Recombinant III-type humanized collagen as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicine, in particular to recombinant III-type humanized collagen as well as a preparation method and application thereof. In one embodiment, the recombinant III type humanized collagen prepared by the invention has an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 4. The recombinant III-type humanized collagen prepared by the invention has good biological activity, can promote collagen synthesis related gene expression, promote skin fibroblast proliferation, increase the content of skin collagenous fibers and elastic fibers, and improve skin elasticity and compactness; in addition, the expression of osteogenesis related genes is promoted, and the proliferation of osteoblasts is promoted. The results show that the recombinant III-type humanized collagen prepared by the invention has an extremely wide application prospect in developing related medical and cosmetic products for delaying skin aging and promoting wound repair and bone tissue healing.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically refers to a recombinant type III humanized collagen and a preparation method and application thereof. Background Art

[0002] Human collagen α1(III) chain is composed of COL3A1 Genetically encoded, three α1 chains polymerize to form type III collagen molecules with a right-handed triple helix structure, accounting for about 5% to 20% of the total collagen in the human body. Type III collagen has attracted much attention because of its important role in tissue repair, skin elasticity maintenance and cell signal transduction.

[0003] Recombinant collagen has good water solubility, low immune rejection, high stability and easy absorption, and is gradually being used in the fields of cosmetics, food, medical devices, etc. However, recombinant technology also has its shortcomings. On the one hand, although the prokaryotic expression system (such as Escherichia coli) has high expression efficiency, it lacks a post-translational modification mechanism and cannot achieve key modifications such as hydroxylation of collagen, resulting in insufficient biological activity; on the other hand, although the eukaryotic expression system (such as yeast and mammalian cells) can provide a modification environment closer to the human body, it has problems such as low expression efficiency, high production cost, and difficulty in forming a triple helix structure.

[0004] Most domestic recombinant collagen products are artificially designed non-natural sequence analogs or selected fragments, which are difficult to achieve the structural strength of natural collagen and have potential biocompatibility and safety risks. In addition, existing research lacks systematic development of specific functional areas, making it difficult to meet the needs of medical cosmetology, tissue engineering and other fields for efficient and stable recombinant collagen.

[0005] Therefore, developing a recombinant humanized collagen that can be efficiently expressed, retains biological activity and has specific functions and a preparation method thereof is a technical challenge that needs to be solved urgently. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a novel type III recombinant humanized collagen and a preparation method and application thereof.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions: In a first aspect, the present invention provides the following type III recombinant humanized collagen.

[0008] The present invention provides a recombinant humanized collagen A, the structure of which is X n Y; wherein X is a fragment of positions 1158 to 1199 of type III collagen α1 chain, and Y is a fragment of positions 411 to 639 of type III collagen α1 chain; 1≤ n ≤30.

[0009] The present invention also provides a recombinant humanized collagen B, the structure of which is X n YZ; wherein X is a fragment of type III collagen α1 chain at positions 1158 to 1199, Y is a fragment of type III collagen α1 chain at positions 411 to 639, and Z is an amino acid sequence of type III collagen α1 chain at positions 1381 to 1440; 1≤ n ≤30.

[0010] Preferably, 1≤n≤20, more preferably, 1≤n≤10, and even more preferably, 1≤n≤5.

[0011] Most preferably, n=2.

[0012] Also preferably, n is an integer.

[0013] The amino acid sequence of the recombinant humanized collagen of the present invention includes any one of the following (I) to (III): (I) the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 4; or (II) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (I); or (III) A sequence having a homology of more than 90% with the amino acid sequence shown in (I).

[0014] In a second aspect, the present invention provides a method for preparing the recombinant humanized collagen described in the first aspect: S1. constructing a plasmid containing a nucleic acid encoding a fragment at positions 1158 to 1199, a fragment at positions 411 to 639, and / or a fragment at positions 1381 to 1440 of type III collagen α1 chain; S2, constructing an expression vector for prolyl-4-hydroxylase α subunit and / or β subunit; S3, co-transforming a plasmid containing nucleic acid encoding fragments 1158 to 1199, 411 to 639 and / or 1381 to 1440 of type III collagen α1 chain and an expression vector for prolyl-4-hydroxylase into a host cell to obtain a co-expression strain / cell; and S4. Ferment and culture the co-expression strain / cell to obtain the recombinant humanized collagen.

[0015] Specifically, the plasmid containing the nucleic acid encoding the fragments 1158 to 1199, 411 to 639 and / or 1381 to 1440 of the type III collagen α1 chain in step S1 includes a backbone vector and a promoter, a nucleic acid encoding the recombinant humanized collagen and a terminator in sequence; The promoter is selected from any one of T7 promoter, sCMV promoter, Lac promoter, tac promoter, IPL promoter, araB promoter, AOX1 promoter, trc promoter or trp promoter; The terminator is selected from any one of a T7 terminator, an rrnB T1 terminator, an AOX1 terminator, a p-independent terminator or a p-dependent terminator.

[0016] In some embodiments, the plasmid containing the nucleic acid encoding the fragments 1158 to 1199, the fragments 411 to 639 and / or the fragments 1381 to 1440 of the type III collagen α1 chain includes, in sequence, a PcDNA3.1 vector or a pPICZα series vector, an AOX1 promoter, a nucleic acid encoding the recombinant humanized collagen described in the first aspect above, and an AOX1 terminator.

[0017] Specifically, the host cell in step S3 is selected from any one or more of Pichia pastoris, Saccharomyces cerevisiae, and mammalian cells.

[0018] In some specific embodiments, the host cell is Pichia pastoris.

[0019] The present invention provides a method for preparing the recombinant humanized collagen described in the first aspect above, comprising fermenting and culturing the host cell described in the present invention. Specifically, the successfully constructed co-expression strain is inoculated into a YPD medium with a total culture volume of 3 L, the dissolved oxygen is maintained at about 40%, and the culture is performed for 18 to 96 hours, and the cell supernatant is taken and lysed to obtain the recombinant humanized collagen.

[0020] The present invention provides recombinant humanized collagen, which is prepared by the above preparation method.

[0021] In a third aspect, the present invention further provides use of the recombinant humanized collagen described in the first aspect in preparing products.

[0022] Specifically, the products include medical cosmetic products, wound repair materials, bone tissue healing materials, artificial tissue scaffolds, biocomposite materials, drug sustained-release preparations, cosmetics or skin care products.

[0023] The beneficial effects of the present invention are: (1) It overcomes the shortcoming of the eukaryotic expression system that lacks appropriate post-translational processing mechanisms, allowing the expression product to be correctly folded and modified, thus ensuring its biological activity; (2) Provides a solution to the difficulty in synthesizing recombinant humanized type III collagen; (3) The recombinant humanized collagen provided by the present invention has good biological activity, and has the functions of promoting fibroblast collagen synthesis, improving skin elasticity, promoting bone tissue healing and other biological activities; (4) Improving the expression efficiency of recombinant collagen by selecting key fragments for repeated splicing; (5) The recombinant humanized collagen provided by the present invention has the potential to be widely used in the medical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention.

[0025] In the attached picture: Figure 1 : SDS-PAGE electrophoresis of the recombinant humanized collagen of the present invention; Figure 2 : The recombinant humanized collagen of the present invention increases the expression of genes related to collagen synthesis in human skin fibroblasts; Figure 3 : EVG staining results of elastic fibers; Figure 4 :Collagen fiber Masson staining results; Figure 5 :Results of fibroblast and osteoblast proliferation experiments; Figure 6 : The recombinant humanized collagen described in the present invention improves the expression of osteoblast-related genes in mouse osteoblasts. DETAILED DESCRIPTION

[0026] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0027] Example 1: Preparation of recombinant humanized collagen (recombinant collagen A (whose structure is X n Y, 1≤n≤30, in this embodiment, n=2 is taken as an example) 1.1 Construction of recombinant plasmid for co-expression of recombinant humanized collagen and proline hydroxylase (1) Construction of recombinant plasmid: The nucleotide sequences of the 1158-1199 fragment (i.e., X) and the 411-639 fragment (i.e., Y) of the α1 chain of type III collagen (SEQ.ID NO.2) and the human P4Hα1 gene sequence (SEQ.ID NO.3) were respectively ligated to the vector of the eukaryotic expression system (pPICZα A) by double restriction digestion to construct a recombinant plasmid for transformation of Pichia pastoris recombinant strains.

[0028] Amino acid sequence of the 1158-1199 and 411-639 fragments of the α1 chain of type III collagen (SEQ.IDNO.1): GPIGPPGPRGNRGERGSEGSPGHPGQPGPPGPPGAPGPCCGGGPIGPPGPRGNRGERGSEGSPGHPGQPGPPGPPGAPGPCCGGGARGPPGPAGANGAPGLRGGAGEPGKNGAKGEPGPRGERGEAGIPGVPGAKGEDGKDGSPGEPGANGLPGAAG ERGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQG.

[0029] Nucleotide sequence of the 1158-1199th and 411-639th fragments of the α1 chain in type III collagen (SEQ.IDNO.2): GGTCCCATTGGACCACCAGGGCCTCGAGGTAACAGAGGTGAAAGAGGATCTGAGGGCTCCCCAGGCCACCCAGGGCAACCAGGCCCTCCTGGACCTCCTGGTGCCCCTGGTCCTTGCTGTGGTGGTGGTCCCATTGGACCACCAGGGCCTCGAGGTAACAGAGGTGAAAGAGGATCTGAGGGCTCCCCAGGCCACCCAGGGCAACCAGGCCCTCCTGGACCTCCTGGTGCCCCTGGTCCTTGCTGTGGTGGTGGAGCCCGGGGTCCTCCAGGACCAGCCGGTGCTAATGGTGCTCCTGGACTGCGAGGTGGTGCAGGTGAGCCTGGTAAGAATGGTGCCAAAGGAGAGCCCGGACCACGTGGTGAACGCGGTGAGGCTGGTATTCCAGGTGTTCCAGGAGCTAAAGGCGAAGATGGCAAGGATGGATCACCTGGAGAACCTGGTGCAAATGGGCTTCCAGGAGCTGCAGGAGAAAGGGGTGCCCCTGGGTTCCGAGGACCTGCTGGACCAAATGGCATCCCAGGAGAAAAGGGTCCTGCTGGAGAGCGTGGTGCTCCAGGCCCTGCAGGGCCCAGAGGAGCTGCTGGAGAACCTGGCAGAGATGGCGTCCCTGGAGGTCCAGGAATGAGGGGCATGCCCGGAAGTCCAGGAGGACCAGGAAGTGATGGGAAACCAGGGCCTCCCGGAAGTCAAGGAGAAAGTGGTCGACCAGGTCCTCCTGGGCCATCTGGTCCCCGAGGTCAGCCTGGTGTCATGGGCTTCCCCGGTCCTAAAGGAAATGATGGTGCTCCTGGTAAGAATGGAGAACGAGGTGGCCCTGGAGGACCTGGCCCTCAGGGTCCTCCTGGAAAGAATGGTGAAACTGGACCTCAGGGACCCCCAGGGCCTACTGGGCCTGGTGGTGACAAAGGAGACACAGGACCCCCTGGTCCACAAGGA。

[0030] Nucleotide sequence of hydroxylase P4Hα1 (SEQ.ID NO.3):

[0031] (2) Recombinant plasmid amplification: Take 2~5 μL of the above recombinant plasmid and transfer it into 30~60 μL of E. coli competent cells DH5α. Mix well and place on ice for 30 min. Then place the suspension in a dry thermostat at 42℃ for 90 s. Take it out and place on ice for 3 min. Add 500 μL of LB medium without antibiotics and culture it in a constant temperature shaker at 160~200 rpm and 37℃ for 2~3 h. Take 20~40 μL of bacterial solution and spread it evenly on the LB plate containing 5~20 μg / mL Zecion antibiotics. Culture it in a constant temperature incubator at 37℃ for 16~18 h. Pick the colony on the plate and inoculate it in 10 mL of LB medium containing 10 μg / mL Zecion antibiotics. Culture it at 160~200 rpm and 37℃ for 13~16 h.

[0032] (3) Extraction of recombinant plasmid: Use a plasmid extraction kit to extract the plasmid from the above culture medium, obtain the recombinant plasmids respectively, and measure the concentration.

[0033] (4) Plasmid linearization and purification: The plasmid was double-digested using enzymes. The reaction system (50 μL) is shown in Table 1.

[0034] Table 1: Double enzyme digestion reaction system

[0035] Place the prepared reaction system on a PCR instrument, digest at 37°C for 1-3 h, and then adjust the temperature to 60-80°C for 10 min to inactivate. After digestion and linearization, purify the plasmid using the WizardSV Geland PCR Clean-Up System cleaning kit according to the operating instructions.

[0036] 1.2 Recombinant plasmid transformation to obtain co-expression strain (1) Preparation of competent cells: Take 100-200 μL of GS115 Pichia pastoris strain and inoculate it into 200 mL YPD medium. Incubate overnight at 200-250 rpm and 30°C until OD 600nm Between 1.2 and 1.5; place the bacterial solution in an ice bath for 30 min, centrifuge at 4℃ 1500rpm for 5 min, resuspend the bacteria with 20 mL of sterile water, and repeat twice; then centrifuge the suspension at 4℃ 1500 rpm for 5 min, resuspend the bacteria with 500 μL of 1M sorbitol, and repeat twice.

[0037] (2) Plasmid electroporation: a) Add 80 μL competent yeast and 20 μL purified linearized recombinant plasmid to the electroporation cup, place on ice for 10 min and then electroporate. The electroporation parameters are set as follows: voltage 1.5 kV; capacitance 25 μF; resistance 200~400 W, electroporation 10 msec; b) After the electroporation, add 2 mL of 1 M sorbitol solution precooled at 4°C, gently pipette to mix, and transfer to a 2.5 mL EP tube to obtain the electroporation solution; c) Take 200 μL of the electroporated bacterial solution and spread it on a YPD plate containing Zecion antibiotics. Incubate the plate at 30°C for 48 hours until a single colony appears.

[0038] 1.3 Acquisition and identification of recombinant humanized collagen (1) Select the activated engineered bacteria containing the recombinant plasmid and inoculate them into 20 mL of YPD medium. Culture them at 220 rpm and 30°C for 24 h. (2) Take the engineered bacteria cultured in YPD at a 2% inoculum and add it to YPG medium. Cultivate at 220 rpm and 30°C for 24 h until the OD 600 nm =2.1; (3) The above culture medium was centrifuged at 3000 rpm for 10 min to collect the cells, resuspended in 50 mL YPM and placed in a 250 mL Erlenmeyer flask, and cultured at 220 rpm and 30°C for 48 h. The obtained fermentation liquid was centrifuged at 10000-15000 rpm for 5 min, and the supernatant was obtained to obtain the recombinant humanized collagen solution. The identification was performed by SDS-PAGE, and the identification results were as follows: Figure 1 As shown, recombinant humanized collagen A was successfully expressed with a molecular weight of about 70 kDa.

[0039] Example 2: Preparation of recombinant humanized collagen (recombinant collagen B (whose structure is X n YZ, 1≤n≤30, in this embodiment, n=2 is taken as an example) 2.1 Construction of recombinant plasmid for co-expression of recombinant humanized collagen and proline hydroxylase (1) Construction of recombinant plasmid: The nucleotide sequences of the fragments at positions 1158-1199 (i.e., X), 411-639 (i.e., Y), and 1381-1440 (i.e., Z) of the α1 chain of type III collagen (SEQ.ID NO.5) and the human P4Hα1 gene sequence (SEQ.ID NO.3) were ligated to the vector of the eukaryotic expression system (pPICZα A) by double restriction digestion to construct a recombinant plasmid for transformation of Pichia pastoris recombinant strains.

[0040] Amino acid sequences of the fragments at positions 1158-1199, 411-639 and 1381-1440 of the α1 chain of type III collagen (SEQ.ID NO.4): GPIGPPGPRGNRGERGSEGSPGHPGQPGPPGPPGAPGPCCGGGPIGPPGPRGNRGERGSEGSPGHPGQPGPPGPPGAPGPCCGGGARGPPGPAGANGAPGLRGGAGEPGKNGAKGEPGPRGERGEAGIPGVPGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAPG PAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGQASGNVKKALKLMGSNEGEFKAEGNSKFTYTVLEDGCTKHTGEWSKTVFEYRTRKAVRLP.

[0041] Nucleotide sequences of the fragments at positions 1158-1199, 411-639 and 1381-1440 of the α1 chain in type III collagen (SEQ.ID NO.5):

[0042] (2) The steps of amplification and extraction of the recombinant plasmid are the same as those in Example 1.1; (3) Plasmid linearization and purification: Use enzymes to perform double digestion of the plasmid. The reaction system (50 μL) is the same as Table 1. Place the prepared reaction system on a PCR instrument, digest at 37°C for 1-3 h, and then adjust the temperature to 60-80°C for 10 min to inactivate. The plasmid after digestion and linearization was transformed with WizardSV Geland PCR Clean-2.2 to obtain a co-expression strain The steps are the same as in Example 1.2.

[0043] 2.3 Acquisition and identification of recombinant humanized collagen (1) Select the activated engineered bacteria containing the recombinant plasmid and inoculate them into 20 mL of YPD medium. Culture them at 220 rpm and 30°C for 24 h. (2) Take the engineered bacteria cultured in YPD at a 2% inoculum and add it to YPG medium. Cultivate at 220 rpm and 30°C for 24 h until the OD 600 nm =2.1; (3) The above culture medium was centrifuged at 3000 rpm for 10 min to collect the cells, resuspended in 50 mL YPM and placed in a 250 mL Erlenmeyer flask, and cultured at 220 rpm and 30°C for 48 h. The obtained fermentation liquid was centrifuged at 10000-15000 rpm for 5 min, and the supernatant was obtained to obtain the recombinant humanized collagen solution. The identification was performed by SDS-PAGE, and the identification results were as follows: Figure 1 As shown, recombinant humanized collagen B was successfully expressed with a molecular weight of about 80 kDa.

[0044] Comparative Example 1: Preparation of recombinant humanized collagen Recombinant humanized collagen was prepared according to the preparation method of CN 116925207 A as a positive control.

[0045] Application Example 1: Recombinant humanized collagen promotes the expression of genes related to collagen synthesis in human skin fibroblasts 1.1 Human skin fibroblast (HSF) culture Human skin fibroblasts (purchased from Sunncel) were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. The cell culture conditions were 37°C and 5% CO 2, humidity greater than 90%. After three cell passages, the fibroblasts were divided into 4 groups, a blank control group, a positive control group (5 mg / mL recombinant humanized collagen prepared in Comparative Example 1), an experimental group A (5 mg / mL recombinant humanized collagen A prepared in Example 1) and an experimental group B (5 mg / mL recombinant humanized collagen B prepared in Example 2), and inoculated into 6-well plates, with 3 replicates in each group. After incubation for 24 hours, the medium was changed and the samples were added, and the culture was continued for 48 hours.

[0046] 1.2 Total RNA extraction (1) Aspirate the original culture medium, wash twice with PBS, add 1 mL of Trizol solution to the culture plate to lyse the cells, pipette to mix, and let stand for 5 min; (2) Add 200 μL of chloroform to each well, shake vigorously for 30 seconds to allow the aqueous phase and organic phase to fully contact, and let stand at room temperature for 3 minutes; (3) Centrifuge at 14,000 rpm for 15 min at 4°C. The tube can be separated into three layers. RNA is in the upper aqueous phase. Transfer the mixture to another new RNase-free EP tube. (4) Precipitate RNA: Add an equal volume of isopropanol, mix gently, and let stand at room temperature for 15 min. (5) Centrifuge at 14,000 rpm for 10 min at 4°C to collect the RNA precipitate and discard the supernatant; (6) Wash twice with 1 mL of 75% ethanol, centrifuge at 14,000 rpm for 5 min at 4°C, discard the supernatant, and air-dry in a clean bench; (7) Add 20 μL of DEPC water to dissolve the precipitate.

[0047] 1.3 Total RNA purity and integrity testing (1) Purity test: Take 1 μL RNA sample and dilute it 50 times. Measure the OD value on the BioPhotometer plus Eppendorf nucleic acid protein analyzer. 260 / OD 280 The ratio is greater than 1.8, indicating that the prepared RNA is relatively pure and has no protein contamination.

[0048] (2) Total RNA integrity test: Take 3 μL of RNA sample and run 1.5% agarose gel electrophoresis at 160 V × 15 min. Use a gel imaging system to observe the 5s rRNA, 18s rRNA and 28s rRNA bands of the total RNA. If the three bands are intact, it proves that the total RNA extraction is relatively complete.

[0049] 1.4 Reverse transcription (1) Prepare the extracted RNA solution according to the instructions of the RevertAid First Strand cDNA Synthesis Kit (purchased from Thermo scientific); (2) Keep the above reaction system solution at 25°C for 10 min; 42°C for 30 min; and 85°C for 5 s.

[0050] 1.5 Quantitative PCR (1) The primer sequences used for PCR are shown in Table 2.

[0051] Table 2: Primer sequences and product sizes used for PCR. Purification was performed according to the instructions of the Up System Clean-up Kit.

[0052]

[0053] (2) Use a quantitative PCR instrument (ABI PRISM® 7500 Sequence Detection System). The reaction system is shown in Table 3. The reaction program is: 95°C for 5 min; 95°C for 15 s, 60°C for 32 s, a total of 40 cycles, analyze the melting curve, record its threshold cycle number (Ct value), and use the internal reference GAPDH The Ct value of the target gene was corrected based on the difference, and the relative gene expression analysis was performed using 2 -∆ ∆CT Method calculation.

[0054] Table 3: PCR reaction system

[0055] The results of the expression levels of genes related to collagen synthesis are as follows Figure 2 As shown, It means that the data of this group are statistically different from those of the control group. P <0.01, P <0.001. COL1 gene and COL3 gene encode type I and type III collagen, respectively, which are key components for maintaining tissue structure and function. Compared with the blank control group, the collagen synthesis-related genes in the positive control group, experimental group A and experimental group B COL1A1 , COL3A1 The expression levels of the two groups were significantly upregulated, and the upregulation effect of the experimental groups (A, B) (48.9%, 47.5%) was better than that of the positive control group (31.5%).

[0056] Application Example 2: Effect of recombinant humanized collagen on the ratio of type I and type III collagen 2.1 Grouping and Dosing The groups and dosing schedules are shown in Table 4.

[0057] Table 4: Grouping and dosing regimen

[0058] 2.2 Experimental methods 18 adult female New Zealand rabbits weighing about 3 kg were selected and purchased from Jilin Jintai Meidi Biotechnology Co., Ltd. The rabbits were randomly divided into 3 groups, namely, a blank control group, a positive control group and an experimental group (Example 1, recombinant collagen A); the backs of the rabbits in each group were shaved, and then the skin of the shaved area was cleaned, and 200 μL of physiological saline and recombinant collagen prepared by different methods (Comparative Example 1 and Example 1) were intradermally injected into the center of the back of the rabbits in each group. Four weeks after the injection, the tissues of the injection site of the rabbits were taken, EVG staining and Masson staining were performed, and the proportion of elastic fibers and collagen fibers was observed and analyzed under a microscope.

[0059] 2.3 EVG staining (1) Preparation of paraffin sections: The dorsal skin tissue fixed in paraformaldehyde for 48 h was dehydrated, paraffin-embedded and sectioned; (2) Dewaxing and rehydrating paraffin sections: Dewax the sections in xylene I and xylene II for 30 min each, then in anhydrous ethanol I, anhydrous ethanol II, 95% alcohol, 90% alcohol, 80% alcohol, and 70% alcohol for 5 min each, and rinse with tap water. (3) EVG staining: alcohol hematoxylin, ferric chloride and iodine solution were mixed in a ratio of 5:2:2 to form EVG staining solution. The sections were placed in the EVG staining solution for 30 min and then rinsed with tap water. (4) Background differentiation: Differentiate in ferric chloride differentiation solution for a few seconds, rinse with tap water, and repeat this step to control the degree of differentiation until the elastic fibers appear purple-black and the background appears gray-white under a microscope.

[0060] (5) Re-staining with VG: Mix saturated picric acid and acid fuchsin in a ratio of 9:1 to form a VG staining solution, stain for 1-3 min. Wash quickly with water; (6) Dehydration and transparent sealing: Soak the sections in 95% alcohol I, 95% alcohol II, anhydrous ethanol I, and anhydrous ethanol II for 5 min to dehydrate, then put them in xylene I and xylene II for transparentization for 5 min, and seal the sections with sealing solution; (7) Microscope examination and image acquisition.

[0061] EVG staining results are as follows Figure 3 As shown, elastic fibers are purple-black, collagen fibers are red, and the background is yellow. The results were analyzed using image processing software, and the results are shown in Table 5. It means that the data of this group are statistically different from those of the control group. P <0.01, P <0.001. The proportion of elastic fibers in the control group was 0.51%, the proportion of elastic fibers in the positive control group was 0.72%, and the proportion of elastic fibers in the experimental group was 1.16%, which was significantly higher than that in the control group and significantly better than that in the positive control group.

[0062] Table 5: EVG staining and Masson staining results

[0063] 2.4 Masson staining (1) Dewaxing and rehydrating paraffin sections: same as step 2.3 (2); (2) Hematoxylin staining: Immerse the sections in hematoxylin staining solution for 5 min, then rinse with tap water. (3) Ponceau-Acid Fuchsin Staining: The sections were immersed in Ponceau-Acid Fuchsin staining solution for 5 min and then rinsed with 0.2% glacial acetic acid. (4) Phosphomolybdic acid differentiation: Place the slices in phosphomolybdic acid differentiation solution for a few seconds and rinse with tap water; (5) Aniline blue staining: Place the sections in aniline blue staining solution for 5 min and rinse with 0.2% glacial acetic acid; (6) Dehydration and transparent mounting: Same as step 2.3 (6); (7) Microscope examination and image acquisition.

[0064] Masson staining is a classic method for collagen fiber staining. Figure 4 As shown, collagen fibers are blue, muscle fibers, cellulose and red blood cells are red, and cell nuclei are blue-black. The results were analyzed using image processing software, and the results are shown in Table 5. The proportion of collagen fibers in the control group was 40.83%, the proportion of collagen fibers in the positive control group was 59.57%, and the proportion of collagen fibers in the experimental group was 67.17%, which was significantly higher than that in the control group and better than that in the positive control group.

[0065] Application Example 3: Recombinant humanized collagen promotes the proliferation of fibroblasts and osteoblasts (1) Mouse dermal fibroblasts and mouse osteoblasts (purchased from Pricella) were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. The cell culture conditions were 37°C and 5% CO 2 , greater than 90% humidity. After the cells were passaged three times, cell proliferation experiments were performed.

[0066] (2) Cells were cultured at 2×10 3 The cells were inoculated at a density of 100 cells / well in a 96-well culture plate and cultured overnight in a cell culture incubator. Subsequently, the culture medium was aspirated, and 100 μL of 2 mg / mL of recombinant collagen A prepared in Example 1 was added to the wells inoculated with cells as experimental group A, 100 μL of 2 mg / mL of recombinant collagen B prepared in Example 2 was added as experimental group B, and 100 μL of 2 mg / mL of recombinant collagen prepared in Comparative Example 1 was added as a positive control group, and an equal amount of cell-free basal culture medium was used as a blank control group. At the same time, only basal culture medium was added to the wells without cells inoculated as a blank group, and 100 μL was added to each well, and culture was continued in a cell culture incubator for 24 hours.

[0067] (3) Aspirate the culture medium, add 20 μL of MTT solution (concentration of 5 mg / mL) and 100 μL of basal culture medium to each well, and continue to culture in the cell culture incubator for 4 hours. After the culture is completed, add 150 μL of DMSO to each well and incubate at 37°C for 10 minutes. Finally, use a microplate reader to measure the absorbance (OD value) at a wavelength of 570 nm, and calculate the relative viability of the experimental group and the positive control group cells according to the following formula: Cell viability (%) = (OD experimental / positive group-OD blank group) / (OD blank control group-OD blank group) × 100. The experimental results are shown in Table 6.

[0068] Table 6: Relative viability of cells in the positive control group and experimental group

[0069] The results were statistically analyzed using Graphpad Prism software. Figure 5 As shown, the results of the fibroblast proliferation experiment showed that compared with the blank control group, the cell viability of the positive control group increased by 62.51%, the cell viability of the experimental group A increased by 70.95%, and the cell viability of the experimental group B increased by 71.75%; the results of the osteoblast proliferation experiment showed that compared with the blank control group, the cell viability of the positive control group increased by 15.62%, the cell viability of the experimental group A increased by 25.24%, and the cell viability of the experimental group B increased by 23.21%, and all had significant statistical differences (P<0.001). The above results show that the recombinant collagen of the present invention can significantly promote the proliferation of fibroblasts and osteoblasts, and is better than the positive control group.

[0070] Application Example 4: Recombinant humanized collagen promotes the expression of osteoblast-related genes in mouse osteoblasts 4.1 Mouse osteoblast culture Mouse osteoblasts (purchased from Pricella) were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. The cell culture conditions were 37°C and 5% CO 2 , humidity greater than 90%. After three cell passages, the osteoblasts were divided into 4 groups, a blank control group, a positive control group (5 mg / mL recombinant humanized collagen prepared in Comparative Example 1), an experimental group A (5 mg / mL recombinant humanized collagen A prepared in Example 1) and an experimental group B (5 mg / mL recombinant humanized collagen B prepared in Example 2), and inoculated into 6-well plates, with 3 replicates in each group. After incubation for 24 hours, the medium was changed and the samples were added, and the culture was continued for 48 hours.

[0071] 4.2 Total RNA extraction Same as step 1.2.

[0072] 4.3 Total RNA purity and integrity testing Same as step 1.3.

[0073] 4.4 Reverse transcription Same as step 1.4.

[0074] 4.5 Quantitative PCR (1) The primer sequences used for PCR are shown in Table 7.

[0075] Table 7: Primer sequences used for PCR, product sizes

[0076] (2) Use a quantitative PCR instrument (ABI PRISM® 7500 Sequence Detection System). The reaction system is shown in Table 7. The reaction program is: 95°C for 5 min; 95°C for 15 s, 60°C for 32 s, a total of 40 cycles, analyze the melting curve, record its threshold cycle number (Ct value), and use the internal reference Gapdh The Ct value of the target gene was corrected based on the difference, and the relative gene expression analysis was performed using 2 -∆ ∆CT Method calculation.

[0077] Table 8: PCR reaction system

[0078] The results of the expression levels of genes related to collagen synthesis are as follows Figure 6 As shown, It means that the data of this group are statistically different from those of the control group. P <0.01, P <0.001.

[0079] BMP2 (bone morphogenetic protein 2), as a key member of the TGF-β superfamily, can induce mesenchymal stem cells to differentiate into osteoblasts, thereby promoting the formation of bone and cartilage tissue. SPP1 (secretory phosphoprotein 1, also known as osteopontin) is a non-collagenous bone matrix glycoprotein that is widely distributed in bone tissue due to its high affinity for hydroxyapatite and participates in processes such as mineralization of bone matrix and cell adhesion.

[0080] In this experiment, compared with the blank control group, the osteogenesis-related genes in the positive control group and the experimental group Bmp2 , Spp1 The expression levels of α-glucose and β-glucose in the experimental groups (A and B) were significantly upregulated, and the upregulation effect was better than that in the positive control group. Bmp2 The expression levels of experimental groups A and B were increased by 70.3% and 66.7%, respectively, which were better than those of the positive control group (up 42.1% compared with the blank control group). Spp1 The expression levels were increased by 122.1% and 116.9%, respectively, which were better than the positive control group (upgraded by 100% compared with the blank control group).

[0081] In summary, the recombinant humanized collagen prepared by the present invention has good biological activity, can promote the expression of genes related to collagen synthesis, promote the proliferation of skin fibroblasts, increase the content of skin collagen fibers and elastic fibers, and improve skin elasticity and firmness; in addition, it can also promote the expression of genes related to osteogenesis and promote the proliferation of osteoblasts. The above results show that the recombinant humanized collagen prepared by the present invention has extremely broad application prospects in the development of medical and beauty products related to delaying skin aging, promoting wound repair and bone tissue healing.

Claims

1. A recombinant humanized collagen, characterized in that: The recombinant humanized collagen is recombinant humanized collagen A, and its structure is X n Y; wherein X is a fragment of positions 1158 to 1199 of type III collagen α1 chain, and Y is a fragment of positions 411 to 639 of type III collagen α1 chain; 1≤ n ≤30.

2. The recombinant humanized collagen according to claim 1, characterized in that: The amino acid sequence of the recombinant humanized collagen includes any one of the following (I) to (III): (I) the amino acid sequence shown in SEQ ID NO: 1; (II) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (I); or (III) A sequence having a homology of more than 90% with the amino acid sequence shown in (I).

3. A recombinant humanized collagen, characterized in that: The recombinant humanized collagen is recombinant humanized collagen B, and its structure is X n YZ; wherein X is a fragment of type III collagen α1 chain at positions 1158 to 1199, Y is a fragment of type III collagen α1 chain at positions 411 to 639, and Z is an amino acid sequence of type III collagen α1 chain at positions 1381 to 1440; 1≤ n≤30.

4. The recombinant humanized collagen according to claim 3, characterized in that: The amino acid sequence of the recombinant humanized collagen includes any one of the following (I) to (III): (I) the amino acid sequence shown in SEQ ID NO: 4; (II) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (I); or (III) A sequence having a homology of more than 90% with the amino acid sequence shown in (I).

5. The recombinant humanized collagen according to claim 1 or claim 3, characterized in that: 1≤ n ≤20。 6. The method for preparing the recombinant humanized collagen according to any one of claims 1 to 5, characterized in that: The preparation method comprises the following steps: S1. constructing a plasmid containing a nucleic acid encoding a fragment at positions 1158 to 1199, a fragment at positions 411 to 639, and / or a fragment at positions 1381 to 1440 of type III collagen α1 chain; S2, constructing an expression vector for prolyl-4-hydroxylase α subunit and / or β subunit; S3, co-transforming a plasmid containing nucleic acid encoding fragments 1158 to 1199, 411 to 639 and / or 1381 to 1440 of type III collagen α1 chain and an expression vector for prolyl-4-hydroxylase into a host cell to obtain a co-expression strain / cell; and S4. Ferment and culture the co-expression strain / cell to obtain the recombinant humanized collagen.

7. The preparation method according to claim 6, characterized in that: In step S1, the plasmid containing nucleic acid encoding fragments 1158-1199, 411-639 and / or 1381-1440 of type III collagen α1 chain comprises a backbone vector and a promoter, nucleic acid encoding the recombinant humanized collagen and a terminator.

8. The preparation method according to claim 7, characterized in that: The promoter is selected from any one of T7 promoter, sCMV promoter, Lac promoter, tac promoter, IPL promoter, araB promoter, AOX1 promoter, trc promoter or trp promoter; and / or the terminator is selected from any one of T7 terminator, rrnB T1 terminator, AOX1 terminator, p-independent terminator or p-dependent terminator.

9. The preparation method according to claim 6, characterized in that: The host cell in step S3 is selected from any one or more of Pichia pastoris, Saccharomyces cerevisiae, and mammalian cells.

10. Use of the recombinant humanized collagen according to any one of claims 1 to 5 or the recombinant humanized collagen prepared by the preparation method according to any one of claims 6 to 9 in the preparation of medical cosmetic products, wound repair materials, bone tissue healing materials, artificial tissue scaffolds, biocomposite materials, drug sustained-release preparations, cosmetics or skin care products.

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