CN116284339A - Recombinant III type collagen, nucleic acid, expression vector, strain and application thereof - Google Patents
Recombinant III type collagen, nucleic acid, expression vector, strain and application thereof Download PDFInfo
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- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
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Abstract
The invention provides a recombinant III type collagen, nucleic acid, an expression vector, a strain and application thereof, wherein the recombinant III type collagen is obtained by a genetic engineering method, the synthesis and production of the collagen can be realized by using the transformed microorganism chassis cells by adopting a synthetic biological means, and the recombinant collagen produced by adopting the method has high expression quantity, excellent cell proliferation effect, low toxicity, low antigenicity, low immunity, better cell regeneration guiding and biocompatibility, low immune response generated when the recombinant III type collagen is applied to a human body, simple and environment-friendly production and preparation method and the like, and has wide application prospects in the industries of biological medicine, cosmetics, skin care products, food and the like.
Description
Technical Field
The invention relates to the technical field of biology, in particular to recombinant III type collagen, nucleic acid, an expression vector, a strain and application thereof.
Background
Collagen is a protein with the highest content in the body, and accounts for 25% -33% of human protein, is widely distributed in various tissues and organs of the human body, such as skin, bones, cornea, blood vessels and the like, especially in the tissues of the skin and the like, contains a large amount of collagen which is used as an adhesive substance of connective tissues and plays an important role in maintaining the normal physiological functions of cells, tissues and organs. There are 29 types of collagen known at present. Different types of collagen differ in the shape of the globular domain, the spacing of the triple helical structures and the major length of collagen.
About 90% of normal human collagen exists in skin and bone, and collagen of different types and different sites plays an important role in tissue support, intercellular signaling and the like. Traditional collagen production methods use animal skin, tissue or bone materials for extraction. However, this conventional method for extracting and preparing animal-derived collagen has various potential safety hazards, for example, diseases caused by zoonotic agents possibly existing in mad cow disease, foot-and-mouth disease, swine fever and the like; the stability of raw materials in different batches is difficult to ensure; meanwhile, the mode brings a plurality of pollution, and the production mode is not environment-friendly; traditional collagen harvesting approaches based on the above drawbacks limit further applications of collagen.
Disclosure of Invention
The invention mainly aims to provide recombinant type III collagen, nucleic acid, expression vector, strain and application thereof, and aims to provide safe collagen.
To achieve the above object, the present invention provides a recombinant type III collagen, the amino acid sequence of which comprises at least one of the following:
(1)SEQ ID NO：1：GPPGPAGANGAPGLRGGAGEPGKNGAKGEPGP RGERGEAGIPGVPGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAG PNGIPGEKGPAGERGAP；
(2)SEQ ID NO：2：GQPGEKGSPGAQGPPGAPGPLGIAGITGARGLA GPPGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGLPGLAGTAGEPGRDGNPGSDGLPGRDGSPGGKGDRGENGSPGAPGAPGHPGPPGPVGPAGKSGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPRGPVGPSGPPGKDGTSGHPGPIGPPGPRGNRGERGSE；
or (1) or (2) has an amino acid sequence in which 1 or more amino acids are added, substituted or deleted.
In one embodiment, the amino acid sequence comprises 2 to 10 repeats of SEQ ID NO:1, a step of; and/or the amino acid sequence comprises 2-10 repeats of SEQ ID NO:2.
the invention also provides a nucleic acid which codes for the recombinant type III collagen, wherein the amino acid sequence of the recombinant type III collagen comprises at least one of the following:
(1)SEQ ID NO：1：GPPGPAGANGAPGLRGGAGEPGKNGAKGEPGP RGERGEAGIPGVPGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAG PNGIPGEKGPAGERGAP；
(2)SEQ ID NO：2：GQPGEKGSPGAQGPPGAPGPLGIAGITGARGLA GPPGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGLPGLAGTAGEPGRDGNPGSDGLPGRDGSPGGKGDRGENGSPGAPGAPGHPGPPGPVGPAGKSGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPRGPVGPSGPPGKDGTSGHPGPIGPPGPRGNRGERGSE；
or (1) or (2) has an amino acid sequence in which 1 or more amino acids are added, substituted or deleted.
In one embodiment, the nucleic acid sequence is set forth in SEQ ID NO:3 and/or SEQ ID NO:4, wherein SEQ ID NO:3 is as follows:
GGTCCCCCAGGTCCAGCTGGTGCTAACGGAGCCCCAGGTCTGAGAGGTGGTGCCGGTGAACCAGGAAAAAACGGTGCTAAGGGTGAGCCTGGTCCAAGAGGAGAGAGAGGTGAAGCAGGTATTCCTGGTGTTCCAGGTGCTAAGGGAGAAGATGGTAAAGATGGATCTCCAGGTGAACCCGGTGCCAACGGTTTACCAGGTGCTGCCGGAGAAAGAGGAGCACCAGGTTTTAGAGGTCCAGCTGGTCCAAACGGTATTCCAGGAGAAAAGGGACCAGCAGGAGAAAGAGGTGCTCCC；
SEQ ID NO:4 is as follows:
GGACAACCTGGTGAAAAGGGATCTCCTGGTGCCCAAGGACCACCTGGAGCCCCTGGTCCCTTGGGTATTGCTGGAATTACTGGTGCAAGAGGTTTGGCTGGACCACCAGGTATGCCAGGTCCAAGAGGTAGTCCTGGACCACAAGGTGTTAAAGGTGAATCTGGAAAGCCTGGAGCCAACGGTCTGTCAGGAGAGAGAGGACCACCTGGTCCACAAGGACTTCCAGGTTTGGCAGGTACCGCCGGTGAACCTGGAAGAGATGGTAATCCAGGAAGTGACGGTTTGCCAGGTAGAGACGGATCACCAGGAGGAAAGGGAGATAGAGGTGAAAACGGTTCTCCTGGTGCTCCAGGTGCTCCTGGTCATCCCGGTCCACCTGGTCCAGTTGGTCCAGCCGGTAAGTCTGGTGATAGAGGTGAGTCTGGTCCAGCAGGTCCAGCCGGTGCTCCAGGTCCTGCTGGATCTAGAGGTGCTCCTGGACCACAGGGTCCTAGAGGTGATAAGGGTGAGACTGGTGAAAGAGGTGCAGCAGGTATTAAGGGTCATAGAGGTTTTCCAGGTAACCCAGGAGCCCCTGGTAGTCCAGGTCCAGCAGGACAACAAGGTGCTATAGGTAGTCCAGGTCCTGCCGGTCCAAGAGGTCCTGTTGGTCCATCAGGACCACCAGGTAAAGATGGTACCTCCGGTCATCCTGGTCCAATTGGACCACCAGGTCCAAGAGGTAATAGAGGTGAAAGAGGTTCTGAA。
the invention also provides an expression vector, wherein the expression vector is inserted with the nucleic acid, or the expression vector can enable the host cell to express the recombinant type III collagen after the host cell is transfected.
In an embodiment, the expression vector includes any one of pPIC9K, pPICZ a A, GAPZ a, A, pPICZ a B, pPICZ a C, GAPZ a B, GAPZ a C and modified vector thereof.
The invention also provides a strain for expressing the recombinant type III collagen, wherein the strain can express the recombinant type III collagen.
In one embodiment, the strain includes any one of pichia pastoris, saccharomyces cerevisiae and escherichia coli.
In one embodiment, the pichia comprises one or more of SMD1168, KM71H, SMD1163, X33, and GS115.
The invention also provides application of the recombinant type III collagen, the nucleic acid, the expression vector or the strain for expressing the recombinant type III collagen in preparing skin care products, cosmetics, health care products, foods and medicines.
The recombinant type III collagen is obtained by a genetic engineering method, the synthesis production of the collagen can be realized by using the transformed microorganism chassis cells by adopting a synthetic biological means, and the recombinant collagen produced by adopting the method has high expression level, good cell proliferation effect, low toxicity, low antigenicity, low immunity, better cell regeneration guidance and biocompatibility, low immune response when being applied to a human body, simple and environment-friendly production and preparation method and the like, and has wide application prospect in the industries of biological medicine, cosmetics, skin care products, food and the like.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to the structures shown in these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram showing the structure of a CL3.4 expression plasmid according to example 1 of the present invention;
FIG. 2 is a schematic structural diagram of the CL3.8 expression plasmid of example 1 of the present invention;
FIG. 3 is a graph showing the results of the expression level of the collagen shake flask culture protein in example 3 of the present invention;
FIG. 4 is a graph showing the results of the protein expression level of CL3.4 collagen in various chassis cells according to example 4 of the present invention;
FIG. 5 is a graph showing the results of the protein expression level of CL3.8 collagen in various chassis cells according to example 4 of the present invention;
FIG. 6 is a graph showing the results of the protein expression levels of the collagen fermenter culture (0 to 48 hours) in example 5 of the present invention;
FIG. 7 is a graph showing the results of the protein expression levels of the collagen fermenter culture (48 to 72 hours) in example 5 of the present invention;
FIG. 8 is a graph showing the relationship between the absorbance and the standard protein solutions of different concentrations in example 6 of the present invention;
FIG. 9 is a bar chart showing the results of the collagen cell proliferation assay in example 7 of the present invention.
The achievement of the objects, functional features and advantages of the present invention will be further described with reference to the accompanying drawings, in conjunction with the embodiments.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
In a first aspect, the present invention provides a recombinant type III collagen.
In an embodiment of the present invention, the amino acid sequence of the recombinant type III collagen comprises at least one of the following:
(1)SEQ ID NO：1：GPPGPAGANGAPGLRGGAGEPGKNGAKGEPGP RGERGEAGIPGVPGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAG PNGIPGEKGPAGERGAP；
(2)SEQ ID NO：2：GQPGEKGSPGAQGPPGAPGPLGIAGITGARGLA GPPGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGLPGLAGTAGEPGRDGNPGSDGLPGRDGSPGGKGDRGENGSPGAPGAPGHPGPPGPVGPAGKSGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPRGPVGPSGPPGKDGTSGHPGPIGPPGPRGNRGERGSE；
or (1) or (2) has an amino acid sequence in which 1 or more amino acids are added, substituted or deleted.
The recombinant type III collagen is obtained by a genetic engineering method, the synthesis production of the collagen can be realized by using the transformed microorganism chassis cells by adopting a synthetic biological means, and the recombinant collagen produced by adopting the method has high expression level, good cell proliferation effect, low toxicity, low antigenicity, low immunity, better cell regeneration guidance and biocompatibility, low immune response when being applied to a human body, simple and environment-friendly production and preparation method and the like, and has wide application prospect in the industries of biological medicine, cosmetics, skin care products, food and the like.
Aiming at the recombinant type III collagen, the construction mode of the invention is divided into excavation and selection of gene sequences, construction of expression vectors, integration of the expression vectors, construction of collagen expression recombinant strains, chassis cell selection and optimization, amplification fermentation and cell proliferation test. The following will be described in detail for each step.
1. Mining and selection of Gene sequences
The sequence composition and structure of the type III collagen sequence (NP 000081.2) are analyzed by searching the NCBI database to mine the collagen sequence, and a structural domain at each of the N end and the C end is excised in the later protein processing. In addition, it has been shown that sequences containing structural regions of EK and ER, such as GPAGEK, GAPGER and GPAGFR, interact with receptors on the surface of fibroblasts, enhance integrin binding, and modulate cell recognition, adhesion and migration, thereby exerting biological functions.
Based on this, 2 type III collagen sequences were selected, and then their bases were optimized based on codon preference to give the sequence SEQ ID NO:3 and SEQ ID NO:4, the amino acid sequence of which is SEQ ID NO:1 and SEQ ID NO:2.
wherein, SEQ ID NO:1 is as follows:
GPPGPAGANGAPGLRGGAGEPGKNGAKGEPGPRGERGEAGIPGVPGA KGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGIPGEKGPAGERG AP；
SEQ ID NO:2 is as follows:
GQPGEKGSPGAQGPPGAPGPLGIAGITGARGLAGPPGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGLPGLAGTAGEPGRDGNPGSDGLPGRDGSPGGKGDRGENGSPGAPGAPGHPGPPGPVGPAGKSGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPRGPVGPSGPPGKDGTSGHPGPIGPPGPRGNRGERGSE; SEQ ID NO:3 is as follows:
GGTCCCCCAGGTCCAGCTGGTGCTAACGGAGCCCCAGGTCTGAGAGGTGGTGCCGGTGAACCAGGAAAAAACGGTGCTAAGGGTGAGCCTGGTCCAAGAGGAGAGAGAGGTGAAGCAGGTATTCCTGGTGTTCCAGGTGCTAAGGGAGAAGATGGTAAAGATGGATCTCCAGGTGAACCCGGTGCCAACGGTTTACCAGGTGCTGCCGGAGAAAGAGGAGCACCAGGTTTTAGAGGTCCAGCTGGTCCAAACGGTATTCCAGGAGAAAAGGGACCAGCAGGAGAAAGAGGTGCTCCC；
SEQ ID NO:4 is as follows:
GGACAACCTGGTGAAAAGGGATCTCCTGGTGCCCAAGGACCACCTGGAGCCCCTGGTCCCTTGGGTATTGCTGGAATTACTGGTGCAAGAGGTTTGGCTGGACCACCAGGTATGCCAGGTCCAAGAGGTAGTCCTGGACCACAAGGTGTTAAAGGTGAATCTGGAAAGCCTGGAGCCAACGGTCTGTCAGGAGAGAGAGGACCACCTGGTCCACAAGGACTTCCAGGTTTGGCAGGTACCGCCGGTGAACCTGGAAGAGATGGTAATCCAGGAAGTGACGGTTTGCCAGGTAGAGACGGATCACCAGGAGGAAAGGGAGATAGAGGTGAAAACGGTTCTCCTGGTGCTCCAGGTGCTCCTGGTCATCCCGGTCCACCTGGTCCAGTTGGTCCAGCCGGTAAGTCTGGTGATAGAGGTGAGTCTGGTCCAGCAGGTCCAGCCGGTGCTCCAGGTCCTGCTGGATCTAGAGGTGCTCCTGGACCACAGGGTCCTAGAGGTGATAAGGGTGAGACTGGTGAAAGAGGTGCAGCAGGTATTAAGGGTCATAGAGGTTTTCCAGGTAACCCAGGAGCCCCTGGTAGTCCAGGTCCAGCAGGACAACAAGGTGCTATAGGTAGTCCAGGTCCTGCCGGTCCAAGAGGTCCTGTTGGTCCATCAGGACCACCAGGTAAAGATGGTACCTCCGGTCATCCTGGTCCAATTGGACCACCAGGTCCAAGAGGTAATAGAGGTGAAAGAGGTTCTGAA。
the amino acid sequence is not limited to the above-mentioned SEQ ID NO:1 and SEQ ID NO:2, in one embodiment, the sequence compared to SEQ ID NO:1 or SEQ ID NO:2, has an amino acid sequence of 1 or more amino acids added, substituted, deleted, and the like, and retains a similar effect.
In one embodiment, the sequence compared to SEQ ID NO:1, which may comprise 2 to 10 amino acid sequences of SEQ ID NOs: 1 are repeated.
In one embodiment, the sequence compared to SEQ ID NO:2, which may comprise 2 to 10 amino acid sequences of SEQ ID NOs: 2 repeatedly.
2. Construction of expression vectors
The gene sequences CL3.4 and CL3.8 (i.e., the nucleic acid sequences SEQ ID NO:3 and SEQ ID NO: 4) were obtained by PCR amplification, and the expression vector was linearized using EcoRI and NotI endonucleases. And assembling the CL3.4 and CL3.8 gene sequences onto the expression vector by seamless cloning assembly to obtain a CL3.4 expression vector and a CL3.8 expression vector.
The expression vector can be any one of pPIC9K, pPICZ alpha A, GAPZ alpha A, pPICZ alpha B, pPICZ alpha C, GAPZ alpha B, GAPZ alpha C and a modified vector thereof, or a vector modified based on the expression vector. In one embodiment, pPIC9K expression vectors are used, and after assembly, pPIC9K-CL3.4 and pPIC9K-CL3.8 expression vectors are obtained, respectively.
3. Integration of expression vectors and construction of recombinant collagen expression strains
The expression vector constructed above was incubated with SalI enzyme at 37℃for 1 hour, and then recovered by DNA purification to give a linearized vector, which was integrated into the chassis cells.
The chassis cells are coated with an auxotroph culture medium for screening, and the single colonies are subjected to colony PCR verification to obtain the strain integrated with the target vector.
The medium may be: 80ml of water was added to 2g (20 g/L) of agarose and sterilized at 115℃for 20min. Before use, 10 XYNB 10m L, 10 Xglucose 10m L (20 g/L), 500 XBiotin 0.2mL (4X 10) were added to the super clean bench -4 g/L), and the mixture is poured into a flat plate.
Wherein the chassis cell may comprise one of pichia pastoris, saccharomyces cerevisiae and escherichia coli, and in one embodiment, the chassis cell comprises pichia pastoris. Further, in an embodiment, the pichia comprises one or more of SMD1168, KM71H, SMD1163, X33, and GS115. There are various ways of integrating the vector into the cells of the chassis, and either electrotransformation or chemical transformation may be used.
4. Chassis cell selection and optimization
(1) Selection and optimization
Four different chassis strains of pichia pastoris SMD1168, KM71, SMD1163 and GS115 are selected, and pPIC9K-CL3.4 and pPIC9K-CL3.8 are respectively transferred into the strains to obtain recombinant strains SMD1168/CL3.4, KM71/CL3.4, SMD1163/CL3.4 and GS115/CL3.4 which can synthesize collagen CL3.4 and CL3.8 respectively; SMD1168/CL3.8, KM71/CL3.8, SMD1163/CL3.8 and GS115/CL3.8. The expression was analyzed by shake flask culture. It is understood that the above strains can be extended to Saccharomyces cerevisiae chassis cell optimization, E.coli chassis cell optimization.
(2) Shake flask culture
a) The picked colonies were inoculated in 6-7mL of YPD liquid medium (specific component contents are shown in Table 1) and cultured at 30℃and 220rpm for 16 hours.
b) The cells were inoculated in BMGY medium (specific component contents are shown in Table 2, or YPD) at an inoculum size of 10%, and cultured at 30℃and 220rpm for 24 hours.
c) The bacterial liquid cultured for 24 hours is transferred to a 50ml sterile centrifuge tube, centrifuged at 5000rpm for 8min, bacterial cells are collected, and the supernatant is discarded.
d) The cells were washed by resuspension with 40ml of physiological saline (or sterile water) 2 times, and the supernatant was discarded.
e) Adding BMMY culture medium (specific component content is shown in Table 3) into thallus for resuspension, and culturing at 30deg.C and 220 rpm; 1% methanol was added every 24h. Samples were taken at 72h of induction for SDS-PAGE analysis.
TABLE 1 component content of YPD Medium
| Component (A) | Content (g/L) |
| |
10 |
| Tryptone | 20 |
| Glucose (Sterilization alone) | 20 |
TABLE 2 component content of BMGY Medium
| Component (A) | Content (g/L) |
| |
10 |
| Tryptone | 20 |
| KH 2 PO 4 | 11.82 |
| K 2 HPO 4 ·3H 2 O | 3.02 |
| |
10 |
Remarks: 5mL of 10 XYNB, 100. Mu.L of 500 Xbiotin, 5mL of inoculum size were added per 40mL of MGY medium prior to use.
TABLE 3 component content of BMMY Medium
Remarks: to each 45mL of BMMY medium, 5mL of 10 XYNB, 100. Mu.L of 500 Xbiotin, and 0.5mL of methanol were added before use.
The preparation method of the 10 XYNB can be as follows: 13.4g YNB (yeast without amino nitrogen source and containing ammonium sulfate) was dissolved in 100mL deionized water, filtered and sterilized and stored at 4deg.C. The preparation method of 500X biotin can be as follows: 20mg of Biotin was weighed out and dissolved in 100mL of sterile water, which was filtered and sterilized and stored at 4 ℃.
5. Collagen amplified fermentation
The recombinant strain preserved by glycerol at-80 ℃ is streaked on a YPD solid plate and is placed in a constant temperature incubator at 30 ℃ for 2-3 days until single colony is grown. The 2-3 loops were picked and inoculated into 500mL triangular shake flasks containing 50mL YPD liquid medium and incubated at 30℃at 220rpm for 16-18h.
The high density fed-batch fermentation was performed on a 10-L fermenter containing 4.5L BSM medium, the initial fermentation parameters were as follows: temperature 30 ℃, pH 5.0, aeration rate 2.0vvm and rotational speed 200rpm. Ammonia water is added to control the pH value to be 5.0, 500mL of seed liquid is added into the culture medium of the fermentation tank, when dissolved oxygen is reduced to be below 50%, the rotating speed is increased by 50rpm, and finally, 500rpm-600rpm is reached.
Culturing for about 18-20h, rebound of dissolved oxygen, OD (optical density) being larger than 30, feeding glycerol (containing 12 mL/v) with the concentration of 50% (v/v) for 10h, wherein the front 6h is 13.5, 16.2, 19.2, 22.8, 27.2 and 32.4mL/L/h respectively, the back 30mL/L/h, adjusting ventilation rate to 4.0vvm, gradually increasing the rotating speed to 800-900rpm according to the dissolved oxygen, and continuously starving and culturing for 2h after the dissolved oxygen rebounds to the highest point.
And (3) entering a methanol induction stage, wherein the fermentation temperature is reduced to 28 ℃, methanol containing 12mL/L PTM1 is used for fed-batch induction, and the flow rate of the methanol is gradually increased from 1mL/L/h to 6-7mL/L/h. Then, methanol was added at a constant flow rate.
6. Cell proliferation assay
After purifying the obtained collagen, inducing and culturing human skin fibroblasts (fibroblasts) to detect the proliferation effect of the recombinant collagen on the skin fibroblasts. The experimental method may include the steps of:
human skin fibroblast culture conditions: OPTI-MEM medium, 15% serum, 1% triple antibody, 37℃C, 6% CO 2 。
Human skin fibroblasts were plated and seeded at 3X 10 3 Each cell was cultured in 96-well plates at 150uL per well for 24 hours, with 5 96-well plates plated.
The recombinant collagen (namely CL3.4 and CL3.8 proteins) is diluted into a cell culture solution to a final concentration of 50ug/mL, 100ug/mL, 200ug/mL, 400ug/mL and 800ug/mL, and then added into human skin fibroblasts for treatment for 72 hours, and the proliferation of the cells is detected. Meanwhile, commercially available human collagen was used as a control (control).
In a second aspect the invention provides a nucleic acid encoding a recombinant type III collagen as described above.
In one embodiment, the nucleic acid sequence is set forth in SEQ ID NO:3 and/or SEQ ID NO: 4.
The third aspect of the present invention also provides an expression vector into which the above nucleic acid is inserted, or which is capable of causing a host cell to express the above recombinant type III collagen after transfection of the host cell.
In an embodiment, the expression vector includes any one of pPIC9K, pPICZ a A, GAPZ a, A, pPICZ a B, pPICZ a C, GAPZ a B, GAPZ a C and modified vector thereof.
The fourth aspect of the present invention also provides a strain expressing recombinant type III collagen, wherein the strain is capable of expressing the recombinant type III collagen.
In one embodiment, the strain comprises one of pichia pastoris, saccharomyces cerevisiae, and escherichia coli.
In one embodiment, the pichia comprises one or more of SMD1168, KM71H, SMD1163, X33, and GS115.
The fifth aspect of the invention also provides the use of the recombinant type III collagen according to the first aspect, the nucleic acid according to the second aspect, the expression vector according to the third aspect, or the strain expressing the recombinant type III collagen according to the fourth aspect in the preparation of skin care products, cosmetics, health care products, foods and medicines.
Embodiments of the present invention will be described in detail below with reference to specific examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
EXAMPLE 1 construction of expression plasmid
After screening and determining the sequences of the two sections of collagen, codon optimization is carried out, and nucleotide sequences CL3.4 and CL3.8 (shown as SEQ ID NO:3 and SEQ ID NO: 4) are obtained through artificial synthesis. This fragment was amplified by primers CL3.4-F/R and CL3.8-F/R (see Table 4), vector pPIC9K was digested with the fast-cutting enzyme EcoRI/NotI, and amplified fragments CL3.4 and CL3.8 were inserted into digested vector pPIC9K, respectively, to give recombinant plasmids pPIC9K-CL3.4 and pPIC9K-CL3.8. The maps of the recombinant plasmids pPIC9K-CL3.4 and pPIC9K-CL3.8 are shown in FIGS. 1 and 2.
TABLE 4 primers required for engineering bacteria construction
| Primer name | Sequence (5 '-3') |
| CL3.4-F | GGTCCCCCAGGTCCAGCTGGTGCTAAC |
| CL3.4-R | GGGAGCACCTCTTTCTCCTGCTGGTCCC |
| CL3.8-F | GGACAACCTGGTGAAAAGGGATCTCCTGGTG |
| CL3.8-R | TTCAGAACCTCTTTCACCTCTATTACCTCTTGGACCTG |
EXAMPLE 2 construction of strains
The expression vector constructed in example 1 was incubated with SalI enzyme at 37℃for 1 hour, and then recovered by DNA purification to give a linearized vector. The linearized vector is integrated into a chassis cell Pichia pastoris SMD1168 strain in an electrotransformation mode, then an auxotroph culture medium is coated for screening, and a single colony which grows out is subjected to colony PCR verification to obtain a strain integrated with a target vector, namely, an SMD1168/pPIC9K-CL3.4 recombinant strain and an SMD1168/pPIC9K-CL3.8 recombinant strain.
EXAMPLE 3 analysis of collagen Strain by shake flask culture
The picked colonies were inoculated in 6-7mL of YPD liquid medium at 30℃and 220rpm for 16h. The cells were inoculated into BMGY medium at 10% of the amount of inoculation, and cultured at 30℃and 220rpm for 24 hours. Culturing for 24h, transferring the bacterial liquid to a 50mL sterile centrifuge tube, centrifuging at 5000rpm for 8min to collect bacterial cells, and discarding the supernatant. The cells were washed with 40mL of physiological saline in a resuspended state for 2 times, and the supernatant was discarded. Adding BMMY culture medium into thallus for resuspension, and culturing at 30deg.C and 220 rpm; 1% methanol was added every 24h. Samples were taken at 72h of induction for SDS-PAGE analysis.
Referring to FIG. 3, the resulting protein bands were obtained. As can be seen from fig. 3, the collagen CL3.4 and CL3.8 achieve expression.
Example 4 optimization of Chassis cells to increase collagen expression
Four different chassis strains of pichia pastoris SMD1168, KM71, SMD1163 and GS115 are selected, and pPIC9K-CL3.4 and pPIC9K-CL3.8 are respectively transferred into the strains to obtain recombinant strains SMD1168/CL3.4, KM71/CL3.4, SMD1163/CL3.4 and GS115/CL3.4 which can synthesize collagen CL3.4 and CL3.8 respectively; SMD1168/CL3.8, KM71/CL3.8, SMD1163/CL3.8 and GS115/CL3.8.
The collagen expression was analyzed by shake flask culture of example 3, as shown in FIGS. 4 and 5.
In FIG. 4, lane 1 shows the protein expression corresponding to strain SMD1168/CL3.4, lanes 2-4 show the protein expression corresponding to strain KM71/CL3.4, lanes 5-6 show the protein expression corresponding to strain SMD1163/CL3.4, and lane 7 shows the protein expression corresponding to strain GS115/CL 3.4. As a result, it was found that the best-suited chassis strain for CL3.4 (nucleic acid SEQ ID NO: 3) was Pichia pastoris KM71.
In FIG. 5, lane 1 shows the protein expression corresponding to strain SMD1168/CL3.8, lanes 2-4 show the protein expression corresponding to strain KM71/CL3.8, lanes 5-7 show the protein expression corresponding to strain SMD1163/CL3.4, and lane 8 shows the protein expression corresponding to strain GS115/CL 3.4. As can be seen, for CL3.8 (nucleic acid SEQ ID NO: 4), the most suitable chassis strain for expression is Pichia pastoris KM71 or Pichia pastoris GS115.
It should be noted that, taking FIG. 5 as an example, lanes 2-4 correspond to 3 strains screened from the same plate, and the expression levels of the strains are very different; lanes 5-7 are 3 strains screened from the same plate, and the situation that the expression quantity is very different also occurs; the result shows that when the strains are transformed to select positive clones, a plurality of strains are selected to perform fermentation verification simultaneously, so that false positive events are avoided.
EXAMPLE 5 production of collagen by fermentation in 10-L fermenter
The optimized strains KM71/CL3.4 and KM71/CL3.8 obtained by the optimization are taken, recombinant strains stored in glycerol tubes at the temperature of-80 ℃ are streaked on YPD solid plates, and the YPD solid plates are placed in a constant temperature incubator at the temperature of 30 ℃ for culturing for 2-3 days until single colonies grow. The 2-3 loops were picked and inoculated into 500mL triangular shake flasks containing 50mL YPD liquid medium and incubated at 30℃and 220rpm for 16-18h.
The high density fed-batch fermentation was performed on a 10-L fermenter containing 4.5L BSM medium, the initial fermentation parameters were as follows: temperature 30 ℃, pH 5.0, aeration rate 2.0vvm and rotational speed 200rpm. Ammonia water is added to control the pH value to be 5.0, 500mL of seed liquid is added into the culture medium of the fermentation tank, when dissolved oxygen is reduced to be below 50%, the rotating speed is increased by 50rpm, and finally, 500rpm-600rpm is reached.
Culturing for about 18-20h, rebound of dissolved oxygen, OD (optical density) being larger than 30, feeding glycerol (containing 12 mL/v) with the concentration of 50% (v/v) for 10h, wherein the front 6h is 13.5, 16.2, 19.2, 22.8, 27.2 and 32.4mL/L/h respectively, the back 30mL/L/h, adjusting ventilation rate to 4.0vvm, gradually increasing the rotating speed to 800-900rpm according to the dissolved oxygen, and continuously starving and culturing for 2h after the dissolved oxygen rebounds to the highest point.
And (3) entering a methanol induction stage, wherein the fermentation temperature is reduced to 28 ℃, methanol containing 12mL/L PTM1 is used for fed-batch induction, and the flow rate of the methanol is gradually increased from 1mL/L/h to 6-7mL/L/h. Then adding methanol at a constant flow rate, and ending the final fermentation by monitoring the state of the fermentation tank in real time and selecting a proper time point.
The results of the real-time sampling test performed during fermentation are shown in FIGS. 6 and 7. As can be seen from fig. 6 and 7, the yields of collagen CL3.4 and CL3.8 in the fermenter were significantly improved compared with the shake flask culture, and the yields of collagen CL3.4 and CL3.8 were continuously accumulated from 0h to 72h, and the protein expression level was maintained in a stable state during the late stage. The expression level of collagen CL3.8 is significantly higher than that of collagen CL 3.4.
EXAMPLE 6 collagen production analysis
Standard substance solutions with different concentrations and a light absorption value measuring method are prepared: and (3) purifying the collagen sample obtained by fermentation, and quantitatively analyzing by using a biuret method. Preparing a 10mg/ml standard protein solution by using standard crystalline Bovine Serum Albumin (BSA); taking 12 test tubes, adding 0, 0.2, 0.4, 0.6, 0.8 and 1.0ml of standard protein solution respectively, supplementing 1ml of water respectively, and then adding 4ml of biuret reagent. After shaking thoroughly, the mixture was left at room temperature (20 to 25 ℃) for 30 minutes, and colorimetric measurement was carried out at 540 nm.
Average of two sets of measurementsThe values, the absorbance values, and the protein content are plotted on the abscissa and the absorbance values are plotted on the ordinate, and the results are shown in FIG. 8. The standard curve is y= 0.0456x-0.0138, R 2 =0.9994, where x is the protein concentration and y is the absorbance at 540 nm.
By adopting the preparation method and the absorbance measurement method, 1ml of collagen CL3.4 and CL3.8 are respectively added into a test tube, 4ml of biuret reagent is added, and after being fully and evenly shaken, the mixture is placed at room temperature (20-25 ℃) for 30 minutes, and colorimetric measurement is carried out at 540 nm.
The absorbance values of CL3.4 and CL3.8 are 0.150 and 0.223, respectively, and the corresponding contents of the collagen CL3.4 and CL3.8 are 3.6g/L and 5.2g/L, respectively, calculated according to the prepared standard curve.
EXAMPLE 7 collagen cell proliferation assay
After purifying the obtained collagen, inducing and culturing human skin fibroblasts (fibroblasts) to detect the proliferation effect of the recombinant collagen on the skin fibroblasts. The experimental method may include the steps of:
human skin fibroblast culture conditions: OPTI-MEM medium, 15% serum, 1% triple antibody, 37℃C, 6% CO 2 。
Human skin fibroblasts were plated and seeded at 3X 10 3 Each cell was cultured in 96-well plates at 150uL per well for 24 hours, with 5 96-well plates plated. The recombinant collagen (namely CL3.4 and CL3.8 proteins) is diluted into a cell culture solution to a final concentration of 50ug/mL, 100ug/mL, 200ug/mL, 400ug/mL and 800ug/mL, and then added into human skin fibroblasts for treatment for 72 hours, and the proliferation of the cells is detected. Meanwhile, purchased human collagen was used as a control.
The detection results are shown in FIG. 9, and the detection results show that the concentration of the recombinant collagen is in the range of 50ug/mL to 800ug/mL, and the collagen CL3.4 and the collagen CL3.8 can both effectively promote cell proliferation; and the cell proliferation rate gradually increases with increasing concentration. In particular to collagen CL3.4, which has a particularly obvious effect of promoting cell proliferation. In the control group, the cell proliferation rate did not increase as the concentration increased. Therefore, the collagen can effectively promote the proliferation of cells, can be used in the fields of biological medicine, cosmetics, skin care products, foods and the like, and has important strategic significance for popularization and further development of collagen synthesis.
The foregoing description is only of the optional embodiments of the present invention, and is not intended to limit the scope of the invention, and all the equivalent structural changes made by the description of the present invention and the accompanying drawings or the direct/indirect application in other related technical fields are included in the scope of the invention.
Claims (10)
1. A recombinant type III collagen, characterized in that its amino acid sequence comprises at least one of the following:
(1)SEQ ID NO：1：GPPGPAGANGAPGLRGGAGEPGKNGAKGEPGP RGERGEAGIPGVPGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPA GPNGIPGEKGPAGERGAP；
(2)SEQ ID NO：2：GQPGEKGSPGAQGPPGAPGPLGIAGITGARGLA GPPGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGLPGLAGTAGEPGRDGNPGSDGLPGRDGSPGGKGDRGENGSPGAPGAPGHPGPPGPVGPAGKSGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPRGPVGPSGPPGKDGTSGHPGPIGPPGPRGNRGERGSE；
or (1) or (2) has an amino acid sequence in which 1 or more amino acids are added, substituted or deleted.
2. The recombinant type III collagen according to claim 1, wherein the amino acid sequence comprises 2 to 10 repeats of SEQ ID NO:1, a step of; and/or the number of the groups of groups,
the amino acid sequence comprises 2-10 repeated SEQ ID NO:2.
3. a nucleic acid encoding the recombinant type III collagen according to claim 1 or 2.
4. A nucleic acid according to claim 3, wherein the nucleic acid sequence is as set forth in SEQ ID NO:3 and/or SEQ ID NO:4, wherein SEQ ID NO:3 is as follows:
GGTCCCCCAGGTCCAGCTGGTGCTAACGGAGCCCCAGGTCTGAGAGGTGGTGCCGGTGAACCAGGAAAAAACGGTGCTAAGGGTGAGCCTGGTCCAAGAGGAGAGAGAGGTGAAGCAGGTATTCCTGGTGTTCCAGGTGCTAAGGGAGAAGATGGTAAAGATGGATCTCCAGGTGAACCCGGTGCCAACGGTTTACCAGGTGCTGCCGGAGAAAGAGGAGCACCAGGTTTTAGAGGTCCAGCTGGTCCAAACGGTATTCCAGGAGAAAAGGGACCAGCAGGAGAAAGAGGTGCTCCC；
SEQ ID NO:4 is as follows:
GGACAACCTGGTGAAAAGGGATCTCCTGGTGCCCAAGGACCACCTGGAGCCCCTGGTCCCTTGGGTATTGCTGGAATTACTGGTGCAAGAGGTTTGGCTGGACCACCAGGTATGCCAGGTCCAAGAGGTAGTCCTGGACCACAAGGTGTTAAAGGTGAATCTGGAAAGCCTGGAGCCAACGGTCTGTCAGGAGAGAGAGGACCACCTGGTCCACAAGGACTTCCAGGTTTGGCAGGTACCGCCGGTGAACCTGGAAGAGATGGTAATCCAGGAAGTGACGGTTTGCCAGGTAGAGACGGATCACCAGGAGGAAAGGGAGATAGAGGTGAAAACGGTTCTCCTGGTGCTCCAGGTGCTCCTGGTCATCCCGGTCCACCTGGTCCAGTTGGTCCAGCCGGTAAGTCTGGTGATAGAGGTGAGTCTGGTCCAGCAGGTCCAGCCGGTGCTCCAGGTCCTGCTGGATCTAGAGGTGCTCCTGGACCACAGGGTCCTAGAGGTGATAAGGGTGAGACTGGTGAAAGAGGTGCAGCAGGTATTAAGGGTCATAGAGGTTTTCCAGGTAACCCAGGAGCCCCTGGTAGTCCAGGTCCAGCAGGACAACAAGGTGCTATAGGTAGTCCAGGTCCTGCCGGTCCAAGAGGTCCTGTTGGTCCATCAGGACCACCAGGTAAAGATGGTACCTCCGGTCATCCTGGTCCAATTGGACCACCAGGTCCAAGAGGTAATAGAGGTGAAAGAGGTTCTGAA。
5. an expression vector into which the nucleic acid of claim 3 or 4 has been inserted, or which is capable of allowing a host cell to express the recombinant type III collagen of claim 1 or 2 after transfection of the host cell.
6. The expression vector of claim 5, wherein the expression vector comprises any one of pPIC9K, pPICZ a A, GAPZ a A, pPICZ a B, pPICZ a C, GAPZ a B, GAPZ a C and a modified vector thereof.
7. A strain expressing recombinant type III collagen, wherein the strain is capable of expressing recombinant type III collagen according to claim 1 or 2.
8. The recombinant type III collagen-expressing strain of claim 7, wherein the strain comprises any one of pichia pastoris, saccharomyces cerevisiae, and escherichia coli.
9. The recombinant type III collagen expressing strain of claim 8, wherein the pichia pastoris comprises one or more of SMD1168, KM71H, SMD1163, X33, and GS115.
10. Use of the recombinant type III collagen according to claim 1 or 2, or the nucleic acid according to claim 3 or 4, or the expression vector according to claim 5 or 6, or the strain expressing recombinant type III collagen according to any one of claims 7 to 8 for the preparation of skin care products, cosmetics, health care products, food products and pharmaceuticals.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116640206A (en) * | 2023-07-19 | 2023-08-25 | 山东福瑞达生物股份有限公司 | Recombinant humanized III type collagen and preparation method and application thereof |
| CN116874590A (en) * | 2023-08-16 | 2023-10-13 | 医械妆(广州)技术服务有限公司 | Recombinant III type collagen and preparation method thereof |
| CN117025655A (en) * | 2023-07-11 | 2023-11-10 | 山东丰金美业科技有限公司 | Gene expression vector, genetically engineered bacterium and method for improving yield of humanized III type recombinant collagen and application |
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2022
- 2022-12-29 CN CN202211741953.4A patent/CN116284339A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117025655A (en) * | 2023-07-11 | 2023-11-10 | 山东丰金美业科技有限公司 | Gene expression vector, genetically engineered bacterium and method for improving yield of humanized III type recombinant collagen and application |
| CN117025655B (en) * | 2023-07-11 | 2024-02-27 | 山东丰金美业科技有限公司 | Gene expression vector, genetically engineered bacterium and method for improving yield of humanized III type recombinant collagen and application |
| CN116640206A (en) * | 2023-07-19 | 2023-08-25 | 山东福瑞达生物股份有限公司 | Recombinant humanized III type collagen and preparation method and application thereof |
| CN116640206B (en) * | 2023-07-19 | 2023-10-10 | 山东福瑞达生物股份有限公司 | Recombinant humanized III type collagen and preparation method and application thereof |
| CN116874590A (en) * | 2023-08-16 | 2023-10-13 | 医械妆(广州)技术服务有限公司 | Recombinant III type collagen and preparation method thereof |
| CN116874590B (en) * | 2023-08-16 | 2024-06-07 | 医械妆(广州)技术服务有限公司 | Recombinant III type collagen and preparation method thereof |
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