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

Abstract

The invention relates to the technical field of recombinant protein expression, in particular to recombinant human-derived III-type collagen as well as a preparation method and application thereof. The invention provides a recombinant human source III type collagen. The amino acid sequence of the recombinant human source III type collagen is shown as SEQ ID NO. 1. The recombinant human-derived III-type collagen prepared by the invention can be better applied to a human body, better exerts biological functions of the collagen, avoids repulsive interaction, is high in safety, can effectively promote cell proliferation and oxidation resistance, and is high in expression yield, stable in batches and good in quality; the recombinant human III type collagen prepared by the invention has the potential of being applied to preparation and production of biological medicines and cosmetics.

Description

Technical Field

[0001] The present invention relates to the technical field of recombinant protein expression, and in particular to a recombinant human type III collagen and a preparation method and application thereof. Background Art

[0002] Collagen is one of the most important and abundant proteins in mammals, accounting for about 30% of the total protein in the human body. Collagen is a structural protein and the main component of the extracellular matrix. Type III collagen is a triple helix consisting of three peptide chains curled to the right. It can provide tensile strength and structural integrity for many organs and participate in regulating basic activities such as cell adhesion, proliferation, migration, and differentiation. As a natural protein in the body, type III collagen is highly favored in the fields of biomedicine, food, and daily chemicals due to its good biocompatibility, weak antigenicity, high affinity to the skin, and controllable biodegradability.

[0003] Natural collagen is an insoluble fibrin protein, and its molecular sequence contains a unique (Gly-XY)n highly repetitive sequence. It is subject to strict post-translational modification and complex self-assembly during the synthesis process. Therefore, it has always faced the problems of difficult expression, low expression, difficult secretion and complex purification, which are the main reasons for the current small variety and high price of type III collagen. Because type III collagen coexists with other types of collagen and has a low content, there are severe problems such as difficulty, low purity and high cost in extracting type III collagen from animal tissues. The collagen prepared by recombinant expression has the advantages of single molecular weight, high purity and no hidden dangers of virus transmission. In addition, the expression amount, hydrophilicity and stability of collagen can be improved by optimizing the amino acid sequence of collagen, and the molecular weight of collagen can be optimized; thus, the prepared collagen has good tissue compatibility, skin permeability and safety.

[0004] The expression systems of recombinant collagen mainly include prokaryotes (mainly Escherichia coli), yeast, plants, baculovirus, and mammalian cell expression systems. At this stage, Escherichia coli and yeast are the main expression systems, but the efficient Escherichia coli, yeast and other expression systems lack the post-translational modification of collagen in animal cells, and the corresponding recombinant enzymes need to be added. The invention patent (application number 201910088754.X) proposes a method for preparing recombinant human type III collagen α1 chain using Pichia pastoris. This method can prepare recombinant human type III collagen, but the protein prepared by the Pichia pastoris expression system is still somewhat different from that of human sources.

[0005] There is an urgent need to provide a recombinant human type III collagen that can be expressed using mammalian cells. Summary of the invention

[0006] The purpose of the present invention is to provide a recombinant human type III collagen that can be better applied to the human body, better exert its biological function, and avoid rejection, and has high safety.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a recombinant human type III collagen, the amino acid sequence of the recombinant human type III collagen is shown as SEQ ID NO.1.

[0009] Preferably, the recombinant human type III collagen comprises, from the amino terminus, an amino terminus signal peptide, a mature peptide chain of human type III collagen, and a carboxyl terminus affinity purification tag;

[0010] The amino acid sequence of the amino-terminal signal peptide is shown in SEQ ID NO.2; the carboxyl-terminal affinity purification tag is a His tag.

[0011] The present invention also provides a gene encoding the recombinant human type III collagen, and the nucleotide sequence of the gene is shown in SEQ ID NO.3.

[0012] The invention also provides a recombinant expression vector, comprising the gene and the transposon vector.

[0013] Preferably, the transposon vector is a pPB transposon vector containing a transposase system.

[0014] The present invention also provides a recombinant cell, comprising the recombinant expression vector and a host cell.

[0015] Preferably, the host cell is a CHO cell.

[0016] The present invention also provides a method for preparing recombinant human type III collagen, comprising the following steps:

[0017] (1) connecting the gene to a transposon vector to obtain a recombinant expression vector;

[0018] (2) transfecting the recombinant expression vector into host cells to obtain recombinant cells;

[0019] (3) Cultivating, screening, and domesticating the recombinant cells to obtain a monoclonal cell line;

[0020] (4) The monoclonal cell line is expanded and purified to obtain recombinant human type III collagen.

[0021] Preferably, in step (3), the culture temperature is 30-40°C; the culture time is 40-50h;

[0022] In step (4), the temperature of the expanded culture is 30-40° C.; and the purification method is nickel ion column affinity chromatography.

[0023] The present invention also provides the use of the recombinant human type III collagen, the gene, the recombinant expression vector, the recombinant cell or the recombinant human type III collagen prepared by the preparation method in the preparation of biological preparations that promote cell proliferation and / or anti-oxidation.

[0024] Beneficial effects of the present invention:

[0025] The present invention selects the natural full-length mature peptide chain of human type III collagen, removes the propeptides at both ends, and the expression level is much higher than the gene containing the propeptide; different specific affinity purification tags are designed at both ends of the collagen, which is beneficial to the purification of the full-length protein and the identification of the collagen.

[0026] In the present invention, the transposase system is introduced into the vector, which can improve the stable expression of recombinant human type III collagen; in addition, the present invention selects a CHO cell expression system with humanized post-translational modification, which solves the problems of unstable structure of type III collagen, incomplete hydroxylation modification, etc.; and also makes the expressed recombinant human type III collagen have a complete triple helix structure and good thermal stability, so that the collagen can be better applied to the human body and better exert its biological function, while avoiding rejection, with high safety, and can be safely applied to the fields of biomedical materials and cosmetics.

[0027] The recombinant human type III collagen prepared by the present invention has the functions of type III collagen, can effectively promote cell proliferation and anti-oxidation, and has high expression yield, batch-to-batch stability, and good quality; the recombinant human type III collagen prepared by the present invention has the potential to be applied to the preparation and production of biomedicine and cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of recombinant expression vector, wherein A is recombinant expression vector 1, and B is recombinant expression vector 2;

[0029] Figure 2 Agarose electrophoresis results of recombinant expression vectors, wherein A is an agarose electrophoresis result of recombinant expression vector 1, lane 1 is an agarose electrophoresis result of recombinant expression vector 1, lane 2 is an agarose electrophoresis result of recombinant expression vector 1 after being digested with XhoI, and B is an agarose electrophoresis result of recombinant expression vector 2, lane 1 is recombinant expression vector 2, and lane 2 is an agarose electrophoresis result of recombinant expression vector 2 after being digested with XhoI;

[0030] Figure 3 Comparison of recombinant human type III collagen expression; Lane 1 is monoclonal cell line 1, and Lane 2 is monoclonal cell line 2;

[0031] Figure 4 Recombinant human type III collagen expression identification diagram;

[0032] Figure 5 The results of recombinant human type III collagen promoting cell proliferation, where A is the 24h cell proliferation result and B is the 48h cell proliferation result;

[0033] Figure 6 The results of the antioxidant activity of recombinant human type III collagen, where A is the result of rhCOL3A1ABTS free radical scavenging, B is the result of rhCOL3A1 FRAP reducing power assay, C is the result of VC ABTS free radical scavenging, and D is the result of VC FRAP reducing power assay. DETAILED DESCRIPTION

[0034] The present invention provides a recombinant human type III collagen, the amino acid sequence of which is shown in SEQ ID NO.1;

[0035]

[0036] In the present invention, the recombinant human type III collagen comprises, from the amino terminus, an amino terminus signal peptide, a mature peptide chain of human type III collagen, and a carboxyl terminus affinity purification tag;

[0037] The amino acid sequence of the amino-terminal signal peptide is shown in SEQ ID NO.2: KWLLLLLLLLLLLLLLLLVT; the carboxyl-terminal affinity purification tag is a His tag.

[0038] The present invention also provides a gene encoding the recombinant human type III collagen, the nucleotide sequence of which is shown in SEQ ID NO.3;

[0039]

[0040] The invention also provides a recombinant expression vector, comprising the gene and the transposon vector.

[0041] In the present invention, the transposon vector is a pPB transposon vector containing a transposase system;

[0042]

[0043] The present invention also provides a recombinant cell, comprising the recombinant expression vector and a host cell.

[0044] In the present invention, the host cell is a CHO cell (purchased from the cell bank of the Chinese Academy of Sciences).

[0045] The present invention also provides a method for preparing recombinant human type III collagen, comprising the following steps:

[0046] (1) connecting the gene to a transposon vector to obtain a recombinant expression vector;

[0047] (2) transfecting the recombinant expression vector into host cells to obtain recombinant cells;

[0048] (3) Cultivating, screening, and domesticating the recombinant cells to obtain a monoclonal cell line;

[0049] (4) The monoclonal cell line is expanded and purified to obtain recombinant human type III collagen.

[0050] In the present invention, in step (3), the culture temperature is 30-40°C, preferably 33-37°C, and more preferably 35°C; the culture time is 40-50h, preferably 43-47h, and more preferably 45h;

[0051] The screening includes pressure screening and monoclonal screening;

[0052] The method used for monoclonal screening is the limiting dilution method;

[0053] In step (4), the temperature of the expanded culture is 30-40°C, preferably 33-37°C, and more preferably 35°C; the purification method is nickel ion column affinity chromatography.

[0054] The present invention also provides the use of the recombinant human type III collagen, the gene, the recombinant expression vector, the recombinant cell or the recombinant human type III collagen prepared by the preparation method in the preparation of biological preparations that promote cell proliferation and / or anti-oxidation.

[0055] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0056] Example 1 Preparation of recombinant human type III collagen

[0057] (1) Using the codon-optimized nucleotide sequence of recombinant human type III collagen (SEQ ID NO. 3) as a template, gene synthesis was performed, and the gene was connected to a pCHO1.0 vector without a transposase system (recombinant expression vector 1) and a pPB vector containing a transposase system (SEQ ID NO. 4) (recombinant expression vector 2) by seamless cloning technology, respectively, to obtain recombinant expression vector 1 and recombinant expression vector 2 (such as Figure 1 As shown) [The expression recombinant vectors were artificially synthesized by General Biotechnology Co., Ltd. (Chuzhou, China)];

[0058] The recombinant expression vector 1 and the recombinant expression vector 2 were digested with Xho I restriction endonuclease (Takara Company). The digestion system was: expression vector 1 / expression vector 2 10 μL (0.6 μg / μL), 10×Buffer 5 μL, Xho I restriction endonuclease (10 U / μL) 2.0 μL each, and water was added to 50 μL; the digestion conditions were: 37°C, digestion for 3 h; then agarose gel electrophoresis was performed to identify the digestion results. Figure 2 As shown, the results showed that the recombinant expression vector was successfully constructed.

[0059] (2) The recombinant expression vector 1 and the recombinant expression vector 2 successfully constructed in step (1) are respectively transfected into Chinese hamster ovary cells (CHO cells, purchased from the cell bank of the Chinese Academy of Sciences) to obtain recombinant cells.

[0060] (3) The recombinant cells in step (2) were cultured in a 37°C, 5% CO2 cell culture incubator for 48 h, and then subjected to pressure screening, the specific steps being as follows: the six-well plate cells were removed from the 37°C incubator, the supernatant culture medium was discarded, 2 mL of DMEM / F12 (containing 10% serum + 15 μg / mL blasticidin) was added, and screening was performed until the negative control cells were basically dead; the blasticidin concentration was reduced to 10 μg / mL, and the culture was continued to obtain cell pools 1 and 2 expressing recombinant type III collagen; then, a limiting dilution method was used for monoclonal screening to obtain monoclonal cell lines 1 and 2 expressing recombinant type III collagen, respectively, the specific steps being as follows: after cell pools 1 and 2 were fully grown in the six-well plate, the supernatant culture medium in the six-well plate was discarded, the cells were washed once with 1 mL of 37°C pre-warmed PBS, and the PBS was discarded; 0.4 mL of trypsin was added to each well, and 1 mL of lysine was added when the cells were observed to shrink and round under a microscope and to be single cells. DMEM / F12 (containing 10% serum) was used to terminate the digestion, and the cells were blown off with a pipette; the digested cells were transferred to a 2 mL centrifuge tube, centrifuged at 1000 rpm for 5 min; the cells were suspended with 1 mL of DMEM / F12 (containing 10% serum) medium and counted; the cells were gradually diluted to 10 cells / mL with DMEM / F12 (containing 10% serum), 100 μL of the diluted cells were inoculated into a 96-well plate, and observed under a microscope the next day, and 96 wells with 1 cell per well were picked for marking for subsequent culture until they were expanded to a 6-well plate, and untamed monoclonal cell line 1 and untamed monoclonal cell line 2 were obtained respectively; when the untamed monoclonal cell line 1 and untamed monoclonal cell line 2 in the 6-well plate were full, the supernatant medium in the 6-well plate was discarded, and the cells were washed once with 1 mL of PBS pre-warmed at 37°C, and the PBS was discarded; 0.4 mL of trypsin was added to each well, and 1 mL of The digestion was terminated with DMEM / F12 (containing 10% serum) and the cells were dispersed with a pipette; the digested cells were transferred to a 2 mL centrifuge tube and centrifuged at 1000 rpm for 5 min; the cells were resuspended with 1 mL CHO serum-free medium (DC1003, Henan Punuoyi Biological Products Research Institute Co., Ltd.) and counted; the cells were diluted to 5 × 10 510 cells / mL were inoculated into 4 mL of culture medium in a 6-well plate to obtain monoclonal cell line 1 and monoclonal cell line 2; cell density and viability were counted every 24 hours; after culturing for 7 days, 1 mL of supernatant was taken into a 1.5 mL centrifuge tube, centrifuged at 1000 rpm for 5 min, and the supernatant was taken; the supernatant was put back into a new 1.5 mL centrifuge tube, centrifuged at 12000 rpm for 5 min; 80 μL of supernatant was taken and added to a new 1.5 mL centrifuge tube, and then 20 μL of 5× protein loading buffer (BL529A, Biosharp) was added and the mixture was boiled in water for 10 min; the supernatant was taken for electrophoresis to compare the expression effects of monoclonal cell line 1 and monoclonal cell line 2. The results are shown in the figure. Figure 3 As shown;

[0061] from Figure 3 It can be seen that the recombinant human type III collagen expressed by the recombinant expression vector 2 is significantly more effective than that expressed by the recombinant expression vector 1;

[0062] (4) The monoclonal cell line 2 in step (3) was placed in a 37°C, 5% CO2 cell culture incubator for expansion culture; after the cells were fully grown, the cells were removed from the 37°C incubator, the supernatant culture medium was discarded, the cells were washed once with 5 mL PBS pre-warmed at 37°C, and the PBS was discarded; 2 mL of trypsin was added to each 10 cm cell culture dish, and when the cells were observed to shrink and become round under a microscope and to be single cells, 4 mL of DMEM / F12 (containing 10% serum and 10 μg / mL blasticidin) was added to terminate the digestion, and the cells were blown off with a pipette; the digested cells were transferred to a 15 mL centrifuge tube, centrifuged at 1000 rpm for 5 min; the cells were suspended with CHO serum-free medium and counted; the cells were diluted to 5×10 5 cells / mL, inoculated 30mL culture medium in 125mL cell culture flasks, and a total of 5 flasks were inoculated; the cell culture flasks were placed on an orbital shaker in a 37°C, 5% CO2 cell culture incubator for culture at 120rpm; the biosafety cabinet counter was wiped and disinfected with 75% alcohol and irradiated with ultraviolet light for 30min; the cell density and viability were counted every 24h; after 7d of culture, the supernatant in the culture medium was purified by Ni-NTA Sepharose: 2mL Ni-NTASepharose slurry (50% suspension) filler was loaded on the column, equilibrated with PBS (pH7.4) for 5 column volumes, loaded with the sample, equilibrated again, and eluted with 2mL of eluents with different concentrations of imidazole (100mM, 500mM, 750mM, 1M and 2M), respectively, equilibrated again after elution, and washed with deionized water for 5 times the column volume to obtain the eluate, and the imidazole in the eluate was removed to obtain the recombinant human type III collagen solution.

[0063] Example 2 Identification of recombinant human type III collagen using western blot

[0064] After culturing for 7 days in step (4) of Example 1, the supernatant in the culture medium was separated by polyacrylamide gel electrophoresis and then the protein was transferred to a PVDF membrane by wet transfer method, and blocked with 5% skim milk powder at 25°C for 90 minutes; diluted Anti-HISTag Antibody (HRP-66005, proteintech) was added dropwise to a final concentration of 0.4 μg / mL, and incubated on a shaker at 4°C for 12 hours; the membrane was washed with TBST buffer for 10 minutes × 3 times, and Mouse Anti-Human IgG (S0002, Affinity) dilution was added to a final concentration of 0.25 μg / mL and incubated at room temperature for 1 hour; the membrane was washed with TBST buffer for 10 minutes × 3 times, and then the luminescent solution in the ECL luminescent ultrasensitive kit [36208ES76, Yisheng Biotechnology (Shanghai) Co., Ltd.] was prepared, and the luminescent solution A and luminescent solution B were mixed in a ratio of 1:1 according to the reagent instructions and used immediately. In the gel imaging system, 1 mL of the prepared luminescent solution was evenly added to the washed membrane, and then exposed and developed. The imaging results are shown as follows Figure 4 As shown;

[0065] from Figure 4 It can be seen that the molecular weight of recombinant human type III collagen is about 150kDa, and the theoretical molecular weight of recombinant human type III collagen is about 98kDa. The difference between the two is due to the post-translational modification of recombinant human type III collagen in CHO cells.

[0066] Example 3 Protein concentration determination:

[0067] The amount of protein obtained in the supernatant of 0.15 L of Example 1 step (4) after culturing for 7 days was determined using a BCA kit (P0010S, Bio-Tech Biotechnology Co., Ltd.) to be 150 μg. Therefore, the amount of protein in 1 L of culture supernatant was 1 mg, and the protein yield was 1 mg / mL.

[0068] Example 4

[0069] Umbilical cord mesenchymal stem cells (UCMSCs, from the Laboratory of Tissue Engineering and Regeneration Clinical Medicine Center of Xinxiang Medical College) grown to 80% of the culture dish area were obtained, digested with trypsin, and made into a cell density of 1×10 5 / mL cell suspension; 100 μL of the cell suspension was inoculated into a 96-well culture plate and placed in an incubator at 37°C and 5% CO2 saturated humidity; after culturing for 24 hours, the complete culture medium was aspirated and replaced with culture medium containing samples of different concentrations: the recombinant human type III collagen (rhCOL3A1) in Example 1 was added to a high-glucose DMEM culture medium containing 10% fetal bovine serum, and the final concentrations of the recombinant human type III collagen were 0.05 mg / mL, 0.1 mg / mL, and 0.2 mg / mL, respectively; Commercially available recombinant human type III collagen (commercially available collagen) was added to high-glucose DMEM medium containing 10% fetal bovine serum, and the final concentrations of recombinant human type III collagen were 0.05 mg / mL, 0.1 mg / mL, and 0.2 mg / mL, respectively; the high-glucose DMEM medium without cells was used as the blank group, and the high-glucose DMEM medium without collagen (containing 10% serum) was used as the control group; the cells were cultured in an incubator at 37°C and 5% CO2 saturated humidity for 24 h, 48 h, 72 h, and 96 h, respectively. After culturing for 24 h, 48 h, 72 h, and 96 h, 10 μL of CCK-8 reagent was added to each group, incubated in a cell culture incubator for 3 h, and the absorbance value (OD value) of each well was measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay; the relative proliferation rate (RGR) of the cells was calculated according to the following formula based on the mean absorbance of each group, and the calculation results are as follows: Figure 5 As shown;

[0070]

[0071] from Figure 5 It can be seen that the recombinant human type III collagen (rhCOL3A1) described in Example 1 has a better effect on promoting the proliferation of UCMSCs than commercially available recombinant human type III collagen.

[0072] Example 5

[0073] The recombinant human type III collagen prepared in Example 1 was prepared into collagen solutions with concentrations of 0.02 mg / mL, 0.05 mg / mL, 0.08 mg / mL, 0.1 mg / mL, and 0.15 mg / mL using PBS, and the antioxidant activity of the samples was determined using total antioxidant capacity (ABTS method, FRAP method) test kits (AS632164 and AS632166, purchased from Shanghai Fusheng Industrial Co., Ltd.); both methods used water-soluble vitamin C as a control system to quantify the antioxidant capacity of antioxidant substances, and the test results are as follows: Figure 6 As shown;

[0074] The ABTS method is a relatively accurate method for determining in vitro antioxidant activity; the ABTS free radical scavenging method can determine the antioxidant status of multi-component mixtures and can be used to measure the overall antioxidant activity; ABTS free radicals can react with antioxidants to remove cations, and the absorbance value also changes linearly;

[0075] The FRAP method can determine the total antioxidant level of samples under acidic conditions. Antioxidants reduce Fe 3+ Tripyridine triazine (Fe 3+ -TPTZ) produces blue Fe 2+ -TPTZ reflects antioxidant capacity;

[0076] from Figure 6 It can be seen that the recombinant human type III collagen prepared in Example 1 has a good linear relationship between ABTS clearance rate and reducing power, and the antioxidant capacity increases accordingly with the increase of concentration; therefore, the recombinant human type III collagen prepared in Example 1 has certain antioxidant properties.

[0077] It can be seen from the above embodiments that the present invention provides a recombinant human type III collagen and its preparation method and application. The recombinant human type III collagen prepared by the present invention can be better applied to the human body, better exert its biological function, and avoid rejection, with high safety, can effectively promote cell proliferation and anti-oxidation, and has high expression yield, batch-to-batch stability, and good quality; the recombinant human type III collagen prepared by the present invention has the potential to be applied to the preparation and production of biomedicine and cosmetics.

[0078] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A recombinant human type III collagen, characterized in that: The amino acid sequence of the recombinant human type III collagen is shown in SEQ ID NO.

1.

2. The recombinant human type III collagen according to claim 1, characterized in that: The recombinant human type III collagen protein comprises, from the amino terminal, an amino terminal signal peptide, a mature peptide chain of human type III collagen, and a carboxyl terminal affinity purification tag; The amino acid sequence of the amino-terminal signal peptide is shown in SEQ ID NO.2; the carboxyl-terminal affinity purification tag is a His tag.

3. The gene encoding the recombinant human type III collagen according to claim 1, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.

3.

4. A recombinant expression vector, characterized in that: Comprising the gene and transposon vector according to claim 3.

5. The recombinant expression vector according to claim 4, characterized in that The transposon vector is a pPB transposon vector containing a transposase system.

6. A recombinant cell, characterized in that Comprising the recombinant expression vector and host cell according to claim 4 or 5.

7. The recombinant cell according to claim 6, characterized in that The host cell is a CHO cell.

8. A method for preparing recombinant human type III collagen, characterized in that: The steps include: (1) connecting the gene described in claim 3 to a transposon vector to obtain a recombinant expression vector; (2) transfecting the recombinant expression vector into host cells to obtain recombinant cells; (3) Cultivating, screening, and domesticating the recombinant cells to obtain a monoclonal cell line; (4) The monoclonal cell line is expanded and purified to obtain recombinant human type III collagen.

9. The preparation method according to claim 8, characterized in that: In step (3), the culture temperature is 30-40° C. and the culture time is 40-50 h. In step (4), the temperature of the expanded culture is 30-40° C.; and the purification method is nickel ion column affinity chromatography.

10. Use of the recombinant human type III collagen described in claim 1 or 2, the gene described in claim 3, the recombinant expression vector described in claim 4 or 5, the recombinant cell described in claim 6 or 7, or the recombinant human type III collagen prepared by the preparation method described in claim 8 or 9 in the preparation of biological preparations that promote cell proliferation and / or anti-oxidation.

Images