A recombinant type III collagen, nucleic acid, expression vector, strain and its application

Abstract

This invention provides a recombinant type III collagen, nucleic acid, expression vector, strain, and its applications. A recombinant type III collagen is obtained through genetic engineering. Using synthetic biology techniques, collagen can be synthesized and produced using modified microbial chassis cells. The recombinant collagen produced in this way exhibits high expression levels, excellent cell proliferation effects, low toxicity, low antigenicity, low immunogenicity, the ability to guide cell regeneration, and good biocompatibility. When applied to humans, it produces a low immune response. The production method is simple, environmentally friendly, and has broad application prospects in the biopharmaceutical, cosmetic, and skincare industries.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a recombinant type III collagen, nucleic acid, expression vector, strain, and their applications. Background Technology

[0002] Collagen is the most abundant protein in the body, accounting for approximately 25% to 33% of total human protein. It is widely distributed throughout various tissues and organs, such as skin, bones, cornea, and blood vessels, with particularly high concentrations in tissues like the skin. As a binding substance in connective tissues, collagen plays a vital role in maintaining the normal physiological functions of cells, tissues, and organs. Currently, 29 types of collagen are known. Different types of collagen differ in the shape of their globular domains, the spacing of their triple helix structures, and the length of their macrophages.

[0003] In healthy individuals, approximately 90% of collagen is found in the skin and bones. Different types and locations of collagen play crucial roles in tissue support and intercellular signal transduction. Traditional collagen production methods involve extraction from animal skin, tissues, or bones. However, this traditional method of extracting animal-derived collagen carries numerous safety risks. For example, it carries the risk of zoonotic diseases due to pathogens such as mad cow disease, foot-and-mouth disease, and swine fever. Furthermore, the stability of different batches of raw materials is difficult to guarantee. This method also generates significant pollution and is environmentally unfriendly. These drawbacks limit the further application of collagen in the traditional collagen production process. Summary of the Invention

[0004] The main objective of this invention is to provide a recombinant type III collagen, nucleic acid, expression vector, strain, and its application, with the aim of providing a safe collagen.

[0005] To achieve the above objectives, the recombinant type III collagen proposed in this invention has an amino acid sequence comprising at least one of the following:

[0006] (1) SEQ ID NO: 1: GPPGPAGANGAPGLRGGAGEPGKNGAKGEPGP RGERGEAGIPGVPGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAP;

[0007] (2) SEQ ID NO: 2: GQPGEKGSPGAQGPPGAPGPLGIAGITGARGLA GPPGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGLPGLAGTAGEPGRDGNPGSDGLPGRDGSPGGKGDRGENGSPGAPGAPGHPGPPGPVGPAGKSGDRGES GPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPRGPVGSGPPGKDGTSGHPGPIGPPGPRGNRGERGSE;

[0008] Or compared with (1) or (2), it has an amino acid sequence with one or more amino acids added, substituted, or deleted.

[0009] In one embodiment, the amino acid sequence comprises 2 to 10 repeats of SEQ ID NO: 1; and / or, the amino acid sequence comprises 2 to 10 repeats of SEQ ID NO: 2.

[0010] The present invention also proposes a nucleic acid encoding a recombinant type III collagen as described above, wherein the recombinant type III collagen has an amino acid sequence comprising at least one of the following:

[0011] (1) SEQ ID NO: 1: GPPGPAGANGAPGLRGGAGEPGKNGAKGEPGP RGERGEAGIPGVPGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAP;

[0012] (2) SEQ ID NO: 2: GQPGEKGSPGAQGPPGAPGPLGIAGITGARGLA GPPGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGLPGLAGTAGEPGRDGNPGSDGLPGRDGSPGGKGDRGENGSPGAPGAPGHPGPPGPVGPAGKSGDRGES GPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPRGPVGSGPPGKDGTSGHPGPIGPPGPRGNRGERGSE;

[0013] Or compared with (1) or (2), it has an amino acid sequence with one or more amino acids added, substituted, or deleted.

[0014] In one embodiment, the nucleic acid sequence is as shown in SEQ ID NO: 3 and / or SEQ ID NO: 4, wherein SEQ ID NO: 3 is as follows:

[0015] GGTCCCCCAGGTCCAGCTGGTGCTAACGGAGCCCCAGGTCTGAGAGGTGGTGCCGGTGAACCAGGAAAAAACGGTGCTAAGGGTTGAGCCTGGTCCAAGAGGAGAGAGAGGTGAAGCAGGTATTCCTGGTGTTCCAGGTGCTAAGGGAGA AGATGGTAAAGATGGATCTCCAGGTGAACCCGGTGCCAACGGTTTACCAGGTGCTGCCGGAGAAAGAGGAGCACCAGGTTTTAGAGGTCCAGCTGGTCCAAACGGTATTCCAGGAGAAAAGGGACCAGCAGGAGAAAGAGGTGCTCCC;

[0016] SEQ ID NO: 4 is as follows:

[0017] .

[0018] The present invention also proposes an expression vector, wherein the expression vector is inserted with the above-mentioned nucleic acid, or the expression vector is capable of enabling the host cells to express the above-mentioned recombinant type III collagen after transfection.

[0019] In one embodiment, the expression vector includes any one of pPIC9K, pPICZαA, GAPZαA, pPICZαB, pPICZαC, GAPZαB, GAPZαC, and their modified vectors.

[0020] The present invention also proposes a strain that expresses recombinant type III collagen, wherein the strain is capable of expressing the above-mentioned recombinant type III collagen.

[0021] In one embodiment, the strain includes any one of Pichia pastoris, Saccharomyces cerevisiae, and Escherichia coli.

[0022] In one embodiment, the Pichia pastoris includes one or more of SMD1168, KM71, KM71H, SMD1163, X33 and GS115.

[0023] The present invention also proposes the use of the recombinant type III collagen as described above, or the nucleic acid as described above, or the expression vector as described above, or the strain expressing recombinant type III collagen as described above, in the preparation of skin care products, cosmetics and pharmaceuticals.

[0024] This invention provides a recombinant type III collagen protein obtained through genetic engineering. Using synthetic biology techniques, collagen can be synthesized and produced using modified microbial chassis cells. The recombinant collagen produced in this way exhibits high expression levels, excellent cell proliferation effects, low toxicity, low antigenicity, low immunogenicity, the ability to guide cell regeneration, and good biocompatibility. When applied to the human body, it produces a low immune response. The production method is simple, environmentally friendly, and has broad application prospects in the biopharmaceutical, cosmetic, and skincare industries. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0026] Figure 1 This is a schematic diagram of the structure of the CL3.4 expression plasmid in Example 1 of the present invention;

[0027] Figure 2 This is a schematic diagram of the structure of the CL3.8 expression plasmid in Example 1 of the present invention;

[0028] Figure 3 This is a graph showing the results of collagen expression levels in shake-flask culture in Example 3 of the present invention.

[0029] Figure 4 This is a graph showing the protein expression levels of CL3.4 collagen in different basal cells in Example 4 of the present invention.

[0030] Figure 5This is a graph showing the protein expression levels of CL3.8 collagen in different basal cells in Example 4 of the present invention.

[0031] Figure 6 This is a graph showing the protein expression levels during collagen fermentation (0-48 hours) in Example 5 of this invention.

[0032] Figure 7 This is a graph showing the protein expression levels during collagen fermentation (48-72 hours) in Example 5 of this invention.

[0033] Figure 8 This is a graph showing the relationship between absorbance values ​​and different concentrations of standard protein solutions in Example 6 of the present invention.

[0034] Figure 9 This is a bar graph showing the results of the collagen cell proliferation experiment in Example 7 of the present invention.

[0035] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0036] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0037] The first aspect of this invention proposes a recombinant type III collagen.

[0038] In this embodiment of the invention, the recombinant type III collagen has an amino acid sequence including at least one of the following:

[0039] (1) SEQ ID NO: 1: GPPGPAGANGAPGLRGGAGEPGKNGAKGEPGP RGERGEAGIPGVPGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAP;

[0040] (2) SEQ ID NO: 2: GQPGEKGSPGAQGPPGAPGPLGIAGITGARGLA GPPGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGLPGLAGTAGEPGRDGNPGSDGLPGRDGSPGGKGDRGENGSPGAPGAPGHPGPPGPVGPAGKSGDRGES GPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPRGPVGSGPPGKDGTSGHPGPIGPPGPRGNRGERGSE;

[0041] Or compared with (1) or (2), it has an amino acid sequence with one or more amino acids added, substituted, or deleted.

[0042] This invention provides a recombinant type III collagen protein obtained through genetic engineering. Using synthetic biology techniques, collagen can be synthesized and produced using modified microbial chassis cells. The recombinant collagen produced in this way exhibits high expression levels, excellent cell proliferation effects, low toxicity, low antigenicity, low immunogenicity, the ability to guide cell regeneration, and good biocompatibility. When applied to the human body, it produces a low immune response. The production method is simple, environmentally friendly, and has broad application prospects in the biopharmaceutical, cosmetic, and skincare industries.

[0043] For this recombinant type III collagen, the construction method of this invention is divided into gene sequence mining and selection, expression vector construction, expression vector integration and construction of recombinant collagen expression strains, chassis cell selection and optimization, scale-up fermentation, and cell proliferation testing. The following will elaborate on each step.

[0044] I. Gene Sequence Mining and Selection

[0045] Collagen sequences were searched and mined using the NCBI database. A type III collagen sequence (NP_000081.2) was selected, and its sequence composition and structure were analyzed. It was found that both the N-terminus and C-terminus contain domains that are cleaved during later protein processing. Furthermore, studies have shown that sequences containing EK and ER structural regions, such as GPAGEK, GAPGER, and GPAGFR, can interact with receptors on the surface of fibroblasts, enhancing integrin binding and regulating cell recognition, adhesion, and migration, thereby exerting biological functions.

[0046] Based on this, two type III collagen sequences were selected, and their bases were optimized based on codon preference to obtain sequences SEQ ID NO:3 and SEQ ID NO:4, whose amino acid sequences are SEQ ID NO:1 and SEQ ID NO:2.

[0047] Among them, SEQ ID NO: 1 is as follows:

[0048] GPPGPAGANGAPGLRGGAGEPGKNGAKGEPGPRGERGEAGIPGVPGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAP;

[0049] SEQ ID NO: 2 is as follows:

[0050] GQPGEKGSPGAQGPPGAPGPLGIAGITGARGLAGPPGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGLPGLAGTAGEPGRDGNPGSDGLPGRDGSPGGKGDRGENGSPGAPGAPGHPGP PGPVGPAGKSGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPRGPVGSGPPGKDGTSGHPGPIGPPGPRGNRGERGSE;

[0051] SEQ ID NO: 3 is as follows:

[0052] GGTCCCCCAGGTCCAGCTGGTGCTAACGGAGCCCCAGGTCTGAGAGGTGGTGCCGGTGAACCAGGAAAAAACGGTGCTAAGGGTTGAGCCTGGTCCAAGAGGAGAGAGAGGTGAAGCAGGTATTCCTGGTGTTCCAGGTGCTAAGGGAGA AGATGGTAAAGATGGATCTCCAGGTGAACCCGGTGCCAACGGTTTACCAGGTGCTGCCGGAGAAAGAGGAGCACCAGGTTTTAGAGGTCCAGCTGGTCCAAACGGTATTCCAGGAGAAAAGGGACCAGCAGGAGAAAGAGGTGCTCCC;

[0053] SEQ ID NO: 4 is as follows:

[0054] .

[0055] The amino acid sequence is not limited to SEQ ID NO: 1 and SEQ ID NO: 2 above. In one embodiment, an amino acid sequence with added, substituted, or deleted one or more amino acids has a similar effect compared to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0056] In one embodiment, the amino acid sequence may comprise 2 to 10 repeats of SEQ ID NO: 1, compared to the amino acid sequence shown in SEQ ID NO: 1.

[0057] In one embodiment, the amino acid sequence may comprise 2 to 10 repeats of SEQ ID NO:2, compared to the amino acid sequence shown in SEQ ID NO:2.

[0058] II. Construction of the Expression Carrier

[0059] The gene sequences of CL3.4 and CL3.8 (i.e., nucleic acid sequences SEQ ID NO: 3 and SEQ ID NO: 4) were obtained by PCR amplification, and the expression vectors were linearized using EcoRI and NotI restriction enzymes. The CL3.4 and CL3.8 gene sequences were assembled into the expression vectors through seamless cloning assembly to obtain the CL3.4 expression vector and the CL3.8 expression vector.

[0060] The expression vector can be any one of pPIC9K, pPICZαA, GAPZαA, pPICZαB, pPICZαC, GAPZαB, GAPZαC, and their modified vectors, or a vector modified based on the above expression vectors. In one embodiment, the pPIC9K expression vector is used, and after assembly, pPIC9K-CL3.4 and pPIC9K-CL3.8 expression vectors are obtained, respectively.

[0061] III. Integration of Expression Vectors and Construction of Recombinant Collagen Expression Strains

[0062] The constructed expression vector was incubated with SalI enzyme at 37°C for 1 h, and then the linearized vector was obtained by DNA purification and recovery. The vector was then integrated into the chassis cells.

[0063] Chassis cells were screened by coating them on auxotrophic medium, and the resulting single colonies were verified by colony PCR to obtain strains that integrated the target vector.

[0064] The culture medium can be prepared by adding 2 g (20 g / L) agarose to 80 ml of water and sterilizing at 115°C for 20 min. Before use, add 10 mL of 10×YNB, 10 mL of 10×glucose (20 g / L), and 0.2 mL of 500×biotin (4×10⁻⁶) to a clean bench. - 4 Mix well and pour into a plate (g / L).

[0065] The chassis cells may include one of Pichia pastoris, Saccharomyces cerevisiae, and Escherichia coli. In one embodiment, the chassis cells include Pichia pastoris. Further, in one embodiment, the Pichia pastoris includes one or more of SMD1168, KM71, KM71H, SMD1163, X33, and GS115. There are various methods for integrating the vector into the chassis cells, including electroporation and chemical transformation.

[0066] IV. Chassis Cell Selection and Optimization

[0067] (1) Selection and optimization

[0068] Four different Pichia pastoris strains—SMD1168, KM71, SMD1163, and GS115—were selected. pPIC9K-CL3.4 and pPIC9K-CL3.8 were transformed into these strains, yielding recombinant strains SMD1168 / CL3.4, KM71 / CL3.4, SMD1163 / CL3.4, and GS115 / CL3.4; and SMD1168 / CL3.8, KM71 / CL3.8, SMD1163 / CL3.8, and GS115 / CL3.8, respectively, capable of synthesizing collagen CL3.4 and CL3.8. Their expression was analyzed through shake-flask culture. It is understandable that these strains can be extended for optimization of Saccharomyces cerevisiae and Escherichia coli chassis cells.

[0069] (2) Shake flask culture

[0070] a) Pick one loopful of colonies and inoculate them into 6-7 mL of YPD liquid medium (see Table 1 for specific component contents), and incubate at 30℃ and 220 rpm for 16 h.

[0071] b) Transfer the inoculum to BMGY medium (see Table 2 for specific component contents, or YPD) at a 10% inoculum and incubate at 30°C and 220 rpm for 24 h.

[0072] c) Transfer the bacterial culture after 24 h to a 50 ml sterile centrifuge tube, centrifuge at 5000 rpm for 8 min, collect the bacterial cells, and discard the supernatant.

[0073] d) Add 40 ml of physiological saline (or sterile water) to resuspend and wash the bacterial cells twice, and discard the supernatant.

[0074] e) The bacterial cells were resuspended in BMMY medium (specific component contents are shown in Table 3) and cultured at 30℃ and 220 rpm; 1% methanol was added every 24 h. Samples were taken at 72 h of induction for SDS-PAGE analysis.

[0075] Table 1 Component content of YPD medium

[0076]

[0077] Table 2 Component content of BMGY culture medium

[0078]

[0079] Note: For every 40 mL of BMGY medium, add 5 mL of 10×YNB, 100 μL of 500×biotin, and 5 mL of inoculum before use.

[0080] Table 3 Component content of BMMY medium

[0081]

[0082] Note: Before use, add 5 mL of 10×YNB, 100 μL of 500×biotin, and 0.5 mL of methanol to every 45 mL of BMMY medium.

[0083] The preparation method for 10×YNB is as follows: Dissolve 13.4g of YNB (yeast without amino nitrogen source, containing ammonium sulfate) in 100 mL of deionized water, filter to remove bacteria, and store at 4℃. The preparation method for 500×biotin is as follows: Weigh 20mg of Biotin, dissolve it in 100 mL of sterile water, filter to remove bacteria, and store at 4℃.

[0084] V. Collagen Fermentation Scale-up

[0085] Take the recombinant bacterial strain preserved in glycerol tubes at -80℃, streak it on a YPD solid plate, and incubate it in a 30℃ incubator for 2-3 days until single colonies grow. Pick 2-3 loops and inoculate them into a 500 mL Erlenmeyer flask containing 50 mL of YPD liquid medium, and incubate at 30℃ and 220 rpm for 16-18 h.

[0086] High-density fed-batch fermentation was performed in a 10-L fermenter containing 4.5 L of BSM medium. Initial fermentation parameters were as follows: temperature 30°C, pH 5.0, aeration rate 2.0 vvm, and turbine rotation speed 200 rpm. Ammonia was added to maintain the pH at 5.0. 500 mL of seed culture was inoculated into the fermenter medium. When dissolved oxygen dropped below 50%, the turbine rotation speed was increased by 50 rpm, eventually reaching 500-600 rpm.

[0087] After culturing for approximately 18-20 hours, dissolved oxygen rebounded, and OD exceeded 30. 50% (v / v) glycerol (containing 12 mL / L PTM1) was fed for 10 hours, with the concentrations being 13.5, 16.2, 19.2, 22.8, 27.2, and 32.4 mL / L / h for the first 6 hours, and 30 mL / L / h thereafter. The aeration rate was adjusted to 4.0 vvm, and the aeration speed was gradually increased to 800-900 rpm based on dissolved oxygen levels. After the dissolved oxygen rebounded to its highest point, the culture was further starved for 2 hours.

[0088] During the methanol induction phase, the fermentation temperature was lowered to 28℃, and methanol containing 12 mL / L PTM1 was used for fed-batch induction. The methanol flow rate was gradually increased from 1 mL / L / h to 6-7 mL / L / h. Then, methanol was continuously added at this constant flow rate.

[0089] VI. Cell Proliferation Experiment

[0090] After purifying the obtained collagen, human skin fibroblasts (i.e., fibroblasts) were induced and cultured. The effect of recombinant collagen on the proliferation of skin fibroblasts was then detected. This experimental method may include the following steps:

[0091] Human skin fibroblast primary cell culture conditions: OPTI-MEM medium, 15% serum, 1% triple antibody, 37℃, 6% CO2.

[0092] Human skin fibroblasts were plated and seeded at a density of 3 × 10⁶ cells / year. 3 Cells were seeded into 150 μL per well in 96-well plates and cultured for 24 hours. Five 96-well plates were then seeded.

[0093] Recombinant collagen (i.e., CL3.4 and CL3.8 proteins) was diluted with cell culture medium to final concentrations of 50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, and 800 μg / mL, and added to human skin fibroblast primary cells for 72 h. Cell proliferation was then assessed. Commercially available human collagen was used as a control.

[0094] A second aspect of the present invention provides a nucleic acid that encodes recombinant type III collagen as described above.

[0095] In one embodiment, the nucleic acid sequence is as shown in SEQ ID NO: 3 and / or SEQ ID NO: 4.

[0096] A third aspect of the present invention also provides an expression vector, wherein the expression vector is inserted with the above-mentioned nucleic acid, or the expression vector is capable of enabling the host cell to express the above-mentioned recombinant type III collagen after transfection.

[0097] In one embodiment, the expression vector includes any one of pPIC9K, pPICZαA, GAPZαA, pPICZαB, pPICZαC, GAPZαB, GAPZαC, and their modified vectors.

[0098] A fourth aspect of the present invention also provides a strain expressing recombinant type III collagen, wherein the strain is capable of expressing the above-mentioned recombinant type III collagen.

[0099] In one embodiment, the strain includes one of Pichia pastoris, Saccharomyces cerevisiae, and Escherichia coli.

[0100] In one embodiment, the Pichia pastoris includes one or more of SMD1168, KM71, KM71H, SMD1163, X33 and GS115.

[0101] The fifth aspect of the present invention also proposes the use of recombinant type III collagen as described in the first aspect, or nucleic acid as described in the second aspect, or expression vector as described in the third aspect, or strain expressing recombinant type III collagen as described in the fourth aspect, in the preparation of skin care products.

[0102] The embodiments of the present invention will be described in detail below with reference to specific examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0103] Example 1: Construction of expression plasmid

[0104] After screening and determining the sequences of two collagen proteins, codon optimization was performed, and the nucleotide sequences CL3.4 and CL3.8 (as shown in SEQ ID NO: 3 and SEQ ID NO: 4) were artificially synthesized. These fragments were amplified using primers CL3.4-F / R and CL3.8-F / R (see Table 4). The vector pPIC9K was digested with the rapid digestion enzymes EcoRI / NotI, and the amplified fragments CL3.4 and CL3.8 were inserted into the digested vector pPIC9K, respectively, to obtain the recombinant plasmids pPIC9K-CL3.4 and pPIC9K-CL3.8. Please refer to the diagrams of the recombinant plasmids pPIC9K-CL3.4 and pPIC9K-CL3.8. Figure 1 and Figure 2 .

[0105] Table 4 Primers required for engineered bacteria construction

[0106]

[0107] Example 2 Construction of strain

[0108] The expression vector constructed in Example 1 was incubated with SalI enzyme at 37°C for 1 h, and then purified and recovered to obtain a linearized vector. This linearized vector was integrated into the Pichia pastoris SMD1168 strain in a chassis cell via electroporation. The cells were then screened on auxotrophic medium, and single colonies were verified by colony PCR to obtain strains that had integrated the target vector, namely, the recombinant strains SMD1168 / pPIC9K-CL3.4 and SMD1168 / pPIC9K-CL3.8.

[0109] Example 3: Analysis of Collagen Strains in Shake Flask Culture

[0110] One loopful of colonies was inoculated into 6-7 mL of YPD liquid medium and incubated at 30°C and 220 rpm for 16 h. The colonies were then transferred to BMGY medium at a 10% inoculation rate and incubated at 30°C and 220 rpm for 24 h. After 24 h of incubation, the bacterial culture was transferred to a 50 mL sterile centrifuge tube and centrifuged at 5000 rpm for 8 min to collect the bacterial cells, discarding the supernatant. The cells were resuspended in 40 mL of physiological saline and washed twice, discarding the supernatant again. The cells were then resuspended in BMMY medium and incubated at 30°C and 220 rpm; 1% methanol was added every 24 h. Samples were taken at 72 h of induction for SDS-PAGE analysis.

[0111] Please see Figure 3 The obtained protein bands. From Figure 3 It can be seen that both CL3.4 and CL3.8 of this collagen were expressed.

[0112] Example 4: Optimization of chassis cells to enhance collagen expression

[0113] Four different chassis strains of Pichia pastoris SMD1168, KM71, SMD1163, and GS115 were selected. pPIC9K-CL3.4 and pPIC9K-CL3.8 were transformed into these strains, respectively, to obtain recombinant strains SMD1168 / CL3.4, KM71 / CL3.4, SMD1163 / CL3.4, and GS115 / CL3.4; SMD1168 / CL3.8, KM71 / CL3.8, SMD1163 / CL3.8, and GS115 / CL3.8, which can synthesize collagen CL3.4 and CL3.8, respectively.

[0114] The collagen expression was analyzed through shake-flask culture in Example 3. Figure 4 and Figure 5 .

[0115] exist Figure 4In the diagram, lane 1 shows the protein expression of strain SMD1168 / CL3.4, lanes 2-4 show the protein expression of strain KM71 / CL3.4, lanes 5-6 show the protein expression of strain SMD1163 / CL3.4, and lane 7 shows the protein expression of strain GS115 / CL3.4. Therefore, it can be concluded that for CL3.4 (nucleic acid SEQ ID NO: 3), the most suitable chassis strain for expression is Pichia pastoris KM71.

[0116] exist Figure 5 In the diagram, lane 1 shows the protein expression of strain SMD1168 / CL3.8, lanes 2-4 show the protein expression of strain KM71 / CL3.8, lanes 5-7 show the protein expression of strain SMD1163 / CL3.4, and lane 8 shows the protein expression of strain GS115 / CL3.4. Therefore, for CL3.8 (nucleic acid SEQ ID NO: 4), the most suitable chassis strains for expression are Pichia pastoris KM71 or Pichia pastoris GS115.

[0117] It should be noted that, with Figure 5 For example, lanes 2-4 correspond to three strains screened from the same plate, and their expression levels show very obvious differences; lanes 5-7 are also three strains screened from the same plate, and their expression levels also show very large differences. This result indicates that when selecting positive clones through strain transformation, it is necessary to select several strains for fermentation verification at the same time to avoid false positive events.

[0118] Example 5: Fermentation culture of collagen in a 10-L fermenter for production

[0119] The optimal strains KM71 / CL3.4 and KM71 / CL3.8 obtained through the above optimization were used. Recombinant strains stored in glycerol tubes at -80℃ were streaked onto YPD solid plates and incubated at 30℃ for 2-3 days until single colonies appeared. Two to three loops were then inoculated into 500 mL Erlenmeyer flasks containing 50 mL of YPD liquid medium and incubated at 30℃ and 220 rpm for 16-18 hours.

[0120] High-density fed-batch fermentation was performed in a 10-L fermenter containing 4.5 L of BSM medium. Initial fermentation parameters were as follows: temperature 30°C, pH 5.0, aeration rate 2.0 vvm, and turbine rotation speed 200 rpm. Ammonia was added to maintain the pH at 5.0. 500 mL of seed culture was inoculated into the fermenter medium. When dissolved oxygen dropped below 50%, the turbine rotation speed was increased by 50 rpm, eventually reaching 500-600 rpm.

[0121] After culturing for approximately 18-20 hours, dissolved oxygen rebounded, and OD exceeded 30. 50% (v / v) glycerol (containing 12 mL / L PTM1) was fed for 10 hours, with the concentrations being 13.5, 16.2, 19.2, 22.8, 27.2, and 32.4 mL / L / h for the first 6 hours, and 30 mL / L / h thereafter. The aeration rate was adjusted to 4.0 vvm, and the aeration speed was gradually increased to 800-900 rpm based on dissolved oxygen levels. After the dissolved oxygen rebounded to its highest point, the culture was further starved for 2 hours.

[0122] Entering the methanol induction stage, the fermentation temperature was lowered to 28℃, and methanol containing 12 mL / L PTM1 was used for fed-batch induction. The methanol flow rate was gradually increased from 1 mL / L / h to 6-7 mL / L / h. Then, methanol was continuously added at this constant flow rate, and the fermentation was terminated at an appropriate time point by real-time monitoring of the fermenter's status.

[0123] Real-time sampling and testing were performed during the fermentation process; the results can be found in [link to relevant documentation]. Figure 6 and Figure 7 .from Figure 6 and Figure 7 The results show that, compared with shake flask culture, the yields of collagen CL3.4 and CL3.8 were significantly increased in the fermenter. From 0 h to 72 h of fermentation, the yields of both collagen CL3.4 and CL3.8 accumulated continuously, and their expression levels remained stable in the later stages. Furthermore, the expression level of collagen CL3.8 was significantly higher than that of collagen CL3.4.

[0124] Example 6: Collagen Production Analysis

[0125] Preparation of standard solutions of different concentrations and methods for absorbance determination: After purification of the collagen sample obtained from fermentation, quantitative analysis was performed using the biuret method. A 10 mg / ml standard protein solution was prepared using standard crystalline bovine serum albumin (BSA). Twelve test tubes were divided into two groups, with 0, 0.2, 0.4, 0.6, 0.8, and 1.0 ml of the standard protein solution added respectively. Water was added to bring the volume to 1 ml, and then 4 ml of biuret reagent was added. After thorough mixing, the tubes were incubated at room temperature (20–25 °C) for 30 minutes, and absorbance was measured at 540 nm.

[0126] The average values ​​of the two sets of measurements were taken, and a standard curve was plotted with protein content on the x-axis and absorbance on the y-axis. The results are shown in the figure. Figure 8 Its standard curve is y = 0.0456x - 0.0138, R0 2 =0.9994, where x is the protein concentration and y is the absorbance at 540 nm.

[0127] Using the above preparation method and absorbance measurement method, 1 ml of collagen CL3.4 and CL3.8 were added to test tubes, 4 ml of biuret reagent was added, and the mixture was shaken thoroughly. After being placed at room temperature (20~25℃) for 30 minutes, the absorbance was measured at 540 nm.

[0128] The absorbance values ​​of CL3.4 and CL3.8 were 0.150 and 0.223, respectively. Based on the prepared standard curve, the contents of collagen CL3.4 and CL3.8 were calculated to be 3.6 g / L and 5.2 g / L, respectively.

[0129] Example 7: Collagen Cell Proliferation Experiment

[0130] After purifying the obtained collagen, human skin fibroblasts (i.e., fibroblasts) were induced and cultured. The effect of recombinant collagen on the proliferation of skin fibroblasts was then detected. This experimental method may include the following steps:

[0131] Human skin fibroblast primary cell culture conditions: OPTI-MEM medium, 15% serum, 1% triple antibody, 37℃, 6% CO2.

[0132] Human skin fibroblasts were plated and seeded at a density of 3 × 10⁶ cells / year. 3 Five cells were seeded into five 96-well plates, 150 μL per well, and cultured for 24 hours. Recombinant collagen (CL3.4 and CL3.8 proteins) was diluted with cell culture medium to final concentrations of 50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, and 800 μg / mL, and added to primary human skin fibroblasts for 72 hours. Cell proliferation was then assessed. Commercially available human collagen was used as a control.

[0133] The test results are as follows: Figure 9 As shown in the results, within the recombinant collagen concentration range of 50 μg / mL to 800 μg / mL, both collagen CL3.4 and CL3.8 effectively promoted cell proliferation; and the cell proliferation rate gradually increased with increasing concentration. Collagen CL3.4, in particular, showed a significantly stronger effect on cell proliferation. In the control group, however, the cell proliferation rate did not increase with increasing concentration. Therefore, this demonstrates that the collagen of this application can effectively promote cell proliferation and can be used in biomedicine, cosmetics, skincare products, and other fields, holding significant strategic importance for the promotion and further development of collagen synthesis.

[0134] The above description is merely an optional embodiment of the present invention and does not limit the patent scope of the present invention. All equivalent structural transformations made using the contents of the present invention's specification and drawings under the inventive concept of the present invention, or direct / indirect applications in other related technical fields, are included within the patent protection scope of the present invention.

Claims

1. A recombinant type III collagen, characterized in that, Its amino acid sequence is shown in any of the following: (1) SEQ ID NO: 1: GPPGPAGANGAPGLRGGAGEPGKNGAKGEPGPRGERGEAGIPGVPGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAP; (2) SEQ ID NO: 2: GQPGEKGSPGAQGPPGAPGPLGIAGITGARGLAGPPGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGLPGLAGTAGEPGRDGNPGSDGLPGRDGSPGGKGDRGENGSPGAPGAPGHPGP PGPVGPAGKSGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPRGPVGSGPPGKDGTSGHPGPIGPPGPRGNRGERGSE.

2. A nucleic acid, characterized in that, The nucleic acid encodes the recombinant type III collagen as described in claim 1.

3. The nucleic acid as described in claim 2, characterized in that, The nucleic acid sequence is shown in SEQ ID NO: 3 or SEQ ID NO: 4, wherein SEQ ID NO: 3 is as follows: GGTCCCCCAGGTCCAGCTGGTGCTAACGGAGCCCCAGGTCTGAGAGGTGGTGCCGGTGAACCAGGAAAAAACGGTGCTAAGGGTTGAGCCTGGTCCAAGAGGAGAGAGAGGTGAAGCAGGTATTCCTGGTGTTCCAGGTGCTAAGGGAGA AGATGGTAAAGATGGATCTCCAGGTGAACCCGGTGCCAACGGTTTACCAGGTGCTGCCGGAGAAAGAGGAGCACCAGGTTTTAGAGGTCCAGCTGGTCCAAACGGTATTCCAGGAGAAAAGGGACCAGCAGGAGAAAGAGGTGCTCCC; SEQ ID NO: 4 is as follows: .

4. An expression carrier, characterized in that, The expression vector is inserted with the nucleic acid as described in claim 2 or 3, or the expression vector is capable of causing the host cell to express the recombinant type III collagen as described in claim 1 after transfection.

5. The expression vector as described in claim 4, characterized in that, The expression vector includes any one of pPIC9K, pPICZαA, pGAPZαA, pPICZαB, pPICZαC, pGAPZαB, pGAPZαC, and their modified vectors.

6. A bacterial strain expressing recombinant type III collagen, characterized in that, The strain is capable of expressing the recombinant type III collagen as described in claim 1.

7. The strain expressing recombinant type III collagen as described in claim 6, characterized in that, The strains include any one of Pichia pastoris, Saccharomyces cerevisiae, and Escherichia coli.

8. The strain expressing recombinant type III collagen as described in claim 7, characterized in that, The Pichia pastoris includes one or more of SMD1168, KM71, KM71H, SMD1163, X33 and GS115.

9. The use of the recombinant type III collagen as described in claim 1 in the preparation of skin care products.

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