Preparation method and application of recombinant humanized III-type collagen

Abstract

The invention discloses a preparation method and application of recombinant humanized III-type collagen, and belongs to the technical field of genetic engineering. According to the invention, three segments with good hydrophilicity and high solubility are screened from a humanized III-type collagen sequence through a bioinformatics method, and coding genes of the three segments are integrated into a pichia pastoris genome in a multi-copy form after being subjected to pichia pastoris codon preference optimization, so that a recombinant strain of the humanized III-type collagen is obtained. The recombinant strain secretes and expresses collagen, the purity can reach 95% or above only through one-step purification, the downstream separation and purification difficulty is obviously reduced, and the production process is easy to amplify. The prepared three recombinant human-derived III-type collagen proteins all have the activity of promoting cell migration and the activity of promoting cell proliferation, and have good application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and particularly relates to a preparation method and application of recombinant humanized type III collagen. Background Art

[0002] Collagen is the most abundant and widely distributed functional protein in mammals, mainly present in tissues such as skin, ligaments, cartilage, blood vessels, teeth, and bones, accounting for about 30% of the total human protein and 70 - 80% in bones and connective tissues. The triple helix structure of collagen is formed by the winding of three α-peptide chains, and each α-chain contains a large number of repeated Gly-Xaa-Yaa characteristic sequences (where X and Y are mostly proline and hydroxyproline). Different arrangements and glycosylation modifications of the three peptide chains result in a variety of types. So far, 29 different types of collagen have been reported successively, among which type I collagen and type III collagen account for the largest proportion, together accounting for more than 90% of the total collagen in the human body. Type III collagen has a high content in blood vessels and connective layers, and is remarkable for promoting wound repair and activating coagulation factors to promote blood coagulation.

[0003] Currently, the collagen used is generally of animal origin, prepared from animal connective tissues by traditional methods such as acid hydrolysis, alkali hydrolysis, or enzymatic hydrolysis. This extraction process not only consumes a large amount of raw materials, but also has the risks of immunogenicity and pathogen contamination in clinical applications.

[0004] With the development of synthetic biology, the biosynthesis of collagen has become a hot research direction. Compared with the extraction of collagen from animal sources, the recombinant collagen products produced by genetic engineering methods have the advantages of high biocompatibility, low immunogenicity, and short production cycle. At the same time, they can also improve the hydrophilicity and processability of collagen, thus solving most of the disadvantages of traditional extraction methods. However, the current biosynthesis of collagen still has certain limitations. The gene sequence of type III collagen is relatively long, and due to the highly repetitive (Gly-X-Y) n sequence of collagen, the subsequent self-assembly process is extremely complex, and it has always faced problems such as difficult expression, low expression level, difficult secretion, and complex purification. Summary of the Invention

[0005] To solve the above technical problems, the object of the present invention is to provide a method for preparing and applying recombinant humanized type III collagen. The present invention conducts bioinformatics analysis on the human type III collagen sequence, screens out three truncated protein sequences COL1 / 2 / 3 with high hydrophilicity and good solubility, and successfully expresses them in Pichia pastoris; by adding the α-factor signal peptide, the collagen is secreted extracellularly, greatly reducing the separation and purification cost; and further through techniques such as screening of high-copy integration strains and optimization of the fermentation process engineering, efficient secretion expression of recombinant type III humanized collagen is achieved.

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

[0007] In the first aspect, the present invention provides a group of recombinant humanized type III collagen, and the amino acid sequences of the humanized type III collagen are shown as SEQ ID NO.1-3.

[0008] In the second aspect, the present invention provides a gene encoding the above humanized type III collagen.

[0009] Based on the above technical solution, further, the nucleotide sequence of the gene is shown as SEQ ID NO.4-6.

[0010] In the third aspect, the present invention provides an expression vector into which the above gene is inserted.

[0011] Based on the above technical solution, further, the expression vector is pPIC9K.

[0012] In the fourth aspect, the present invention provides a host cell carrying the above expression vector, and the host cell can express humanized type III collagen.

[0013] Based on the above technical solution, further, the chassis cell of the host cell is Pichia pastoris KM71.

[0014] Based on the above technical solution, further, the recombinant type III humanized collagen is expressed driven by the AOX1 strong inducible promoter.

[0015] In the fifth aspect, the present invention provides a method for producing the above recombinant humanized type III collagen, including the following steps:

[0016] (1) After activating and expanding the above host cell, inoculate it into a fermentation medium for culture;

[0017] (2) Collect the supernatant of the fermentation broth, carry out separation and purification, dialysis to remove salts, and lyophilize to obtain recombinant humanized type III collagen.

[0018] Based on the above technical solution, further, the activated culture medium in step (1) is YPD culture medium, and its composition is: yeast extract powder 10 g / L, peptone 20 g / L, glucose 20 g / L, and the balance is water.

[0019] Based on the above technical solution, further, the culture medium for expansion culture in step (1) is BMGY culture medium, and its composition is: yeast extract powder 10 g / L, peptone 20 g / L, YNB 13.4 g / L, peptone 20 g / L, K2HPO4 3.012 g / L, KH2PO4 11.813 g / L, glycerol 10 mL / L, biotin 400 μg / L, and the balance is water.

[0020] Based on the above technical solution, further, the culture medium for fermentation in step (1) is BMMY culture medium, and its composition is: yeast extract powder 10 g / L, peptone 20 g / L, YNB 13.4 g / L, peptone 20 g / L, K2HPO4 3.012 g / L, KH2PO4 11.813 g / L, methanol 5 mL / L, biotin 400 μg / L, and the balance is water.

[0021] Based on the above technical solution, further, the conditions for fermentation culture in step (1) are: culturing at 25 - 30 °C and 100 - 300 rpm for 90 - 144 h, and during this period, methanol with a volume percentage of 1% is supplemented every 20 - 28 h.

[0022] Based on the above technical solution, further, the method for separation and purification in step (2) includes one or a combination of two or more of salting - out, ion - exchange chromatography, affinity chromatography, hydrophobic chromatography, acid - base precipitation, and membrane separation; preferably affinity chromatography.

[0023] In the sixth aspect, the present invention provides the application of the above - mentioned humanized type III collagen in the preparation of daily cosmetics, biomedical materials, and drugs for promoting the proliferation and migration of skin cells.

[0024] Based on the above technical solution, further, the biomedical materials include filling materials, repair materials, hemostatic dressings, wound dressings, and drug - release carriers.

[0025] The beneficial effects of the present invention compared with the prior art are as follows:

[0026] The recombinant strain of the present invention has successfully achieved the highly soluble extracellular expression of three truncated humanized type III collagens, with a molecular weight not exceeding 30 kDa, 100% human origin, and high hydrophilicity. It is more easily absorbed by the human body, has good biocompatibility, a simple preparation method, low production cost, and can promote the proliferation and migration of skin cells, accelerate the repair process of damaged skin, have a certain repair effect on skin sensitivity, post-inflammatory damage, etc., and enhance the skin barrier function. It is rich in hydrophilic amino acids, can absorb a large amount of water, increase the water content of the skin stratum corneum, keep the skin moist and smooth, and improve skin dryness problems. It can increase the elasticity and toughness of the skin, reduce the generation of wrinkles, improve the firmness and smoothness of the skin, and make the skin more youthful. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention, the accompanying drawings related to the embodiments will be briefly introduced below.

[0028] Figure 1 It is a graph of the evaluation results of protein hydrophilicity and solubility predicted by bioinformatics means.

[0029] Figure 2 It is a graph of the single and double enzyme digestion verification results of the pPIC9K-COL1 / 2 / 3 recombinant plasmid; among them, lanes 1-2 are the double and single enzyme digestion bands of pPIC9K-COL1 respectively; lanes 3-4 are the double and single enzyme digestion bands of pPIC9K-COL2 respectively; lanes 5-6 are the double and single enzyme digestion bands of pPIC9K-COL3 respectively; M is DNA Marker (DL 15000).

[0030] Figure 3 It is a genomic PCR graph of the recombinant strain KM71 / pPIC9K-COL1 / 2 / 3; among them, lanes 1-5 are the genomic PCR bands of the KM71 / pPIC9K-COL1 transformant; lanes 6-10 are the genomic PCR bands of the KM71 / pPIC9K-COL2 transformant; lanes 11-15 are the genomic PCR bands of the KM71 / pPIC9K-COL3 transformant; M is DNA Marker (DL 10000).

[0031] Figure 4 It is an SDS-PAGE detection graph of the recombinant humanized type III collagen; among them, lane 1 is the negative control; lanes 2-3 are the supernatant and purified bands of the COL1 fermentation broth respectively; lanes 4-5 are the supernatant and purified bands of the COL2 fermentation broth respectively; lanes 6-7 are the supernatant and purified bands of the COL3 fermentation broth respectively; M is the protein Marker.

[0032] Figure 5SDS-PAGE detection chart for optimizing the induction temperature during the fermentation process of recombinant humanized type III collagen; among them, lane 1 is at 22 °C; lane 2 is at 25 °C; lane 3 is at 28 °C; lane 4 is at 30 °C; M is the protein Marker.

[0033] Figure 6 SDS-PAGE detection chart for optimizing the induction time during the fermentation process of recombinant humanized type III collagen; among them, lane 1 is 24 h; lane 2 is 48 h; lane 3 is 72 h; lane 4 is 96 h; lane 5 is 120 h; lane 6 is 144 h; M is the protein Marker.

[0034] Figure 7 SDS-PAGE detection chart for optimizing the inducer concentration during the fermentation process of recombinant humanized type III collagen; among them, lane 1 is 0.25%; lane 2 is 0.5%; lane 3 is 0.75%; lane 4 is 1%; lane 5 is 1.5%; lane 6 is 2%; M is the protein Marker.

[0035] Figure 8 Result chart of recombinant humanized type III collagen COL1 / 2 / 3 promoting cell migration. Detailed implementation mode

[0036] The present invention will be described in detail below in conjunction with the embodiments, but the implementation modes of the present invention are not limited thereto. Obviously, the embodiments described below are only partial embodiments of the present invention. For those skilled in the art, without creative efforts, obtaining other similar embodiments will fall within the protection scope of the present invention.

[0037] Example 1 Construction of recombinant vector pPIC9K-COL1 / 2 / 3

[0038] According to the prediction on an online website, the hydrophilicity and solubility of the three sequences derived from the human natural type III collagen α1 chain are higher than those of the truncated humanized type III collagen COL1 / 2 / 3. The amino acid sequences of COL1 / 2 / 3 are shown in SEQ ID NO.1-3 respectively. After codon optimization of the gene of the truncated humanized type III collagen COL1 / 2 / 3, it was synthesized by Jiutian Gene Technology (Tianjin) Co., Ltd. into the pET-29a vector (the nucleotide sequences are shown in SEQ ID NO.4-6 respectively). Using pET-29a-COL1 / 2 / 3 as templates and F1 / 2 / 3-R1 as upstream and downstream primers respectively, gene amplification was carried out. At the same time, using pPIC9K as a template and F4-R4 as upstream and downstream primers, inverse PCR amplification was carried out to linearize the vector. Subsequently, after gel extraction and recovery of the amplified vector and the amplified fragment, they were ligated under the action of seamless ligase at a ratio of V / I = 1 / 8 (the reaction system is shown in Table 1) and transferred into DH5α competent cells. Positive transformants were screened using LB plates with Kana resistance, and colony PCR was performed. After single enzyme digestion verification with SalI and double enzyme digestion verification with BamHI and SalI ( Figure 2 ), the transformants with correct sequencing verification were subcultured and plasmid was extracted to obtain the pPIC9K-COL1 / 2 / 3 recombinant vector.

[0039] The upstream and downstream primers used for amplification are as follows:

[0040] F1: 5’-AGAAAAGAGAGGCTGAAGCTGGTCCTCAAGGTTTACAAGGTTTGC-3’;

[0041] F2: 5’-GAAAAGAGAGGCTGAAGCTGGTTTTAGAGGTCCAGCAGGC-3’;

[0042] F3: 5’-AGAAAAGAGAGGCTGAAGCTGGTCCGGAAGGTGGTAAAGG-3’;

[0043] R1: 5’-AACAGTCATGTCTAAGGTCAGCTTTGTTAGCAGCCGGATCTCA-3’;

[0044] F4: 5’-TGACCTTAGACATGACTGTTCCTCAGTTC-3’;

[0045] R4: 5’-AGCTTCAGCCTCTCTTTTCTCGAG-3’.

[0046] Table 1 Homologous recombination enzyme ligation system

[0047]

[0048] Example 2: Construction and Screening of Recombinant Strain KM71 / pPIC9K-COL1 / 2 / 3

[0049] Construction of recombinant strain KM71 / pPIC9K-COL1 / 2 / 3: The recombinant plasmid pPIC9K-COL1 / 2 / 3 extracted in Example 1 was linearized by SalI digestion, recovered by gel extraction, and then mixed with competent cells of Pichia pastoris KM71 in a 2 mm electroporation cuvette and incubated on ice for 10 min. After electroporation at 1500 V for 5 ms, 1 mL of pre-cooled D-sorbitol solution with a concentration of 1 M was immediately added, transferred to a centrifuge tube, incubated at 30 °C and 180 rpm for 1 - 2 h, and then spread on an RDB (glucose regeneration solid medium) plate and incubated upside down at 30 °C for 2 d.

[0050] Screening of recombinant high-copy strain KM71 / pPIC9K-COL1 / 2 / 3: The transformants on the RDB plate were picked into 1 mL of ddH2O, diluted by a certain multiple, and then spread on a G418 antibiotic plate containing 0.2 mg / mL, and incubated upside down at 30 °C for 2 d. And the concentration of G418 was gradually increased to 0.5, 1.0, 2.0, 4.0 mg / mL. Finally, positive transformants were picked from the G418 antibiotic plate containing 4.0 mg / mL for genomic PCR verification ( Figure 3 ) and fermentation expression verification, and the strains with correct verification and higher expression levels were selected for strain preservation.

[0051] Example 3: Fermentation Culture of KM71 / pPIC9K-COL1 / 2 / 3

[0052] The glycerol bacteria ice crystals preserved in Example 2 were picked into 3 mL of YPD medium (10 g / L yeast extract powder, 20 g / L peptone, 20 g / L glucose, and the balance was water), cultured at 30 °C and 180 rpm for 16 - 24 h, and then inoculated into 50 mL (250 mL Erlenmeyer flask) of BMGY medium (10 g / L yeast extract powder, 20 g / L peptone, 13.4 g / L YNB, 20 g / L peptone, 3.012 g / L K2HPO4, 11.813 g / L KH2PO4, 10 mL / L glycerol, 400 μg / L biotin, and the balance was water) at an inoculation amount of 1%, and cultured at 30 °C and 220 rpm until the OD 620Around 0 to 10; Centrifuge to collect the bacterial cells and wash them twice with ddH2O, then resuspend them in 50 mL (250 mL Erlenmeyer flask) of BMMY medium (10 g / L yeast extract, 20 g / L peptone, 13.4 g / L YNB, 20 g / L peptone, 3.012 g / L K2HPO4, 11.813 g / L KH2PO4, 5 mL / L methanol, 400 μg / L biotin, the rest is water), culture at 28 °C and 250 rpm for 72 h, and supplement 1% methanol every 24 h during this period.

[0053] Take samples of the fermentation broth supernatant for SDS-PAGE analysis and detection ( Figure 4 ), and it is found that all three truncated collagens can be soluble and secreted, and the expression level of the target protein can reach more than 80% of the total extracellular protein.

[0054] Example 4: Isolation and purification of recombinant humanized type III collagen

[0055] Recombinant humanized type III collagen was purified by Ni column (His Trap excel 5 ml) affinity chromatography. First, replace the 20% ethanol in the chromatography column and the system with ddH2O until the UV 280 and conductivity tend to be stable. After loading the fermentation broth supernatant of Example 3, wash away impurities with PBS buffer (10 mM) containing 20 mM imidazole, then elute the target protein with PBS buffer (10 mM) containing 200 mM imidazole, and then regenerate the chromatography column with PBS buffer containing 500 mM imidazole. After one-step purification, it is found by ImageJ gray-scale analysis that the purity of the purified protein is 95%.

[0056] Load the eluted target protein into a dialysis bag for desalting, with the ratio of the content to pure water being 1:100, and change the pure water every 3 - 4 hours for a total of 4 times. Freeze-dry the dialyzed target protein to make a pure collagen product for subsequent SDS-PAGE analysis and detection.

[0057] Example 5: Optimization of fermentation conditions for recombinant humanized type III collagen

[0058] Taking the recombinant strain KM71 / pPIC9K-COL3 as an example, optimize the fermentation conditions by culturing at three levels of ① fermentation temperature; ② inducer addition concentration; ③ induction time. The specific parameters are as follows:

[0059] The temperature parameters are: 22 °C, 25 °C, 28 °C, and 30 °C.

[0060] The inducer addition amounts are: 0.25%, 0.5%, 0.75%, 1%, 1.5%, and 2% of the medium volume, and supplement it every 24 h.

[0061] The induction times were: 24 h, 48 h, 72 h, 96 h, 120 h, 144 h.

[0062] The single - factor fermentation conditions of the recombinant strain KM71 / pPIC9K - COL3 were optimized according to the above parameters respectively, and the supernatant of the fermentation broth was taken for SDS - PAGE to detect the expression level of the target protein. The results were as Figures 5 - 7 shown. The results showed that the conditions of 28 °C, an inducer addition amount of 1%, supplemented every 24 h, and fermented for 120 h were the most suitable for the expression of collagen.

[0063] Example 6: Promoting cell migration activity experiment of recombinant humanized type III collagen

[0064] Use a marker pen to draw a horizontal line at the bottom of the 6 - well plate as a mark. After collecting NIH / 3T3 cells in good growth state and counting them, inoculate them into a 6 - well cell culture plate at 5×10 5 cells / well, and place them in an incubator at 37 °C and 5% CO2. When the cell density reaches 95%, use a 10 - μl pipette tip to vertically draw a scratch downward along the ruler against the well plate. Rinse the cells 3 times with PBS to remove the scratched cells. Then add 2 ml of medium containing recombinant humanized type III collagen COL1 / 2 / 3 samples at a concentration of 1 mg / ml respectively. Use PBS buffer (10 mM) as the control group, culture in an incubator at 37 °C and 5% CO2, and take pictures and observe under a microscope at the intersection of the horizontal and vertical scratches at 0 h and after 24 h. Use Image J software to calculate the scratch area at the beginning and after 24 h of treatment, and calculate the cell migration rate (%)=(A 给药前 - A 给药后 ) / scratch area at 24 h.

[0065] The results were as Figure 8 shown. All three recombinant humanized type III collagens had the ability to promote cell migration. Among them, COL3 had the highest activity, and its migration rate was 10 times higher than that of the blank control group, indicating its good application prospects as cosmetics and biomedical materials.

[0066] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A group of recombinant humanized type III collagen, characterized in that, The amino acid sequence of the humanized type III collagen is shown in SEQ ID NO.1-3.

2. A gene encoding the humanized type III collagen according to claim 1.

3. The gene according to claim 2, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO.4-6.

4. An expression vector into which the gene according to claim 2 or 3 is inserted.

5. The expression vector according to claim 4, characterized in that, The expression vector is pPIC9K.

6. A host cell carrying the expression vector according to claim 4 or 5, characterized in that, The host cell can express humanized type III collagen.

7. The host cell according to claim 6, wherein The chassis cell of the host cell is Pichia pastoris KM71.

8. A method for producing the recombinant humanized type III collagen according to claim 1, characterized in that, Comprising the following steps: (1) After activating and expanding the host cell according to claim 6 or 7, inoculate it into a fermentation medium for culture; (2) Collect the supernatant of the fermentation broth, perform separation and purification, dialyze to remove salts, and lyophilize to obtain recombinant humanized type III collagen.

9. The method according to claim 8, wherein The activation medium in step (1) is YPD medium, and its composition is: yeast extract powder 10 g / L, peptone 20 g / L, glucose 20 g / L, and the balance is water; The expansion medium in step (1) is BMGY medium, and its composition is: yeast extract powder 10 g / L, peptone 20 g / L, YNB 13.4 g / L, peptone 20 g / L, K2HPO4 3.012 g / L, KH2PO4 11.813 g / L, glycerol 10 mL / L, biotin 400 μg / L, and the balance is water; The fermentation medium in step (1) is BMMY medium, and its composition is: yeast extract powder 10 g / L, peptone 20 g / L, YNB 13.4 g / L, peptone 20 g / L, K2HPO4 3.012 g / L, KH2PO4 11.813 g / L, methanol 5 mL / L, biotin 400 μg / L, and the balance is water; The conditions for fermentation culture in step (1) are: culture at 25-30 °C and 100-300 rpm for 90-144 h, and supplement methanol with a volume percentage of 1% every 20-28 h during this period; The separation and purification method in step (2) includes one or a combination of two or more of salting out, ion exchange chromatography, affinity chromatography, hydrophobic chromatography, acid-base precipitation, and membrane separation; preferably affinity chromatography.

10. Use of the humanized type III collagen according to claim 1 in the preparation of daily-use cosmetics, biomedical materials, and drugs for promoting the proliferation and migration of skin cells.

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