Recombinant collagen as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to recombinant collagen as well as a preparation method and application thereof. The amino acid sequence of the recombinant collagen is as shown in SEQ ID NO: 1. The invention has the beneficial effects that the expression system formed based on the optimized recombinant collagen sequence and the expression vector provided by the invention can stably express the recombinant collagen with a stable triple helix structure, has high expression quantity, is suitable for industrial mass production of the recombinant collagen, and has wide application prospects. One-time fermentation can be completed within 20-30 hours, and the production cost is low. The recombinant collagen does not generate immunological rejection when being applied to a human body, and can be widely applied to the medical beauty industry.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and in particular relates to a recombinant collagen protein and a preparation method and application thereof. Background Art

[0002] Collagen is a type of trimeric macromolecule formed by three polypeptide chains called α chains entangled with each other. Each chain contains a typical (GXY)n amino acid repeating sequence, where G represents glycine, the unknown X is usually represented by proline (Pro), and Y is usually hydroxyproline (Hyp). The three α chains (which can be the same or different, depending on the characteristic structure of collagen) are intertwined like cables to form a right-handed triple helix structure, which has become the qualitative characteristic structure of all collagen (Sylvie Ricard-Blum, Florence Ruggiero. The collagen superfamily: from the extracellular matrix to the cell membrane [J] Pathologie Biologie 2005, 53: 430-442.; Xia Xianming, Liu Ning. Recent research on collagen [J]. Journal of Basic and Clinical General Surgery, 1995, 2 (4): 237-239). It is known that hydroxyproline in the triple helix structure of collagen can effectively ensure the stability of the triple helix structure, but the mechanism of its stability has not been clearly studied (Gelse K, Poschl E, Aigner'T, Collagen-structure, funetion, and biosynthesis [J]. Advanced Drug Delivery Reviews, 2003, 55 (20): 1531-1546.).

[0003] The collagen family is the most abundant protein in the extracellular matrix and includes many family members. According to the latest reports, at least 29 different types of collagen have been discovered. These proteins are assembled from 43 different α-peptide chains (Chung E, Rhodes K. Isolation of three collagenous components of probable basement membrane origin from several tissues[J], Biochem, Biophys, 1976, 71: 1167~1174.). Although the members of the collagen family present in the cell matrix vary greatly in size, function and tissue distribution, they all contain at least one triple helical domain (COL) and most of them can form aggregate structures (Furuto DK, Miller EJ, Isolation of a unique collagenous fraction from limited pepsin digests of human placental tissue: Characterization of one of the constituent polypeptide chains. [J]. Biol. Chem, 1980. 255: 290-295.; Furuto DK, Miller EJ. Characterization of a unique collagenous fraction from limited pepsin digests of human placental tissue: molecular organization native aggregate [J]. Biochemistry, the 1981. 20: 1635~1640.).

[0004] According to the different collagen structures and supramolecular organizations, collagen can be roughly divided into 8 types: fibrillar collagen, microfibrillar collagen, fibrillar-like collagen, reticular collagen, anchored collagen, transmembrane collagen, matrix membrane collagen, and other special functional collagens. Among them, 90% are fibrillar collagens, which are widely distributed in tissues. Collagen plays an extremely important role in maintaining the integrity of the tissue and organ structure of organisms. Among them, it plays an important role in connective tissue, especially bone and cartilage tissue (Adaehi T, Tomita M, Shimizu K, et al. Generation of hybrid transgenic silkworms that express Bombyxmori Prolyl-hydroxylase a-subunits and human collagens in posterior silk glands: Production of cocoons that contained collagens with hydroxylated praline residues [J]. Journal of Biotechnology, 2006126 (2): 205~219.). The stable triple helix structure of collagen gives it good mechanical stability and thermal stability, and it can be used in materials such as food packaging films. Its antioxidant properties have a good preservation effect on food. In addition, collagen is easy to digest and absorb, contains rich and diverse amino acids, is nutritious, can enhance human immunity, and delay aging (Patrizia Sabatelli, Francesca Gualand, Sudheer Kumar Gara, et al. Expression of collagen VI a5 and a6 chains in human muscle and in Duchenne muscular dystrophy-related musele fibrosis [J]. Matrix Biology. 2012, 31: 187-196.).Collagen is called "soft gold for the skin". It contains a large number of polar groups in its molecules and is a good moisturizing factor. It also contains a variety of amino acids, among which tyrosine can prevent the formation of melanin in the skin (Engel J, Furthmayr H, Odermatt E, et al. Structure and macromolecular organization of type Vl collagen [J]. Ann N Y Acad Sci, 1985, 460: 25-37.). These characteristics make it have the functions of natural moisturizing, whitening, and skin repair, and it has great application in beauty and skin care cosmetics. In addition, collagen also has good applications in health care functions such as weight loss, blood pressure reduction, blood lipid reduction, and calcium supplementation. In addition, collagen has good biocompatibility, hemostasis, and low immunogenicity in addition to its own good stability. Moreover, there is no safety problem in the process of human digestion and absorption, and it can be used as a medical material in biomedicine (Camacho Vamegas O, Bertini E, Zhang RZ, et al. Ulrichseleroatonic muscular dystrophy is caused by recessive mutations in collagentype Vl[J]. Proc Natl Acad Sci USA, 2001: 98: 7516~7521.).

[0005] Recombinant collagen is produced by using genetic engineering technology to reverse transcribe a segment of mRNA of collagen to obtain its cDNA sequence, select a suitable expression vector, and after double enzyme digestion, transfer it to Escherichia coli or other expression systems to induce expression. The obtained protein is separated and purified to obtain the expressed recombinant collagen. Compared with collagen extracted by traditional methods, human recombinant collagen has the advantages of high activity, no virus risks, low immune rejection reaction, strong processability and can be applied to medicine, food and other fields. However, the currently known methods for preparing recombinant collagen have the problems of low expression level, inability to stably express the triple helix structure, unstable triple helix structure, long fermentation time and high production cost. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present invention provides a recombinant collagen that can stably express a triple helix structure with a stable triple helix structure, has a high expression level, and is suitable for industrial mass production of recombinant collagen, as well as a preparation method and application thereof.

[0007] In a first aspect, the present invention provides a recombinant collagen, the amino acid sequence of which is shown in SEQ ID NO:1.

[0008] In a second aspect, the present invention provides a recombinant collagen gene encoding the aforementioned recombinant collagen.

[0009] In some embodiments, the nucleotide sequence of the recombinant collagen gene is shown as SEQ ID NO:2.

[0010] In a third aspect, the present invention provides a recombinant collagen expression vector comprising the aforementioned recombinant collagen gene.

[0011] In a fourth aspect, the present invention provides a recombinant collagen engineering bacterium, which comprises the aforementioned recombinant collagen gene or recombinant collagen expression vector.

[0012] In a fifth aspect, the present invention provides a method for preparing recombinant collagen, comprising: fermenting the aforementioned recombinant collagen engineering bacteria to induce the expression of recombinant collagen; and, after the fermentation is completed, recovering and purifying the expressed recombinant collagen.

[0013] In some embodiments, the fermentation medium comprises the following components: 10.6 g / L potassium dihydrogen phosphate, 4.00 g / L diammonium hydrogen phosphate, 1.7 g / L citric acid, 1.23 g / L magnesium sulfate heptahydrate, 25.0 g / L glucose, 4.4 mg / L thiamine hydrochloride, 5.0 mL / L complex trace elements (4 g / L copper sulfate pentahydrate, 0.16 g / L sodium iodide, 6 g / L manganese sulfate monohydrate, 1 g / L calcium sulfate dihydrate, 44 g / L ferrous sulfate heptahydrate, 5 g / L cobalt dichloride hexahydrate, 14 g / L zinc chloride, 0.4 g / L sodium molybdate dihydrate, 0.04 g / L boric acid, 1 mL / L sulfuric acid), 600 g / L glucose, and 60 g / L yeast extract.

[0014] In some embodiments, when the OD600 reaches 50-60 under the conditions of temperature of 36.5-37.5°C, pH of 6.5-7.5, dissolved oxygen of 20%-40%, and pressure of 0.02-0.5 bar, isopropyl thiogalactoside is added for induction, and the fermentation is continued at a temperature of 18-22°C and a pH of 6.5-7.5 until the 20th-30th hour.

[0015] In some embodiments, purification includes collecting bacterial cells from the fermentation product, followed by disruption, centrifugation to obtain the supernatant, affinity chromatography, ion exchange chromatography, and concentration.

[0016] In a sixth aspect, the present invention provides a composition, including but not limited to a cosmetic product, comprising the aforementioned recombinant collagen.

[0017] The beneficial effects of the present invention are:

[0018] The expression system formed based on the optimized recombinant collagen sequence and expression vector provided by the present invention can stably express recombinant collagen with a triple helix structure and a stable triple helix structure, with a high expression amount, suitable for industrial mass production of recombinant collagen, and a fermentation can be completed in 20 to 30 hours, with low production cost. The obtained recombinant collagen was experimented on animals, and the experimental results showed that the recombinant collagen failed to cause immune rejection reactions in animals. Therefore, it can be preliminarily inferred that the recombinant collagen will not produce immune rejection reactions in the human body. Therefore, the recombinant collagen will not produce immune rejection reactions when applied to the human body, and can be widely used in the medical beauty industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is the pColdIII vector map, pCold III vector sequence and pColdIII vector sequence multiple cloning site information diagram;

[0020] Figure 2 is the circular dichroism (CD) absorption spectrum;

[0021] Figure 3 This is the SDS-PAGE profile of collagen;

[0022] Figure 4 This is the HPLC purity determination spectrum of collagen;

[0023] Figure 5 This is a standard curve of BSA protein concentration. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention is clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] Example 1 Construction of engineering bacteria for recombinant collagen expression system

[0026] ① Select the optimized DNA sequence fragment of recombinant collagen, the nucleotide sequence of the gene is shown in the sequence shown in SEQ ID NO: 2, and the protein sequence encoded by the gene is shown in SEQ ID NO: 1. Use PCR to amplify the DNA sequence of the target gene. The PCR reaction system is: 10×PCR reaction buffer 5μL, dNTP 2μL, forward primer 0.5μL, reverse primer 0.5μL, DNA template 2μL, Taq enzyme 0.4μL, ddH2O 39.6μL; reaction conditions are; 94℃5min; 94℃30s, 55℃30s, 72℃30s (30 cycles); 72℃7min, and then use restriction endonuclease endonuclease and pCold III plasmid to construct the recombinant collagen expression vector

[0027] ② The constructed recombinant collagen expression vector pCold III (such as Figure 1 coli engineering strain, and then the recombinant collagen engineering bacteria containing collagen DNA fragments were obtained by PCR amplification method and blue-white screening method, which were used as strains for subsequent experiments.

[0028] Table 1 Sequence information

[0029] Sequence number (SEQ ID NO:) describe 1 Amino acid sequence of recombinant collagen 2 Nucleotide sequence of recombinant collagen

[0030] SEQ ID NO: 1: GSPGLPGPRGEQGPTGPTGPAGPRGLQGLQGLQGERG EQGPTGPAGPRGLQGERGEQGPTGLAGKAGEAGAKGETGPAGPQGPRGEQGPQGLPGKDGEAGAQGPAGPMGPAGERGEKGEPGTQGAKGDRGETGPVGPR GERGEAGPAGKDGERGFPGERGVEGQNGQDGLPGKDGKDGQNGKDGLPGKDGKDGQNGKDGLPGKDGKDGQDGKDGLPGKDGKDGLPGKDGKDGQPGKPGKY

[0031] SEQ ID NO: 2: GGCAGCCCGGGCCTGCCGGGCCCGCGCGGCGAACA GGGCCCGACCGGCCCGACCGGCCCGGCGGGCCCGCGCGGCCTGCAGGGCCTGCAGGGCCTGCAGGGCGAACGCGGCGAACAGGGCCCGACCGGCCCGGCGGGCCCGCGCGGCCTGCAGGGCGAACGCGGCGAACAGGGCCCGACCGGCCTGGCGGG CAAAGCGGGCGAAGCGGGCGCGAAAGGCGAAACCGGCCCGGCGGGCCCGCAGGGCCCGCGCGGCGAACAGGGCCCGCAGGGCCTGCCGGGCAAAGATGGCGAAGCGGGCGCAGGGCCCGGCGGGCCCGATGGGCCCGGCGGGCGAACGCGGCGA AAAAGGCGAACCGGGCACCCAGGGCCGAAAGGCGATCGCGGCGAAACCGGCCCGGTGGGCCCGCGCGGCGAACGCGGCGAAGCGGGCCCGGCGGGCAAAGATGGCGAACGCGGCTTTCCGGGCGAACGCGGCGTGGAAGGCCAGAACGGCCAGGA TGGCCTGCCGGGCAAAGATGGCAAAGATGGCCAGAACGGCAAAGATGGCCTGCCGGGCAAAGATGGCAAAGATGGCCAGAACGGCAAAGATGGCCTGCCGGGCAAAGATGGCAAAGATGGCCAGGATGGCAAAGATGGCCTGCCGGGCAAAGATGGC AAAGATGGCCTGCCGGGCAAAGATGGCAAAGATGGCCAGCCGGGCAAACCGGGCAAATAT

[0032] Example 2 Fermentation and culture of recombinant collagen engineering bacteria

[0033] Step 1: Use 2×YT medium to revive and expand the recombinant collagen engineering bacteria in a constant temperature shaker at 37°C and 220rpm;

[0034] Step 2: The recombinant collagen bacterial solution that has completed the expansion culture is inoculated into the fermentation tank at an inoculation ratio of 10%. The fermentation medium composition is (referred to as fermentation medium A): 10.6 g / L potassium dihydrogen phosphate, 4.00 g / L diammonium hydrogen phosphate, 1.7 g / L citric acid, 1.23 g / L magnesium sulfate heptahydrate, 25.0 g / L glucose, 4.4 mg / L thiamine hydrochloride, 5.0 mL / L complex trace elements (4 g / L copper sulfate pentahydrate, 0.16 g / L sodium iodide, 6 g / L manganese sulfate monohydrate, 1 g / L calcium sulfate dihydrate, 4 g / L ferrous sulfate heptahydrate) 4g / L, cobalt dichloride hexahydrate 5g / L, zinc chloride 14g / L, sodium molybdate dihydrate 0.4g / L, boric acid 0.04g / L, sulfuric acid 1mL / L), glucose 600g / L, yeast extract 60g / L; fermentation culture was carried out at 37°C, pH=7.0, dissolved oxygen was 30%, and the tank pressure was maintained at 0.1bar, and when the OD600 in the fermenter reached 50-60, IPTG was added to the fermenter to induce it at a final concentration of 1mmol / L, and culture was continued at 20°C, pH=7.0 until the 22nd hour.

[0035] Step 3: Collect the fermentation liquid of the recombinant collagen engineering bacteria in the fermentation tank, centrifuge the fermentation liquid of the recombinant collagen engineering bacteria, and collect the bacteria.

[0036] Example 3 Recombinant collagen purification

[0037] Step 1: Use PBS-20mM imidazole solution to resuspend the recombinant collagen engineering bacteria cells, and use a high-pressure homogenizer to break the resuspended recombinant collagen engineering bacteria cells;

[0038] Step 2: centrifuge the recombinant collagen fragmentation solution and collect the supernatant; then clarify the supernatant;

[0039] Step 3: Use PBS-20 mm imidazole to balance the affinity chromatography column, and then use affinity chromatography and ion exchange chromatography to purify the collagen in the clear solution to obtain recombinant collagen solution 1. Then, the recombinant collagen 1 solution is ultrafiltered and concentrated to obtain a recombinant collagen 1 solution concentrate. Then, the recombinant collagen 1 solution concentrate is purified again using ion exchange chromatography to obtain a recombinant collagen 2 solution;

[0040] Step 4: The recombinant collagen 2 solution is concentrated and replaced with an ultrafiltration membrane package, and finally a high-concentration, injection-grade, triple-helix recombinant collagen solution with a concentration of 4 mg / mL is obtained.

[0041] 1. Collect the circular dichroism (CD) absorption spectra of the recombinant collagen solution in the far ultraviolet (190-260nm) and near ultraviolet (250-340nm), and analyze its secondary structure through software. The results show that the recombinant collagen prepared by the present invention has a positive peak at a wavelength near 221nm in the far ultraviolet region. The circular dichroism spectrum data is predicted and calculated by CDNN analysis software, and the secondary structure content is calculated as follows: Figure 2 As shown, there is an obvious negative peak near 191nm and an obvious positive peak at 221nm. The data show that the helix is ​​2.5%, the antiparallel β-fold is 5.3%, the parallel β-fold is 1.4%, the β-fold is 33.1%, and the random coil is 61.6%. In summary, the above results show that the recombinant collagen has a triple helical structure.

[0042] 2. Use SDS-PAGE and HPLC to analyze the purity of recombinant collagen. The SDS-PAGE spectrum is as follows: Figure 3 As shown, the HPLC purity determination spectrum is as follows Figure 4 The HPLC purity determination data are shown in Table 2.

[0043] Table 2 HPLC purity determination data

[0044]

[0045] From SDS-PAGE, we can see that the target band is clearly visible and the impurity protein band is faintly visible; HPLC results show that the purity of recombinant collagen can reach 99.7%.

[0046] 3. According to the same method as Example 2, a fermentation medium with a changed composition (referred to as fermentation medium B) was used to ferment and culture the recombinant collagen engineering bacteria, and the recombinant collagen was purified according to the same method as Example 3. The obtained recombinant collagen solution and the recombinant collagen solution obtained in Example 3 were diluted 4 times respectively, and the protein content of the recombinant collagen was determined using a BCA protein content determination kit to examine the effect of the fermentation medium composition on the expression level of the recombinant collagen. Fermentation medium B includes the following components: potassium dihydrogen phosphate 10.6 g / L, diammonium hydrogen phosphate 4.00 g / L, citric acid 1.7 g / L, magnesium sulfate heptahydrate 1.23 g / L, glucose 25.0 g / L, thiamine hydrochloride 4.4 mg / L, complex trace elements 5.0 mL / L (copper sulfate pentahydrate 4 g / L, sodium iodide 0.16 g / L, manganese sulfate monohydrate 6 g / L, calcium sulfate dihydrate 1 g / L, ferrous sulfate heptahydrate 44 g / L, cobalt dichloride hexahydrate 5 g / L, zinc chloride 14 g / L, sodium molybdate dihydrate 0.4 g / L, boric acid 0.04 g / L, sulfuric acid 1 mL / L), glucose 600 g / L, BSA protein concentration standard curve is as shown in Figure 5 The measurement results are shown in Table 3.

[0047] Table 3 Recombinant collagen content

[0048] Fermentation medium <![CDATA[OD 562 ]]> <![CDATA[OD 562 ]]> <![CDATA[OD 562 ]]> Protein concentration (mg / mL) Fermentation medium A 0.558 0.560 0.561 2.4 Fermentation medium B 0.322 0.393 0.389 1.47

[0049] The results of the BCA method showed that the fermentation medium A in the embodiment of the present invention can harvest 2.4 g / L of recombinant collagen, which is 63.26% higher than the 1.47 g / L of recombinant collagen harvested by the fermentation medium B. This indicates that the fermentation medium with optimized composition is used in the present invention, so that the expression of recombinant collagen of the strain is further improved.

[0050] The above results confirm that the method of the present invention can produce high-purity collagen, and the expression level of collagen is high, so that collagen can be produced in large quantities.

[0051] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. A recombinant collagen, characterized in that: Its amino acid sequence is shown in SEQ ID NO:

1.

2. A recombinant collagen gene, characterized in that: It encodes the recombinant collagen according to claim 1.

3. The recombinant collagen gene according to claim 2, characterized in that: Its nucleotide sequence is shown in SEQ ID NO:

2.

4. A recombinant collagen expression vector, characterized in that: It comprises the recombinant collagen gene according to claim 2 or 3.

5. A recombinant collagen engineering bacterium, characterized in that: It comprises the recombinant collagen gene according to claim 2 or 3 or the recombinant collagen expression vector according to claim 4.

6. A method for preparing recombinant collagen, characterized in that: The preparation method comprises: Fermenting the recombinant collagen engineering bacteria according to claim 5 to induce the expression of recombinant collagen; and After the fermentation is completed, the expressed recombinant collagen is recovered and purified.

7. The preparation method according to claim 6, characterized in that: The fermentation medium comprises the following components: 10.6 g / L potassium dihydrogen phosphate, 4.00 g / L diammonium hydrogen phosphate, 1.7 g / L citric acid, 1.23 g / L magnesium sulfate heptahydrate, 25.0 g / L glucose, 4.4 mg / L thiamine hydrochloride, 5.0 mL / L complex trace elements, 600 g / L glucose, and 60 g / L yeast extract; The composite trace elements include the following components: 4 g / L copper sulfate pentahydrate, 0.16 g / L sodium iodide, 6 g / L manganese sulfate monohydrate, 1 g / L calcium sulfate dihydrate, 44 g / L ferrous sulfate heptahydrate, 5 g / L cobalt dichloride hexahydrate, 14 g / L zinc chloride, 0.4 g / L sodium molybdate dihydrate, 0.04 g / L boric acid, and 1 mL / L sulfuric acid.

8. The preparation method according to claim 6, characterized in that: When the OD600 reaches 50-60 under the conditions of temperature of 36.5-37.5°C, pH of 6.5-7.5, dissolved oxygen of 20%-40% and pressure of 0.02-0.5 bar, isopropyl thiogalactoside is added for induction, and the fermentation is continued at a temperature of 18-22°C and a pH of 6.5-7.5 until the 20th-30th hour.

9. The preparation method according to claim 6, characterized in that: The purification includes collecting bacterial cells from the fermentation product, and then sequentially performing crushing, centrifugation to obtain supernatant, affinity chromatography, ion exchange chromatography and concentration.

10. A composition, characterized in that The composition comprises the recombinant collagen according to claim 1.

Images