the collagen (G-X-Y)n triple repeat have not been changed, and they still maintain the physical and chemical properties and biological activities similar to the original collagen.

[0023] (2) Constructing a recombinant expression vector Ligate the synthesized DNA into the expression vector pPIC9K to construct two recombinant expression vectors, pPIC9K-COL1A1M1 expressing recombinant α1(I)M1 collagen and pPIC9K-COL2A1M6 expressing recombinant α1(II)M6 collagen, respectively.

[0024] (3) Constructing a recombinant engineering strain, inducing expression, and screening the strain Linearize the recombinant expression vector with Sac I, electrotransform it into competent cells of Pichia pastoris, transfer it to an MD plate for preliminary screening, screen it with YPD plates containing different concentrations of G418, prick the colonies and inoculate them into BMGY medium, induce expression in BMMY medium, and screen for highly expressing engineering strains. The above-screened highly expressing engineering bacteria are Pichia pastoris, and the deposit numbers are CGMCC NO.21891 and CGMCC NO.21892, respectively.

[0025] (4) Fermenting and culturing at high density The highly expressing engineering bacteria identified by protein expression are fermented and cultured at high density using a fermenter.

[0026] (5) Purifying the protein The fermentation supernatant was purified by primary cation exchange chromatography, and high-purity α1(I)M1 and α1(II)M6 collagens were obtained by lyophilization.

[0027] The α1(I)M1 and α1(II)M6 recombinant collagens expressed in Pichia pastoris obtained by the present invention were analyzed by characterizing the properties of the protein and in vitro experiments. The proteins obtained in the present invention conform to the structural characteristics of the recombinant collagen, have cell adhesion activity, and are basically consistent with commercially available human collagen. More importantly, the structural characteristics and cell adhesion activities of the two mutant proteins and the collagen before mutation are similar or identical.

[0028] The present invention further provides a composition comprising the α1 chain of the above recombinant collagen or the α1 chain of the collagen prepared by the above method.

[0029] The present invention further provides a product comprising the α1 chain of the above recombinant collagen, the α1 chain of the collagen prepared by the above method, or the above composition. The above products include, but are not limited to, pharmaceuticals, pharmaceutical compositions, medical devices, biomaterials, tissue engineering products, cosmetics, or health care products.

[0030] Furthermore, the above products include materials that adhere to cells, provide space for support, growth and migration, or materials that are channels for transporting nutrients and metabolites.

[0031] Furthermore, the above product is a collagen hydrogel.

[0032] The present invention further provides the use for preparing finished products such as the α1 chain of the above recombinant collagen, nucleotides, recombinant expression vectors, engineering bacteria, and compositions, including but not limited to the use for pharmaceuticals, medical devices, biomaterials, tissue engineering products, cosmetics or health care products.

[0033] The present invention further provides the use of the α1 chain of the recombinant collagen, nucleotide, recombinant expression vector, engineering bacterium or composition for preparing a product for promoting wound repair or tissue regeneration. Further, the above product is a collagen hydrogel.

Advantages of the Invention

[0034] (1) The α1 chain variant of collagen in the present invention has a very small proportion of amino acid sites to be changed in the original natural sequence (the proportion of mutated amino acids is less than 1%, and the homology of amino acid sequences before and after mutation is 99% or more). While obtaining the completely recombinant α1 chain of collagen, without changing the properties (physical and chemical properties, biological activities) of the original protein itself, when prepared into related products, it has the same properties, biological activities, etc. as the recombinant protein of the natural sequence. α1(I) M1 has similar physical, chemical properties and biological activities compared to α1(I), and α1(II) M6 has similar physical, chemical properties and biological activities compared to α1(II), and they have the value to be used in the field of biomedical materials. By conducting cell adhesion experiments on α1(I) M1, α1(II) M6, α1(I), and α1(II), it is found that there is no significant difference in cell adhesion activity between α1(I) and α1(I) M1, and between α1(II) M6 and α1(II), and the cell adhesion activities of α1(I) M1 and α1(II) M6 are basically the same as those of commercially available human collagen. Collagen hydrogels were prepared using α1(I) M1, α1(II) M6, α1(I), and α1(II) collagen expressed in Pichia pastoris, and the liquid mechanical properties of the hydrogels were detected. It is found that there is no significant difference in viscosity, elastic modulus, and swelling degree between the hydrogels prepared from α1(I) and α1(I) M1, and between the hydrogels prepared from α1(II) M6 and α1(II). Freeze-dried collagen hydrogels were scanned using a scanning electron microscope, and all of them have a porous network structure with a pore size range of 100 - 200 μm, indicating that they may be used in the field of biomedical materials. Four collagen hydrogels and NIH / 3T3 cells were co-cultured in vitro. When calcein AM was added, live cells that adhered to and grew on the hydrogels and emitted green fluorescence were detected. In addition to MTT detection, blue-violet crystals formed by the adhesion, migration, and growth of live cells within the hydrogels could be observed.

[0035] (2) In the present invention, SDS-PAGE electrophoresis and Western Blot were used to identify the expressed proteins. As a result, for the full-length α1(I) chain and α1(II) chain, when expressed in Pichia pastoris, the main degradation bands (main degradation products) that basically occupy the same ratio as the target band (target product) of the full-length α1 chain were removed for α1(I)M1 and α1(II)M6, and the yield of the target product was improved. Then, a high-density fermentation experiment was carried out in a fermenter, and the fermentation product was detected by SDS-PAGE electrophoresis. α1(I)M1 and α1(II)M6 can maintain the integrity of the target band even under high-density fermentation conditions, and the main degradation bands are still not generated. Recombinant human α1(I) and α1(II) produced by fermentation under the same high-density fermentation conditions generate obvious main degradation bands.

[0036] Furthermore, the recombinant collagen of the present invention can purify the fermentation supernatant by performing cation exchange chromatography in one step, and obtain a freeze-dried sponge of high-purity α1(I)M1 and α1(II)M6 collagen by freeze-drying. In SDS-PAGE electrophoresis detection, there are no main degradation bands, and only a single band of the target product is detected. Therefore, a high-purity target product can be obtained, and the purification cost can also be reduced.

[0037] For α1(I) and α1(II), when performing cation exchange chromatography in one step, only a purified protein in which the target band (target product, full-length α1 chain) and the main degradation band (main degradation product) are mixed can be obtained. Therefore, a second-step affinity chromatography is required to obtain high-purity α1(I) and α1(II) collagen.

Brief Description of the Drawings

[0038]

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Embodiments for Carrying Out the Invention

[0039] To enable those skilled in the art to better understand the technical solutions of the present invention, the preferred embodiments of the present invention will be described in detail below. However, the following embodiments do not limit the scope of the present invention. In the embodiments of the present invention, conventional molecular biology experimental methods are used without particularly explaining them. Processes such as PCR, enzyme digestion, ligation, and codon optimization included in the embodiments can all be understood and easily implemented by those skilled in the art based on the product specifications or basic knowledge in the field, so they will not be described in detail.

[0040] Example 1. Design and Synthesis of Amino Acid Sequences The amino acid sequence of the α1 chain of human type I collagen (denoted as α1(I)) refers to the portion from 162 to 1218 (PRO_0000005720) in the Uniprot database P02452-1 (https: / / www.uniprot.org / uniprot / P02452) sequence, and it is the amino acid sequence of the α1 chain of mature human type I collagen. It does not contain the portions that are processed and shed in the α1(I) precursor protein such as the signal peptide, C-terminal propeptide, and N-terminal propeptide, and its sequence is shown in SEQ ID NO:1.

[0041] SEQ ID NO:1:

[0042] As a result of long-term experimental research, a mutant of the α1 chain of recombinant human type I collagen designated as α1(I)M1 was obtained. Compared with α1(I), α1(I)M1 has four amino acid changes in the amino acid sequence, and these four amino acids are mutated to proline (abbreviated as Pro, P). M at position 106, R at position 109, M at position 190, and R at position 193 in the amino acid sequence shown in SEQ ID NO:1 are changed to P, and the remaining amino acid sequence remains unchanged. The homology between α1(I)M1 and α1(I) is 99.6%.

[0043] The full length of the altered amino acid sequence (α1(I)M1) is 1057 AA, and the sequence is shown in SEQ ID NO:2.

[0044] SEQ ID NO:2:

[0045] The differences between α1(I)M1 and the α1(I) amino acid sequence are shown by the gray background and bold amino acids in Figure 1.

[0046] The gene encoding α1(1)M1 shown in SEQ ID NO:2 (designated COL1A1M1) DNA sequence is shown in SEQ ID NO:3.

[0047] SEQ ID NO:3:

[0048] The amino acid sequence of the α1 chain of human type II collagen (denoted as α1(II)) refers to the portion from 182 to 1241 (PRO_0000005730) in the Uniprot database P02458 (https: / / www.uniprot.org / uniprot / P02458) sequence, and it is the amino acid sequence of the α1 chain of mature human type II collagen. It does not contain the processed and shed portions in the α1(II) precursor protein such as the signal peptide, C-terminal propeptide, and N-terminal propeptide, and its sequence is shown in SEQ ID NO:4.

[0049] SEQ ID NO:4:

[0050] As a result of long-term experimental research, a mutant of the α1 chain of recombinant human type II collagen designated as α1(II)M6 was obtained. Compared with α1(II), α1(II)M6 has nine amino acid changes in its amino acid sequence, and these nine amino acids are mutated to proline (abbreviated as Pro, P). V at position 67, M at position 68, M at position 72, M at position 75, R at position 78, M at position 108, R at position 111, M at position 162, and R at position 165 in the amino acid sequence shown in SEQ ID NO:4 are changed to P, and the remaining amino acid sequence remains unchanged. The homology between α1(II)M6 and α1(II) is 99.2%.

[0051] The full length of the modified sequence (α1(II)M6) is 1060 AA, and the sequence is shown in SEQ ID NO:5.

[0052] SEQ ID NO:5:

[0053] The differences between α1(II)M6 and the α1(II) amino acid sequence are shown by the gray background and bold amino acids in Figure 2.

[0054] The DNA sequence of the gene encoding α1(II)M6 shown in SEQ ID NO:5 (designated as COL2A1M6) is shown in SEQ ID NO:6.

[0055] SEQ ID NO:6:

[0056] At both ends of the DNA sequence encoding α1(I)M1, a DNA sequence encoding a Strep-Tag II tag was added to the amino terminus and a DNA sequence encoding a 6×His Tag tag was added to the carboxy terminus. After α1(I)M1 was finally expressed, what was obtained was a tag-containing protein, which had a total of 1071 amino acids and was shown in SEQ ID NO:7.

[0057] SEQ ID NO:7:

[0058] After the optimized design, the DNA sequence of the gene encoding the SEQ ID NO:7 amino acid sequence (α1(I)M1), designated as COL1A1M1, is shown in SEQ ID NO:8.

[0059] SEQ ID NO:8:

[0060] At both ends of the DNA sequence encoding α1(II)M6, a DNA sequence encoding a Strep-Tag II tag was added to the amino terminus and a DNA sequence encoding a 6×His Tag tag was added to the carboxyl terminus. After α1(II)M6 was finally expressed, the resulting protein was a tagged protein with a total of 1076 amino acids, and the sequence is shown in SEQ ID NO:9.

[0061] SEQ ID NO:9:

[0062] After the optimized design, the DNA sequence of the gene encoding the SEQ ID NO:9 (α1(II)M6) amino acid sequence (designated as COL2A1M6) is shown in SEQ ID NO:10. SEQ ID NO:10:

[0063] The synthesis of the DNA sequence was completed upon request from Nanjing Jinsirui Science & Technology Biology Corp., and DNA fragments of two genes, SEQ ID NO:8 and SEQ ID NO:10, were synthesized.

[0064] Example 2. Construction of recombinant expression vectors and strain screening (1) Construction of recombinant expression vectors The SEQ ID NO:8 and SEQ ID NO:10 of the synthesized gene fragments were recombined into the pPIC9K empty vector (purchased from Thermo Fisher Scientific, Inc.), and the target fragments were accurately inserted into the reading frame of the secretory vector containing the secretion signal α-factor, obtaining two recombinant expression vector plasmids, pPIC9K-COL2A1M6 expressing α1(II)M6 and pPIC9K-COL1A1M1 expressing α1(I)M1.

[0065] The pPIC9K-COL2A1M6 and pPIC9K-COL1A1M1 plasmids were transformed into competent Escherichia coli DH5α (purchased from Sangon Biotech (Shanghai) Co., Ltd.), and positive clones were screened on LB resistant plates containing ampicillin. The recombinant plasmids were extracted, and the sequences were determined by identification (completed by Sangon Biotech (Shanghai) Co., Ltd.) and accurately verified. The plasmid patterns of pPIC9K-COL1A1M1 and pPIC9K-COL2A1M6 are shown in Figure 3 and Figure 4 respectively.

[0066] (2) Strain screening 10 μg of the above recombinant expression vector plasmid was digested overnight at 37°C with SacI (purchased from TaKaRa, Dalian, China; specific operations were carried out according to the kit instructions) to linearize it, and the linearized plasmid was recovered using a PCR product purification kit (purchased from Shengong Biological Engineering (Shanghai) Co., Ltd.), and the volume was adjusted to about 10 μL.

[0067] The linearized plasmid was electrotransformed into competent cells of the host yeast Pichia pastoris SMD1168 (purchased from Thermo Fisher Scientific). The electrotransformed bacterial solution was coated on one MD plate in amounts of 100 μL - 200 μL each, left at room temperature for 10 minutes, and then reversely cultured at 30°C for 2 - 5 days until single colonies (positive transformants) appeared.

[0068] 2 mL of sterile double-distilled water was added to the surface of the MD plate, and the His + transformants on the plate surface were gently scraped off and transferred to a 50 mL centrifuge tube. The bacterial suspension was diluted with sterile double-distilled water, and 10 5 cells were coated on YPD plates containing 0.5 mg / mL G418 and reversely cultured at 30°C for 3 - 4 days until single colonies appeared. Colonies were picked from the YPD plates and transferred to sterile 96-well plates (200 μL of YPD / well), mixed uniformly, cultured at 30°C for 48 hours, the bacterial solution in the wells was mixed well, and 10 μL of it was inoculated into each well of a new sterile 96-well plate and cultured at 30°C for 24 hours. Then this operation was repeated again. After 24 hours, 1 μL was taken from the third 96-well plate and inoculated onto YPD plates containing 1.0 mg / mL and 4 mg / mL G418 respectively, and the culture was continued at 30°C for 96 - 120 hours. If the Pichia pastoris transformants can grow on plates containing high-concentration (4 mg / mL) G418, it means that the transformants contain multiple copies of the target gene, that is, multiple recombinant fragments have entered the yeast and been integrated into the yeast chromosome by the same recombination. Through the screening at this stage, a high-copy, high-expression recombinant yeast engineering strain was obtained.

[0069] Two engineering bacteria samples containing pPIC9K-COL1A1M1 and pPIC9K-COL2A1M6 were constructed and deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms.

[0070] The engineering bacteria expressing recombinant α1(I)M1 collagen containing the recombinant expression vector pPIC9K-COL1A1M1 was deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms. The deposit number is CGMCC NO.21891. The address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is March 11, 2021. The classification and naming is Pichia pastoris.

[0071] The engineering bacteria expressing recombinant α1(II)M6 collagen containing the recombinant expression vector pPIC9K-COL2A1M6 was deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms. The deposit number is CGMCC NO.21892. The address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is March 11, 2021. The classification and naming is Pichia pastoris.

[0072] Example 3. Induced expression and identification of recombinant collagen The recombinant engineering bacteria expressing α1(I)M1 and α1(II)M6 obtained in Example 2 were taken respectively. The engineering strains of Pichia pastoris expressing the full-length type I collagen α1-chain protein in existing patents and the engineering strains of Pichia pastoris expressing the full-length type II collagen α1-chain protein were used as controls. The two control engineering strains are both the existing research results of the inventor team. The expressed full-length collagen α1-chain was similarly added with a Strep-Tag II tag at the amino terminus of the peptide chain and a 6×His Tag tag at the carboxyl terminus. The rest is from the patents of Application No. 201911135958.0 (the name is Yeast Recombinant Human Type I Collagen α1-chain Protein, Synthesis Method and Its Use. The engineering bacterial species of Pichia pastoris expressing the full-length α1(I) chain in the patent was deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, and the deposit number is CGMCC NO.17150), Application No. 201911088025.0 (the name is Method for Preparing Recombinant Human Type II Collagen Single Chain by Pichia pastoris. The engineering bacterial species of Pichia pastoris expressing the full-length α1(II) chain in the patent was deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, and the deposit number is CGMCC NO.17149). The four engineering bacteria were placed in a 100 mL Erlenmeyer flask containing 10 mL of BMGY medium and cultured at 28 - 30 °C and 220 rpm until the OD600 reached 2 - 6 (16 - 18 days). Centrifuged at room temperature at 1500 - 3000 g for 5 minutes to collect the bacterial cells. The bacterial cells were resuspended in BMMY medium until the OD600 reached about 2, placed on a shaker at 28 - 30 °C and 220 rpm, and continuously grown for 3 days. 100% methanol was added to the medium every 24 hours until the final concentration of methanol in the medium reached 1.0%. After inducing with methanol for 16 hours or more, 1 mL of the bacterial liquid sample was collected, placed in a 1.5 mL EP tube, centrifuged at 4 °C and 12000 g for 5 minutes, and the expression supernatant was collected. The samples to be detected for later use were stored at -80 °C.

[0073] 5× loading buffer (250 mM Tris-HCl, pH 6.8, 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5% β-mercaptoethanol) was added to the obtained expression supernatant, and it was placed in a metal bath at 100 °C and heated for 10 minutes for SDS-PAGE detection. Since the expressed target protein has an Srtep-Tag II tag at the amino terminus and a 6×His Tag tag at the carboxyl terminus, Western Blot detection was performed using anti-Srtep-Tag II and anti-6×His Tag antibodies (purchased from Nanjing Genscript Biotech Corporation) (specific operations are carried out with reference to the specification).

[0074] The SDS-PAGE of the expression supernatant is shown in Figure 5 below. α1(I)M1, α1(II)M6, α1(I), and α1(II) can all be efficiently secreted and expressed in the extracellular expression supernatant by induced expression for 24 hours. α1(I) and α1(II) show the expected target band (>116 kDa) and distinct main degradation bands (<116 kDa). The α1(I)M1 and α1(II)M6 of the present invention only have the expected target band (>116 kDa).

[0075] The results calculated using Image Lab software (Bio-Rad Gel Doc XR+ imager) are as follows. (1) The apparent molecular weight of the α1(I)M1 target band (116.3 kDa) is basically the same as that of the α1(I) target band (116.4 kDa), and the apparent molecular weight of the α1(II)M6 target band (118.2 kDa) is basically the same as that of the α1(II) target band (118.1 kDa), which is significantly more than the apparent molecular weight of the main degradation band of α1(I) (104.5 kDa) and the apparent molecular weight of the main degradation band of α1(II) (106.9 kDa). (2) The ratio of the target band to the main degradation band in the α1(I) electrophoresis result is 51.5%:48.3%, and the ratio of the target band to the main degradation band in the α1(II) electrophoresis result is 52.1%:47.8%. The main degradation products are basically the same as the proportion of the target products.

[0076] As can be seen from the ECL chemiluminescence color development results in Figure 6 (the fully automatic chemiluminescence image analysis system Tanon 5200 synthesizes the protein molecular mass standard into the image), the Srtep-TagII tag can be detected at the amino terminus, and the 6×His Tag tag can be detected at the carboxyl terminus. The target band is the same as the apparent molecular weight size in SDS-PAGE. The two recombinant collagens, α1(I)M1 and α1(II)M6, can efficiently secrete and express the full-length sequence. The expression of the target band is as expected. The target band sequences of the two collagens, α1(I) and α1(II), are full-length, but the main degradation band lacks the amino-terminal sequence, and only the carboxyl-terminal 6×His Tag tag was detected.

[0077] The target bands of α1(I)M1 and α1(II)M6 in SDS-PAGE, and the target bands and main degradation bands of α1(I) and α1(II) in SDS-PAGE were excised, digested with trypsin, and the tryptic digestion peptide fragments of the recombinant collagen were detected by Nano-HPLC-MS / MS mass spectrometry (completed by entrusting Suzhou Putai Bio-Technology Co., Ltd.). The detected peptide fragments were compared in sequence (Uniprot database). The comparison results of the data and the superposition diagram of the identified peptide fragments and the natural sequence (the part with the gray background color is the part where the peptide fragments identified by mass spectrometry in the band are exactly the same as the natural sequence) are shown in Figures 7 and 8, and the results are as follows. (1) The peptide fragments detected after enzymatic digestion in the target bands of α1(I)M1, α1(I), and the main degradation bands of α1(I) all belong to the sequence of the α1 chain of type I collagen. (2) The peptide fragments detected after enzymatic digestion at the target bands of α1(II)M6 and α1(II) and the main degradation bands of α1(II) all belong to the sequence of the α1 chain of human type II collagen.

[0078] From the above results, α1(I)M1 and α1(II)M6 can be expressed in the same way as α1(I) and α1(II), and belong to the recombinant collagens of the α1 chain of human type I collagen and the α1 chain of human type II collagen respectively. However, when α1(I) and α1(II) are expressed, degradation occurs, and the main degradation bands also belong to the corresponding types of collagen.

[0079] Example 4. Fermentation and purification at high density (1) Fermenting the genetically engineered bacteria at high density Recombinant α1(I)M1 and α1(II)M6 collagens were expressed and produced on a large scale, and a fermentation broth containing recombinant collagen was obtained. Seed medium YPG (containing 10 g / L yeast powder, 20 g / L yeast peptone, and 10 g / L anhydrous glycerol), fermentation medium (NH 4 H 2 PO 4 190.4 g / L, KH 2 PO 4 10.06 g / L, CaSO 4 ·2H 2 O 1.18 g / L, K 2 SO 4 18.2 g / L, MgSO 4 ·7H 2 O 14.9 g / L, containing 40 g / L glycerol), feed medium (containing 50% W / V glycerol, with 12 mL of PTM1 trace elements added per liter), induction medium (containing 100% methanol, with 12 mL of PTM1 trace elements added per liter). PTM1 was filtered and sterilized with a 0.22 μm filter membrane and stored at 4°C. After the fermentation medium was sterilized at high temperature and cooled to room temperature, PTM1 was added and the pH was adjusted to 5.0 with aqueous ammonia.

[0080] The batch culture conditions and inducible expression conditions of the engineering strain are as follows. The fed-batch culture method is adopted, and the culture temperature is 30°C. The engineered bacteria were inoculated into a 1-L shaking flask containing the seed-containing medium YPG and cultured at 220 rpm and 30 °C for 18 - 20 hours until OD600 reached 2 - 10. Using a 5-L fermenter (Guangzhou Baoxing Biological Technology Co., Ltd.), 2 L of the fermentation medium (containing 2% glycerol) was separately sterilized and adjusted to 300 rpm in rotational speed, 4 L / min in aeration rate, 30 °C in temperature, and pH 4.5 with an alkaline solution prepared with concentrated ammonia water before inoculation. Then, 0.9 mL of PTM1 was inoculated, and the prepared 200-mL seed solution was inoculated into the tank (inoculation by flame ring). The 100 calibration of the dissolved oxygen electrode was clicked, and fermentation was started after 100 calibration. When the growth dissolved oxygen first dropped to 30%, the growth dissolved oxygen was maintained at 30% by the dissolved oxygen / rotational speed cascade function. When the glycerol was consumed, the dissolved oxygen rebounded, and when the dissolved oxygen exceeded 70% (OD600 value about 20), the cascade speed control of the dissolved oxygen was released, the stirring was adjusted to 650 rpm, and glycerol was continuously supplied at 30% with a supply amount of 80 mL. After stopping the replenishment of glycerol and when the dissolved oxygen rebounded above 70%, the pH was set to 4 and the temperature to 29 °C, and induction culture was carried out using a mixed carbon source of methanol and glycerol (methanol: 50% glycerol = 7:3). Manually add 5 mL of the above mixed carbon source. After the dissolved oxygen rebounded above 70%, the supply rate was set to 8 mL / h, increased to 10 mL / h after 1 hour, and then increased to 20 mL / h again after 1 hour. When the value of the dissolved oxygen was less than 30%, the supply was stopped. When the dissolved oxygen rebounded and the dissolved oxygen returned to 30%, continuous supply was carried out. After induction for 40 - 60 hours, when the protein concentration did not increase significantly or decreased as measured by UV, it was put into the tank. The quantitative formula for the protein by UV is C (mg / mL) = 0.144 * (A215 - A225), where A215 < 1.5.Meanwhile, engineering strains of Pichia pastoris expressing the full-length α1(I) chain (the deposit number of the strain at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms is CGMCC NO. 17150) and engineering strains of Pichia pastoris expressing the full-length α1(II) chain (the deposit number of the strain at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms is CGMCC NO. 17149) were respectively obtained and fermented at high density.

[0081] The results are shown in Table 1. After 48 hours of induction, for α1(I) compared with α1(I)M1 and for α1(II)M6 compared with α1(II), there are no significant differences in the three indicators of cell concentration (OD600), wet cell weight, and UV quantification of the concentration of the expressed protein in the fermentation broth. However, the results of collecting the fermentation supernatant and detecting by SDS-PAGE electrophoresis are shown in Figure 9. Under the conditions of high-density fermentation, the main degradation bands of α1(I) and α1(II) are extremely obvious, which is no different from the induced expression by the shaking flask. The most main products of α1(I)M1 and α1(II)M6 are still their target bands. Since the main degradation bands do not appear, it means that the removal effect of the main degradation bands (main degradation products) of α1(I)M1 and α1(II)M6 is effectively maintained even under the conditions of high-density fermentation.

[0082]

Table 1

[0083] (2) Purification of collagen Buffer A: 20 mM KH 2 PO 4 with a pH of 4.0. Buffer B: 20 mM KH 2 PO 4 0.5 M NaCl, with a pH of 4.0. The fermentation broth was collected and centrifuged at 2000 g for 30 min at 4°C to separate the cells and the fermentation supernatant. After balancing the cation exchange medium (the chromatography packing material was UniGel-80sp manufactured by SUZHOU NANOMICRO TECHNOLOGY CO., LTD. loaded onto a GCC-50-400 chromatography column manufactured by Lisure Science (Suzhou) Co., Ltd., and using a GE AKTA Pure protein separation, chromatography, and purification system) with Buffer A, it was set at a flow rate of 40 us / cm for loading. The sample volume was 0.5 L per run. When it was detected that the absorbance value of ultraviolet A215 increased, the sampling of the sample was started. After the loading was completed, the sampling of the sample was stopped, and the cation chromatography medium was balanced with Buffer A. When the absorbance value of A215 decreased, the ultraviolet and conductivity stopped changing until they reached the minimum value. The eluate was collected, the components were detected and quantified individually, then dialysis (the dialysis solution was ultrapure water) was performed, followed by concentration and freeze-drying to collect the freeze-dried collagen sponge. The purified freeze-dried sponge was dissolved in ultrapure water and subjected to SDS-PAGE electrophoresis, as shown in Figure 10. After purifying α1(I)M1 and α1(II)M6 by one-step ion exchange, most of the impurity proteins, small degradation bands, etc. were removed, and high-purity single target proteins were obtained (calculated using Image Lab software, the purity of α1(I)M1 was 90.1%, and the purity of α1(II)M6 was 88.3%).After performing the same purification process on α1(I) and α1(II), the main degradation bands still appeared and could not be removed. Since the main degradation products are similar in size and properties to the target products, it is difficult to separate them by one-step purification. According to the contents of the patents with application numbers 201911135958.0 and 201911088025.0, to obtain the products of single-target full-length α1(I) chain and full-length α1(II) chain, there are Srtep-TagII tags at the amino group terminals of the full-length α1(I) chain and full-length α1(II) chain, and 6×His tags at the carboxyl terminals. Due to this property, it can only be achieved by performing double-affinity purification using Ni-NTA and Strep-Tactin affinity chromatography media. In SDS-PAGE, the main degradation products of the main degradation bands are discarded, the biosynthetic resources of the bacterial cells are wasted, the purification process is increased, and the yield of the target product is decreased.

[0084] Example 5. Detection of Recombinant Collagen (1) Analysis of Fourier Transform Infrared Spectrum (FT-IR) In the test, a small amount of freeze-dried samples of α1(I)M1, α1(II)M6, α1(I), and α1(II) collagen after purification were taken and mixed with potassium bromide (KBr) respectively, ground into powder and tableted, and scanned in the range of 4000~400 cm -1 at room temperature (Thermo Scientific, Nicolet TM iS TM 10 FT-IR spectrometer), and the analysis of the method and results refers to (Jeong, H.J. Venkatesan and S. Kim, Isolation and characterization of collagen from marine fish (Thunnus obesus). Biotechnology and Bioprocess Engineering, 2013. 18(6): p. 1185-1191.).

[0085] The infrared spectra obtained by scanning the protein purification samples of α1(I)M1, α1(II)M6, α1(I), and α1(II) are shown in Figures 11 and 12. The characteristic absorption average wavenumbers are amide A (3299 cm -1 approx.), amide B (3081 cm -1 approx.), amide I (1650 cm -1 approx.), amide II (1530 - 1550 cm -1 approx.), amide III (1240 cm -1 approx.), which are consistent with the structural characteristics of recombinant collagen. It shows that the amino acid mutations in α1(I)M1 and α1(II)M6 do not affect the properties of collagen itself (see References [1]. Chen Jingtao et al., Study on the infrared spectra of recombinant collagen and bovine type I collagen. Materials Review, 2008(03): pp. 119 - 121. [2]. Doyle, B.B., E.G. Bendit and E.R. Blout, Infrared spectroscopy of collagen and collagen‐like polypeptides. Biopolymers, 1975. 14(5): p. 937 - 957. [3]. Zhou Aimei et al., Separation, purification and structural characteristics of recombinant human collagen. Food and Fermentation Industries, 2015(03): pp. 46 - 52.).

[0086] (2) Detection of the cell adhesion activity of recombinant collagen For the method of detecting the cell adhesion activity of recombinant collagen, refer to the literature Juming Yao, Satoshi Yanagisawa, Tetsuo Asakura. Design, Expression and Characterization of Collagen-Like Proteins Based on the Cell Adhesive and Crosslinking Sequences Derived from Native Collagens, J Biochem. 136, 643 - 649 (2004). It was completed by entrusting the Functional Nanomaterials and Biomedicine Test Research Laboratory of the School of Pharmacy, Changzhou University.

[0087] The specific implementation method is as follows: Normal cultured NIH / 3T3 cells (purchased from the Cell Bank of the Chinese Academy of Sciences, product number GNM6, and the culture and subculture methods are carried out according to the cell specification). Recombinant α1(I)M1, α1(II)M6, α1(I), α1(II) collagen freeze-dried sponges, control human collagen (purchased from Sigma, product number C7774), and bovine serum albumin (BSA, purchased from Shanghai Shengong Biological Engineering Co., Ltd.) were taken and dissolved (in ultrapure water or 1M HCl solution), and the protein concentration was measured according to the experimental formula for UV protein quantification C (mg / mL) = 0.144×(A215 - A225), and diluted to 0.5 mg / mL with PBS (pH 7.4). 100 μL of various protein solutions and control blank PBS solutions were added to a 96-well cell culture plate, left at room temperature for 60 minutes, and 10 5 cells of well-cultured NIH / 3T3 cells were added to each well, and incubated at 37 °C and 5% CO 2 for 60 minutes. The cells in the wells were washed 4 times with PBS. The absorbance value at OD492nm was detected using an LDH detection kit (Roche, 04744926001) (the specific operation was carried out according to the specification).

[0088] The characteristics corresponding to the absorbance at OD492nm can represent the cell adhesion activity of the collagen sample. The higher the absorbance at OD492nm, the more protein adheres to more cells, and the higher the adhesion activity, the more it helps the cells to adhere to the wall or adhere to the extracellular matrix in a short time, contributing to the construction of a better extracellular environment. The results are shown in Figure 13. The collagens of recombinant α1(I)M1, α1(II)M6, α1(I), and α1(II) all have cell adhesion activity similar to that of commercially available natural human collagen, and are all higher than the control group. The cell adhesion activity of α1(I)M1 is basically the same as that of α1(I), and the cell adhesion activity of α1(II)M6 is basically the same as that of α1(II), with no significant difference.

[0089] (3) Preparation and detection of recombinant collagen hydrogel Take the collagens of recombinant α1(I)M1, α1(II)M6, α1(I), and α1(II), dissolve them in water for injection at a concentration of 10%, adjust the pH to the range of 4 - 6, filter and sterilize with a 0.22 μm sterile filter, add 0.1 g of a 10% (w / w) sterile N-hydroxysuccinimide (NHS) solution per 1 g of the collagen dry powder and mix uniformly, then add 0.13 g of a 50% (w / w) sterile 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) solution per 1 g of the collagen dry powder, leave it at room temperature (20 - 30 °C) and react for 2 - 6 hours to generate a hydrogel. Dialyze the hydrogel against a sterile PBS solution (8.5 g / L NaCl, 2 HPO 4 0.5 g / L, NaH 2 PO 4 0.15 g / L, pH 7.2) at a gel:PBS dialysate ratio of 1:6 (m / m) for 120 hours continuously, and completely change the dialysate every 24 hours to remove the residues of NHS and EDC. Put the dialyzed hydrogel into a sterile container and leave it at room temperature.

[0090] Freeze-dry the hydrogel to remove moisture, weigh the freeze-dried hydrogel, leave it in the sterile PBS solution again for 24 hours, after it completely absorbs water and swells, take out the hydrogel, absorb the surface moisture with absorbent paper and weigh it. According to the method in the literature, the swelling ratio: Q 膨潤率 =(W 吸水膨潤質量 -W 乾燥ゲル重量 ) / W 乾燥ゲル重量 was calculated. Use a rheometer (Discovery HR-2) to measure the elastic modulus (storage elastic modulus, small amplitude frequency scan, 25 °C, stress 0.5%, 0.1 - 100.0 rad / s), dynamic viscosity (maintain at the flow peak, 25 °C, shear rate 2.0 s -1) was detected. The lyophilized hydrogel was rapidly frozen in liquid nitrogen and broken, and the surface of the lyophilized hydrogel was scanned using a scanning electron microscope (Hitachi's TM3030PLUS). The results of the elastic modulus, kinematic viscosity, and swelling ratio are shown in Table 2. α1(I)M1 compared with α1(I), and α1(II)M6 compared with α1(II), the hydrodynamic properties of the hydrogels prepared under the same conditions are basically the same and have not changed significantly.

[0091]

Table 2

[0092] As shown in Figures 14 and 15, the hydrogels prepared with α1(I)M1 and α1(II)M6, like the hydrogels prepared with α1(I) and α1(II), both have a porous network structure. The pore size range gathers at 100 - 200 μm, has good permeability, has a spatial structure base for maintaining a large amount of water, and may be used in the field of biomedical materials as a space for cells to adhere, support, grow and migrate, or as a channel for transporting nutrients and metabolites.

[0093] (4) Detection of cells in the recombinant collagen hydrogel The aseptically stored hydrogel was placed in a 24-well cell culture plate. Normal cultured NIH / 3T3 cells (purchased from the Cell Bank of the Chinese Academy of Sciences, product number GNM6, the culture and subculture methods were carried out according to the cell specifications) were taken, washed with PBS, digested with trypsin, added with medium, pipetted uniformly and counted, and inoculated into the cell culture plate containing the hydrogel at 10 5 cells / well, and co-cultured for 24 - 72 hours to observe the adhesion and proliferation status of the cells on the hydrogel.

[0094] (1) A 24-well plate was taken, and 1 mM calcein AM (purchased from Beyotime Biotechnology Inc.) was prepared with DMSO and diluted to a 50 μM calcein working solution with D-PBS. The medium in the well was aspirated, washed multiple times with PBS, 1 mL of serum-containing DMEM medium was added, 100 μL of calcein AM solution (1 / 10 of the medium) was added, incubated for 30 minutes to stain the cells, the growth medium was replaced, cultured for 30 minutes, the hydrogel was gently taken out, placed in a new culture well, and photographed with a fluorescence microscope (the maximum excitation wavelength is 494 nm and the maximum emission wavelength is 514 nm).

[0095] (2) Another 24-well plate was taken, 200 μL of MTT solution (purchased from Beyotime Biotechnology Inc.) was added, and after culturing NIH / 3T3 cells for 4 hours, the formation status of purple crystals in the cells was observed. The medium was discarded, the hydrogel was washed with PBS, the hydrogel was cut longitudinally, placed in a new culture well, and photographed with a microscope.

[0096] The experiment in this example was completed upon request by the Functional Nanomaterials and Biomedicine Laboratory of the School of Pharmacy, Changzhou University. The results are shown in Figures 16 and 17. When observed with a bright-field microscope, the morphology of NIH / 3T3 cells in the hydrogels prepared with α1(I)M1 and α1(II)M6 is normal and typical of fibroblast morphology, similar to that of the hydrogels prepared with α1(I) and α1(II). When NIH / 3T3 cells adhered and grew on the hydrogels were stained with calcein AM, green fluorescence was detected (the bright parts in the photos). When MTT was added to NIH / 3T3 cells growing on the hydrogels, purple crystals were formed (the black parts in the photos). The green fluorescence and purple crystals are only formed by living cells, indicating that NIH / 3T3 cells can adhere, grow, and migrate normally within the hydrogels. The hydrogels prepared with α1(I)M1 and α1(II)M6 have biological functions similar to those of the hydrogels prepared with the natural sequences of α1(I) and α1(II) and can be applied to fields such as wound repair and tissue regeneration as novel biomedical devices.

Claims

1. An α1 chain of recombinant collagen that is α1(I)M1 or α1(II)M6, wherein the α1(I)M1 is obtained by amino acid mutation of the natural full-length amino acid sequence of the α1 chain of human type I collagen, and the α1(II)M6 is obtained by amino acid mutation of the natural full-length amino acid sequence of the α1 chain of human type II collagen, and the sequence of the α1(I)M1 is shown in SEQ ID NO: 2, and the sequence of the α1(II)M6 is shown in SEQ ID NO:

5. An α1 chain of recombinant collagen, characterized in that.

2. A nucleotide encoding the α1 chain of the recombinant collagen according to Claim 1.

3. The nucleotide sequence encoding α1(I)M1 is shown in SEQ ID NO: 3, and the nucleotide sequence encoding α1(II)M6 is shown in SEQ ID NO:

6. The nucleotide according to Claim 2, characterized in that.

4. A recombinant expression vector containing the nucleotide according to Claim 2.

5. An engineering bacterium, which is Pichia pastoris containing the recombinant expression vector according to Claim 4.

6. An engineering bacterium, which is Pichia pastoris expressing the α1 chain of the recombinant collagen according to Claim 1.

7. The engineering bacterium is deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, and the deposit number is CGMC NO. 21891 or CGMC NO. 21892. Among them, the engineering bacterium with the deposit number CGMC NO. 21891 expresses the α1 chain of recombinant α1(I)M1 collagen, and the engineering bacterium with the deposit number CGMC NO. 21892 expresses the α1 chain of recombinant α1(II)M6 collagen. The engineering bacterium according to Claim 5, characterized in that.

8. The aforementioned engineered bacteria are deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, and the deposit numbers are CGMC NO. 21891 or CGMC NO. 21892. Among them, the engineered bacteria with the deposit number of CGMC NO. 21891 express the α1 chain of recombinant α1(I)M1 collagen, and the engineered bacteria with the deposit number of CGMC NO. 21892 express the α1 chain of recombinant α1(II)M6 collagen. The engineered bacteria according to claim 6 are characterized in that.

9. Use of the recombinant expression vector according to claim 4 or the engineered bacteria according to any one of claims 5 to 8 for preparing the α1 chain of the recombinant collagen.

10. A composition characterized by containing the α1 chain of the recombinant collagen according to claim 1.

11. A product, which is a pharmaceutical, a medical device, a biomaterial, a tissue engineering product or a cosmetic, and is characterized by containing the α1 chain of the recombinant collagen according to claim 1 or the composition according to claim 10.

12. The product according to claim 11, which is a material that adheres to cells, provides a space for support, growth and migration, or is a material that is a channel for transporting nutrients and metabolites.

13. The product according to claim 12, which is characterized in that the product is a collagen hydrogel.

14. Use of the α1 chain of the recombinant collagen according to claim 1, the nucleotide according to any one of claims 2 to 3, the recombinant expression vector according to claim 4, the engineered bacteria according to any one of claims 5 to 8, or the composition according to claim 10 for preparing a pharmaceutical, a medical device, a biomaterial, a tissue engineering product, or a cosmetic.

15. Use of the α1 chain of the recombinant collagen according to claim 1, the nucleotide according to any one of claims 2 to 3, the recombinant expression vector according to claim 4, the engineered bacteria according to any one of claims 5 to 8, or the composition according to claim 10 for preparing a product for promoting wound repair or tissue regeneration.

16. The use according to claim 14, wherein the pharmaceutical, the medical device, the biomaterial, the tissue engineering product, or the cosmetic is a collagen hydrogel.

17. The use according to claim 15, wherein the product is a collagen hydrogel.

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