Preparation method and application of CHO (Chinese hamster ovary) cell expression triple helix structure recombinant humanized collagen

Abstract

The invention discloses a preparation method and application of CHO cell expression triple helix structure recombinant humanized collagen, and relates to the technical field of biology. The preparation method comprises the following steps: fermenting and purifying recombinant engineering cells to obtain the recombinant humanized collagen with the triple-helix structure; the construction method of the recombinant engineering cell comprises the following steps: transforming a recombinant plasmid for expressing alpha1 (III) T1 and a recombinant plasmid for expressing hP4H alpha1 and hP4HB into a host cell, and constructing to obtain the recombinant engineering cell. The recombinant humanized collagen with the triple helix structure and high proline hydroxylation rate can be prepared by utilizing the preparation method, can be widely applied to the fields of cosmetics, medical cosmetology, medical instruments, biomedical materials and the like, and has important market application value.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a preparation method and application of CHO cells expressing triple-helix structure recombinant humanized collagen. Background Art

[0002] Collagen is the most abundant protein in the body and the main component of the extracellular matrix (ECM). It plays an important role in maintaining the normal physiological functions and damage repair of cells, tissues and organs. In terms of molecular structure, each collagen peptide chain is mainly composed of Gly-XY (X, Y are any amino acid residues other than Gly) triplet repeats. This unique structure is necessary for the formation of the advanced structure of collagen fibers. It determines the excellent biocompatibility and low immunogenicity of collagen, and has been widely used in the pharmaceutical, health care and cosmetics industries.

[0003] In the prior art, collagen can be divided into two categories: animal-derived and recombinant collagen. Animal-derived collagen has a higher risk of immunogenicity. Recombinant collagen refers to the production of collagen by fermentation expression after human collagen gene is subjected to specific sequence design, enzyme cutting and splicing, and connected to a vector through genetic engineering technology. Compared with animal sources, recombinant collagen has the advantages of higher biocompatibility, lower immunogenicity, better processability (such as water solubility and emulsification properties), and no cytotoxicity. At present, the recombinant expression of collagen mainly uses Escherichia coli and yeast as host bacteria, but the recombinant collagen obtained by Escherichia coli expression has the problem of difficult purification and the risk of high endotoxin content. In addition, some amino acid sequence fragments in natural collagen are expressed by Escherichia coli or yeast systems without hydroxylation modification, and almost no higher-level structure of protein. Although expression systems such as Escherichia coli and yeast are more common, due to the high consistency of mammalian cell and human cell structures, the collagen expressed by the mammalian cell expression system has the advantages of high protein activity, closer to natural protein and correct higher-level structure.

[0004] The present invention intends to express and prepare recombinant humanized collagen with a high proline hydroxylation rate and a triple helix structure by adopting a CHO cell expression system that is closer to humans. Summary of the invention

[0005] The purpose of the present invention is to provide a preparation method of recombinant humanized collagen with triple helical structure expressed by CHO cells and its application, so as to solve the problems existing in the above-mentioned prior art. The preparation method can be used to prepare recombinant humanized collagen with triple helical structure and high proline hydroxylation rate, which can be widely used in the fields of cosmetics, medical cosmetology, medical devices, biomedical materials, etc., and has important market application value.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a method for constructing a recombinant engineering cell expressing triple helical recombinant humanized collagen, comprising the steps 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 engineering cell;

[0008] The amino acid sequences of the α1(III)T1, the hP4Hα1 and the hP4HB are shown in SEQ ID NOs. 1-3, respectively;

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

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

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

[0012] Furthermore, the recombinant plasmid expressing hP4Hα1 and hP4HB comprises a hP4Hα1 encoding gene and a 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 comprises the following steps:

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

[0017] The hP4HB coding gene was connected to the expression vector PEE6.4 to construct the recombinant plasmid PEE6.4-hP4HB;

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

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

[0020] The present invention also provides the use of the above-mentioned recombinant engineering cells in the preparation of triple helical structure recombinant humanized collagen.

[0021] The present invention also provides a method for preparing a triple-helical recombinant humanized collagen expressed by CHO cells, comprising the steps of obtaining the triple-helical recombinant humanized collagen by fermentation and purification using the above-mentioned recombinant engineering cells.

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

[0023] The present invention also provides the use of the triple helical structure recombinant humanized collagen in the preparation of functional biomaterials, cosmetics or medical devices.

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

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

[0026] The triple helical type III recombinant human collagen prepared by the present invention can be widely used in the fields of cosmetics, medical cosmetology, medical devices, biomedical materials, etc., and has important market application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

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

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

[0030] Figure 3 is the map of the recombinant plasmid PEE6.4-hP4HB;

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

[0032] Figure 5 This is a Western Blot detection image of α1(Ⅲ)T1 expression supernatant;

[0033] Figure 6 This is a Western Blot detection diagram of hP4Hα1 and hP4HB protein expression;

[0034] Figure 7 This is the circular dichroism detection result diagram;

[0035] Figure 8 This is the result of transmission electron microscopy. DETAILED DESCRIPTION

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

[0037] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0038] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0039] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

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

[0041] Example 1

[0042] 1. Sequence synthesis

[0043] The full-length sequence of type III collagen α1 chain was optimized to obtain the truncated variant sequence of the α1 chain functional region α1(III)T1 (amino acid sequence as shown in SEQ ID NO.1). The nucleic acid sequence SEQ ID NO.4 was obtained by reverse translation according to the Pichia codon table and optimization of 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, among which 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. Construction of recombinant expression vector:

[0053] The α1(Ⅲ)T1 DNA sequence (SEQ ID NO.4) was connected to the expression vector PEE12.4, the hP4Hα1 DNA sequence (SEQ ID NO.5) was connected to 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 synthetase) as a screening marker), and the hP4HB DNA sequence (SEQ ID NO.6) was connected to the expression vector PEE6.4, to construct the recombinant plasmid PEE12.4-α1(Ⅲ)T1 expressing α1(Ⅲ)T1, the recombinant plasmid PEE12.4-Puro-hP4Hα1 expressing hP4Hα1 and the recombinant plasmid PEE6.4-hP4HB expressing hP4HB.

[0054] The recombinant plasmids PEE12.4-α1(Ⅲ)T1, PEE12.4-Puro-hP4Hα1 and PEE6.4-hP4HB were transformed into competent Escherichia coli DH5α, and positive clones were screened on LB resistance plates containing Amp. The recombinant plasmids were extracted and sequenced to verify their correctness. Figure 1-Figure 3 shown.

[0055] Take 3 μg of recombinant plasmids PEE12.4-Puro-hP4Hα1 and PEE6.4-hP4HB, double digest with NotⅠ and BamHⅠ, digest at 37℃ for 1h, and recover using SanPrep column DNA gel recovery kit. Connect the two recovered bands with T4DNALigase. After connection, transfer to DH5α competent strain, screen positive clones on LB resistance plate containing Amp, extract recombinant plasmids for sequencing identification, verify correctness, and obtain recombinant plasmid PEE12.4-Puro-hP4Hα1-hP4HB. The relevant plasmid map is shown in Figure 4 .

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

[0057] Take 50 μg of the recombinant plasmid PEE12.4-α1(Ⅲ)T1, cut it with PvuⅠ (37℃ for 1h to linearize it, and then use the PCR product purification kit to recover the linearized plasmid. The linearized plasmid was electrotransformed into empty host CHO-K1 cells, and the electrotransformed cells were transferred into a T175 culture flask, placed in a 37℃, 5% carbon dioxide incubator, and allowed to stand for 48h. After 48 hours, the cells in the culture flask were gently blown off, the supernatant was removed by centrifugation, and the pressure medium (basal medium + 25μM L-MSX) (L-MSX is L-methionine sulfoximide), the cell suspension was inoculated into a 24-well plate, placed in a 37°C, 5% carbon dioxide incubator for static culture, and the cell medium was changed every 7 days. When the medium was changed for the fourth time, the cell supernatant of each well was collected for Elisa detection, and the 10 cell pools with the best growth conditions were selected. The 10 cell pools were screened by flow-batch culture, and the live cell density and viability were recorded every day. The supernatant was harvested on the seventh day for Elisa detection. The three cell pools with the highest OD were selected.

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

[0059] The above cells were cultured to the logarithmic growth phase, 0.8 cell / well / 200μL was plated, and the monoclonal wells were marked under a microscope and grown for 14 days. The monoclonal cells were removed from the wells and transferred to 24-well plates for expansion culture. After the cells covered the bottom of the wells, ELISA detection was performed. Monoclonal cell lines with higher OD values ​​were selected for expansion culture, and cell lines with poor growth were eliminated. 11 cell lines were screened by flow-batch culture, and the live cell density and viability were recorded every day. The supernatant was harvested on the seventh day for Western Blot detection. The Western Blot detection results of the expression supernatant are shown as follows: Figure 5 As shown, 7 monoclonal cell lines successfully and efficiently secreted and expressed the full-length sequence of α1(Ⅲ)T1 recombinant collagen.

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

[0061] Take 50 μg of the recombinant plasmid PEE12.4-Puro-hP4Hα1-hP4HB, digest it with PvuⅠ at 37℃ for 1h 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 screened in step 4. The electroporated cells were transferred into a T175 culture flask, placed in a 37° C., 5% carbon dioxide incubator, and allowed to stand for 48 hours.

[0063] After 48 hours, the cells in the culture flask were gently blown down, the supernatant was removed by centrifugation, and the cells were resuspended in pressure culture medium (basal culture medium + 30μM L-MSX + 4μg / mL Puromycin), and the cell density was adjusted to 80,000 cells / 600μL. The cell suspension was inoculated into a 24-well plate and placed in a 37°C, 5% carbon dioxide incubator for static culture. The cell medium was changed every 2 days, and the cell viability was tested every day. When all negative cells died, the Puromycin concentration was reduced to 2μg / mL, and the cell viability was waited for to recover and fill 24 wells. The cells were expanded and cultured, and the cell strain 5D-053 with the best growth condition was selected. The cells were taken for Western Blot detection, and the results were as follows: Figure 6 As shown. Figure 6 As can be seen from the results, hP4Hα1 and hP4HB can 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] According to the monoclonal cell line construction method described in step 4, 5D-053 was screened for monoclonal cells to obtain a high-yield monoclonal strain, and Fed-Batch fermentation culture was carried out in a bioreactor. After the fermentation, the bioreactor fermentation liquid was collected and centrifuged at 4°C to remove the precipitate. The supernatant was filtered through a 0.45 μm filter membrane again to keep the original liquid clear.

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

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

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

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

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

[0071] (6) Washing: Wash with 20 mM imidazole for 2 column volumes at a flow rate of 30 mL / min. Add 1 bottle per column volume.

[0072] (7) Elution: 250 mM imidazole for 5 column volumes, flow rate 30 mL / min, and one bottle for each column volume.

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

[0074] (9) Equilibration: 15 column volumes of ddH2O, flow rate 30 mL / min.

[0075] (10) Storage: Rinse with 20% ethanol for 3 column volumes and store the filler in 20% ethanol.

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

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

[0078] The α1(Ⅲ)T1 freeze-dried powder was reconstituted and then acid hydrolyzed. The sample was analyzed by liquid chromatography-mass spectrometry. The peak area of ​​the targeted data was calculated using MassLynx quantitative software. The identification result was obtained using the standard curve method. The results showed that the hydroxyproline content reached more than 30% of the total proline.

[0079] The α1(Ⅲ)T1 freeze-dried powder was dissolved in water and tested by circular dichroism spectrometer. The test results are as follows Figure 7 As shown, it is consistent with the circular dichroism spectral characteristics of triple-helical collagen.

[0080] The α1(Ⅲ)T1 freeze-dried powder was negatively stained and then tested by transmission electron microscopy. The test results are as follows Figure 8 Shown are the typical structural features of collagen microfibrils with alternating light and dark characteristics.

[0081] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for constructing a recombinant engineered cell expressing triple helical recombinant humanized collagen, characterized in that: The method comprises the steps of transforming a host cell with a recombinant plasmid expressing α1(III)T1 and a recombinant plasmid expressing hP4Hα1 and hP4HB to construct the recombinant engineered cell; The amino acid sequences of the α1(III)T1, the hP4Hα1 and the hP4HB are shown in SEQ ID NOs. 1-3, respectively; The host cell is CHO-K1 cell.

2. The construction method according to claim 1, characterized in that: The recombinant plasmid of α1(III)T1 contains the α1(III)T1 encoding gene, and the nucleotide sequence of the α1(III)T1 encoding gene is shown in SEQ ID NO.

4.

3. The construction method according to claim 2, characterized in that: The recombinant plasmid of α1(III)T1 is constructed by connecting the α1(III)T1 coding gene into the expression vector PEE12.

4.

4. The construction method according to claim 1, characterized in that: The recombinant plasmid expressing hP4Hα1 and hP4HB comprises a hP4Hα1 encoding gene and a 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.

5. The construction method according to claim 4, characterized in that: The method for constructing the recombinant plasmid expressing hP4Hα1 and hP4HB comprises the following steps: The hP4Hα1 encoding gene is connected to the PEE12.4 expression vector carrying an antibiotic selection marker to construct a recombinant plasmid PEE12.4-Puro-hP4Hα1; The hP4HB coding gene was connected to the expression vector PEE6.4 to construct the recombinant plasmid PEE6.4-hP4HB; The recombinant plasmid PEE12.4-Puro-hP4Hα1 and the recombinant plasmid PEE6.4-hP4HB were double-digested with NotⅠ and BamHI, and then ligated to obtain the recombinant plasmid expressing hP4Hα1 and hP4HB.

6. A recombinant engineered cell expressing triple-helical recombinant humanized collagen constructed according to the construction method according to any one of claims 1 to 5.

7. Use of the recombinant engineered cell as claimed in claim 6 in the preparation of triple helical recombinant humanized collagen.

8. A method for preparing a triple-helical recombinant humanized collagen expressed by CHO cells, characterized in that: The method comprises the step of obtaining the triple helical recombinant humanized collagen by fermentation and purification using the recombinant engineered cells as described in claim 6.

9. A recombinant humanized collagen with a triple helix structure prepared according to the preparation method of claim 8.

10. Use of the triple helical recombinant humanized collagen as claimed in claim 9 in the preparation of functional biomaterials, cosmetics or medical devices.

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