Self-assembling recombinant collagen, preparation method and use thereof

Abstract

This patent application discloses a self-assembled recombinant collagen, a preparation method and use thereof. The amino acid sequence of the self-assembled recombinant collagen of the present invention is shown in SEQ ID No. 1, and it can self-assemble in an aqueous solution to form a heptamer structure. The self-assembled recombinant collagen has excellent long-term storage stability, effectively avoiding the problem of protein degradation during storage.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a novel self-assembling recombinant collagen, a preparation method thereof and application thereof. Background Art

[0002] Collagen is a biological macromolecular protein, the main component of animal connective tissue, and the most abundant and widely distributed functional protein in mammals, accounting for 25% to 30% of the total protein. It is closely related to the formation and maturation of tissues, the transmission of information between cells, joint lubrication, wound healing, calcification, blood coagulation and aging. It is one of the most critical raw materials in the biotechnology industry and is widely used in medical materials, cosmetics, and food industries.

[0003] The collagen currently used in industry is mainly extracted from animal skin or bones through acid, alkali or enzymatic methods. Its main source is animal connective tissue. However, collagen extracted from animal tissues has risks such as animal-derived diseases. At the same time, large-scale preparation will cause excessive demand for animals, causing tremendous pressure on animal breeding on the supply side.

[0004] With the large-scale application of genetic engineering technology, genetic engineering recombinant expression of collagen has successfully solved the bottleneck of large-scale preparation of collagen by using patterned expression hosts to express exogenous proteins. Compared with traditional animal collagen extraction, genetic engineering recombinant preparation of collagen has the following advantages: 1. There are many types of collagen produced, which can be collagen from common sources such as cattle, pigs, and fish. Human collagen can also be produced, which has better safety and immune efficacy in the fields of medical devices. 2. It can effectively avoid animal-derived diseases. Recombinant collagen usually uses simple prokaryotic or eukaryotic cells as expression hosts. Its cell pathogens cannot be transmitted to each other due to the huge difference in cell structure with humans. 3. The production cycle is short, cost-saving, can be quickly amplified, and is suitable for industrial large-scale production. Compared with the long animal culture of 3-5 months, the microbial culture cycle only takes 2 or 3 days and the culture is simple. Large-scale culture can be carried out by providing simple C source and N source, which is easy to prepare at industrial level. Therefore, the preparation of recombinant collagen, especially recombinant human collagen, is currently a hot spot in collagen production research.

[0005] At present, the recombinant collagen prepared on a large scale mostly uses microorganisms such as Escherichia coli and yeast as expression hosts. However, microbial cells have obvious disadvantages compared with human cells in protein post-translational modification, and can only achieve simple modifications such as glycosylation, methylation, and acetylation. Therefore, the prepared recombinant protein cannot form higher-level micelles and tube structures, and mostly exists in low-level structures such as linear, α-helical, and β-folded. Therefore, it generally cannot form a stable and complex spatial structure like natural collagen. It is easy to degrade and locally aggregate during production and storage, which increases the difficulty of purification and storage. Cross-linking agents can be used to cross-link recombinant collagen to increase its stability, but cross-linking agents are toxic or immunogenic to the human body, so the use of cross-linking agents is not recommended for safety considerations. In short, there is currently no good solution to the problem of recombinant collagen degradation. Summary of the invention

[0006] The amino acid sequence determines the spatial structure of recombinant collagen. The inventor accidentally discovered in the process of scientific research practice that by adding specific amino acid sequences to both ends of 52 repetitions of the amino acid sequence of human type I collagen GAPGAPGLQGAPGSQ, a macromolecular recombinant collagen with a polymer structure can be obtained. The recombinant collagen of the present invention exhibits excellent stability during long-term storage in aqueous solution, because a more stable polymer is formed.

[0007] Specifically, the present invention includes:

[0008] 1. A recombinant collagen, the amino acid sequence of which is shown in SEQ ID No.1;

[0009] SEQ ID No.1: MDPVVLQRRDWENPGVTQLNRLAAHPPFASDPM(G AP GAP GSQG AP G LQ) n GAMGSS, where n=52.

[0010] For MDPVVLQRRDWENPGVTQLNRLAAHPPFASDPM(GAPGA PG SQGAPGLQ) nGAMGSS, recombinant collagen with different molecular weights can be constructed by different n values ​​(i.e., different repetition times of GAPGAPGSQ GAP GLQ), wherein n is a positive integer, for example, n=1-20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or any positive integer greater than 101, such as 110, 120, 130, 140, 150, 160, 170, 180, 190, 200. However, the inventors have found that when n=52, the obtained recombinant collagen can self-assemble to form polymers.

[0011] 2. The recombinant collagen according to item 1, wherein the amino acid sequence shown in SEQ ID No. 1 self-assembles to form a polymer (eg, a hexamer and / or a heptamer and / or an octamer) structure.

[0012] 3. The recombinant collagen according to item 1 or 2, further comprising a tag for facilitating purification.

[0013] 4. The recombinant collagen according to item 3, wherein the tag is a His tag, a Flag tag or a c-Myc tag.

[0014] 5. Use of the recombinant collagen according to any one of items 1 to 4 as a tissue engineering material.

[0015] 6. The use according to item 5, wherein the tissue engineering material is selected from subcutaneous fillers, artificial bones, artificial skin, oral absorbable biomembranes, bone implants, vascular scaffolds, interstitial scaffolds and collagen sponges.

[0016] 7. A recombinant collagen aqueous solution comprising the recombinant collagen according to any one of items 1 to 4.

[0017] 8. The recombinant collagen aqueous solution according to item 7, wherein the recombinant collagen has a polymer (eg, hexamer and / or heptamer and / or octamer) structure.

[0018] 9. The recombinant collagen aqueous solution according to item 7, which has been stored at room temperature for more than 3 months, preferably more than 6 months.

[0019] 10. An isolated nucleic acid encoding the recombinant collagen according to any one of items 1 to 4.

[0020] Meanwhile, the present invention provides a method for preparing the recombinant collagen as described above, wherein the recombinant collagen is prepared by efficiently expressing the amino acid sequence provided by SEQ ID No. 1 in an expression host by means of molecular cloning.

[0021] The molecular cloning method mentioned herein refers to a method for efficiently preparing recombinant collagen by utilizing the efficient protein synthesis characteristics of the host cell itself by carrying the gene sequence of the recombinant collagen as described above in the form of a plasmid, integrating it into the host chromosome, etc.; the host cell can be a prokaryotic cell or a eukaryotic cell, including: bacterial hosts such as Escherichia coli, Bacillus subtilis, Bacillus licheniformis, eukaryotic hosts such as Pichia pastoris, Saccharomyces cerevisiae, animal cells, plant cells, etc., preferably Escherichia coli and Pichia pastoris, more preferably Pichia pastoris.

[0022] When expressing proteins by means of molecular cloning, in order to facilitate the identification, purification, quantification and other uses of the proteins, the personnel in this field usually add corresponding tags when constructing the engineering bacteria. The preparation method described in the present invention includes adding tags and small molecule peptides, such as His tags, Flag tags, c-MYC, etc., for the convenience of purification, identification, quantification, etc. without affecting the structure of collagen.

[0023] On the other hand, the present invention also provides a method for preparing self-assembled recombinant collagen, which is achieved by the following technical scheme:

[0024] (1) Obtain the target gene and construct the corresponding expression vector;

[0025] (2) Transforming into an expression host, screening positive transformants, and obtaining a co-expression strain;

[0026] (3) culturing the expression strain obtained in (2) to express the target protein;

[0027] (4) separation and purification to obtain self-assembled recombinant collagen.

[0028] in:

[0029] In step (1), the target gene can be obtained by PCR amplification or whole gene synthesis.

[0030] In step (1), the corresponding plasmid refers to a plasmid capable of high-throughput constitutive expression or inducible expression of collagen genes in a selected expression system.

[0031] The expression in step (3) can be constitutive expression, induced expression, or a combination of the two, wherein the inducer for induced expression can be IPTG (isopropyl-β-D-thiogalactoside), lactose, galactose, methanol and the like.

[0032] In step (4), the separation and purification method of the recombinant collagen can adopt one or a combination of methods such as salting out, ultrafiltration, chromatography, isoelectric precipitation, membrane separation, etc., preferably a combination of chromatography and membrane separation, and more preferably a combination of ion exchange chromatography and membrane separation.

[0033] In addition, the present invention also provides the use of the recombinant collagen as described above in skin care products, health care products, tissue materials, medical devices, etc., preferably in tissue materials and medical devices.

[0034] The beneficial effects of the present invention are: providing an amino acid sequence of a recombinant collagen protein that can maintain stable self-assembly properties in an aqueous solution, a preparation method thereof and an application thereof; the recombinant collagen protein after self-assembly has excellent long-term storage stability, and effectively avoids the problem of protein degradation during storage. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 It is a graph showing the results of size exclusion chromatography of NHLC protein and reference substance.

[0036] Figure 2 The figure shows the results of SDS-PAGE electrophoresis detection of NHLC protein.

[0037] Figure 3 Transmission electron microscopy image showing the self-assembly properties of NHLC protein.

[0038] Figure 4 Transmission electron microscopy image showing that HLC protein does not have self-assembly properties. DETAILED DESCRIPTION

[0039] The present invention will be described in detail below through specific embodiments. It should be particularly pointed out that these descriptions are merely exemplary descriptions and do not constitute limitations on the scope of the present invention.

[0040] Example 1: Preparation of recombinant collagen in large intestine expression system

[0041] 1. Construction of pKK223-3-HLC expression strain

[0042] The amino acid sequence of SEQ ID No. 1 was optimized according to the codon preference of Escherichia coli, synthesized by whole gene synthesis, and directly connected to the pKK223-3 plasmid to obtain the pKK223-3-NHLC plasmid. The pKK223-3-NHLC was transferred into DE3 competent cells by heat shock, coated on a resistance plate, and a single colony on the plate was picked as the expression strain.

[0043] 2. Expression of Target Protein

[0044] (1) Pick a single colony that has been successfully constructed by colony PCR verification, inoculate it into a test tube containing 3.0 ml of LB medium (with ampicillin antibiotics), and culture it at 37°C for 10-12 hours to obtain seed liquid.

[0045] (2) Inoculate the cultured seed solution into 100 ml LB liquid medium at a 1% inoculum volume.

[0046] 37°C, 220 rpm, culture until OD 600 =2-3, add IPTG with a final concentration of 0.15 mM, cool to 28°C for induction, and collect the bacteria by centrifugation at 9000 rpm for 10 min after 10-12 h of induction.

[0047] (3) A 10% (g wet bacteria / mL PBS) bacterial suspension was prepared with a PBS buffer solution at pH 6.0, and the suspension was homogenized at 800 bar for 3 min. The suspension was centrifuged at 9000 rpm for 10 min, and the supernatant was collected as the crude protein solution.

[0048] 3. Isolation and purification of target protein

[0049] (1) Add NaCl with a final concentration of 60% to the crude protein supernatant collected by centrifugation, stir to dissolve, let stand at room temperature for 2 hours, centrifuge at 9000 rpm for 10 minutes, and collect the supernatant. The collected supernatant is concentrated and desalted by a 30KD ultrafiltration membrane.

[0050] (2) The pH of the concentrated and desalted protein supernatant was adjusted to 6.5 with phosphoric acid, loaded onto a cation exchange resin, and the target protein was obtained after elution with 0.5 M NaCl;

[0051] (3) The collected target protein solution was desalted by a 30KD ultrafiltration membrane, then placed in a -20°C refrigerator for pre-freezing for 4 hours, and then transferred to a vacuum freeze dryer for freeze-drying. After 48 hours, the freeze-dried protein was collected, which was the NHLC protein.

[0052] Example 2: Preparation of control protein in large intestine expression system

[0053] The amino acid sequence obtained by removing the linker sequence from the amino acid sequence shown in SEQ ID No.1 (i.e., GAPGAPGLQGAPGSQ repeated 52 times) was optimized according to the codon preference of Escherichia coli, synthesized by whole gene synthesis, and the protein was prepared by referring to the method described in Example 1. The obtained protein was named HLC protein.

[0054] Example 3 Protein molecular weight and structure confirmation

[0055] 1. NHLC protein self-assembly performance test

[0056] Take 5 mg of the purified lyophilized protein in Example 1, add it to 10 mL of ultrapure water, and dissolve it completely. Then, detect it through a superdex 200increase molecular exclusion chromatography column, the mobile phase is ultrapure water, the flow rate is 0.75 mL / min, and the sample volume is 500 μL. At the same time, control tests of known proteins of different molecular weights are carried out under the same conditions.

[0057] like Figure 1 As shown, the NHLC protein peaks at a retention time of 10 min ( Figure 1 The corresponding molecular weight of the known protein with the same peak time is about 670KD ( Figure 1 Therefore, the molecular weight of NHLC protein is about 670KD.

[0058] Figure 2 As shown, the molecular weight of the NHLC protein monomer is about 90 kD.

[0059] Combination Figure 1 and Figure 2 The test results show that the NHLC protein self-assembles and exists in the form of a multimer (heptamer).

[0060] 2. Transmission electron microscopy observation of the self-assembly characteristics of NHLC protein

[0061] The proteins prepared in Examples 1 and 2 were prepared into aqueous solutions with a concentration of 1.0 g / L, and the morphology of the proteins in the solutions was observed by transmission electron microscopy. Figure 3 and Figure 4 As shown. Figure 3 and Figure 4 It can be seen that the NHLC protein prepared in Example 1 can self-assemble in aqueous solution, while the HLC protein prepared in Example 2 does not have this property.

[0062] Example 4 Accelerated protein solution stability test

[0063] The recombinant collagen prepared in Examples 1 and 2 was prepared into a protein solution with a protein concentration of 1 mg / mL using ddH2O. After filtering with a 0.22 μm sterile filter in an ultra-clean workbench, the solution was dispensed into sterile centrifuge tubes and sealed. The tubes were placed at 40°C±2°C and a relative humidity of 75%±5%. Samples were taken at 1 month, 2 months, 3 months, and 6 months, with 3 tubes taken each time, and the purity of the protein solution (protein purity was determined by high performance liquid chromatography) was detected. The results are shown in Table 1.

[0064] Table 1 Results of accelerated protein solution stability test

[0065]

[0066]

[0067] From the test results in Table 1, it can be seen that the purity change of NHLC protein within 6 months is less than 1%, and it is stable under the conditions of long-term storage stability accelerated test, and basically no degradation occurs; while the purity of HLC protein as a control is only 78.35% after storage for 6 months, and degradation is obvious.

Claims

1. A recombinant collagen, the amino acid sequence of which is shown in SEQ ID No.1; SEQ ID No.1: MDPVVLQRRDWENPGVTQLNRLAAHPPFASDPM(G A P GAP G S Q G A P G LQ) n GAMGSS, where n = 52.

2. The recombinant collagen according to claim 1, further comprising a tag for facilitating its purification.

3. The recombinant collagen according to claim 2, wherein The tag is a His tag, a Flag tag or a c-Myc tag. A recombinant collagen aqueous solution comprising the recombinant collagen according to any one of claims 1 to 3.

5. The recombinant collagen aqueous solution according to claim 4, wherein The recombinant collagen has a polymer structure.

6. The recombinant collagen aqueous solution according to claim 4, which has been stored at room temperature for more than 3 months.

7. The recombinant collagen aqueous solution according to claim 4, which has been stored at room temperature for more than 6 months.

8. An isolated nucleic acid encoding the recombinant collagen according to any one of claims 1 to 3.

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