CN118791593A - Preparation process, product and application of human-like collagen - Google Patents

Abstract

The invention relates to the field of biotechnology, in particular to a preparation process, a product and application of human-like collagen, wherein the amino acid sequence of the human-like collagen is shown as SEQ ID NO.1, or the amino acid sequence is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown as SEQ ID NO. 1. The invention obtains a new sequence of human-like collagen, which is different from the known sequence, expands a new reference sequence source, can obviously promote human skin fibroblast proliferation, has excellent skin repair capability, and lays a solid foundation for large-scale production and application.

Description

Preparation process, product and application of human-like collagen

Technical Field

The present invention relates to the field of biotechnology, and in particular to a preparation process, product and application of human-like collagen.

Background Art

Collagen is the most abundant protein in the human body. It is mainly found in human skin, bones, cartilage, teeth, tendons, ligaments and blood vessels. It is an extremely important structural protein in connective tissue, supporting organs and protecting the body. According to the function and the structural characteristics of the tissues it constitutes, it can be divided into: fibrous collagen, reticular collagen, fiber-related collagen located on the fiber surface, beaded collagen, basement membrane-fixed fiber collagen, and collagen with transmembrane structure.

Human-like collagen is a high-molecular biological protein produced by reverse transcribing human collagen mRNA into cDNA through bioengineering technology, and then recombining a segment of the gene after enzyme cutting into E. coli (Escherichia coli) through high-density fermentation, separation, renaturation and purification processes.

Prior art CN 115252881A discloses a human-like collagen composition and a preparation method thereof, wherein the human-like collagen composition comprises the following raw materials: 5-15 parts by weight of human-like collagen; 10-20 parts by weight of cross-linkable powder; 1-2 parts by weight of a cross-linking agent; 20-25 parts by weight of fish glue extract and 50-80 parts by weight of chitosan. The human-like collagen composition finally obtained has good structural stability, water absorption, comfort, human compatibility and hemostatic effect.

The prior art CN114085394B discloses a recombinant collagen two-phase gel and its preparation method and application, which relates to the technical field of biomedical materials; through a synchronous emulsification and cross-linking method, during the emulsification process, a cross-linking agent is simultaneously dripped into the emulsifying system, and part of the emulsion droplets directly wrap the cross-linking agent into the interior of the collagen emulsion particles. The internal cross-linking degree of the emulsion particles is high, and the internal cross-linking degree of some emulsion droplets that are not wrapped with the cross-linking agent or wrapped with a small amount of cross-linking agent is small, thereby forming emulsion particles with multiple cross-linking degree distributions. Through a single emulsification and cross-linking reaction, collagen microspheres with different cross-linking degrees are simultaneously prepared, so that the cross-linking degree distribution of the collagen microspheres in the recombinant collagen two-phase gel is different, and gradient degradation can be achieved.

Prior art CN117327170B discloses a method for preparing human-like collagen and its application in cosmetics, which can effectively promote the proliferation activity of fibroblasts, lubricate the stratum corneum, repair the brick wall structure, and promote the growth of epithelial cells and fibroblasts. It can well promote fibroblasts to generate Elastin, increase skin tightness, make the skin firm, and give it elasticity and tenderness. The human-like collagen of the present invention has the functions of protecting the skin's water barrier, improving the skin's microenvironment, moisturizing, clearing and not sticky. It protects the skin barrier, resists microorganisms and enzymes, regulates the cell growth microenvironment, and resists external stimuli.

Prior art CN116286914B discloses a transmembrane protein complex gene and its application in the preparation of recombinant human-like collagen by biological fermentation, wherein the transmembrane protein complex gene comprises three segments of genes, the nucleotide sequence of the first segment of the gene is shown in SEQ ID NO.1, the nucleotide sequence of the second segment of the gene is shown in SEQ ID NO.2, and the third segment of the gene is selected from a human collagen gene fragment capable of expressing a human-like collagen with a molecular weight of 6-12 kDa. The third segment of the target gene human-like collagen is secreted and expressed into the fermentation broth or the periplasmic space of Escherichia coli using the transmembrane protein complex of Escherichia coli, centrifuged and the periplasmic protein is extracted, and then purified to obtain the fermentation broth or Escherichia coli.

The above-mentioned prior arts either focus on the selection of cross-linking components and ratios, or focus on gene cloning, but both cannot achieve large-scale production.

Based on the defects of the prior art, the present invention will develop a preparation process and product of human-like collagen.

Summary of the invention

The present invention first provides a human-like collagen, the amino acid sequence of which is shown as SEQ ID NO.1.

In certain embodiments, the amino acid sequence is a human-like collagen obtained by substituting and/or deleting and/or adding one or more amino acid residues of 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) a nucleic acid molecule encoding the above-mentioned human-like collagen;

B2) an expression cassette containing the nucleic acid molecule described in B1);

B3) a recombinant vector containing the nucleic acid molecule described in B1),

B4) a recombinant vector containing the expression cassette described in B2);

B5) a recombinant microorganism containing the nucleic acid molecule described in B1);

B6) a recombinant microorganism containing the expression cassette described in B2);

B7) a recombinant microorganism containing the recombinant vector described in B3);

B8) a recombinant microorganism containing the recombinant vector described in B4);

Optionally, the vector comprises a yeast expression vector;

Optionally, the recombinant microorganism is a yeast cell.

In certain embodiments, the nucleic acid molecule sequence is shown as SEQ ID No.2.

The present invention also provides the use of the human-like collagen in preparing medicines for skin repair, wound healing and scar reduction.

The present invention also provides the use of the above-mentioned biomaterial in preparing human-like collagen.

The present invention also provides a process for preparing human-like collagen, which comprises the following steps: utilizing the above-mentioned biological material to obtain human-like collagen.

In certain embodiments, the sequence of the human-like collagen is shown as SEQ ID No.1.

In some embodiments, the biological material contains the nucleic acid molecule described in SEQ ID No.2.

Finally, the present invention provides a product containing human-like collagen, which includes the above-mentioned protein or the above-mentioned biomaterial, and a cross-linking agent; optionally, the cross-linking agent is at least one of 1,4-butanediol diglycidyl ether, glutaraldehyde, NHS, and genipin.

Compared with the prior art, the present invention has at least the following beneficial effects:

(1) The present invention obtains a new human-like collagen sequence, which is different from the known sequence and expands the source of new reference sequences;

(2) The present invention obtains a new human-like collagen that can significantly promote the proliferation of human skin fibroblasts and has excellent skin repair ability, laying a solid foundation for large-scale production and application.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Comparison of proliferation rates.

DETAILED DESCRIPTION

In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

Example 1 Preparation of collagen

1. Genetic design

The present invention uses the amino acid sequence of human type III collagen α1 chain published in the NCBI database as a template, optimizes the nucleotide sequence of the amino acid sequence to obtain the amino acid sequence of SEQ ID No.1 and the nucleotide sequence of SEQ ID No.2.

SEQ ID No.1:

IAG I GAMGSH;

SEQ ID No.2:

GGTGCACTTGGCTCCGCCGGAGTGCTGGCCCAAAGGGAGATGCCGGACAACCTGGTGAAAAGGGTTCACCTGGTGCTCAGGGACCACCTGGAGCTCCAGGTCCTCTGGGCATCGCTGGAATCACCGGTGCTAGGGGATTAGCAGGTCCACCTGGAATGCCTGGACCAAGAGGATCCCCTGGTCCTCAAGGTGTTAAGGGTGAGTCAGGTAAGCCTGGAGCCAACGGTTTGTCTGGTGAGCGAGGTCCTCCAGGT CCACAAGGTCTGCCAGGTTTAGCAGGAACTGCTGGTGAGCCTGGTCGAGATGGTAACCCAGGTTCAGATGGTTTGCCA GGCAGGGATGGTTCCCCAGGAGGAAAGGGACAGGGGTGAGAATGGTTCACCAGGAGCCCCAGGTGCCCCAGGACATCCCGGACCTCCTGGTCCTGTTGGTCCTGCCGGCAAATCTGGAGATCGTGGAGAAAGTGGACCTGCTGGTCCTGCAGGAGCTCCTGGTCCTGCTGGAAGTAGAGGAGCTCCTGGTCCTCAGGGTCCTAGGGGTGACAAAGGAGAAACTGGAGAACGTGGTGCCGCCGGTATCAAAGG ACACAGAGGTTTCCCTGGAAACCCAGGCGCACCAGGTTCACCAGGTCCAGCCGGACAACAGGGTGCCATGGGTTCACAC;

Qingke Biotechnology Company was commissioned to synthesize SEQ ID No.2 into the pUC57 vector. Then, the upstream and downstream primers with EcoRI and Not I restriction sites were used for gene amplification. After the amplified product was purified and recovered, the amplified product and the expression vector pPI C9k were double-digested by EcoRI and Not I restriction endonucleases. The pPI C9K vector backbone and the amplified product with sticky ends were obtained by agarose gel electrophoresis and gel cutting recovery. Then, T4 ligase was used for ligation reaction, and the ligated product was transformed into JM109 competent cells by heat shock. Positive transformants were screened using LB plates with Amp resistance, and the transformants with correct colony PCR, double restriction digestion, and sequencing verification were selected for cultivation and the recombinant plasmid was extracted using the Sangon Plasmid Extraction Kit, thereby obtaining a sufficient amount of recombinant plasmid for electroporation of Pichia pastoris.

The plasmid was linearized using Sal I restriction endonuclease, and then the linearized product was obtained by isopropanol precipitation and pure water resolubilization. Then, the linearized product was electrotransformed into Pichia pastoris GS115 electrotransformation competent cells and coated on MD plates. The positive transformants grown on the above MD plates were transferred to G418 resistance plates with concentration gradients of 2.0, 3.0, 4.0, and 5.0 mg/mL, respectively. The strains that grew well on the plates at each concentration were selected for cultivation, and the genome was extracted and PCR verification and sequencing verification were performed to successfully obtain Pichia pastoris engineered strains capable of expressing human-like collagen.

After the above-mentioned recombinant Pichia yeast engineering bacteria were streaked and activated, a single colony was picked and inoculated into 10mL/50mL YPD medium, cultured at 30°C and 220rpm for 20h, inoculated into 25mL/250mL initial medium with an inoculum of 1% and cultured for 24h, the bacteria were collected and centrifuged at 4°C and 5000rpm for 15min, the supernatant was discarded, the bacteria were washed twice with pre-cooled ddH2O and resuspended in fresh 25mL/250mL induction medium, fermented and cultured at 30°C and 220rpm, 0.75% filtered and sterilized methanol was supplemented to the medium every 12h, and the collagen expression was measured every 24h. By sampling the fermentation broth supernatant and performing SDS-PAGE protein electrophoresis analysis, a band of about 20kDa appeared, that is, type III human collagen secretion expression was successful. 8000rpm, 15min after collecting the supernatant, after desalting, the fermentation supernatant was chromatographed through a cation exchange column, and then eluted using a 0-500mM sodium chloride gradient, and the elution peak was collected. Then, SDS-PAGE was used for identification. The results showed that the purity of the human-like collagen obtained by the present invention exceeded 96%.

Example 2 Effect Test

Human skin fibroblasts HSF in logarithmic growth phase were inoculated on a 96-well plate at 5×10 ⁵ /mL per well, with 5 replicate wells in each group. After the cells adhered to the wall, DMEM complete medium and CCK-8 solution were mixed at a volume ratio of 100:1 and the cells were incubated for 2 hours. The supernatant was aspirated and the absorbance at a wavelength of 450 nm was detected by a microplate reader. The cells in the 96-well plate were washed once with PBS. The experimental group was cultured with DMEM medium containing recombinant human type I and II collagen of Example 1 at different concentrations (0 ug/mL, 50 ug/mL, 100 ug/mL, 200 ug/mL), and the control group was cultured with only DMEM complete medium. The absorbance at a wavelength of 450 nm was measured after 96 hours of culture. The results are shown in FIG1 , indicating that the human-like collagen of Example 1 can promote the proliferation of human skin fibroblasts.

The above is 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 principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims (10)

1. A human-like collagen, characterized in that its amino acid sequence is shown in SEQ ID NO.1. 2. A human-like collagen, characterized in that its amino acid sequence is obtained by replacing and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No.1. 3. A biomaterial related to the human-like collagen according to claim 1 or 2, which is any one of the following B1) to B8): B1) a nucleic acid molecule encoding the human-like collagen of claim 1; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1), B4) a recombinant vector containing the expression cassette described in B2); B5) a recombinant microorganism containing the nucleic acid molecule described in B1); B6) a recombinant microorganism containing the expression cassette described in B2); B7) a recombinant microorganism containing the recombinant vector described in B3); B8) a recombinant microorganism containing the recombinant vector described in B4); The vector comprises a yeast expression vector; The recombinant microorganism is a yeast cell. 4. The biomaterial according to claim 3, characterized in that the nucleic acid molecule sequence is as shown in SEQ ID No.2. 5. Use of the human-like collagen according to claim 1 in the preparation of drugs for skin repair, wound healing and scar reduction. 6. Use of the biomaterial according to claim 3 in the preparation of human-like collagen. 7. A process for preparing human-like collagen, characterized in that it comprises the following steps: using the biomaterial according to claim 3 to obtain human-like collagen. 8. The preparation process according to claim 7, characterized in that the sequence of the human-like collagen is as shown in SEQ ID No. 1. 9. The preparation process according to claim 7, characterized in that the biological material contains the nucleic acid molecule as described in SEQ ID No. 2. 10. A product containing human-like collagen, characterized in that the product comprises the protein according to any one of claims 1-2 or the biomaterial according to claim 3, and a cross-linking agent; the cross-linking agent is at least one of 1,4-butanediol diglycidyl ether, glutaraldehyde, NHS, and genipin.