Recombinant Humanized Collagen and Its Preparation Method and Application

Abstract

The present invention relates to the field of genetic engineering technology, and specifically relates to recombinant humanized collagen, its preparation method and application. The present invention provides a method for co-expressing prolyl-4-hydroxylase and recombinant humanized collagen. The recombinant strain constructed by using the method provided by the present invention can successfully express recombinant humanized collagen with a triple helix structure, overcomes the difficulty of macromolecule synthesis, retains the biological activity of collagen, and lays a foundation for the development of the functional region of collagen.

Description

[0001] This application claims the priority of a Chinese patent application titled "Recombinant Humanized Collagen and Its Preparation Method and Application" with the application number 202310638280.8 and filed with the Chinese Patent Office on May 31, 2023. The entire content thereof is incorporated herein by reference. Technical Field

[0002] The present invention relates to the field of genetic engineering technology, and particularly to recombinant humanized collagen and its preparation method and application. Background Art

[0003] Collagen is the most abundant protein in the human body, accounting for 25% - 30% of the total protein. As the most abundant protein in the extracellular matrix, collagen maintains the integrity of cell structure and various physiological functions. Collagen is the main structural component of all connective tissues and is also present in the interstitial tissues of almost all parenchymal organs. In the past decade, people's understanding of collagen has been continuously increasing, and the members of the collagen family have also increased rapidly. Through research, it has been found that more than 20 different types of collagen have been discovered in the human body, which can be roughly divided into the following categories: fibril-forming collagen, basement membrane collagen, microfibril collagen, anchoring fibril, transmembrane region collagen, and incompletely characterized collagen. Type III collagen is a fibrillar collagen that is widely present in the skin and blood vessels of newborns. It can strengthen the strength and elasticity of microvessels, provide sufficient nutrients for cells, and maintain the fullness, smoothness, and luster of the skin. It is also known as baby protein. However, some studies have found that it has other functions. In tissues, the diameter of type III collagen fibers is smaller than that of type I collagen. When type I and type III collagens appear in the same collagen fiber, type III collagen is responsible for regulating the fibril diameter. Type III collagen is also present in adult cartilage. Some studies believe that during tissue healing, type III collagen acts as a modifier of the fiber network composed of type II collagen and other small collagens. In addition, type III collagen is the main structural component of hollow organs such as large blood vessels, uterus, and intestines. As an extracellular matrix protein, it maintains the shape and structure of the skin and tissues and organs. Type III collagen also interacts with platelets in the coagulation cascade reaction and is also an important signaling molecule for wound healing.

[0004] Currently, international research only focuses on the Gly-Xaa-Yaa recombinant region of type III collagen, or a segment of the amino acid sequence of type III collagen, or the a1 synthesis method of type III collagen, without developing the sequence of the specific functional region of type III collagen. The 411-518 sequence of the a1 chain in type III collagen is the active triple helix active site in COL3A1, which plays an important role in cell adhesion and ligand recombination. This sequence also contains the Glu-Lys-Gly and Glu-Arg-Gly triplets, enhancing the cell adhesion in this region. Moreover, the recombinant protein containing multiple tandem repeats of the triple helix fragment makes it have a more stable helical conformation or a more favorable ligand binding configuration, making the triple helix region highly flexible, which is conducive to ligand binding, cell membrane attachment and adhesion, or other biological activities. By constructing a human prolyl hydroxylase and co-expressing it with the specific functional region of type III collagen, humanized collagen was successfully synthesized and formed a correct triple helix structure, which can fill the gap in the international field where peptide segments cannot form a correct helix structure. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide recombinant humanized collagen and its preparation method and application. The present invention uses a eukaryotic expression system to co-express prolyl hydroxylase and a specific region of type III collagen to obtain recombinant humanized collagen.

[0006] The present invention provides recombinant humanized collagen, and its structure is X n Y;

[0007] wherein, X is a fragment from the 411th to 639th positions of the a1 chain of type III collagen, and Y is a fragment from the 1158th to 1199th positions of the a1 chain of type III collagen; 1≤n≤30.

[0008] The amino acid sequence of the humanized collagen described in the present invention includes any one of the following I to III:

[0009] (I), the amino acid sequence shown in SEQ ID NO: 1; or

[0010] (II), a sequence obtained by substituting, deleting, adding, and / or replacing one or more amino acids on the basis of the amino acid sequence shown in (I); or

[0011] (III), a sequence having a homology of more than 90% with the amino acid sequence shown in (I).

[0012] The present invention provides a preparation method of the recombinant humanized collagen, including the following steps:

[0013] S1. Construct a plasmid containing the coding nucleic acid of the fragment at positions 411 - 639 and the fragment at positions 1158 - 1199 of the α1 chain of type III collagen;

[0014] S2. Construct an expression vector for the prolyl - 4 - hydroxylase α subunit and / or β subunit;

[0015] S3. Co - transform the plasmid containing the coding nucleic acid of the fragment at positions 411 - 639 and the fragment at positions 1158 - 1199 of the α1 chain of type III collagen and the expression vector of prolyl - 4 - hydroxylase into a host cell to obtain a co - expression strain;

[0016] S4. Ferment and culture the co - expression strain to obtain the recombinant humanized collagen.

[0017] Specifically, in step S1, the plasmid containing the coding nucleic acid of the fragment at positions 411 - 639 and the fragment at positions 1158 - 1199 of the α1 chain of type III collagen sequentially includes a backbone vector and a promoter, the nucleic acid encoding the recombinant humanized collagen as claimed in claim 1 or 2, and a terminator;

[0018] The promoter is selected from any one of the T7 promoter, sCMV promoter, Lac promoter, tac promoter, IPL promoter, araB promoter, AOX1 promoter, trc promoter, or trp promoter;

[0019] The terminator is selected from any one of the T7 terminator, rrnB T1 terminator, AOX1 terminator, ρ - independent terminator, or ρ - dependent terminator.

[0020] In some specific embodiments, the plasmid containing the coding nucleic acid of the fragment at positions 411 - 639 and the fragment at positions 1158 - 1199 of the α1 chain of type III collagen sequentially includes the PcDNA3.1 vector or pPICZα series vectors, the AOX1 promoter, the nucleic acid encoding the recombinant humanized collagen as claimed in claim 1 or 2, and the AOX1 terminator.

[0021] Specifically, in step S2, the expression vector for the prolyl - 4 - hydroxylase α subunit and / or β subunit includes the nucleic acid encoding prolyl - 4 - hydroxylase and the PcDNA3.1 vector.

[0022] Specifically, the host cell in step S3 is selected from any one or more of Escherichia coli, Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae, and mammalian cells. In some specific embodiments, the host cell is Pichia pastoris and / or CHO cells.

[0023] The present invention provides an expression unit, which includes a promoter, the nucleic acid encoding collagen as described in the present invention, and a terminator.

[0024] Furthermore, the expression unit includes an expression unit composed of the nucleic acid of the present invention in a single or multiple tandem forms, the promoter, and the terminator, and the present invention does not make any limitation thereto.

[0025] The present invention also provides a transcription unit containing the nucleic acid or the expression module described above. The transcription unit refers to a DNA sequence starting from a promoter to the end of a terminator. Regulatory fragments may also be included on both sides or between the promoter and the terminator. The regulatory fragments may include a promoter, an enhancer, a transcription termination signal, a polyadenylation sequence, an origin of replication, a nucleic acid restriction site, and a homologous recombination site operably linked to the nucleic acid sequence, such as an enhancer of the promoter, a poly(A) signal, etc. The promoter in the present invention is selected from any one of a T7 promoter, an sCMV promoter, a Lac promoter, a tac promoter, an IPL promoter, an araB promoter, a trc promoter, or a trp promoter; the terminator is selected from any one of a T7 terminator, an rrnB T1 terminator, a rho-independent terminator, or a rho-dependent terminator.

[0026] Specifically, in some embodiments, the expression unit of the present invention may further include an enhancer, an intron, a cofactor, a transcription element, or other specific elements. For example, the expression unit of the present invention may include a promoter, an enhancer, a transcription element, any one of the nucleic acids encoding collagen of the present invention, and a terminator. The present invention does not make any limitation thereto, and any expression unit containing the nucleic acid encoding collagen of the present invention is within the protection scope of the present invention.

[0027] In some other embodiments, the expression unit of the present invention sequentially includes an AOX1 promoter, a nucleic acid encoding collagen, and an AOX1 terminator. Compared with other promoters and terminators, the AOX1 promoter and the AOX1 terminator used in the present invention have high specificity, can increase the transcription rate of genes, and accurately control the transcription termination of DNA.

[0028] The present invention provides an expression vector, which includes a backbone vector and the nucleic acid encoding collagen of the present invention; or includes a backbone vector and the expression unit of the present invention. The backbone vector includes pUC series vectors, pCAMBIA series vectors, pPICZα series vectors, pSC series vectors, pET series vectors. It may be a shuttle vector, a phage, or a viral vector, and the present invention does not make any limitation thereto. Through screening of the backbone vector, it is found that when the nucleic acid or the expression unit of the present invention is constructed onto a pPICZα series vector, its expression level is higher. Therefore, in the embodiments of the present invention, the backbone vector is preferably pPICZαB.

[0029] The expression vector described in the present invention refers to a nucleic acid vector, which is a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences or elements essential for the expression of the operably linked coding gene in a specific host organism. The nucleic acid sequences or elements necessary for expression in bacteria include promoters, ribosome binding sites, and possibly other sequences. The expression vectors described in the present invention include plasmid vectors, which can be linear or circular, and can be single-stranded or double-stranded, and the present invention does not limit this. The expression vector described in the present invention contains an expression unit of the collagen-encoding nucleic acid as described above. Further, the expression vector described in the present invention further includes an expression unit of the prolyl-4-hydroxylase-encoding nucleic acid.

[0030] If the prolyl-4-hydroxylase-encoding nucleic acid and the expression unit provided by the present invention are not in the same expression vector, the present invention also provides a plasmid combination, which includes the plasmid vector encoding the collagen nucleic acid described in the present invention and a recombinant plasmid containing the prolyl-4-hydroxylase-encoding nucleic acid. The recombinant plasmid containing the prolyl-4-hydroxylase-encoding nucleic acid includes the PcDNA3.1 vector, the pPICZα series vectors, and the nucleotide sequence shown in any one of SEQ ID NO: 2-6.

[0031] The prolyl-4-hydroxylase-encoding nucleic acid includes the nucleic acid of P4Hα and / or the nucleic acid of P4Hβ. The two nucleic acids can be in the same expression unit or in two expression units. When in the same expression unit, the nucleic acid encoding P4Hα and the nucleic acid encoding P4Hβ share the same promoter and terminator.

[0032] The nucleic acid encoding prolyl-4-hydroxylase is an optimized nucleic acid sequence with Pichia pastoris preference, its codon adaptation index increases, and the GC content is between 41% and 43%, which can be stably expressed in the host body. Compared with other coding nucleic acids of prolyl-4-hydroxylase, the nucleic acid provided by the present invention has a higher expression level and higher activity of the expression product in the eukaryotic expression system.

[0033] The present invention provides a host, which includes at least one of the following I) or II):

[0034] I), integrating the nucleic acid encoding collagen or the expression unit described in the present invention into the genome;

[0035] II), transfecting or transforming the expression vector or plasmid combination described in the present invention.

[0036] In the present invention, an expression vector or plasmid combination is transfected or transformed into a host; the transformation methods include: chemical transformation and electroporation; the transfection methods include calcium phosphate co-precipitation, artificial liposome method, viral transfection. The viral transfection includes adenovirus transfection, adeno-associated virus transfection, lentivirus transfection, etc. In the embodiments of the present invention, the plasmid combination enters the host by electroporation or chemical transfection.

[0037] Furthermore, the host in the present invention includes bacteria, fungi, viruses or animals. The bacteria include Gram-positive bacteria and Gram-negative bacteria; the Gram-positive bacteria include, but are not limited to, Escherichia coli. The fungi include molds, yeasts, and mushrooms; the yeasts include Saccharomyces cerevisiae, Saccharomyces cerevisiae, Pichia pastoris, and Candida spp. The viruses include, but are not limited to, adenovirus, adeno-associated virus, lentivirus, prion. The animals include humans, mice, rabbits, pigs, zebrafish, etc.

[0038] Specifically, in some embodiments, the host in the present invention is selected from any one or more of Escherichia coli, Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae, mammalian cells. Preferably, it is Pichia pastoris and CHO cells.

[0039] The present invention provides the application of the expression unit, expression vector, plasmid combination or host in the preparation of type III collagen.

[0040] The present invention provides a method for preparing recombinant humanized collagen, including fermenting and culturing the host cells of the present invention. Specifically, the successfully constructed recombinant strain is inoculated into YPD medium, the total culture volume is 3 L, the dissolved oxygen is maintained at about 40%, cultured for 18 - 96 h, and the cell supernatant is taken and lysed to obtain recombinant humanized collagen.

[0041] The present invention provides recombinant humanized collagen, which is obtained by the above preparation method.

[0042] The present invention also provides the application of the recombinant humanized collagen in products with repair function filling and support, drug sustained-release preparations, vaccine protectants and stabilizers or drugs. The products include foods, cosmetics, drugs or health products, etc.; the cosmetic raw materials include raw materials for whitening, anti-aging, repair, anti-wrinkle, firming skin, etc.; the drugs include drugs for treating diseases such as hemorrhoids, sclerosis, etc.

[0043] The recombinant humanized collagen provided by the present invention can also be used to prepare materials that can be used as tissue engineering materials and medical beauty materials, including filling tissue depressions, such as forehead wrinkles, crow's feet, nasolabial folds, neck wrinkles, and wrinkles on the whole body skin, repairing damaged skin, fading fine lines, whitening, anti-aging, and tightening the skin; for preparing biological scaffold materials, sustained and controlled release materials for drugs, products for treating skin problems, vaccine protectants or stabilizers, and hemostatic materials. Using this raw material as a wound dressing can repair damaged skin, promote cell adhesion, proliferation, and migration, thereby avoiding scar formation. Using this raw material as a hydrodermabrasion needle can promote collagen regeneration, whiten, and moisturize. When this raw material is compounded with artificial bone, it can improve biocompatibility and promote the growth and crawling of bone cells.

[0044] The present invention found the sequence 411-639 of the specific function of human type III collagen a1 chain, spliced the specific functional region repeatedly n times, 1≤n≤30, and then added the functional sequence 1158-1199 of type III collagen a1 chain. By co-expressing prolyl hydroxylase with the target protein, a stable target protein with a triple helix structure was obtained. This protein is beneficial to ligand binding, cell membrane attachment and adhesion, has good mechanical properties, and promotes cell proliferation and regeneration or other biological activities. The synthesis method of the specific functional region of type III collagen provided by the present invention overcomes the shortcomings of the eukaryotic expression system and the problem of difficult synthesis of recombinant humanized type III collagen. It makes the degree of proline hydroxylation higher, solving the problem that proline cannot be hydroxylated or the degree of hydroxylation is low. The collagen synthesized by using the present invention can play a role in cell adhesion, migration, proliferation and differentiation through the interaction with the cell surface receptor integrin, and can be widely applied in the medical field. Brief Description of the Drawings

[0045] Figure 1 Showing the optimization result of the nucleic acid sequence of P4Hα1;

[0046] Figure 2 Showing the optimization result of the nucleic acid sequence of P4Hβ;

[0047] Figure 3 Showing the SDS-PAGE identification diagram of the crude rhP4H enzyme solution;

[0048] Figure 4 Showing the identification diagram of recombinant humanized type III collagen;

[0049] Figure 5 Showing the cell state staining diagram after electroporation;

[0050] Figure 6 Showing the triple helix structure detection diagram;

[0051] Figure 7 Showing the biological activity detection diagram;

[0052] Figure 8 Process flow diagram is shown;

[0053] Figure 9 Linear regression equation for determination of hydroxyproline content is shown. Detailed implementation mode

[0054] The present invention provides recombinant humanized collagen and its preparation method and application. Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously make changes or appropriate modifications and combinations to the methods and applications in this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0055] The test materials used in the present invention are all ordinary commercially available products and can be purchased in the market.

[0056] The following further elaborates the present invention in conjunction with embodiments:

[0057] Example 1 Pichia pastoris expression system

[0058] Any one or both of P4Hα1 and P4Hβ are digested with double enzymes and ligated into the vector of the eukaryotic expression system to construct a recombinant plasmid for transformation of Pichia pastoris recombinant strains; where P4Hα and P4Hβ include all sublines.

[0059] 1.1 Construction of recombinant expression plasmids of the α subunit (C-P4Hα) and β subunit (P4Hβ) of prolyl-4-hydroxylase

[0060] The human P4Hα gene sequence and P4Hb gene sequence are respectively selected on NCBI as follows:

[0061] Amino acid sequence of hydroxylase P4Hα1 (SEQ.ID NO.5):

[0062] MIWYILIIGILLPQSLAHPGFFTSIGQMTDLIHTEKDLVTSLKDYIKAEEDKLEQIKKWAEKLDRLTSTATKDPEGFVGHPVNAFKLMKRLNTEWSELENLVLKDMSDGFISNLTIQRQYFPNDEDQVGAAKALLRLQDTYNLDTDTISKGNLPGVKHKSFLTAEDCFELGKVAYTEADYYHTELWMEQALRQLDEGEISTIDKVSVLDYLSYAVYQQGDLDKALLLTKKLLELDPEHQRANGNLKYFEYIMAKEKDVNKSASDDQSDQKTTPKKKGVAVDYLPERQKYEMLCRGEGIKMTPRRQKKLFCRYHDGNRNPKFILAPAKQEDEWDKPRIIRFHDIISDAEIEIVKDLAKPRLRRATISNPITGDLETVHYRISKSAWLSGYENPVVSRINMRIQDLTGLDVSTAEELQVANYGVGGQYEPHFDFARKDEPDAFKELGTGNRIATWLFYMSDVSAGGATVFPEVGASVWPKKGTAVFWYNLFASGEGDYSTRHAACPVLVGNKWVSNKWLHERGQEFRRPCTLSELE

[0063] Amino acid sequence of hydroxylase P4Hα2 (SEQ.ID NO.6):

[0064] mklwvsallmawfgvlscvqaefftsighmtdliyaekelvqslkeyilveeaklskikswankmealtsksaadaegylahpvnayklvkrlntdwpaledlvlqdsaagfianlsvqrqffptdedeigaakalmrlqdtyrldpgtisrgelpgtkyqamlsvddcfgmgrsaynegdyyhtvlwmeqvlkqldageeatttksqvldylsyavfqlgdlhralelt rrllsldpsheraggnlryfeqlleeerektltnqteaelatpegiyerpvdylperdvyeslcrgegvkltprrqkrlfcryhhgnrapqlliapfkeedewdsphivryydvmsdeeierikeiakpklaratvrdpktgvltvasyrvsksswleedddpvvarvnrrmqhitgltvktaellqvanygvggqyephfdfsrrpfdsglktegnrlatflnymsdveaggatvfpdlgaaiwpkkgtavfwynllrsgegdyrtrhaacpvlvgckwvsnkwfhergqeflrpcgstevd

[0065] Amino acid sequence of hydroxylase P4Hα3 (SEQ.ID NO.7):

[0066] mgpgarlaallavlalgtgdperaaargdtfsaltsvaralaperrllgllrrylrgeearlrdltrfydkvlslhedsttpvanpllaftlikrlqsdwrnvvhsleaseniralkdgyekveqdlpafedlegaaralmrlqdvymlnvkglargvfqrvtgsaitdlyspkrlfsltgddcfqvgkvaydmgdyyhaipwleeavslfrgsygewktedeasledaldhlafayfragnvscalslsrefllyspdnkrmarnvlkyerllaespnhvvaeaviqrpniphlqtrdtyeglcqtlgsqptlyqipslycsyetnsnaylllqpirkevihlepyialyhdfvsdseaqkirelaepwlqrsvvasgekqlqveyrisksawlkdtvdpklvtlnhriaaltgldvrppyaeylqvvnygigghyephfdhatspssplyrmksgnrvatfmiylssveaggatafiyanlsvpvvrnaalfwwnlhrsgegdsdtlhagcpvlvgdkwvankwiheygqefrrpcsssped

[0067] Amino acid sequence of hydroxylase P4Hα4 (SEQ.ID NO.8):

[0068] mgpgarlaallavlalgtgdperaaargdtfsaltsvaralaperrllgllrrylrgeearlrdltrfydkvlslhedsttpvanpllaftlikrlqsdwrnvvhsleaseniralkdgyekveqdlpafedlegaaralmrlqdvymlnvkglargvfqrvtgsaitdlyspkrlfsltgddcfqvgkvaydmgdyyhaipwleeavslfrgsygewktedeasledaldhlafayfragnvscalslsrefllyspdnkrmarnvlkyerllaespnhvvaeaviqrpniphlqtrdtyeglcqtlgsqptlyqipslycsyetnsnaylllqpirkevihlepyialyhdfvsdseaqkirelaepwlqrsvvasgekqlqveyrisksawlkdtvdpklvtlnhriaaltgldvrppyaeylqvvnygigghyephfdhatspssplyrmksgnrvatfmiylssveaggatafiyanlsvpvvrhcfggtctgvvkgtvthfmlavlswweisgwptsgymsmdrnsadpaapalktellaerswwspvafqrsqepkagvgeekaeqppgrrpcqlclclanqrqgrgcyqgtlrmyi

[0069] Amino acid sequence of hydroxylase P4Hβ (SEQ.ID NO.9):

[0070] MLRRALLCLAVAALVRADAPEEEDHVLVLRKSNFAEALAAHKYLLVEFYAPWCGHCKALAPEYAKAAGKLKAEGSEIRLAKVDATEESDLAQQYGVRGYPTIKFFRNGDTASPKEYTAGREADDIVNWLKKRTGPAATTLPDGAAAESLVESSEVAVIGFFKDVESDSAKQFLQAAEAIDDIPFGITSNSDVFSKYQLDKDGVVLFKKFDEGRNNFEGEVTKENLLDFIKHNQLPLVIEFTEQTAPKIFGGEIKTHILLFLPKSVSDYDGKLSNFKTAAESFKGKILFIFIDSDHTDNQRILEFFGLKKEECPAVRLITLEEEMTKYKPESEELTAERITEFCHRFLEGKIKPHLMSQELPEDWDKQPVKVLVGKNFEDVAFDEKKNVFVEFYAPWCGHCKQLAPIWDKLGETYKDHENIVIAKMDSTANEVEAVKVHSFPTLKFFPASADRTVIDYNGERTLDGFKKFLESGGQDGAGDDDDLEDLEEAEEPDMEEDDDQKAVKDEL

[0071] According to the principle of codon preference, the nucleic acid sequences of hydroxylase P4Hα1 and hydroxylase P4Hβ were optimized in the 5' region (translation initiation efficiency), DNA repeat sequences, mRNA secondary structure, GC content, SD sequence, and excluding designated restriction enzyme sites, etc., to optimize into a DNA sequence with Pichia pastoris preference. Restriction enzymes were selected for full-length synthesis based on the sequence information of P4Hα1 and P4Hβ.

[0072] The nucleic acid sequence of hydroxylase P4Hα1 (SEQ.ID NO.3):

[0073]

[0074] Nucleic acid sequence of hydroxylase P4Hβ (SEQ.ID NO.4):

[0075]

[0076] As Figures 1-2 shown, after the nucleic acid sequence of P4Hα1 was optimized, the CAI increased from 0.77 to 0.84, and the GC content changed from 41.62% to 42.07%. After the nucleic acid sequence of P4Hβ was optimized, the CAI increased from 0.69 to 0.83, and the GC content changed from 56.25% to 41.93%.

[0077] Subsequently, the optimized DNA sequences were respectively double-digested and ligated to the plasmid of the eukaryotic expression system to construct recombinant plasmids.

[0078] 1.2 Obtaining of rhC-P4H recombinant Pichia pastoris engineering bacteria

[0079] (1) Amplification of recombinant plasmid

[0080] Take 2 - 5 μL of the above plasmid and transfer it into 30 - 60 μL of Escherichia coli competent cell DH5α (placed on ice). Mix well and incubate on ice for 30 min. Place the mixed suspension in a dry bath thermostat, set the temperature to 42°C for 90 s, take it out and incubate on ice for another 3 min. Add 500 μL of LB medium without antibiotics, and culture it in a constant temperature shaker at 160 - 200 rpm and 37°C for 2 - 3 h. Take 20 - 40 μL of the bacterial solution and evenly spread it on an LB plate containing 5 - 20 μg / mL Zecion antibiotic, and culture it in a constant temperature incubator at 37°C for 16 - 18 h. Pick a single colony on the plate and inoculate it into 10 mL of LB medium containing 10 μg / mL Zecion antibiotic, and culture it at 160 - 200 rpm and 37°C for 13 - 16 h.

[0081] (2) Extraction, identification and linearization of recombinant plasmid

[0082] Use a plasmid extraction kit to extract the plasmid from the above culture solution to obtain recombinant plasmids respectively, and measure the concentration. Design upstream and downstream primers according to the gene sequence, and perform 3 - time sequencing alignment in both forward and reverse directions.

[0083] If the identification is successful, use enzymes to double-digest the plasmid. The enzyme digestion reaction system is as follows:

[0084] 10X CutSmart@Buffer 5 μL Recombinant plasmid co-expressing recombinant humanized collagen and prolyl hydroxylase 4 μL <![CDATA[ddH2O]]> 39 μL BamH I 1 μL Not I 1 μL

[0085] Place the prepared reaction solution on a PCR instrument, and perform enzyme digestion at 37°C for 1 - 3 h. Then adjust the temperature to 60 - 80°C for 10 min to inactivate. The digested and linearized plasmid is purified using the Wizard SV Gel and PCR Clean-Up System cleaning kit according to the operation instructions. Keep a small amount for agarose gel electrophoresis identification, and store the rest at -20°C for standby.

[0086] (3) Preparation of competent cells of Saccharomyces cerevisiae

[0087] Take 100 - 200 μL of X33 expression bacteria or GS115 expression strain or other yeast strains and inoculate them into 200 mL of YPD medium. Incubate overnight at 250 - 30 °C with shaking at 200 - 250 rpm until OD600nm = 1.2 - 1.5. Centrifuge the cell culture at 4 °C and 1500 rpm for 5 min, and resuspend it with 20 mL of sterile water. Repeat this process twice for the cell mass. Then centrifuge the resuspended solution at 4 °C and 1500 rpm for 5 min, resuspend the cell mass with 5 mL of sterile water, and add 50 - 70 μL of DTT, mix well and let it stand at room temperature for 20 min. Subsequently, centrifuge the resuspended solution at 4 °C and 1500 rpm for 5 min, resuspend the cell mass with 500 μL of 1 M sorbitol, and repeat twice.

[0088] (4) Electrotransformation of recombinant plasmid to prepare competent cells. The method is as follows:

[0089] a) Immerse the electroporation cuvette in 75% ethanol for more than 20 min, irradiate it with ultraviolet light for 20 min, and dry it for later use;

[0090] b) Add 20 - 100 μL of X competent cells and no less than 4 μg of linearized recombinant plasmid into the electroporation cuvette. After standing in an ice bath for 10 min, perform electroporation. The electroporation conditions are set as follows: voltage: 1.5 KV; capacitance: 25 μF; resistance: 200 - 400 W, and electroporate for 10 msec;

[0091] c) Add 2 mL of 1 M sorbitol solution pre - cooled at 4 °C, gently mix the cell mass with a pipette tip, and transfer it to a 2.5 mL EP tube;

[0092] d) Spread the cell resuspension on a YPD plate containing Zecion antibiotic, and incubate at a constant temperature for 48 h until single colonies appear.

[0093] 1.3 Expression and identification of specific amino acid sequences of type III collagen in Pichia pastoris strains co - expressing recombinant human collagen and prolyl hydroxylase

[0094] Amino acid sequences (SEQ.ID NO.1) of the 411 - 639 and 1158 - 1199 fragments of the α1 chain in type III collagen:

[0095] (gargppgpagangapglrggagepgkngakgepgprgergeagipgvpgakgedgkdgspgepganglpgaagergapgfrgpagpngipgekgpagergapgpagprgaagepgrdgvpggpgmrgmpgspggpgsdgkpgppgsqgesgrpgppgpsgprgqpgvmgfpgpkgndgapgkngerggpggpgpqgppgkngetgpqgppgptgpggdkgdtgppgpqg) n +gpigppgprgnrgergsegspghpgqpgppgppgapgpccgg(1 ≤ n ≤ 30) (In this embodiment, n = 2 is taken as an example for construction.)

[0096] Nucleotide sequences of the fragments at positions 411 - 639 and 1158 - 1199 of the a1 chain in type III collagen (SEQ.ID NO.2):

[0097] (GGAGCCCGGGGTCCTCCAGGACCAGCCGGTGCTAATGGTGCTCCTGGACTGCGAGGTGGTGCAGGTGAGCCTGGTAAGAATGGTGCCAAAGGAGAGCCCGGACCACGTGGTGAACGCGGTGAGGCTGGTATTCCAGGTGTTCCAGGAGCTAAAGGCGAAGATGGCAAGGATGGATCACCTGGAGAACCTGGTGCAAATGGGCTTCCAGGAGCTGCAGGAGAAAGGGGTGCCCCTGGGTTCCGAGGACCTGCTGGACCAAATGGCATCCCAGGAGAAAAGGGTCCTGCTGGAGAGCGTGGTGCTCCAGGCCCTGCAGGGCCCAGAGGAGCTGCTGGAGAACCTGGCAGAGATGGCGTCCCTGGAGGTCCAGGAATGAGGGGCATGCCCGGAAGTCCAGGAGGACCAGGAAGTGATGGGAAACCAGGGCCTCCCGGAAGTCAAGGAGAAAGTGGTCGACCAGGTCCTCCTGGGCCATCTGGTCCCCGAGGTCAGCCTGGTGTCATGGGCTTCCCCGGTCCTAAAGGAAATGATGGTGCTCCTGGTAAGAATGGAGAACGAGGTGGCCCTGGAGGACCTGGCCCTCAGGGTCCTCCTGGAAAGAATGGTGAAACTGGACCTCAGGGACCCCCAGGGCCTACTGGGCCTGGTGGTGACAAAGGAGACACAGGACCCCCTGGTCCACAAGGA) n +GGTCCCATTGGACCACCAGGGCCTCGAGGTAACAGAGGTGAAAGAGGATCTGAGGGCTCCCCAGGCCACCCAGGGCAACCAGGCCCTCCTGGACCTCCTGGTGCCCCTGGTCCTTGCTGTGGTGGT(1≤n≤30)(In this example, n = 2 is used for construction)

[0098] The DNA sequence of this amino acid sequence is digested with two enzymes and ligated into a Pichia pastoris strain containing prolyl hydroxylase to construct a strain co-expressing recombinant humanized collagen and proline hydroxylase.

[0099] 1.4. Obtaining of the strain co-expressing recombinant humanized collagen and proline hydroxylase

[0100] (1) Amplification of recombinant plasmid

[0101] Take 2 - 5 μL of the above plasmid and transfer it into 30 - 60 μL of competent Escherichia coli cells DH5α (placed on ice). Mix well and incubate on ice for 30 min. Place the mixed suspension in a dry bath incubator at a temperature set to 42°C for 90 s. After taking it out, incubate on ice for another 3 min. Add 500 μL of LB medium without antibiotics and culture it in a constant temperature shaker at 160 - 200 rpm and 37°C for 2 - 3 h. Take 20 - 40 μL of the bacterial solution and evenly spread it on an LB plate containing 5 - 20 μg / mL Zecion antibiotic. Incubate it in a constant temperature incubator at 37°C for 16 - 18 h. Pick monoclonal colonies on the plate and inoculate them into 10 mL of LB medium containing 10 μg / mL Zecion antibiotic. Culture it at 160 - 200 rpm and 37°C for 13 - 16 h.

[0102] (2) Extraction, identification and linearization of recombinant plasmid

[0103] Use a plasmid extraction kit to extract plasmids from the above culture solution to obtain recombinant plasmids respectively, and measure the concentration. Design upstream and downstream primers according to the gene sequence and perform 3 - time sequencing alignment in both forward and reverse directions.

[0104] If the identification is successful, use enzymes to perform double digestion on the plasmid. The enzyme digestion reaction system is as follows:

[0105] 10X CutSmart@Buffer 5 μL Recombinant plasmid co-expressing recombinant humanized collagen and prolyl hydroxylase 4 μL <![CDATA[ddH2O]]> 39 μL BamH I 1 μL Not I 1 μL

[0106] Place the prepared reaction solution on a PCR instrument at PCR temperature, and perform enzyme digestion at 37°C for 1 - 3 h. Then adjust the temperature to 60 - 80°C for 10 min to inactivate. The digested and linearized plasmid is purified using the Wizard SV Gel and PCR Clean - Up System cleaning kit according to the operation instructions. Keep a small amount for agarose gel electrophoresis identification, and store the rest at - 20°C for standby.

[0107] (3) Preparation of competent yeast cells

[0108] Inoculate 100 - 200 μL of the X33 expression strain or GS115 expression strain or other yeast strains into 200 mL of YPD medium, and culture overnight at 200 - 250 rpm and 25℃ - 30℃ until OD600nm = 1.2 - 1.5. Centrifuge the cell culture at 4℃ and 1500 rpm for 5 min, and resuspend it with 20 mL of sterile water. Repeat this process twice for the cells. Then, centrifuge the resuspended solution at 4℃ and 1500 rpm for 5 min, resuspend the cell pellet with 5 mL of sterile water, and add 50 - 70 μL of DTT, mix well, and let it stand at room temperature for 20 min. Subsequently, centrifuge the resuspended solution at 4℃ and 1500 rpm for 5 min, resuspend the cell pellet with 500 μL of 1M sorbitol, and repeat this twice.

[0109] (4) Preparation of competent cells for electrotransformation of the recombinant plasmid is as follows:

[0110] a) Immerse the electroporation cuvette in 75% alcohol for more than 20 min, irradiate it with ultraviolet light for 20 min, and dry it for later use.

[0111] b) Add 20 - 100 μL of X competent cells and no less than 4 μg of linearized recombinant plasmid into the electroporation cuvette, let it stand in an ice bath for 10 min, and then perform electroporation. The electroporation conditions are set as follows: voltage: 1.5 KV; capacitance: 25 μF; resistance: 200 - 400 W, and electroporate for 10 sec.

[0112] c) Add 2 mL of 1M sorbitol solution pre - cooled to 4℃, gently mix the cell pellet with a pipette tip, and transfer it to a 2.5 mL EP tube.

[0113] d) Spread the cell resuspension on a YPD plate containing Zecion antibiotic, and incubate at 25℃ - 30℃ for 48 h until single colonies appear.

[0114] Culture the engineering bacteria containing the recombinant plasmid, and the specific implementation plan is as follows:

[0115] (1) Separately pick the activated engineering bacteria containing the recombinant plasmid and inoculate them into 20 mL of YPD medium, and culture at 200 - 260 rpm and 25℃ - 30℃ for 18 - 24 h. Pipette the culture solution at an inoculation amount of 2% into 25 mL of YPG medium, and culture at 200 - 260 rpm and 25℃ - 30℃ for 18 - 20 h until OD600 = 2.1.

[0116] (2) Centrifuge the above culture medium at 2000 - 4000 rpm for 10 min to collect the bacteria. Resuspend the bacteria with 20 - 50 mL of YPM and place them in a 250 mL Erlenmeyer flask. Incubate at 200 - 260 rpm, 25℃ - 30℃ for 36 - 72 h. Centrifuge the obtained fermentation broth at 10000 - 15000 rpm for 5 min, and take the supernatant to obtain the crude rhP4H enzyme solution. Identify by SDS - PAGE, and the identification results are as Figure 3 shown.

[0117] Example 2 CHO expression system

[0118] 2.1. Construction of plasmids

[0119] Construct the optimized nucleic acid sequences of hydroxylase P4Hα1, hydroxylase P4Hβ and the nucleic acid sequence of specific amino acids in type III collagen onto the vectors of mammalian cells respectively. The steps are as follows:

[0120] Double - digest P4Hα and P4Hβ and ligate them onto the mammalian vector. Then, construct the nucleic acid sequence SEQ.ID NO.1 of specific amino acids in type III collagen onto the mammalian cell vector by double - digestion.

[0121] 2.2. Transform the two plasmids into the CHO cell expression system at a ratio of 1:5 to 1:20. The steps are as follows:

[0122] (1) Immerse the electroporation cuvette in 75% alcohol for more than 20 min, irradiate with ultraviolet lamp for 20 min, and dry for use;

[0123] (2) The cell density is 1x10^6 - 5x10^8 cells / ml, the plasmid dosage is 2 - 20 μg, the volume of electroporation solution is 50 - 100 μl, the voltage is 50V - 150V, and the capacitance is 600 μF - 950 μF.

[0124] (3) After electroporation, place the electroporation cuvette in a constant - temperature incubator for 8 - 12 min to allow the nucleic acid to fully enter the cells.

[0125] (4) Take out the electroporation cuvette from the constant - temperature incubator, inoculate the cell suspension into the pre - warmed medium, pipette up and down evenly, and then place it in the incubator for normal culture.

[0126] (5) Culture normally for 4 h. After the cells adhere, change the cells to fresh medium to remove the dead cells on the upper layer.

[0127] 2.3. CHO cell resuscitation, sub - culture, transformation, culture, protein identification

[0128] 2.3.1. Cell resuscitation

[0129] (1) Take out the cell cryopreservation tube from liquid nitrogen, quickly place it in a 37 °C water bath to thaw until there is no crystal in the cryopreservation tube, and wipe the outer wall of the cryopreservation tube with 75% alcohol;

[0130] (2) Transfer the cells in the cryopreservation tube to a 15 mL centrifuge tube containing 6 mL of complete medium, and centrifuge at 1000 - 2000 rpm for 5 - 10 min;

[0131] (3) Discard the supernatant, resuspend the precipitate with 6 mL of complete medium, and inoculate it into a 25 cm 2 culture flask, and culture it in a 37 °C, 5% CO2 cell culture incubator;

[0132] 2.3.2. Subculture

[0133] Adherent cells:

[0134] (1) When the cells grow to cover 80% - 90% of the area of the culture flask, discard the culture medium in the 25 cm 2 culture flask, and wash the cells once with PBS;

[0135] (2) Add about 1 mL - 3 mL of 0.25% trypsin digestion solution to the culture flask, observe under an inverted microscope, and add 5 mL of complete culture medium to terminate digestion after the cells shrink and become round, then gently pipette the cells to make them detach,

[0136] Transfer the suspension to a 15 mL centrifuge tube, and centrifuge at 1000 rpm for 5 min;

[0137] (3) Discard the supernatant, resuspend the precipitated cells with 1 - 2 mL of complete medium, and subculture them by splitting into new flasks at a ratio of 1:2,

[0138] After adding additional medium, place it in a 37 °C, 5% CO2 cell culture incubator for culture;

[0139] Suspension cells:

[0140] When the cells reach about 1x10 ^6 / ml to 1x10 ^9 / ml, the following methods can be used for medium change or subculture.

[0141] Method ①: Collect the cells, centrifuge at 1000 rpm - 1500 rpm for 5 min, discard the supernatant, add 1 - 2 mL of culture medium and pipette evenly, and divide the cell suspension into new flasks containing medium at a ratio of 1:2 to 1:5.

[0142] Method ②: Place the culture flask upright, discard the upper half of the medium after the cells precipitate, and divide the cell suspension into new flasks containing medium at a ratio of 1:2 to 1:5.

[0143] 2.3.3. Transformation

[0144] (1) CHO cells were cultured in DMEM medium containing 10%-20% fetal bovine serum and subcultured at a ratio of 1:3 to 1:10 48 h to 96 h before electroporation. Fresh medium was changed 24 h before electroporation. Cells were examined microscopically before electroporation, and logarithmic growth phase cells with 80%-90% confluence adherent to the culture dish were digested with trypsin, centrifuged at 1000 r / min for 10 min, and the precipitate was washed and centrifuged with serum-free DMEM medium. Meanwhile, cell counting was performed, and the number of cells in each experimental group was adjusted to reach 2×10 6 ~2×10 9 .

[0145] (2) For the electro-sensitivity experiment of CHO cells, the pH range of the low ionic strength Tris-Cl buffer was 6-8, the electric field strength was 500-800 V / cm, and the current was 25-100 uF. The cells were suspended in the electroporation buffer (the optimized parameters of the protocol are shown in Table 1), ice-bathed for 10-20 min, pulsed 2-5 times, with an interval of ice-bathing for 1-3 min, and ice-bathed for 10 min after electroporation. The cell suspension was diluted 10-fold with DMEM medium containing 10% fetal bovine serum and cultured at 37°C and 5% CO2 for 6 h. Viable cells were counted by the trypan blue staining method (the trypan blue staining results are as Figure 4 shown). The results were analyzed by one-way ANOVA with a nested design using SPSS 13.0 software. The medium was changed every 3 days. After the appearance of resistant clones, DMEM maintenance medium was changed, and the number of resistant clones was observed and counted daily (the results are shown in Table 2).

[0146] Table 1

[0147]

[0148]

[0149] Table 2

[0150]

[0151] 2.3.4. Cell culture

[0152] Suspension cells

[0153] (1) After 2-3 days of shake flask culture, the cell density increases and the nutrients are exhausted. Calculate the scale-up volume based on the cell density. In the laminar flow hood, inoculate the shake flask cells into the shake flask, and aseptically weld the inoculation shake flask pipeline and the reactor pipeline with an aseptic welding machine. First, use a pump to drain the PBS in the tank, and pay attention to maintaining sterility during this process. After the PBS is drained, pump in a certain amount of culture medium, adjust the parameters of this batch of cell culture. After the pH, Temperature, dO2, Stirrer, and readings are stable, inoculate a certain amount of cells into the bioreactor, and finally supplement the culture medium to the culture volume designed for this experiment. After inoculating the cells, turn on the bioreactor monitoring system Bio Xpert to monitor the culture status in real time until the end of this cell culture.

[0154] The cells are cultured in a bioreactor at a cell density of 500,000 cells per milliliter, and the total culture volume is 3 L. During the day, the dissolved oxygen dO2 is automatically controlled to be about 40%. When the reactor is overnight, the method of passing air through the surface of the culture solution is adopted to prevent cell hypoxia. During the whole culture process, cell counting is performed every 12 h, 1 mL of cell supernatant is taken for detection, and recombinant humanized type III collagen is identified.

[0155] Adherent cells:

[0156] After 2-3 days of culture, the cell density increases and the nutrients are exhausted. Calculate the scale-up volume based on the cell density. In the laminar flow hood, inoculate the shake flask cells into the culture flask, and aseptically weld the inoculation shake flask pipeline and the reactor pipeline with an aseptic welding machine. First, use a pump to drain the PBS in the tank, and pay attention to maintaining sterility during this process. After the PBS is drained, pump in a certain amount of culture medium, adjust the parameters of this batch of cell culture. After the pH, Temperature, dO2, Stirrer, and readings are stable, inoculate a certain amount of cells into the culture flask, and finally supplement the culture medium to the culture volume designed for this experiment. During the whole culture process, cell counting is performed at regular intervals, and recombinant humanized type III collagen is identified through cell supernatant and cell lysate. The identification results are as Figure 5 shown, and the target protein is successfully expressed.

[0157] Example 3 Detection of Target Protein

[0158] 3.1 Detection of Hydroxyproline Content

[0159] Hydroxyproline is a specific amino acid contained in collagen, and its content is relatively stable. The sample is hydrolyzed into hydroxyproline under the action of 105°C and c(HCI)=6 mol / L hydrochloric acid. After hydroxyproline is oxidized by chloramine T, it reacts with p-dimethylaminobenzaldehyde to form a red compound, and colorimetric determination is carried out at a wavelength of 560 nm.

[0160] The specific operation steps are as follows:

[0161] Reagents (all reagents are of analytical grade unless otherwise specified)

[0162] A. L-Hydroxyproline reference standard: National reference standard

[0163] B. Hydrochloric acid solution, c(HCl) = 6 mol / L: Mix analytical grade hydrochloric acid and water in equal volumes

[0164] C. pH = 6.0 buffer solution: Weigh 57 g of sodium acetate trihydrate, 37.5 g of trisodium citrate, 5.5 g of citric acid monohydrate, 385 mL of isopropanol, add 500 mL of water, adjust the pH to 6.0 with citric acid monohydrate, and dilute to 1000 mL with water

[0165] D. Chloramine T solution: Weigh 3.5 g of chloramine T and dilute to 50 mL with water. Prepare it freshly before use

[0166] E. Oxidant solution: Mix the chloramine T solution and the pH = 6.0 buffer solution in a ratio of 1:4

[0167] F. 60% Perchloric acid solution: Measure 43 mL of perchloric acid and dilute to 50 mL with water

[0168] G. p-Dimethylaminobenzaldehyde solution: Weigh 10 g of p-dimethylaminobenzaldehyde and dissolve it in 15 mL of 60% perchloric acid solution

[0169] H. Color reagent: Measure 15 mL of p-dimethylaminobenzaldehyde solution and dissolve it in 65 mL of isopropanol

[0170] I. Sodium hydroxide solution, c(NaOH) = 6 mol / L: Weigh 24 g of sodium hydroxide and dilute to 100 mL with water

[0171] Precisely measure 0.5 mL of blank (water), the L-hydroxyproline reference standard series solutions, and the test solution, add 1 mL of isopropanol and the oxidant solution respectively, mix and let stand at room temperature for 4 min; then add 6.5 mL of the color reagent respectively, mix, place each tube in a 60 °C water bath and heat for 15 min, and then cool. Using the blank as a control, measure the absorbance value at 560 nm. Make a linear regression of the absorbance against the concentration series of the L-hydroxyproline reference standard solution to obtain the linear regression equation ( Figure 9 ), and calculate the content of L-hydroxyproline in the test solution (Table 3). After calculation, the content of the target protein synthesized by this method is between 5% and 15%.

[0172] Table 3

[0173] Concentration μg / mL 0.50 1.00 1.5 2.0 2.5 Absorbance A560 0.134 0.284 0.412 0.547 0.683

[0174] 3.2. Detection of triple helix structure

[0175] The synthesized target protein was detected by circular dichroism spectroscopy, and the steps were as follows:

[0176] The target protein was dissolved in phosphate buffer, then placed in a cuvette and detected by circular dichroism spectrometer.

[0177] The results showed ( Figure 6 ) that the humanized collagen synthesized in the present invention conformed to the characteristics of the triple helix structure.

[0178] 3.3. Detection of biological activity

[0179] After culturing the synthesized target protein in fibroblasts for 14 - 20 days, Calcein-AM staining was performed, and the steps were as follows:

[0180] 3.3.1 (Fibroblasts) culture, amplification and cryopreservation:

[0181] Culture system: RPMI1640 supplemented with 5% - 10% FBS, at 37°C, 5% CO2, pH 6.5. Cultured in culture flasks, the medium was renewed every 2 - 4 days. After cell confluence, subculture was performed, and the cells were passaged to P2 and cryopreserved for subsequent detection.

[0182] 3.3.2 Viable cell staining test

[0183] 3.3.3 Test time point: 14 days after plating.

[0184] Test method: 6 experimental wells were set for each time point in a single group for testing. At the preset test time point, the medium in the wells was gently removed, washed twice with PBS, and then 0.1% - 0.3% concentration of Calcein-AM solution prepared with 1x buffer was added. Incubated at 4°C in the dark for 30 minutes, washed twice with PBS, and then the viable cells stained with green fluorescence were observed under 490 nm excitation light.

[0185] The results showed ( Figure 7 ) that the target protein synthesized in the present invention could stimulate the proliferation of fibroblasts.

[0186] In addition, the amino acid sequences with n = 1 and n = 30 in SEQ.ID NO.1 were also expressed by the same method as in the present invention. Since the selected expression system, test method and test steps were the same as those with n = 2 in the examples, they were not described in detail. The tests showed that within the range of n from 1 to 30, especially for the target proteins with n = 1 and n = 30, the physiological activities were similar to that of the target protein with n = 2.

[0187] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing humanized collagen, characterized in that, The steps include the following: S1. Construct a plasmid containing the coding nucleic acid of humanized collagen; S2. Construct expression vectors for the α subunit and β subunit of prolyl-4-hydroxylase; S3. Co-transform the plasmid containing the coding nucleic acid of humanized collagen and the expression vector of prolyl-4-hydroxylase into a host cell to obtain a co-expression strain; S4. Ferment and culture the co-expression strain to obtain the humanized collagen; The sequence of the coding nucleic acid of the humanized collagen is as follows: (GGAGCCCGGGGTCCTCCAGGACCAGCCGGTGCTAATGGTGCTCCTGGACTGCGAGGTGGTGCAGGTGAGCCTGGTAAGAATGGTGCCAAAGGAGAGCCCGGACCACGTGGTGAACGCGGTGAGGCTGGTATTCCAGGTGTTCCAGGAGCTAAAGGCGAAGATGGCAAGGATGGATCACCTGGAGAACCTGGTGCAAATGGGCTTCCAGGAGCTGCAGGAGAAAGGGGTGCCCCTGGGTTCCGAGGACCTGCTGGACCAAATGGCATCCCAGGAGAAAAGGGTCCTGCTGGAGAGCGTGGTGCTCCAGGCCCTGCAGGGCCCAGAGGAGCTGCTGGAGAACCTGGCAGAGATGGCGTCCCTGGAGGTCCAGGAATGAGGGGCATGCCCGGAAGTCCAGGAGGACCAGGAAGTGATGGGAAACCAGGGCCTCCCGGAAGTCAAGGAGAAAGTGGTCGACCAGGTCCTCCTGGGCCATCTGGTCCCCGAGGTCAGCCTGGTGTCATGGGCTTCCCCGGTCCTAAAGGAAATGATGGTGCTCCTGGTAAGAATGGAGAACGAGGTGGCCCTGGAGGACCTGGCCCTCAGGGTCCTCCTGGAAAGAATGGTGAAACTGGACCTCAGGGACCCCCAGGGCCTACTGGGCCTGGTGGTGACAAAGGAGACACAGGACCCCCTGGTCCACAAGGA)nGGTCCCATTGGACCACCAGGGCCTCGAGGTAACAGAGGTGAAAGAGGATCTGAGGGCTCCCCAGGCCACCCAGGGCAACCAGGCCCTCCTGGACCTCCTGGTGCCCCTGGTCCTTGCTGTGGTGGT, where n = 2.

2. The preparation method according to claim 1, characterized in that, The plasmid in the step S1 sequentially includes a backbone vector, a promoter, a nucleic acid encoding humanized collagen, and a terminator; The promoter is selected from any one of a T7 promoter, an sCMV promoter, a Lac promoter, a tac promoter, an IPL promoter, an araB promoter, an AOX1 promoter, a trc promoter, or a trp promoter; The terminator is selected from any one of a T7 terminator, an rrnB T1 terminator, or an AOX1 terminator.

3. The preparation method according to claim 2, wherein The plasmid sequentially includes a PcDNA3.1 vector or a pPICZα series vector, an AOX1 promoter, a nucleic acid encoding humanized collagen, and an AOX1 terminator.

4. The preparation method according to claim 1, characterized in that, The expression vectors of the prolyl-4-hydroxylase α subunit and β subunit in the step S2 include a nucleic acid encoding prolyl-4-hydroxylase and a PcDNA3.1 vector.

5. The preparation method according to claim 1, characterized in that, The host cell in the step S3 is selected from any one or more of Escherichia coli, Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae, and mammalian cells.

6. The preparation method according to claim 5, wherein, The host cell is Pichia pastoris and / or CHO cells.

7. A humanized collagen, characterized in that, Prepared by the preparation method according to any one of claims 1 to 6.

8. A product with a filling support having a repair function, characterized in that Comprising humanized collagen obtained by the preparation method according to any one of claims 1 to 6.

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