CN118085064A - Recombinant polypeptide and preparation method thereof - Google Patents
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to the technical field of genetic engineering and discloses a recombinant polypeptide and a preparation method thereof, wherein the amino acid sequence of the recombinant polypeptide is shown as SEQ ID No.2, and the fragment is formed by a plurality of repetitions of a core sequence of human III type collagen. The recombinant collagen polypeptide prepared by the invention has very good hydrophilicity and stability, the amino acid composition of the recombinant collagen polypeptide is 100% the same as that of the corresponding part of the amino acid sequence of the natural collagen, the recombinant collagen polypeptide can not generate immune rejection and anaphylactic reaction when being applied to a human body, has good adhesive activity, can enable the function of the natural protein to reduce the immunogenicity of the collagen from animal sources in the human body, can control the risk of pathogenic virus transmission to a certain extent, and can be widely applied to the industries of biological medicines and cosmetics.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a recombinant polypeptide and a preparation method thereof.
Background
Collagen is an important structural protein mainly existing in animal tissues, and has good biocompatibility and biodegradability. Due to its unique properties, collagen has a wide application prospect in many fields of medicine and bioengineering, such as tissue engineering, biomaterials, drug sustained release, etc. However, the traditional collagen extraction method is limited by animal-derived materials, and has the problems of infectious disease transmission risk, unstable quality and the like. Therefore, the research and development of the preparation method of the recombinant collagen with high efficiency, safety and stability has important scientific significance and application value.
Traditional collagen production methods rely mainly on extraction and treatment of animal tissue. This approach has the following limitations: (1) animal derived material limitation: traditional methods rely on animal tissue, such as skin, bone, etc., to extract collagen. However, animal-derived materials are affected by factors such as animal species, growth environment, etc., resulting in large differences in the quality and performance of collagen. (2) risk of transmission of infectious disease: there are potential infectious pathogens in animal tissues, such as viruses, bacteria, etc., and the risk of transmission thereof cannot be completely excluded. (3) low protein purity: in the traditional extraction method, collagen often exists in a mixed mode with other tissue components, the purity is low, and the requirements of some specific applications are difficult to meet. (4) unstable quality: the traditional extraction method is influenced by factors such as animal growth environment, diet and the like, so that the quality and performance of the collagen are unstable.
In order to overcome the limitations of the traditional collagen preparation method, a high-efficiency, safe and stable recombinant collagen preparation method needs to be developed. The method has the following characteristics: (1) high purity: the preparation method of the recombinant collagen can obtain high-purity collagen and meet the requirements of different application fields. (2) controllability: the preparation method should be capable of controlling the structure and properties of the recombinant collagen to meet the requirements of a particular application. (3) biocompatibility: the recombinant collagen should have good biocompatibility to ensure biosafety in vivo. (4) mass production: the preparation method has expandability and can meet the requirement of mass production.
In recent years, researchers have made remarkable research progress on recombinant collagen polypeptides through genetic engineering techniques and biosynthesis methods. The methods utilize gene recombination technology and synthetic biological tools to realize artificial synthesis and expression of collagen polypeptide chains through synthetic gene sequences or biosynthesis pathways. These studies provide a new approach for the customized production and functional regulation of collagen polypeptides. One common approach is to clone the gene sequence of collagen into an expression vector using recombinant DNA technology and express it in a suitable expression host. Researchers can realize the efficient expression and purification of recombinant collagen polypeptides by optimizing expression conditions, such as selecting proper host cells, regulating promoters and regulators of expressed genes, and the like. The method can effectively obtain a large amount of high-purity collagen polypeptide, and provides a basis for further research and application. Another approach is to synthesize collagen polypeptides via synthetic pathways using synthetic biological tools. Researchers can design and synthesize collagen polypeptides with specific sequences and structures, which are synthesized into chains by chemical synthesis or biological synthesis methods. This method allows researchers to precisely control the structure and function of collagen polypeptides, thereby achieving customization of their bioactivity and application properties.
Disclosure of Invention
(One) solving the technical problems
Aiming at the defects of the prior art, the invention provides a recombinant polypeptide and a preparation method thereof.
(II) technical scheme
The amino acid sequence of the recombinant polypeptide is shown as SEQ ID No.2, the fragment consists of a plurality of repetitions of a core sequence of human type III collagen, and the basic repetition unit is GQPGVMGFPGPKGNDG APGKNGERGGPGGPGPQGPPGKNGETGPQ (SEQ ID No. 1).
Preferably, the recombinant polypeptide is C1, comprising 360 amino acids, the amino acid sequence is as follows ：GQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQ(SEQ ID No.2).
Preferably, the DNA sequence of the human collagen polypeptide C1 is as follows ：GGACAACCCGGGGTAATGGGTTTTCCAGGACCGAAAGGAAACGATGGTGCGCCGGGCAAAAATGGCGAGCGCGGTGGCCCGGGTGGGCCAGGTCCGCAAGGTCCACCGGGCAAGAACGGTGAAACGGGTCCGCAAGGTCAACCGGGTGTTATGGGTTTTCCGGGCCCGAAGGGCAACGACGGCGCCCCGGGTAAAAACGGCGAGCGTGGCGGCCCGGGCGGGCCGGGTCCGCAGGGCCCACCGGGTAAAAACGGCGAGACGGGTCCGCAAGGCCAGCCGGGCGTCATGGGCTTCCCGGGTCCGAAGGGTAACGACGGTGCTCCGGGGAAAAATGGCGAGCGTGGTGGCCCGGGCGGGCCTGGACCGCAGGGTCCGCCAGGTAAGAACGGCGAAACCGGCCCACAGGGTCAGCCTGGCGTGATGGGTTTCCCGGGGCCGAAGGGCAATGATGGTGCGCCAGGGAAAAACGGCGAACGTGGTGGTCCGGGCGGACCGGGTCCGCAGGGCCCCCCGGGCAAGAATGGTGAAACCGGTCCGCAGGGCCAACCGGGTGTTATGGGTTTCCCGGGCCCGAAAGGTAATGATGGCGCACCGGGTAAGAACGGTGAACGTGGTGGCCCGGGCGGACCGGGTCCGCAGGGCCCGCCAGGCAAAAACGGTGAAACCGGCCCGCAAGGTCAGCCGGGCGTGATGGGTTTTCCTGGCCCGAAAGGTAATGACGGCGCGCCGGGTAAAAACGGTGAGCGTGGTGGCCCGGGTGGCCCGGGTCCGCAGGGTCCGCCGGGCAAGAACGGTGAAACCGGCCCGCAAGGACAACCGGGAGTTATGGGCTTTCCGGGCCCGAAAGGCAACGACGGTGCTCCGGGTAAGAACGGCGAGCGCGGTGGCCCAGGCGGTCCGGGTCCGCAGGGTCCACCGGGTAAAAATGGCGAGACTGGTCCGCAAGGTCAGCCGGGTGTGATGGGTTTCCCGGGTCCCAAGGGCAACGATGGCGCGCCAGGTAAGAATGGTGAACGCGGTGGTCCGGGCGGTCCGGGTCCGCAGGGTCCGCCTGGCAAGAATGGAGAGACCGGTCCGCAA.
Preferably, the nucleotide sequence of the polynucleotide for encoding the recombinant polypeptide is shown as SEQ ID No.3, and the sequence is a polynucleotide sequence which can be used for expressing escherichia coli after codon optimization.
Preferably, the expression vector comprises the polynucleotide of claim 2.
Preferably, a host cell comprising the expression vector of claim 3.
Preferably, the host cell is E.coli.
Preferably, the recombinant polypeptide is prepared by the following steps:
(1) Amino acids 406-450 of the triple-helix region of the human III-type collagen are subjected to codon optimization, and a complete target gene is obtained through gene synthesis;
(2) Connecting a target gene into a pET-22b plasmid, transferring the plasmid into an escherichia coli Rosetta (DE 3) expression strain, and screening to obtain escherichia coli genetic engineering bacteria;
(3) Culturing engineering bacteria in shake flasks or fermentation tanks and expressing recombinant polypeptides in large quantities;
(2) Harvesting the composition and purifying the polypeptide;
(3) The polypeptide is freeze-dried or made into a solution.
Preferably, the composition comprises the recombinant polypeptide according to claim 1.
Preferably, the composition is useful in medical devices, tissue engineering products, cosmetics or health care products.
Preferably, the recombinant polypeptide is used for promoting cell adhesion.
(III) beneficial technical effects
Compared with the prior art, the invention has the following characteristics:
(1) The III type collagen sequence selected by the invention is obtained through screening, analysis and optimization.
(2) The invention adopts a mature escherichia coli expression system, is suitable for large-scale production, and has the characteristics of low cost and high production efficiency. Meanwhile, the selected gene sequence can further improve the yield of collagen through codon optimization.
(3) The recombinant collagen polypeptide prepared by the invention has very good hydrophilicity and stability, has the amino acid composition 100 percent identical to that of the corresponding part of the amino acid sequence of the natural collagen, can not generate immune rejection and anaphylactic reaction when being applied to human bodies, and can be widely applied to the industries of biological medicine and cosmetics;
(4) The humanized recombinant collagen polypeptide has good adhesive activity on the activity detection surface, and can realize the function of natural proteins in human bodies.
(5) The invention can reduce the immunogenicity of animal-derived collagen as much as possible and can control the risk of pathogenic virus transmission to a certain extent.
Drawings
FIG. 1 is a diagram of protein electrophoresis after purification of recombinant collagen polypeptide C1.
FIG. 2 shows the results of cell adhesion activity assay of recombinant collagen polypeptide C1.
Detailed Description
The technical scheme of the present invention will be further specifically described by means of specific examples, but the present invention is not limited to these examples.
The invention enables the recombinant collagen gene to be expressed in escherichia coli Rosetta (DE 3) strain by synthesizing the humanized type III recombinant collagen gene.
LB medium: 0.5% yeast extract, 1% peptone and 1% sodium chloride (e.g. by preparing solid medium, adding 1.5% agar before sterilizing), and autoclaving at 115℃for 30min.
TB medium: 2.4% yeast extract, 1.2% peptone, 0.4% glycerol, 100mL potassium phosphate buffer salt, and autoclaved at 115℃for 30min.
Example 1: construction and expression of C1 gene expression vector:
(1) The full-length gene sequence of the human collagen polypeptide C1 is subjected to codon optimization and is shown as follows ：ggtttcccaggtatgaaaggtcaccgtggtttcgacggtcgtaacggtgaaaaaggtgaaaccggtgctccgggtctgaaaggtgaaaacggtctgccgggtgaaaacggtgctccgggtccgatgggtccgcgtggcttcccgggtatgaagggccatcgcggtttcgacggccgcaacggcgagaaaggtgaaacaggtgctccgggcctgaaaggtgagaacggtctgccgggcgaaaacggcgctccgggtccgatgggcccgcgcggctttccgggtatgaaaggccatcgtggcttcgacggccgtaacggcgaaaaaggcgaaaccggcgctccgggtctgaagggtgaaaatggtctgccgggtgagaacggtgccccgggtccgatgggcccacgtggcttcccgggcatgaagggtcaccgtggttttgacggtcgtaacggcgaaaagggtgaaaccggtgccccgggtctgaaaggcgaaaacggcctgccgggtgaaaatggtgctccgggcccgatgggcccacgcggctttccgggtatgaaaggccatcgcggcttcgacggccgcaacggtgaaaagggcgaaacaggcgctccgggcctgaaaggcgagaatggtctgccgggcgagaacggcgccccgggtccgatgggcccgcgtggcttcccgggcatgaagggccaccgcggttttgacggtcgcaacggcgagaaaggcgaaaccggcgccccgggtctgaagggcgaaaatggcctgccgggtgagaatggtgccccgggcccgatgggtccgcgcggctttccgggcatgaagggtcatcgtggctttgacggtcgcaacggtgaaaagggtgaaacaggcgccccgggcctgaagggtgagaacggcctgccgggcgaaaatggcgctccgggcccgatgggtccacgtggctttccgggcatgaaaggccaccgcggctttgacggccgtaacggtgagaagggcgaaacaggtgccccgggcctgaagggcgagaatggcctgccgggcgagaatggcgccccgggcccaatgggcccgcgt(SEQ ID No.3).
The full length of the C1 gene is 1080bp, the sequence is synthesized by gene synthesis company and is connected to pET22b plasmid by using enzyme cutting sites of NcoI and XhoI, the plasmid is transformed into escherichia coli Rosetta (DE 3), and positive clones are selected by using ampicillin sodium and chloramphenicol double-resistance-LB plate screening.
(2) E.coli positive clones were picked, cultured overnight in 4mL LB medium, transferred at 1:100, cultured in shake flasks at 37℃until OD600 was 0.4-0.6, induced to express at a final concentration of 0.1mM, cultured at 30℃for 20 hours, and the cells were collected by centrifugation and stored at-20℃or immediately subjected to further purification.
(3) Washing the mixed bacterial precipitate with physiological saline, re-suspending about 400mL of bacterial solution precipitate with a volume of 20-30mL, using lysozyme to assist in bacterial lysis (optional) by matching with Triton X-100, performing ultrasonic sterilization (5 s ultrasonic, 5s intermittent, full length 10min and protection temperature 30 ℃) in an ice-water mixture environment, centrifuging at 12000rpm/min for 20min, and collecting supernatant. At this time, the solution contains a large amount of C-1 recombinant collagen polypeptide.
(4) Washing nickel ion affinity column (Ni-NTA His-Bind Resin) column material with lysis buffer, mixing and incubating the column material with C1 solution, shaking for 30min at room temperature or on ice, loading onto column, washing impurity protein with PBS buffer containing 10mM imidazole, washing target protein C1 with PBS buffer containing 300mM imidazole, ultrafiltering and desalting the obtained product, and lyophilizing to obtain dry powder for use.
(5) The purity of the resulting collagen polypeptide was checked by SDS-PAGE. The specific process is as follows: taking 8 mu L of purified protein solution, adding 2 mu L of protein loading buffer (250 mM Tris-HCl (pH 6.8), 10% SDS,0.5% bromophenol blue, 50% glycerol and mercaptoethanol), placing in boiling water at 100 ℃ for boiling for 3min, adding 10 mu L of each well into SDS-PAGE protein gel, running at 80V for 2h, and then carrying out protein staining for 20min by using coomassie brilliant blue staining solution (0.1% coomassie brilliant blue R-250, 25% isopropanol and 10% glacial acetic acid), and then carrying out decolorization by using protein decolorization solution (10% acetic acid and 5% ethanol). And finally, measuring the protein activity relative to the human natural collagen.
Results:
The electrophoretogram of FIG. 1 shows that C303-1 having an apparent molecular weight of 35.4kDa is obtained, the molecular weight of the polypeptide corresponding to the amino acid sequence of SEQ ID No.2, respectively.
Example 2: cell adhesion Activity assay for recombinant collagen polypeptide C1
The method for detecting the activity of collagen polypeptide can be specifically implemented as follows in reference Juming Yao,Satoshi Yanagisawa,Tetsuo Asakura,Design,Expression and Characterization of Collagen-Like Proteins Based on the Cell Adhesive and Crosslinking Sequences Derived from Native Collagens,J Biochem.136,643-649(2004).:
(1) And detecting the concentration of the protein sample to be detected by using an ultraviolet absorption method, wherein the concentration comprises a control human collagen polypeptide and a recombinant collagen polypeptide C1 sample. Specifically, the ultraviolet light absorption of the sample at 215nm and 225nm was measured, and the protein concentration was calculated using the empirical formula C (ug/mL) =144X (a 215-a 225), taking care of detection under a condition of a215< 1.5. The principle of the method is to measure the characteristic absorption of peptide bonds under far ultraviolet light, the method is not influenced by chromophore content, has few interfering substances and simple and convenient operation, and is suitable for detecting the human collagen and analogues thereof which are not developed by coomassie brilliant blue. After detection of the finished protein concentration (reference Walker JM. The Protein Protocols Handbook, second edition. Humana Press. 43-45.), all tested protein concentrations were adjusted to 0.5mg/mL with PBS.
(2) Mu.L of each protein solution was added to the 96-well plate and left standing overnight at 4℃in comparison with a blank PBS solution.
(3) Each well was washed 2 times with PBS.
(3) 4 X 10 4 well-cultured 3T3 cells were added to each well and incubated at 37℃for 25min.
(4) Each well was washed 2 times with PBS.
(5) Absorbance at OD450 nm was measured with CCK8 kit. From the values of the blank, the degree of cell adherence can be calculated. The cell attachment rate can be used for reflecting the activity of the collagen polypeptide. The higher the activity of the protein, the better the environment can be provided for the cells in a short time, and the cell attachment is assisted.
The results are shown in FIG. 2.
The results in FIG. 2 show that recombinant collagen polypeptide C1 has comparable adhesive activity as compared to commercially available human collagen polypeptides.
The preferred embodiments of the invention disclosed above are intended only to assist in the explanation of the invention. The preferred embodiments are not exhaustive or to limit the invention to the precise form disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best understand and utilize the invention. The invention is limited only by the claims and the full scope and equivalents thereof.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (9)
1. A recombinant polypeptide, characterized in that: the amino acid sequence of the recombinant polypeptide is shown as SEQ ID No.2, and the fragment is formed by multiple repetitions of a core sequence of human III type collagen.
2. A recombinant polypeptide according to claim 1, wherein: the nucleotide sequence of the polynucleotide for encoding the recombinant polypeptide is shown as SEQ ID No.3, and the sequence is a polynucleotide sequence which can be used for expressing escherichia coli after codon optimization.
3. A recombinant polypeptide according to claim 1, wherein: an expression vector comprising the polynucleotide of claim 2.
4. A recombinant polypeptide according to claim 1, wherein: a host cell comprising the expression vector of claim 3.
5. The recombinant polypeptide of claim 4, wherein: the host cell is E.coli.
6. A recombinant polypeptide according to claim 1, wherein: the preparation method of the recombinant polypeptide comprises the following steps:
(1) Amino acids 406-450 of the triple-helix region of the human III-type collagen are subjected to codon optimization, and a complete target gene is obtained through gene synthesis;
(2) Connecting a target gene into a pET-22b plasmid, transferring the plasmid into an escherichia coli Rosetta (DE 3) expression strain, and screening to obtain escherichia coli genetic engineering bacteria;
(3) Culturing engineering bacteria in shake flasks or fermentation tanks and expressing recombinant polypeptides in large quantities;
(2) Harvesting the composition and purifying the polypeptide;
(3) The polypeptide is freeze-dried or made into a solution.
7. A recombinant polypeptide according to claim 1, wherein: the composition comprises the recombinant polypeptide of claim 1.
8. A recombinant polypeptide according to claim 7, wherein: the composition can be used for medical appliances, tissue engineering products, cosmetics or health care products.
9. A recombinant polypeptide according to claim 1, wherein: the use of said recombinant polypeptide for promoting cell adhesion.
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