Expression process of recombinant collagen self-assembled fiber

Abstract

The invention belongs to the technical field of bioengineering, and relates to an expression process of a recombinant collagen self-assembled fiber, the recombinant collagen is a recombinant collagen as shown in a) or b): a) a recombinant collagen with an amino acid sequence of SEQ ID No.1; and b) recombinant collagen obtained by substitution and / or deletion and / or addition of one or more amino acid residues of the amino acid sequence as shown in SEQ ID No.1. According to the invention, the amino acid sequence of the type III collagen alpha1 chain disclosed in an NCBI database is used as a template, and particularly attention is paid to the retention of core functional amino acids. According to the invention, the amino acid sequence is optimally designed, the codon preference of the amino acid sequence is also considered, and the corresponding vector and engineering bacteria are constructed. And carrying out fermentation culture and purification after optimization to finally obtain the high-purity recombinant collagen. The recombinant collagen has an obvious fibrous polymer under a transmission electron microscope, and has excellent biocompatibility.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering and relates to an expression process of recombinant collagen self-assembly fibers. Background Art

[0002] The formation of recombinant collagen self-assembled fibers benefits from the natural biocompatibility and bioactivity of collagen. Collagen is a natural protein widely found in animal connective tissues, which plays a vital role in supporting and protecting body tissues. Through genetic engineering technology, scientists are able to design and produce recombinant collagen with specific sequences, thereby precisely regulating its self-assembly properties.

[0003] The formation process of this self-assembled fiber is that under specific conditions, recombinant collagen molecules spontaneously aggregate through complex interactions to form a highly ordered fiber structure. This process is completely carried out in vitro, allowing scientists to easily prepare collagen fibers with consistency, reproducibility and stable quality.

[0004] Recombinant collagen self-assembling fibers have excellent biocompatibility and can seamlessly integrate with human tissues, thereby reducing the risk of immune rejection. In the field of tissue engineering and regenerative medicine, this fiber is an ideal scaffold material that can provide a suitable environment for cell growth and promote cell adhesion, reproduction and differentiation. By simulating the structure and function of natural tissues, this fiber provides an effective solution for the repair and replacement of damaged tissues.

[0005] In addition to its wide application in tissue engineering and regenerative medicine, recombinant collagen self-assembled fibers also show great potential in drug delivery. This fiber can combine with drugs to form a drug carrier, achieving precise controlled release of drugs. In this way, drugs can be slowly and steadily released to the target site, improving efficacy and reducing side effects. This has brought a revolutionary breakthrough in the field of drug delivery and provided a safer and more effective means for the treatment of various diseases.

[0006] In summary, recombinant collagen self-assembled fibers are leading the development trend in the field of biomedical engineering with their unique properties and broad application prospects. With the continuous advancement of technology and in-depth research, we expect this material to bring more innovations and benefits to human health and quality of life in the future. At the same time, scientists will continue to explore the potential applications of recombinant collagen self-assembled fibers in other fields, providing more possibilities for solving various medical problems. Summary of the invention

[0007] The purpose of the present invention is to address the above-mentioned deficiencies in the prior art. The present invention first provides a recombinant collagen, which is the following recombinant collagen a) or b):

[0008] a) a recombinant collagen having an amino acid sequence of SEQ ID No. 1;

[0009] b) Recombinant 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.

[0010] The present invention also provides a biological material related to the above protein, which is any one of the following B1) to B8):

[0011] B1) a nucleic acid molecule encoding the above protein;

[0012] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0013] B3) a recombinant vector containing the nucleic acid molecule described in B1),

[0014] B4) a recombinant vector containing the expression cassette described in B2);

[0015] B5) a recombinant microorganism containing the nucleic acid molecule described in B1);

[0016] B6) a recombinant microorganism containing the expression cassette described in B2);

[0017] B7) a recombinant microorganism containing the recombinant vector described in B3);

[0018] B8) a recombinant microorganism containing the recombinant vector described in B4);

[0019] Optionally, the vector comprises a yeast expression vector;

[0020] Optionally, the recombinant microorganism comprises an engineered bacterial or yeast cell.

[0021] In certain embodiments, the nucleic acid molecule is a nucleic acid molecule as shown in 1) or 2) or 3) or 4) below:

[0022] 1) The coding sequence is a DNA molecule or cDNA molecule of SEQ ID No. 2 in the sequence list;

[0023] 2) The nucleic acid sequence is the DNA molecule of SEQ ID No. 2 in the sequence list;

[0024] 3) a cDNA molecule or genomic DNA molecule that has 75% or more identity with the nucleotide sequence defined in 1) or 2) and encodes the above-mentioned protein;

[0025] 4) A cDNA molecule or genomic DNA molecule that hybridizes with the nucleotide sequence defined in 1) or 2) under stringent conditions and encodes the above-mentioned protein.

[0026] The present invention also provides the use of the above protein in supporting skin, wound recovery, reducing scars, reducing rejection risk, as a scaffold and drug delivery, and the use is for non-therapeutic purposes.

[0027] The present invention also provides the use of the above-mentioned biomaterial in preparing recombinant collagen self-assembly fibers.

[0028] The present invention also provides a method for preparing a recombinant Escherichia coli engineering strain, comprising the following steps: synthesizing a coding gene for recombinant collagen by gene synthesis, connecting it to a pet20b expression vector, and then electroporating it into an Escherichia coli electroporation competent cell to obtain a recombinant Escherichia coli.

[0029] In certain embodiments, the sequence of the gene encoding the recombinant collagen is shown as SEQ ID No.2.

[0030] The present invention also provides a recombinant collagen self-assembly fiber expression process, comprising the following steps: utilizing the above-mentioned biological material to obtain the recombinant collagen self-assembly fiber.

[0031] The present invention also provides a biosynthetic product for supporting skin, wound recovery and / or reducing scars, the product comprising the above protein or the above biomaterial.

[0032] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0033] The present invention uses the amino acid sequence of the type III collagen α1 chain published in the NCBI database as a template, and pays special attention to the retention of core functional amino acids. In order to adapt to the characteristics of Escherichia coli, we not only optimize the amino acid sequence, but also consider its codon preference, and construct the corresponding vector and engineering bacteria. After optimization, fermentation culture and purification are carried out to finally obtain high-purity recombinant collagen. The recombinant collagen has obvious fibrous polymers under transmission electron microscopy, has excellent biocompatibility, and can be seamlessly combined with human tissues, thereby reducing the risk of immune rejection. In the field of tissue engineering and regenerative medicine, this fiber, as an ideal scaffold material, can provide a suitable environment for cell growth and promote cell adhesion, reproduction and differentiation. By simulating the structure and function of natural tissues, this fiber provides an effective solution for the repair and replacement of damaged tissues. This fiber can be combined with drugs to form a drug carrier to achieve precise controlled release of drugs. In this way, the drug can be slowly and stably released to the target site, improving the efficacy and reducing side effects. This has brought a revolutionary breakthrough in the field of drug delivery and provided a safer and more effective means for the treatment of various diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a schematic diagram of the different components of E. coli expression and purification, where lane 1 is the bacterial solution after disruption, lane 2 is the centrifugal precipitation, lane 3 is the flow through the nickel column, lane 4 is the medium, lane 5 is for impurity removal, lane 6 is elution, lane 7 is the medium after elution, and M is a marker.

[0035] Figure 2 This is a negative staining transmission electron microscopy image.

[0036] Figure 3 It is a schematic diagram of cell adhesion, where the horizontal axis is the amount of protein added at different concentrations, and the vertical axis is the absorption value at 450nm. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical scheme and advantages of the present invention clearer, the specific embodiments of the present invention are further described in detail below in conjunction with specific examples and drawings. If no specific technology or conditions are specified in the examples, the technology or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0038] Example 1. Gene design

[0039] We used the amino acid sequence of type III collagen α1 chain published in the NCBI database as a template, paying special attention to the retention of core functional amino acids. In order to adapt to the characteristics of E. coli, we not only optimized the amino acid sequence, but also considered its codon preference. At the same time, EcoRI and XhoI restriction sites were introduced at the 5' and 3' ends of the gene, respectively. Finally, we commissioned Qingke Biotechnology Company to complete the gene synthesis.

[0040] SEQ ID No.1:

[0041] GCCGPPGCCGYPGPAGPPGPPGPPGTSGHPGSPGSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSDGQPGPPGPPGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGSPGPKGDKGEPGGPGADGVPGKDGPRGPTGPIGPPGPAGQPGDKGEGGAPGLPGIAGPRGSPGERGETGPPGPAGFPGAPGPPGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGLPGLAGTAGEPGRDGNPGSDGLPGRDGSPGFPGNPGAPGSPGPAGQQGAIGSPGPAGPRGPVGPSGPPGKDGTSGHPGPIGPPGPRGNRGERGSEGSPGHPGQPGPPGPPGAPGPCGCCGPPGCC

[0042] SEQ ID No.2:

[0043] ggctgttgcggtccgccgggttgttgtggttatccgggtcctgcgggtccaccgggcccgccgggcccgccgggtac

[0044] ttccggtcacccgggctccccaggctctccgggttatcagggccctccgggcgaaccgggtcaagctggcccgtctg

[0045] gccctccgggcccaccgggcgccatcggtccatccggtccggctggcaaagatggtgagtctggtcgcccgggcc

[0046] gtccgggtgagcgtggtctgccgggtccgccgggcattaagggtccagcgggcatcccgggtttcccaggcatgaa

[0047] gggtcaccgtggcttcgatggtcgtaacggtgaaaagggcgaaaccggtgcaccgggcctgaagggtgagaacgg

[0048] tctgccgggcgaaaatggcgcgccgggcccgatgggcccgcgtggtgcgccgggcgaacgtggtcgtcctggcct

[0049] gccaggtgcagcaggtgcacgtggtaatgatggtgcgcgcggtagcgacggtcagccgggtccgccgggtccgcc

[0050] aggcgcaccgggtttccgcggcccggccggtccgaacggtattccaggtgaaaaaggtccagcgggtgaacgcgg

[0051] tgctccgggcccggccggtccgcgtggcgcagcgggcgaaccaggccgtgacggtgtacctggcggtccgggtat

[0052] gcgcggcatgcctggttctccaggtggtccaggttccgacggtaaaccgggtccacctggctcccagggcgagagc

[0053] ggccgtcctggtccgccgggcccaagcggcccgcgtggtcagccgggcagcccaggcccgaaaggtgataaggg

[0054] tgaaccgggtggtccaggcgccgacggtgtaccgggcaaagatggccctcgtggtccgaccggtccgatcggccc

[0055] gccgggcccggcaggtcagccgggtgataaaggcgaaggtggtgcgccgggcctgccgggtatcgcaggtccgc

[0056] gcggttctccaggtgaacgtggcgaaactggcccgcctggcccggcgggtttcccgggtgcacctggtccgccggg

[0057] tatgccaggcccgcgcggttctccgggtccacagggcgtaaaaggtgaatctggtaagccgggcgctaacggcctgt

[0058] ctggtgaacgcggtccgccaggtcctcagggtctgcctggcctggcgggtactgcaggcgaaccgggccgtgacg

[0059] gtaacccgggcagcgacggcctgccgggccgtgatggctctccgggcttcccaggtaatccgggtgcaccgggctc

[0060] tccgggtccggctggtcagcagggtgcgatcggttctccgggcccggcgggtccgcgtggtccggttggtccgtccg

[0061] gccctccgggtaaagatggcacctccggccatccgggtcctatcggcccgccgggtccgcgtggcaaccgtggtga

[0062] acgcggttccgaaggttctccgggccatccgggtcaaccgggtccgccgggcccaccgggtgctccgggtccgtgt

[0063] ggctgttgcggcccgccgggctgctgt

[0064] Example 2, Vector Construction

[0065] First, we digested the pet20b expression vector and the recombinant humanized type III collagen gene with EcoRI and XhoI enzymes and incubated them at 37°C for 4 hours. After digestion, we successfully obtained the sticky end fragments of the pet20b vector backbone and the target gene. Next, the target gene was ligated to the vector backbone using T4 ligase. The ligation product was then transformed into TOP10 competent cells. These cells were plated on LB plates containing ampicillin and cultured overnight at 37°C. Subsequently, we selected single clones and transferred them to liquid culture medium, and continued to culture them overnight at 37°C and 220rpm. After PCR verification, the correct clones were confirmed to be used for sequencing. Finally, we transferred the verified positive clones to 100mL of liquid LB culture medium for expansion and culture, and used the Sangon Plasmid Extraction Kit to extract the plasmids in preparation for transformation.

[0066] Example 3, E. coli transformation

[0067] First, E. coli is inoculated in a culture medium for culture. After it grows to the logarithmic growth phase, the cells are collected and prepared into competent cells. Then, the recombinant plasmid is mixed with the competent cells, a calcium ion solution is added, and the temperature is lowered for ice bath treatment. Next, the recombinant plasmid is introduced into the E. coli cells by heat shock. Finally, the transformed cells are spread on a selective culture medium containing appropriate antibiotics, and positive clones are screened and verified. Sterile operation must be maintained throughout the experiment to ensure the accuracy of the experimental results.

[0068] Example 4: Induced expression and purification

[0069] The E. coli transformed with the recombinant plasmid was inoculated into 200 mL of LB medium, cultured at 37°C with shaking until the logarithmic growth phase, and induced with 0.2 mM IPTG at 25°C for 16 to 20 hours. After the induction, the cells were collected at 8000 rpm for 10 minutes and the supernatant was discarded. The cells were resuspended in 50 mL of phosphate buffer and ultrasonically disrupted to release intracellular proteins. The supernatant was collected at 14000 rpm for 30 minutes, passed through a Ni-NTA chromatography column, eluted with phosphate buffer containing 200 mM imidazole, and identified by SDS-PAGE.

[0070] Example 5, transmission electron microscopy observation

[0071] 3 μL of recombinant collagen sample (concentration of 0.02 mg / mL) was applied to a glow-discharged 300-mesh carbon support film (China Zhongjing Keyi Thin Film Technology Co., Ltd.) and allowed to stand for one minute, and then the excess liquid was removed with filter paper. Subsequently, 3 μL of 3% phosphotungstic acid was added to the grid and allowed to stand for another minute. The remaining excess liquid was then removed, and this process was repeated three times. Finally, the grid was allowed to dry naturally at room temperature. Micrographs were taken using a Talos L120C operating at 120 kV and equipped with a Ceta Summit electron detector. The imaging magnification was 73,000 times, and the defocus range was set to -1.8 to -2.3 μm, which was determined by Figure 2 It can be clearly seen that the recombinant collagen has obvious fibrous polymers under the transmission electron microscope.

[0072] Example 6: Recombinant humanized type III collagen experiment

[0073] To evaluate the adhesion ability of recombinant humanized type III collagen, 50 μL of collagen at different concentrations was added to a 96-well plate. The well plate was hydrated overnight and then blocked with 1% BSA solution at 37°C for 2 hours. Mesenchymal stem cells (1x104) were then seeded into the same 96-well plate and cultured in a 5% carbon dioxide environment at 37°C for 2 hours. After removing the culture medium, each well was treated with 100 μL of 10% CCK-8 / DMEM (v / v) solution and cultured for another 2 hours. The absorbance of each well at a wavelength of 450 nm was measured using a ThermoFisherScientific spectrophotometer. Statistical analysis was performed using GraphPad Prism 5.01 software to evaluate cell viability. Figure 3 It can be seen that the number of adhered cells increased significantly with increasing protein concentration.

[0074] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A recombinant collagen, which is the recombinant collagen of a) or b) below: a) a recombinant collagen having an amino acid sequence of SEQ ID No. 1; b) Recombinant 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.

2. The biological material related to the protein according to claim 1, which is any one of the following B1) to B8): B1) a nucleic acid molecule encoding the protein according to 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); Optionally, the vector comprises a yeast expression vector; Optionally, the recombinant microorganism comprises an engineered bacterial or yeast cell.

3. The biomaterial according to claim 2, characterized in that: The nucleic acid molecule is the nucleic acid molecule shown in 1) or 2) or 3) or 4) below: 1) The coding sequence is a DNA molecule or cDNA molecule of SEQ ID No. 2 in the sequence list; 2) The nucleic acid sequence is the DNA molecule of SEQ ID No. 2 in the sequence list; 3) a cDNA molecule or genomic DNA molecule that has 75% or more identity with the nucleotide sequence defined in 1) or 2) and encodes the protein of claim 1; 4) A cDNA molecule or genomic DNA molecule that hybridizes with the nucleotide sequence defined in 1) or 2) under stringent conditions and encodes the protein of claim 1.

4. Use of the protein of claim 1 in supporting skin, wound recovery, reducing scars, reducing rejection risk, as a scaffold and drug delivery, wherein the use is for non-therapeutic purposes.

5. Use of the biomaterial according to claim 2 in the preparation of recombinant collagen self-assembled fibers.

6. A method for preparing a recombinant Escherichia coli engineered strain, characterized in that: The method comprises the following steps: synthesizing the coding gene of the recombinant collagen by gene synthesis, connecting it with the pet20b expression vector, and then electrotransforming it into the electrotransformation competent cells of Escherichia coli to obtain the recombinant Escherichia coli.

7. The method according to claim 6, characterized in that: The sequence of the gene encoding the recombinant collagen is shown as SEQ ID No.

2.

8. A process for expressing recombinant collagen self-assembly fibers, characterized in that: The method comprises the following steps: utilizing the biomaterial according to claim 2 to obtain recombinant collagen self-assembled fibers.

9. A biosynthetic product for supporting skin, wound recovery and / or reducing scars, the product comprising the protein of claim 1 or the biomaterial of claim 2.

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