A method for preparing recombinant humanized collagen with a triple-helix structure expressed in CHO cells and its application.

Abstract

This invention discloses a method for preparing recombinant humanized collagen with a triple-helix structure expressed in CHO cells and its applications, relating to the field of biotechnology. The preparation method includes the step of purifying the triple-helix recombinant humanized collagen through fermentation using recombinant engineered cells; the construction method of the recombinant engineered cells includes the step of transforming host cells with a recombinant plasmid expressing α1(Ⅲ)T1 and a recombinant plasmid expressing hP4Hα1 and hP4HB to construct the recombinant engineered cells. This preparation method can produce recombinant humanized collagen with a high proline hydroxylation rate and a triple-helix structure, which can be widely used in cosmetics, medical aesthetics, medical devices, biomedical materials, and other fields, and has significant market application value.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for preparing recombinant humanized collagen with a triple helix structure expressed in CHO cells and its application. Background Technology

[0002] Collagen is the most abundant protein in the body and a major component of the extracellular matrix (ECM), playing a vital role in maintaining the normal physiological functions of cells, tissues, and organs, as well as in repairing damage. Structurally, each collagen peptide chain is primarily composed of repeating Gly-XY (where X and Y are any amino acid residues other than Gly) triplet sequences. This unique structure is essential for the formation of higher-order collagen fiber structures, determining collagen's excellent biocompatibility and low immunogenicity, and leading to its widespread application in the pharmaceutical, health product, and cosmetic industries.

[0003] In existing technologies, collagen can be divided into two main categories: animal-derived collagen and recombinant collagen. Animal-derived collagen carries a higher risk of immunogenicity. Recombinant collagen refers to collagen produced through genetic engineering techniques, where the human collagen gene is designed with specific sequences, digested and spliced ​​with enzymes, ligated into a vector, and then transferred into engineered cells for fermentation expression. Compared to animal-derived collagen, recombinant collagen has advantages such as higher biocompatibility, lower immunogenicity, better processability (e.g., water solubility and emulsification properties), and no cytotoxicity. Currently, recombinant collagen expression mainly uses *E. coli* and yeast as host bacteria. However, recombinant collagen obtained through *E. coli* expression faces challenges in purification and carries a risk of high endotoxin content. Furthermore, expressing only partial amino acid sequences of natural collagen using *E. coli* or yeast systems lacks hydroxylation modification and almost completely lacks the higher-order structure of the protein. Although expression systems such as *E. coli* and yeast are relatively common, due to the high degree of similarity between mammalian and human cell structures, collagen expressed through mammalian cell expression systems has advantages such as high protein activity, closer resemblance to natural proteins, and correct higher-order structure.

[0004] This invention aims to express and prepare recombinant humanized collagen with a high proline hydroxylation rate and a triple helix structure by using a CHO cell expression system that is closer to human cells. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing recombinant humanized collagen with a triple-helix structure expressed in CHO cells and its application, thereby solving the problems existing in the prior art. This preparation method can produce recombinant humanized collagen with a high proline hydroxylation rate and a triple-helix structure, which can be widely used in cosmetics, medical aesthetics, medical devices, biomedical materials, and other fields, and has significant market application value.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a method for constructing recombinant engineered cells expressing triple-helix recombinant humanized collagen, comprising the steps of transforming host cells with a recombinant plasmid expressing α1(Ⅲ)T1 and a recombinant plasmid expressing hP4Hα1 and hP4HB to construct the recombinant engineered cells;

[0008] The amino acid sequences of α1(Ⅲ)T1, hP4Hα1 and hP4HB are shown in SEQ ID NO.1-3, respectively;

[0009] The host cell is a CHO-K1 cell.

[0010] Furthermore, the recombinant plasmid T1 of α1(Ⅲ)T1 contains the α1(Ⅲ)T1 coding gene, and the nucleotide sequence of the α1(Ⅲ)T1 coding gene is shown in SEQ ID NO.4.

[0011] Furthermore, the recombinant plasmid of α1(Ⅲ)T1 is constructed by ligating the α1(Ⅲ)T1 encoding gene into the expression vector PEE12.4.

[0012] Furthermore, the recombinant plasmid expressing hP4Hα1 and hP4HB contains the hP4Hα1 encoding gene and the hP4HB encoding gene;

[0013] The nucleotide sequence of the hP4Hα1 encoding gene is shown in SEQ ID NO.5;

[0014] The nucleotide sequence of the hP4HB encoding gene is shown in SEQ ID NO.6.

[0015] Furthermore, the method for constructing the recombinant plasmid expressing hP4Hα1 and hP4HB includes the following steps:

[0016] The hP4Hα1 encoding gene was ligated into the expression vector PEE12.4-Puro to construct the recombinant plasmid PEE12.4-Puro-hP4Hα1;

[0017] The hP4HB encoding gene was ligated into the expression vector PEE6.4 to construct the recombinant plasmid PEE6.4-hP4HB.

[0018] The recombinant plasmids PEE12.4-Puro-hP4Hα1 and PEE6.4-hP4HB were digested with NotⅠ and BamHI, and then ligated to obtain the recombinant plasmids expressing hP4Hα1 and hP4HB.

[0019] The present invention also provides a recombinant engineered cell expressing a triple-helix recombinant humanized collagen, constructed according to the above-described construction method.

[0020] The present invention also provides the application of the above-mentioned recombinant engineered cells in the preparation of triple-helix recombinant humanized collagen.

[0021] The present invention also provides a method for preparing recombinant humanized collagen with a triple helix structure expressed in CHO cells, comprising the step of obtaining the recombinant humanized collagen with a triple helix structure by fermentation and purification using the above-mentioned recombinant engineered cells.

[0022] The present invention also provides a triple-helix recombinant humanized collagen prepared according to the above preparation method.

[0023] The present invention also provides the application of the above-mentioned triple-helix recombinant humanized collagen in the preparation of functional biomaterials, cosmetics or medical devices.

[0024] The present invention discloses the following technical effects:

[0025] This invention uses a CHO cell expression system that is closer to humans to construct a CHO-K1 cell line that co-expresses α1(Ⅲ) chain truncated variant (abbreviated as α1(Ⅲ)T1), hP4Hα1 and hP4HB, and realizes post-translational modification of recombinant collagen, which can prepare recombinant humanized collagen with a triple helix structure and high proline hydroxylation rate.

[0026] The triple-helix type III recombinant human collagen prepared by this invention can be widely used in cosmetics, medical aesthetics, medical devices, biomedical materials and other fields, and has important market application value. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments 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 these drawings without creative effort.

[0028] Figure 1 The spectrum of the recombinant plasmid PEE12.4-α1(Ⅲ)T1;

[0029] Figure 2 The spectrum of the recombinant plasmid PEE12.4-Puro-hP4Hα1;

[0030] Figure 3 The image shows the recombinant plasmid PEE6.4-hP4HB.

[0031] Figure 4 The spectrum of the recombinant plasmid PEE12.4-Puro-hP4Hα1-hP4HB;

[0032] Figure 5 Western blot analysis of α1(Ⅲ)T1 expression supernatant;

[0033] Figure 6 Western blot analysis of hP4Hα1 and hP4HB protein expression;

[0034] Figure 7 This is a graph showing the results of circular dichroism chromatogram detection.

[0035] Figure 8 This is a diagram showing the results of a transmission electron microscope examination. Detailed Implementation

[0036] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0037] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0038] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0039] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0040] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0041] Example 1

[0042] 1. Sequence synthesis

[0043] The full-length sequence of the type III collagen α1 chain was optimized to obtain the truncated variant sequence α1(Ⅲ)T1 (amino acid sequence shown in SEQ ID NO.1). The nucleic acid sequence SEQ ID NO.4 was obtained by reverse translation according to the Pichia pastoris codon table, optimizing GC content and secondary structure.

[0044] SEQ ID NO.1:

[0045] MMSFVQKGSWLLLALLHPTIILAQ HHHHHH QYDSYDVKSGVAVG ENLYFQG AGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGPLGIAGITGARGLAGPPGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGLPGLAGTAGEPGRDGNPGSDGL PGRDGSPGGKGDRGENGSPGAPGAPGHPGPPGPVGPAGKSGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPA GNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGPLGIAGITGARGLAGPPGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGLPGLAGTAGEPGRDGNPGSDGLPG RDGSPGGKGDRGENGSPGAPGAPGHPGPPGPVGPAGKSGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPCCG ENLYFQGHHHHHHGVGAAAIAGIGGEKAGGFAPYYGDEPMDFKINTDEIMTSLKSVNGQIESLISPDGSRKNPARNCRDLKFCHPELKSGEYWVDPNQGCKLDAIKVFCNMETGETCISANPLNVPRKHWWTDSSAEKKHVWFGESMDGGFQFSYGNPELPEDVLDVHLAFLRLLSSRASQNITYHCKNSIAYMDQASGNVKKALKLMGSNEGEFKAEGNSKFTYTVLEDGCTKHTGEWSKTVFEYRTRKAVRLPIVDIAPYDIGGPDQEFGVDVGPVCFL。

[0046] The amino acid sequence of human prolyl hydroxylase hP4Hα1 is shown in SEQ ID NO.2; the amino acid sequence of human prolyl hydroxylase hP4HB is shown in SEQ ID NO.3.

[0047] SEQ ID NO.2:

[0048] MIWYILIIGILLPQSLAHPGFFTSIGQMTDLIHTEKDLVTSLKDYIKAEEDKLEQIKKWAEKLDRLTSTATKDPEGFVGHPVNAFKLMKRLNTEWSELENLVLKDMSDGFISNLTIQRQYFPNDEDQVGAAKALLRLQDTYNLDTDTISKGNLPGVKHKSFLTAEDCFELGKVAYTEADYYHTELWMEQALRQLDEGEISTIDKVSVLDYLSYAVYQQGDLDKALLLTKKLLELDPEHQRANGNLKYFEYIMAKEKDVNKSASDDQSDQKTTPKKKGVAVDYLPERQKYEmLCRGEGIKMTPRRQKKLFCRYHDGNRNPKFILAPAKQEDEWDKPRIIRFHDIISDAEIEIVKDLAKPRLRRATISNPITGDLETVHYRISKSAWLSGYENPVVSRINMRIQDLTGLDVSTAEELQVANYGVGGQYEPHFDFARKDEPDAFKELGTGNRIATWLFYMSDVSAGGATVFPEVGASVWPKKGTAVFWYNLFASGEGDYSTRHAACPVLVGNKWVSNKWLHERGQEFRRPCTLSELE。

[0049] SEQ ID NO.3:

[0050] MLRRALLCLAVAALVRADAPEEEDHVLVLRKSNFAEALAAHKYLLVEFYAPWCGHCKALAPEYAKAAGKLKAEGSEIRLAKVDATEESDLAQQYGVRGYPTIKFFRNGDTASPKEYTAGREADDIVN WLKKRTGPAATTLPDGAAAESLVESSEVAVIGFFKDVESDSAKQFLQAAEAIDDIPFGITSNSDVFSKYQLDKDGVVLFKKFDEGRNNFEGVTKENLLDFIKHNQLPLVIEFTEQTAPKIFGGEIK THILLFLPKSVSDYDGKLSNFKTAAESFKGKILFIFIDSDHTDNQRILEFFGLKKEECPAVRLITLEEEMTKYKPESEELTAERITEFCHRFLEGKIKPHLMSQELPEDWDKQPVKVLVGKNFEDVA FDEKKNVFVEFYAPWCGHCKQLAPIWDKLGETYKDHENIVIAKMDSTANEVEAVKVHSFPTLKFFPASADRTVIDYNGERTLDGFKKFLESGGQDGAGDDDDLEDLEEAEEPDMEEDDDQKAVKDEL.

[0051] After the above optimization design, DNA sequences encoding α1(Ⅲ)T1, hP4Hα1 and hP4HB were synthesized respectively. The DNA sequence encoding α1(Ⅲ)T1 is shown in SEQ ID NO.4; the DNA sequence encoding hP4Hα1 is shown in SEQ ID NO.5; and the DNA sequence encoding hP4HB is shown in SEQ ID NO.6.

[0052] 2. Constructing a recombinant expression vector:

[0053] The α1(Ⅲ)T1 DNA sequence (SEQ ID NO.4) was ligated into the expression vector PEE12.4, the hP4Hα1 DNA sequence (SEQ ID NO.5) was ligated into the expression vector PEE12.4-Puro (PEE12.4-Puro was constructed by inserting the puro gene into the pEE12.4 vector to replace GS (glutamine synthase) as a selection marker), and the hP4HB DNA sequence (SEQ ID NO.6) was ligated into the expression vector PEE6.4, thereby constructing the recombinant plasmids PEE12.4-α1(Ⅲ)T1 expressing α1(Ⅲ)T1, PEE12.4-Puro-hP4Hα1 expressing hP4Hα1, and PEE6.4-hP4HB expressing hP4HB, respectively.

[0054] Recombinant plasmids PEE12.4-α1(Ⅲ)T1, PEE12.4-Puro-hP4Hα1, and PEE6.4-hP4HB were transformed into competent E. coli DH5α cells. Positive clones were screened on LB agar plates containing Amp, and the recombinant plasmids were extracted and sequenced for identification, confirming their correctness. Related plasmid maps are shown below. Figures 1-3 As shown.

[0055] 3 μg each of recombinant plasmids PEE12.4-Puro-hP4Hα1 and PEE6.4-hP4HB were digested with NotⅠ and BamHI, respectively, at 37℃ for 1 h. The DNA was then recovered using a SanPrep column-based DNA gel extraction kit. The two recovered bands were ligated using T4 DNA Ligase. After ligation, the DNA was transformed into DH5α competent cells. Positive clones were screened on LB accumulator plates containing Amp, and the recombinant plasmid was extracted and sequenced for identification. The recombinant plasmid PEE12.4-Puro-hP4Hα1-hP4HB was confirmed. The plasmid map is shown below. Figure 4 .

[0056] 3. Construction of a recombinant α1(Ⅲ)T1 cell pool

[0057] Take 50 μg of recombinant plasmid PEE12.4-α1(Ⅲ)T1, digest it with PvuI (37℃, 1 h) to linearize it, and then use a PCR product purification kit to recover the linearized plasmid. Electroporate the linearized plasmid into empty host CHO-K1 cells, transfer the electroporated cells into T175 culture flasks, and incubate them at 37℃ in a 5% CO2 incubator for 48 h. After 48 hours, gently blow off the cells from the culture flasks, centrifuge to remove the supernatant, and use pressure medium (basal medium + 25 μM). L-MSX (L-methionine sulfoxide imide) was resuspended in the cells, and the cell suspension was seeded into 24-well plates and incubated statically at 37°C in a 5% CO2 incubator. The medium was changed every 7 days. At the fourth medium change, the cell supernatant from each well was collected for ELISA analysis, and the 10 cell pools with the best growth were selected. These 10 cell pools were then subjected to fed-batch culture for further selection, with daily viable cell density and viability recorded. On day 7, the supernatant was harvested for ELISA analysis. The three cell pools with the highest OD were selected.

[0058] 4. Construction of recombinant α1(Ⅲ)T1 monoclonal cell line

[0059] The cells were cultured to the logarithmic growth phase and seeded at 0.8 cells / well / 200 μL. Single-clone wells were marked under a microscope and cultured for 14 days. Single-clone cells were then removed from the wells and transferred to 24-well plates for expansion culture. ELISA was performed after the cells had reached confluence with the bottom of the wells. Single-clone cell lines with high OD values ​​were selected for further expansion, while poorly growing cell lines were discarded. Eleven cell lines were subjected to fed-batch culture for selection. Viable cell density and viability were recorded daily. On day seven, the supernatant was harvested and Western blot analysis was performed. The Western blot results of the expression supernatant are shown below. Figure 5 As shown, seven monoclonal cell lines successfully and efficiently secreted the full-length sequence of α1(Ⅲ)T1 recombinant collagen.

[0060] 5. Construction of recombinant hP4Hα1-hP4HB cell pool

[0061] Take 50 μg of recombinant plasmid PEE12.4-Puro-hP4Hα1-hP4HB, digest it with PvuⅠ at 37℃ for 1 h to linearize it, and then use a PCR product purification kit to recover the linearized plasmid.

[0062] The linearized plasmid was electroporated into the 1F8 monoclonal cell line obtained in step 4. The electroporated cells were then transferred into T175 culture flasks and placed in a 37°C, 5% CO2 incubator for 48 hours.

[0063] Forty-eight hours later, the cells in the culture flask were gently aspirated, centrifuged to remove the supernatant, and resuspended in pressure medium (basal medium + 30 μM L-MSX + 4 μg / mL Puromycin). The cell density was adjusted to 80,000 cells / 600 μL. The cell suspension was seeded into 24-well plates and incubated at 37°C in a 5% CO2 incubator. The medium was changed every two days, and cell viability was checked daily. When all negative cells died, the Puromycin concentration was reduced to 2 μg / mL. After the cell viability recovered and the cells reached confluence in the 24 wells, the cells were expanded. The best-growing cell line, 5D-053, was selected. Western blot analysis was performed on the cells. The results are shown below. Figure 6 As shown. From Figure 6 The results showed that hP4Hα1 and hP4HB could be detected in the cell lysate of 5D-053, indicating that 5D-053 successfully and efficiently secreted the full-length sequences of hP4Hα1 and hP4HB proteins.

[0064] 6. Purification of recombinant α1(Ⅲ)T1

[0065] Following the monoclonal cell line construction method described in step 4, 5D-053 was screened for monoclonal cells to obtain high-yield monoclonal lines. Fed-Batch fermentation was carried out in a bioreactor. After the fermentation broth was completed, it was collected from the bioreactor, centrifuged at 4°C to remove the precipitate, and the supernatant was passed through a 0.45μm filter membrane again to keep the original solution clear.

[0066] (1) Column packing: 500mL NW ROSE TED FF (purchased from Suzhou Nanomicro Technology Co., Ltd.), medium-pressure glass chromatography column with a size of 50 / 400.

[0067] (2) Column cleaning: 5 column volumes of 0.5M sodium hydroxide were rinsed at a flow rate of 30 mL / min.

[0068] (3) Equilibration: Equilibrate 15 column volumes with 1×PBS at a flow rate of 30 mL / min.

[0069] (4) Sample loading: 10L sample, flow rate 30mL / min.

[0070] (5) Equilibration: Equilibrate 5 column volumes with 1×PBS at a flow rate of 30 mL / min.

[0071] (6) Washing: Wash two column volumes with 20mM imidazole at a flow rate of 30mL / min, and collect one bottle for each column volume.

[0072] (7) Elution: Elute 5 column volumes with 250mM imidazole at a flow rate of 30mL / min, and each column volume is collected in one vial.

[0073] (8) Column cleaning: Rinse 5 column volumes with 0.5M sodium hydroxide at a flow rate of 30 mL / min.

[0074] (9) Equilibrium: Equilibrium with ddH2O for 15 column volumes at a flow rate of 30 mL / min.

[0075] (10) Storage: Rinse three column volumes with 20% ethanol, and finally store the packing material in 20% ethanol.

[0076] (11) The eluent obtained was subjected to electrophoresis to obtain the recombinant α1(Ⅲ)T1 sample, which was dialyzed into ddH2O and then lyophilized for storage.

[0077] 7. Structural characterization of α1(Ⅲ)T1 lyophilized powder

[0078] After reconstitution of α1(Ⅲ)T1 lyophilized powder, acid hydrolysis was performed. The peak area of ​​the target data was calculated by liquid chromatography-mass spectrometry (LC-MS) using MassLynx quantitative software. The identification results were obtained by standard curve method. The results showed that the hydroxyproline content reached more than 30% of the total proline.

[0079] The α1(Ⅲ)T1 lyophilized powder was dissolved in water, and the results were analyzed using a circular dichroism spectrometer. The test results are as follows: Figure 7 As shown, it conforms to the circular dichroism spectral characteristics of triple helix collagen.

[0080] The lyophilized α1(Ⅲ)T1 powder was negatively stained and then examined by transmission electron microscopy. The test results are as follows: Figure 8 As shown, this illustrates the typical structural features of collagen microfibers with alternating light and dark areas.

[0081] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for constructing recombinant engineered cells expressing triple-helix recombinant humanized collagen, characterized in that, The procedure includes the step of transforming a recombinant plasmid expressing α1(Ⅲ)T1 and a recombinant plasmid expressing hP4Hα1 and hP4HB into a host cell to construct the recombinant engineered cell. The amino acid sequences of α1(Ⅲ)T1, hP4Hα1 and hP4HB are shown in SEQ ID NO.1-3, respectively; The host cell is a CHO-K1 cell; The recombinant plasmid expressing α1(Ⅲ)T1 contains the α1(Ⅲ)T1 encoding gene, the nucleotide sequence of which is shown in SEQ ID NO.4; The recombinant plasmid expressing α1(Ⅲ)T1 was constructed by ligating the α1(Ⅲ)T1 encoding gene into the expression vector PEE12.4; The recombinant plasmid expressing hP4Hα1 and hP4HB contains the hP4Hα1 encoding gene and the hP4HB encoding gene. The nucleotide sequence of the hP4Hα1 encoding gene is shown in SEQ ID NO.5; The nucleotide sequence of the hP4HB encoding gene is shown in SEQ ID NO.

6. The method for constructing the recombinant plasmid expressing hP4Hα1 and hP4HB includes the following steps: The hP4Hα1 encoding gene was ligated into the PEE12.4 expression vector carrying an antibiotic selection marker to construct the recombinant plasmid PEE12.4-Puro-hP4Hα1. The hP4HB encoding gene was ligated into the expression vector PEE6.4 to construct the recombinant plasmid PEE6.4-hP4HB. The recombinant plasmids PEE12.4-Puro-hP4Hα1 and PEE6.4-hP4HB were digested with NotⅠ and BamHI, and then ligated to obtain the recombinant plasmids expressing hP4Hα1 and hP4HB.

2. A recombinant engineered cell expressing a triple-helix recombinant humanized collagen, constructed according to the method described in claim 1.

3. The application of the recombinant engineered cells as described in claim 2 in the preparation of triple-helix recombinant humanized collagen.

4. A method for preparing recombinant humanized collagen with a triple-helix structure expressed in CHO cells, characterized in that, The method includes the step of obtaining the triple-helix recombinant humanized collagen by fermentation purification using the recombinant engineered cells described in claim 2.

5. A triple-helix recombinant humanized collagen prepared by the preparation method according to claim 4.

6. The application of the triple-helix recombinant humanized collagen as described in claim 5 in the preparation of functional biomaterials, cosmetics, or medical devices.

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