CN118184766A - Preparation method of recombinant humanized III-type collagen by near-natural extraction of collagen - Google Patents

Abstract

The invention discloses a recombinant humanized III type collagen which is near-natural extracted collagen, which is recombinant humanized III type collagen of the following a) or b): a) The amino acid sequence is the recombinant humanized III type collagen of SEQ ID No. 1; b) The protein with recombinant humanized III type collagen activity is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID No. 1. The invention obtains optimized genes from III type human collagen sequences through screening and transformation, constructs the optimized genes into Pichia pastoris secretion expression plasmids through homologous recombination, and obtains recombinant Pichia pastoris engineering strains with high-expression humanized III type collagen through electric transfer screening. And (3) fermenting, culturing and purifying after optimization to finally obtain the high-purity recombinant humanized III-type collagen. The recombinant humanized III type collagen is closer to natural extracted collagen in the high-level structure, and has potential application prospects in the fields of biomedical materials, cosmetics and the like.

Description

Preparation method of recombinant humanized III-type collagen by near-natural extraction of collagen

Technical Field

The invention belongs to the technical field of bioengineering, and relates to a preparation method of recombinant humanized III-type collagen for near-natural extraction of collagen.

Background

Collagen is a biological polymer material, and is mainly present in connective tissue of animals, accounting for 25% -30% of total protein. It is formed by winding 3 polypeptide chains in a supercoiled mode, and is rich in amino acids such as glycine, proline, hydroxyproline and the like. Collagen plays an important role in maintaining normal physiological functions of cells, tissues, organs and repairing injuries. The variety of collagen is varied, and more than 30 collagen chain encoding genes have been found to form more than 16 collagen molecules. According to their structure, it can be classified into fibrous collagen, basal membrane collagen, microfibril collagen, anchored collagen, hexagonal network collagen, non-fibrous collagen, transmembrane collagen, and the like. Collagen has good biocompatibility, biodegradability and bioactivity, so that the collagen has wide application in the fields of foods, medicines, tissue engineering, cosmetics and the like.

There are four main ways of early collagen acquisition. The first is the physical method, which is the most traditional way, usually by high pressure assist. The second is a chemical method, which includes pretreatment with a solvent combined with extraction at low temperature or hot water, and can be further classified into a hot water extraction method, an acid method, an alkali method, and a salt method according to the solvents. The third is biochemical method, mainly using enzymes to extract collagen. The last is microbiological and enzymatic extraction, which is an emerging extraction modality.

Bioengineering is a method for obtaining collagen by using genetic recombination technology and microbial fermentation technology. In this process, human type I and type III collagen genes are introduced into a microorganism (e.g., E.coli or Pichia pastoris). Then, the recombinant collagen can be obtained by the invention through microbial fermentation and purification technology. The method has the advantages that the method can be produced in large scale, and the obtained recombinant collagen has high purity and good stability, and has excellent biological characteristics of biodegradability, low immunogenicity, promotion of cell adhesion and the like. The technical difficulty of the method is that the unique spiral structure of the collagen is maintained, and the cost is reduced by realizing large-scale mass production. Therefore, bioengineering is a current research hotspot and is widely used in the fields of medicine and tissue engineering. The method can not only avoid the possible virus carried by animal-derived collagen and reduce the potential safety hazard, but also better control the purity, stability and safety of the product. The collagen protein is a novel collagen protein acquisition mode and has great potential and application prospect. However, the collagen obtained in this way is greatly different from the collagen extracted in nature in terms of its higher structure.

Disclosure of Invention

The present invention aims to overcome the above disadvantages of the prior art, and first provides a recombinant humanized type III collagen which is a recombinant humanized type III collagen of the following a) or b) and is obtained by extracting collagen in a near natural way:

a) The amino acid sequence is the recombinant humanized III type collagen of SEQ ID No. 1;

b) The protein with recombinant humanized III type collagen activity is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID No. 1.

The present invention also provides a biological material related to the above protein, which is any one of the following B1) to B8):

B1 Nucleic acid molecules encoding the above proteins;

b2 An expression cassette comprising the nucleic acid molecule of B1);

B3 A recombinant vector comprising the nucleic acid molecule of B1),

B4 A recombinant vector comprising the expression cassette of B2);

B5 A recombinant microorganism comprising the nucleic acid molecule of B1);

B6 A recombinant microorganism comprising the expression cassette of B2);

b7 A recombinant microorganism containing the recombinant vector of B3);

b8 A recombinant microorganism comprising the recombinant vector of B4);

Optionally, the vector comprises a yeast expression vector;

optionally, the recombinant microorganism comprises an engineered bacterium or a yeast cell.

Preferably, the nucleic acid molecule is a nucleic acid molecule as set forth in 1) or 2) or 3) or 4) below:

1) The coding sequence is a DNA molecule or a cDNA molecule of SEQ ID No. 2in a sequence table;

2) The nucleic acid sequence is a DNA molecule of SEQ ID No.2 in a sequence table;

3) A cDNA molecule or a genomic DNA molecule having 75% or more identity to the nucleotide sequence defined in 1) or 2) and encoding the above protein;

4) Hybridizing under stringent conditions with the nucleotide sequence defined in 1) or 2) and encoding a cDNA molecule or genomic DNA molecule of the above protein.

The invention also provides the use of the proteins described above for supporting skin, wound healing and/or reducing scarring, the use being for non-therapeutic purposes.

The invention also provides application of the biological material in preparation of recombinant humanized III-type collagen.

The invention also provides a preparation method of the recombinant pichia pastoris engineering strain, which comprises the following steps: after synthesizing the coding gene of the recombinant humanized III type collagen by a gene synthesis mode, cloning a target gene to the downstream of a secretion signal peptide of a pPIC9K plasmid by utilizing a homologous recombination technology, extracting the plasmid after verification of correctness by PCR and gene sequencing, electrically transferring the plasmid into pichia pastoris electrotransfer competent cells after linearization by using restriction enzymes, and screening by using a gradient G418 to obtain the recombinant pichia pastoris engineering strain of the high-expression recombinant humanized III type collagen.

Preferably, the sequence of the coding gene of the recombinant humanized type III collagen is shown as SEQ ID No. 2.

The invention also provides a preparation method of the recombinant humanized III-type collagen, which comprises the following steps: the recombinant humanized III-type collagen is obtained by using the biological material.

The invention finally provides a biosynthetic product for supporting skin, wound restoration and/or scar reduction, said product comprising a protein as described above or a biomaterial as described above.

Compared with the prior art, the invention has the following advantages and beneficial effects:

according to the invention, through screening and transformation from a III type human collagen sequence, codon optimization is carried out according to pichia pastoris codon preference and shielding restriction enzyme cutting sites, an optimized recombinant humanized III type collagen pichia pastoris expression gene is obtained, the recombinant humanized III type collagen pichia pastoris expression gene is constructed into a pichia pastoris secretion expression plasmid through homologous recombination, and a recombinant pichia pastoris engineering strain with high expression humanized III type collagen is obtained through electrotransformation and G418 screening and screening. And (3) carrying out fermentation culture and purification after optimization to finally obtain the high-purity recombinant humanized III-type collagen. The recombinant humanized III type collagen is closer to natural extracted collagen in the high-level structure, and has potential application prospects in the fields of biomedical materials, cosmetics and the like.

Drawings

FIG. 1 is a schematic of electrophoresis after expression using yeast, wherein lanes 1-5 are schematic of the fermentation supernatants of the different transformants, where M is a protein reference.

FIG. 2 is a circular dichroism spectrum of recombinant humanized type III collagen.

FIG. 3 is a circular dichroism spectrum of recombinant humanized type III collagen expressed by other sequences.

FIG. 4 is a schematic diagram of cell migration, wherein the abscissa indicates the amounts of protein added at different concentrations and the ordinate indicates the number of cells.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the following detailed description of the specific embodiments of the present invention will be given with reference to the accompanying drawings. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

EXAMPLE 1 construction of recombinant humanized type III collagen expression vector

1. Gene design

The invention takes the amino acid sequence (NP-000081.2) of the human III type collagen alpha 1 chain published by NCBI database as a template, and retains core functional amino acid. On the basis, the invention carries out high-throughput screening and optimally designs the nucleotide sequence of the amino acid sequence aiming at the codon preference of pichia pastoris. Meanwhile, a stop codon is introduced at the C end. Next, the present invention introduced EcoRI cleavage site at its 5 'end and NotI cleavage site at its 3' end. Finally, the step of obtaining the product, the invention entrusts the engineering biological technology company to carry out gene synthesis. The corresponding amino acid sequences and nucleotide sequences are as follows:

SEQ ID No.1:

GPPGCCGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGPLGIAGITGARGLAGPPGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGLPGLAGTAGEPGRDGNPGSDGLPGRDGSPGGKGDRGENGSPGAPGAPGHPGPPGPVGPAGKSGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPPGCC

SEQ ID No.2:

ggacctcccggttgttgtggtgcacctggctcccccggagtttctggcccaaagggagatgccggacaacctggtgaaaagggttcacctggtgctcagggaccacctggagctccaggtcctctgggcatcgctggaatcaccggtgctaggggattagcaggtccacctggaatgcctggaccaagaggatcccctggtcctcaaggtgttaagggtgagtcaggtaagcctggagccaacggtttgtctggtgagcgaggtcctccaggtccacaaggtctgccaggtttagcaggaactgctggtgagcctggtcgagatggtaacccaggttcagatggtttgccaggcagggatggttccccaggaggaaagggagacaggggtgagaatggttcaccaggagccccaggtgccccaggacatcccggacctcctggtcctgttggtcctgccggcaaatctggagatcgtggagaaagtggacctgctggtcctgcaggagctcctggtcctgctggaagtagaggagctcctggtcctcagggtcctaggggtgacaaaggagaaactggagaacgtggtgccgccggtatcaaaggacacagaggtttccctggaaacccaggcgcaccaggttcaccaggtccagccggacaacagggtgctataggttcacccggcccagctggaccacctggttgttgc

2. Vector construction

The pPIC9K expression vector and the synthetic recombinant humanized type III collagen gene were double digested simultaneously with EcoRI and NotI and incubated for 4 hours at 37 ℃. Then, the pPIC9K vector backbone and the target gene fragment containing the cohesive end were obtained by agarose gel electrophoresis and tapping recovery. Next, the target gene and the vector backbone were ligated using T4 ligase. The ligation products were transformed into TOP10 competent cells by heat shock and then plated on LB plates containing 100. Mu.g/mL ampicillin and 50. Mu.g/mL kanamycin for overnight culture. Thereafter, the present invention picked a monoclonal and inoculated it into 5mL of LB liquid medium containing 100. Mu.g/mL of ampicillin and 50. Mu.g/mL of kanamycin, and cultured overnight at 37℃and 220 rpm. And 1 mu L of bacterial liquid is taken for PCR verification, and 1mL of bacterial liquid is taken for sequencing after the verification is correct. Finally, positive clones which are verified to be correct are inoculated into 100mL of liquid LB culture medium for expansion culture, and plasmids are extracted by using a plasmid extraction kit, so that plasmids which are sufficient for electrotransformation of pichia pastoris are obtained.

3. Pichia pastoris electric transformation

Linearizing the plasmid by SalI restriction enzyme, and then precipitating with isopropanol and redissolving with pure water to obtain linearization product. This linearized product was then electrotransferred into pichia pastoris GS115 electrotransferred competence and coated onto MD plates. After 3-5 days of observation, the growth of the monoclonal was waited. Then, the monoclonal was selected from the MD plates and spotted onto YPD plates containing 0.25-5 ug/mL G418. By this method, pichia pastoris engineered strains capable of expressing recombinant humanized type III collagen were successfully obtained.

4. Expression purification

The engineering strain expressing the recombinant humanized III type collagen is inoculated into 25mL BMGY culture medium for pre-induction culture. After 16 to 18 hours of culture, when the OD600 reached 2 to 6, the cells were collected by centrifugation. Then, the cells were resuspended using 25mL of BMMY to reach an OD600 of 1, and induction expression was started. During the next 72 hours, 25 μl of filtered sterilized methanol was replenished every 24 hours. Meanwhile, 1mL of the fermentation supernatant was collected every 24 hours, and the supernatant was collected by centrifugation. Finally, expression was identified by SDS-PAGE. The result shows that the recombinant humanized III type collagen engineering strain can express recombinant humanized III type collagen. After collecting the supernatant at 8000rpm for 10min, desalting, the fermented supernatant was subjected to chromatography by a cation exchange column, then eluted with a gradient of 0 to 500mM sodium chloride, and the elution peak was collected. Next, SDS-PAGE was used for identification. Through the purification treatment of the step, the purity of the recombinant humanized III type collagen obtained by the invention is more than 95%. This is an efficient purification step that ensures that the collagen obtained is of high purity.

5. Identification of recombinant humanized III type collagen by circular dichroism

The lyophilized sample was dissolved in 0.05M acetic acid to prepare a collagen solution having a concentration of 0.25 mg/mL. Next, a portion of the solution was removed and transferred to a 0.5 mm cuvette. The cuvette was then placed in a circular dichroism spectrometer for analysis. The spectrometer was scanned three times at 25℃with a wavelength range set to 190-280nm. After each scan was completed, the curve was recorded for later analysis. From the figure, the collagen obtained by expression and purification of the sequence is more similar to the natural extracted collagen.

6. Cell migration experiments

To determine the migration capacity of the cells, the cells were first cultured in serum-free medium at 5% carbon dioxide/95% at 37℃for 24 hours. Then, mesenchymal stem cells (2×10 ⁵ per well) were planted in the upper layer of transwell, and the lower layer was added with complete medium containing 10% fbs and different concentrations of recombinant human type III collagen, and cultured for 24 hours. Thereafter, the transwell chamber was removed, stained with 0.1% crystal violet for 30 minutes, washed three times with PBS, and the upper layer of non-migrating cells was gently removed with a wet cotton swab. The cells were photographed by a microscope and counted. Cell migration data were analyzed using GraphPadPrism 5.01 software. Figure 4 shows that recombinant humanized type III collagen can promote cell migration.

The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.

Claims (9)

1. A recombinant humanized type III collagen that is near-native extracted collagen, which is recombinant humanized type III collagen of either a) or b) as follows:

a) The amino acid sequence is the recombinant humanized III type collagen of SEQ ID No. 1;

b) The protein with recombinant humanized III type collagen activity is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID No. 1.

2. The protein-related biomaterial of claim 1, which is any one of the following B1) to B8):

B1 A nucleic acid molecule encoding the protein of claim 1;

b2 An expression cassette comprising the nucleic acid molecule of B1);

B3 A recombinant vector comprising the nucleic acid molecule of B1),

B4 A recombinant vector comprising the expression cassette of B2);

B5 A recombinant microorganism comprising the nucleic acid molecule of B1);

B6 A recombinant microorganism comprising the expression cassette of B2);

b7 A recombinant microorganism containing the recombinant vector of B3);

b8 A recombinant microorganism comprising the recombinant vector of B4);

Optionally, the vector comprises a yeast expression vector;

optionally, the recombinant microorganism comprises an engineered bacterium or a yeast cell.

3. The biomaterial according to claim 2, characterized in that: the nucleic acid molecule is as shown in the following 1) or 2) or 3) or 4):

1) The coding sequence is a DNA molecule or a cDNA molecule of SEQ ID No. 2in a sequence table;

2) The nucleic acid sequence is a DNA molecule of SEQ ID No.2 in a sequence table;

3) A cDNA molecule or a genomic DNA molecule having 75% or more identity to the nucleotide sequence defined in 1) or 2) and encoding the protein of claim 1;

4) Hybridizing under stringent conditions to the nucleotide sequence defined in 1) or 2) and encoding a cDNA molecule or a genomic DNA molecule of the protein according to claim 1.

4. Use of the protein of claim 1 for supporting skin, wound healing and/or scar reduction, said use being for non-therapeutic purposes.

5. Use of the biomaterial of claim 2 for the preparation of recombinant humanized type III collagen.

6. The preparation method of the recombinant pichia pastoris engineering strain is characterized by comprising the following steps of: after synthesizing the coding gene of the recombinant humanized III type collagen by a gene synthesis mode, cloning a target gene to the downstream of a secretion signal peptide of a pPIC9K plasmid by utilizing a homologous recombination technology, extracting the plasmid after verification of correctness by PCR and gene sequencing, electrically transferring the plasmid into pichia pastoris electrotransfer competent cells after linearization by using restriction enzymes, and screening by using a gradient G418 to obtain the recombinant pichia pastoris engineering strain of the high-expression recombinant humanized III type collagen.

7. The method according to claim 6, wherein: the sequence of the coding gene of the recombinant humanized III type collagen is shown as SEQ ID No. 2.

8. A method for preparing recombinant humanized type III collagen, comprising the steps of: recombinant humanized type III collagen is obtained using the biomaterial of claim 2.

9. A biosynthetic product for supporting skin, wound restoration and/or scar reduction, the product comprising the protein of claim 1 or the biomaterial of claim 2.