Recombinant humanized collagen as well as expression system and application thereof

Abstract

The invention provides recombinant humanized collagen as well as an expression system and application thereof, and belongs to the technical field of active raw material development. The problems that in the prior art, an eukaryotic expression system is low in production efficiency, high in cost and insufficient in biological activity are solved, efficient secretion and soluble expression of the IV type collagen recombinant protein are achieved through the prokaryotic host cell, the production efficiency is remarkably improved, the cost is remarkably reduced, the prepared recombinant protein can effectively inhibit tyrosinase activity, and the recombinant protein can be used for preparing the recombinant protein. The composition provided by the invention can promote HSF cell growth, proliferation, differentiation and migration, improves the content of type I collagen, has obvious whitening, repairing and anti-wrinkle effects, and provides a new efficient active component for food, medicine and cosmetic industries.

Description

Technical Field

[0001] The present invention relates to the technical field of active raw material development, and in particular to recombinant humanized collagen and its expression system and application. Background Art

[0002] Collagen is an early discovered and abundant extracellular matrix protein, widely distributed in connective tissues such as skin, bones, and muscles of humans and animals, and is essential for the physiological functions and damage repair of cells, tissues, and organs. 29 types have been identified, and their molecular structure consists of a repeated Gly-XY sequence (X is mostly proline, and Y is mostly hydroxyproline), forming a stable triple helix. Due to its unique structure, collagen has high affinity and excellent biological activity, compatibility, and degradability, and is widely used in biomedicine, cosmetics, food, and other fields.

[0003] Collagen is divided into fibroblastic collagen and non-fibroblastic collagen. The latter includes various types such as FACIT family and type IV collagen, which play a key role in organisms. In particular, type IV collagen, as the main component of the basement membrane, provides support for tissues, participates in cell signal transduction and regulation, and is essential for maintaining physiological functions. The basement membrane is a dense matrix surrounding cells. Type IV collagen constructs a complex network of living organisms with its various heterotrimeric forms and structural domains. Therefore, its expression and application are increasingly attracting attention.

[0004] Chinese patent CN118852407A discloses humanized type IV collagen, and also discloses the expression vector, preparation method and application of the collagen. Preparation method of collagen: S1 preferably selects the natural humanized type IV collagen nucleotide sequence, optimizes and designs the Pichia codon, and obtains the aat tandem nucleotide sequence; S2 double-digests the vector and the aat tandem nucleotide sequence, connects and transforms the competent Escherichia coli to obtain a plasmid; S3 transforms the plasmid into Pichia and screens to obtain an engineered bacterium; S4 ferments and amplifies the engineered bacterium to obtain a supernatant; S5 purifies the supernatant to obtain humanized type IV collagen. The humanized type IV collagen and preparation method provided by the invention solve the problems of low startup efficiency of the existing constitutive expression system and easy residue caused by the introduction of harmful reagents during the expression process of the inducible expression system. The preparation of the above-mentioned humanized type IV collagen uses the expression system of Pichia, which is complicated to operate and has high cost. When the eukaryotic expression system expresses foreign proteins, the foreign proteins need to go through the process of intracellular sorting. If the folding effect of the foreign proteins after translation is not ideal, they cannot pass the intracellular sorting normally and will be degraded, resulting in the inability to express. Collagen is mainly expressed in microorganisms in the form of single chains, and single chains cannot be folded into a conventional three-dimensional structure, and are easily degraded in Pichia pastoris. However, the prokaryotic expression system has no special requirements for the folding form of the translated sequence. If it cannot be folded normally or is not good for the host, inclusion bodies will be formed in the cell and purified in the form of inclusion bodies. Summary of the invention

[0005] The purpose of the present invention is to provide recombinant humanized collagen and its expression system and application, which have better whitening, repairing and anti-wrinkle effects.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a recombinant humanized type IV collagen. The recombinant humanized type IV collagen is a truncated form of humanized type IV collagen, and the amino acid sequence is shown in SEQ ID NO.1.

[0008] In the present invention, the "truncated body" refers to a protein whose amino acid sequence is missing one or more amino acid residues compared with the natural full-length sequence by deleting or modifying a specific region of the full-length human type IV collagen gene through molecular biological means. Specifically, the amino acid sequence shown in SEQ ID NO.1 is a protein obtained by carefully designing a truncation of the full-length human type IV collagen sequence to optimize its biological properties and application potential. The truncation obtained by the present invention not only inherits the specific biological functions of the original collagen, but also exhibits excellent biological activities, such as whitening and repairing effects, and thus has broad application prospects in the fields of tissue engineering, regenerative medicine, and cosmetic biomaterials.

[0009] In the present invention, the "recombinant humanized type IV collagen" can be understood as a derivative of human type IV collagen, and its amino acid sequence is shown in SEQ ID NO.1. This recombinant protein is an independent entity, different from "a sequence present in the original type IV collagen". It is obtained by precise editing and transformation of the full-length human type IV collagen gene, through intentional truncation, that is, selectively deleting or recombining certain amino acid fragments, thereby generating a new protein entity. Therefore, the "recombinant type IV collagen protein is a truncation of human type IV collagen" here refers to a new recombinant protein that has been artificially designed and constructed and has specific biological functions. It is independent of the original protein sequence, designed to achieve specific application purposes, and can be used independently as a complete protein product.

[0010] The present invention also provides a gene encoding the above-mentioned recombinant type IV humanized collagen, and the nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0011] The present invention also provides a recombinant expression vector, which comprises an initial vector and the gene encoding the recombinant type IV humanized collagen.

[0012] In the present invention, the "recombinant expression vector" refers to a specially designed and constructed DNA molecule, which contains a gene sequence encoding recombinant type IV humanized collagen, and has the basic properties of a cloning vector, that is, it can replicate and stably exist in host cells, and has the ability to express foreign genes, ensuring that foreign genes are effectively transcribed and translated in host cells. In the present invention, the recombinant expression vector accurately inserts the gene sequence encoding recombinant type IV humanized collagen between the regulatory sequences therein, so that the host cell can produce the desired recombinant type IV humanized collagen according to a predetermined regulatory mechanism.

[0013] In the present invention, the "initial vector" refers to the vector used as the basis in the recombination process, which provides the basic framework and replication ability required for inserting foreign genes, contains one or more restriction enzyme cutting sites, allows foreign gene fragments to be inserted through specific enzyme cutting reactions, and provides the necessary physical and functional platform for the insertion, replication and expression of foreign genes. By cloning the gene sequence encoding recombinant type IV humanized collagen into the initial vector, the present invention constructs a recombinant expression vector, thereby achieving efficient expression of the target protein in the host cell.

[0014] Preferably, the initial vector is a pET series vector, a pGEX series vector or a pMAL series vector.

[0015] Preferably, the initial vector is a pET-30a vector.

[0016] The present invention also provides a recombinant strain expressing the recombinant type IV humanized collagen, wherein the recombinant expression vector is transformed into the recombinant strain.

[0017] In the present invention, the "recombinant strain" refers to an engineered microorganism in which exogenous genes are introduced into host cells by genetic engineering technology, and these exogenous genes can stably exist, replicate and express in the host cells; the exogenous gene in the present invention is the gene encoding the above-mentioned recombinant type IV humanized collagen.

[0018] Preferably, the recombinant strain is induced to express by IPTG.

[0019] Preferably, the concentration of IPTG is 0.05-1 mM.

[0020] Preferably, the temperature for inducing expression is 15-20°C;

[0021] The time for inducing expression is 12 to 72 hours.

[0022] The present invention also provides the use of the above-mentioned recombinant humanized type IV collagen, recombinant expression vector or recombinant strain in the preparation of whitening products, repair products or anti-wrinkle products.

[0023] In the present invention, the "whitening products" refer to a series of products used to improve skin color, reduce pigmentation, and brighten skin color; the "repair products" refer to products designed to help the skin return to a healthy state, repair damaged skin barriers, and reduce skin irritation or inflammation, including but not limited to skin damage caused by environmental factors, ultraviolet radiation, chemicals, natural aging or diseases; the "anti-wrinkle products" refer to products designed to reduce skin wrinkles and improve skin elasticity and firmness; the products include but are not limited to related products in the food field, pharmaceutical products, cosmetics or other transdermal care products.

[0024] Beneficial effects of the present invention:

[0025] The recombinant humanized collagen provided by the present invention - recombinant type IV humanized collagen has significant biological activity. Compared with other truncated sequences, the recombinant type IV humanized collagen of the present invention can effectively inhibit the activity of tyrosinase, and this characteristic makes it have obvious advantages in whitening effect. In addition, it can also significantly promote the growth of HSF cells, is conducive to cell proliferation, differentiation and migration, and can significantly increase the content of type I collagen in HSF cells, which may help stimulate collagen regeneration and is of great significance for improving skin elasticity and structure. These biological activities show that recombinant type IV humanized collagen has potential application value in promoting skin health and repair.

[0026] The expression system of the present invention realizes the efficient secretion and soluble expression of recombinant type IV humanized collagen in prokaryotic host cells. This technological breakthrough simplifies the production process and improves production efficiency. The advantage of this expression system is that it can produce recombinant type IV humanized collagen with biological activity on a large scale and at low cost, while avoiding the complex post-translational modification problems that may occur in eukaryotic expression systems. This provides a more efficient and economical solution for the production of recombinant type IV humanized collagen, helps to reduce costs and improve product quality, and thus has broad application prospects in industrial production.

[0027] The recombinant type IV humanized collagen provided by the present invention has broad application prospects in the field of cosmetics, especially in whitening, repairing and anti-wrinkle products. Its ability to inhibit tyrosinase activity makes it an effective whitening ingredient, which helps to reduce pigmentation and brighten skin color. At the same time, due to its ability to promote HSF cell growth and proliferation, type IV collagen recombinant protein also helps skin anti-wrinkle, repair and regeneration, improves skin barrier function, and reduces skin damage. In addition, its potential in increasing the content of type I collagen makes it also valuable in anti-aging and skin anti-wrinkle and firming products, which helps to improve skin elasticity and reduce wrinkles. Therefore, as a new active ingredient, recombinant type IV humanized collagen has brought innovative and efficient possibilities to the cosmetics industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 PCR results (A) and plasmid map (B) for constructing the recombinant expression vector;

[0029] Figure 2 This is the SDS-PAGE result of gradient purification of recombinant humanized type IV collagen;

[0030] Figure 3 Ultrafiltration SDS-PAGE (A) and WB results (B) of recombinant type IV humanized collagen;

[0031] Figure 4This is the result of inhibition of cell tyrosinase activity;

[0032] Figure 5 This is the result diagram of HSF cell proliferation;

[0033] Figure 6 The cell scratch results are shown in Figure A, where Figure A is a schematic diagram of the scratch effect; Figure B is a graph showing the sample scratch migration rate results;

[0034] Figure 7 is the extracellular COL I content of fibroblasts. DETAILED DESCRIPTION

[0035] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0036] Example

[0037] 1. Amino acid sequence of recombinant humanized type IV collagen

[0038] In this example, a human type IV collagen fragment was selected and truncated, and its encoding DNA was obtained by chemical synthesis (commissioned by Suzhou Jinweizhi Biotechnology Co., Ltd.), and named IV-up. IV-up has a large molecular weight and multiple structural domains, and is easy to exert more functions. The amino acid sequence is as follows:

[0039] GTPGPGLPGLQGPVGPPGPPGPPGPPGEKGQMGLSFQGPKGDKGDQGVSGPPGVPGQAQVQEKGDFATKGEKGQKGEPGFQGMPGVGEKGEPGKPGPRGKPGKDGDKGEKGSPGFPGEPGYPGLIGRQGPQGEKGEAGPPGPPGIVIGTGPLGEKGERGYPGTPGPRGEPGPKGFPGLPGQ PGPPGLPVPGQAGAPGFPGERGEKGDRGFPGTSLPGPSGRDGLPGPPGSPGPPGQPGYTNGIVECQPGPPGDQGPPGIPGQPGFIGEIGEKGQKGESCLICDIDGYRGPPGPQGPPGEIGFPGQPGAKGDRGLPGRDGVAGVPGPQGTPGLIGQPGAKGEPGEFYFDLRLKGDKGDPHHHHHH*, as in SEQ Shown as ID NO.1;

[0040] The coding gene sequence is:

[0041]

[0042] 2 Construction of heterologous expression strain BL21(DE3)-pET30a-IV-up

[0043] Synthetic gene sequences:

[0044]

[0045] The pET30a(+) vector was double-digested with restriction endonucleases XhoI and NdeI, and the linearized vector pET30a(+) was homologously recombined with the target gene IV-up using 2×ClonExpress Mix (NoviZan), and the recombinant vector pET30a-IV-up was successfully constructed. To screen positive clones, the homologous recombination products were heat-shock transformed into Escherichia coli DH5α, spread on LB resistance plates containing 100μg / mL kanamycin, and cultured at 37°C overnight. It was verified by colony PCR and sequencing identification (completed by Jinweizhi Biotechnology Co., Ltd.), confirming that the sequencing results were consistent with the designed sequence (1237bp), indicating that the recombinant expression vector was successfully constructed. The PCR results and plasmid map are shown in Figure 2. Figure 1 shown.

[0046] The recombinant plasmid pET30a-IV-up was extracted from DH5α-pET30a-IV-up and heat-shock transformed into the prokaryotic expression host strain E. coli BL21 (DE3) again to successfully obtain the heterologous expression strain BL21 (DE3)-pET30a-IV-up.

[0047] 3 Protein expression and purification of recombinant bacteria BL21(DE3)-pET30a-IV-up

[0048] Pick out the BL21(DE3)-pET30a-IV-up monoclonal strain and inoculate it into 10 mL of LB medium containing 100 μg / mL Kana, and culture it at 37°C, 220 rpm overnight. The next day, take 1% of the overnight culture and transfer it to 1 L of LB medium containing 100 μg / mL Kana, and expand it at 37°C, 220 rpm until the OD 600 =0.6-0.8, and IPTG (isopropylthiogalactoside) was used to induce expression at 18°C ​​for 16h at a final concentration of 0.5mM. The cells were collected by centrifugation at 4°C and 12000rpm for 30min, resuspended in 100mL of pre-cooled Buffer A on ice, and ultrasonically disrupted (break for 5s, stop for 5s, amplitude 50%, resonance 1-3min), centrifuged at 4°C and 12000rpm for 30min, and the supernatant was filtered using a 0.45μm filter membrane and purified by nickel column.

[0049] Affinity chromatography binding buffer Buffer A: 6.017g / L Na2HPO4·12H2O, 0.499g / LNaH2PO4·2H2O, 29.2g / L NaCl, 0.34g / L imidazole, pH=7.5; washing buffer Buffer B: 6.017g / L Na2HPO4·12H2O, 0.499g / L NaH2PO4·2H2O, 29.2g / L NaCl, 34.04g / L imidazole, pH=7.5, AKTA (model: AKTApure 150L (Cytiva)) purification The liquids used were all filtered with a 0.45μm filter membrane.

[0050] Using AKTA protein purifier, the system was cleaned with 20% ethanol and 10 column volumes of pure water in turn, Buffer A was used to balance the nickel column, the sample was loaded with a sample pump, 5-10 column volumes of Buffer A were used to wash away unbound proteins, 0-100% Buffer B (500mM imidazole) gradient elution was set, and the collector was fixed at 2mL / tube, and a total of 25 tubes were collected. All samples were tested by SDS-PAGE, and the results are shown in the following table. Figure 2 shown.

[0051] During protein electrophoresis, the binding of Pro to SDS is weaker than that of other general amino acids, resulting in a lower electrophoresis rate for proteins with a higher Pro content. Therefore, the apparent molecular weight of collagen is 20% to 30% larger than the theoretical molecular weight. The IV-up sequence contains 78 Pros, with a content of 22%. The theoretical molecular weight of IV-up protein is 33.7KDa, and the apparent molecular weight is close to 40.0KDa. The size of the SDS-PAGE gel image is consistent with the apparent molecular weight, indicating that the protein is successfully expressed in a soluble form. The target protein content and purity in tubes 5-11 of the elution collection solution are the highest, and the impurity protein gradually decreases.

[0052] Mix the elution collection solution from tubes 5-11, add them to the 3K ultrafiltration tube, centrifuge at 4°C, 4000rpm horizontal rotor for 30 minutes each time to concentrate the target protein, and collect the filtrate. When the retained target protein is about 2mL, add three times the volume of the elution collection solution of 1×PBS washing solvent, collect the final protein sample for SDS-PAGE detection, and the results are as follows. Figure 3 (A) As shown. The target protein contains a His tag at the carboxyl terminus. Anti-His Tag antibody was used for Western Blot detection. The results are shown in Figure 3 (B) shown.

[0053] 4. Cellular efficacy testing of IV-up recombinant protein

[0054] The IV-up protein samples were fermented in shake flasks and prepared. The protein concentration was determined by the Lowry method. The BSA standard curve was drawn and the sample concentration was determined to be 402.89 ug / mL based on the A750 value of the protein sample.

[0055] The same method was used to select the original type IV collagen sequence with a molecular mass of approximately 20.0 KDa downstream of the IV-up sequence to construct a recombinant expression strain, and the same method was used to shake flask fermentation and prepare (IV-part), and its concentration was measured to be 289.25 μg / mL as a sequence efficacy control.

[0056] 4.1 Inhibition of cellular tyrosinase activity

[0057] Each protein sample was diluted and added to B16 cells. Kojic acid (KA) was used as a standard to detect the inhibition rate of the samples on tyrosinase. The results were as follows: Figure 4 As shown. The IV-up sample showed an inhibitory effect on tyrosinase at a concentration of less than 5% (20.14 μg / mL), while the control sample IV-part showed an inhibitory effect on tyrosinase at a concentration of 20% (57.85 μg / mL). Therefore, compared with other type IV collagen sequence segments (IV-part), the IV-up selected by the present invention has a more significant inhibitory effect on tyrosinase, thereby exerting a potential skin whitening effect.

[0058] 4.2 Cell scratching

[0059] The scratch test was performed on HSF cells. Diluted IV-up and control IV-part protein samples were added to the cells under 1% FBS treatment. The cell safety test results are shown in Figure 5 As shown in the figure, the growth-promoting effect of IV-up protein on HSF at a concentration of 40% (161.16 μg / mL) was close to 200%, showing a concentration-dependency overall, and had a significant ability to promote HSF cell proliferation.

[0060] The control sample IV-part has no obvious proliferation effect on HSF cells, indicating that the IV-up selected in the present invention has a more significant effect on HSF cell proliferation than IV-part, is beneficial to cell proliferation, differentiation and migration, and thus is beneficial to skin anti-aging and repair effects.

[0061] Using DMEM containing 1% FBS as the blank control group, cells migrated slightly 24 hours after scratching; using DMEM containing 10% FBS as the positive control group, more cells migrated 24 hours after scratching. IV-up can reduce the scratch area at a concentration of 5% (20.14μg / mL), which increases the number of migrating cells, while the control sample IV-part only plays a repairing role at a concentration of 20% (57.85μg / mL). Therefore, compared with other amino acid sequence segments of type IV collagen, IV-up protein can play a greater role in skin repair. The results are shown in Figure 2. Figure 6 shown.

[0062] 4.3 Cell type I collagen ELISA detection

[0063] HSF cells were cultured for cell adhesion, and the cell supernatant was collected after 48 hours for ELISA detection. Figure 7 As shown, IV-up protein can significantly increase the secretion of type I collagen at a concentration of 20%, indicating that the IV-up selected in the present invention has the ability to stimulate HSF cells to synthesize and secrete type I collagen, which is beneficial to the anti-wrinkle effect on the skin.

[0064] 5 Conclusion

[0065] In summary, compared with other amino acid sequence segments of type IV collagen, the recombinant collagen IV-up in the present invention can significantly promote the growth of HSF cells, is beneficial to cell proliferation, differentiation and migration, and is beneficial to the anti-aging and repair effects of the skin; and IV-up can inhibit the activity of tyrosinase and has potential skin whitening effects. At the same time, IV-up can significantly increase the type I collagen content in HSF cells, which may have the purpose of stimulating collagen regeneration and play an anti-wrinkle effect on the skin. Therefore, the truncated IV-up recombinant collagen prepared by the present invention has obvious biological activity, is beneficial to promoting skin whitening, repair and anti-wrinkle effects, and has broad application prospects in the future cosmetics field.

[0066] The above is only 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 principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A recombinant type IV humanized collagen, characterized in that: The recombinant humanized type IV collagen is a truncated form of human type IV collagen, and the amino acid sequence is shown in SEQ ID NO.

1.

2. The gene encoding the recombinant humanized type IV collagen according to claim 1, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

3. A recombinant expression vector, characterized in that: The recombinant expression vector comprises an initial vector and the gene encoding type IV collagen recombinant protein according to claim 2.

4. The recombinant expression vector according to claim 3, characterized in that The initial vector is a pET series vector, a pGEX series vector or a pMAL series vector.

5. The recombinant expression vector according to claim 4, characterized in that The initial vector is pET-30a vector.

6. A recombinant strain expressing the recombinant humanized type IV collagen according to claim 1, characterized in that: The recombinant strain is transformed with the recombinant expression vector according to any one of claims 3 to 5.

7. The recombinant strain according to claim 6, characterized in that The recombinant strain was induced to express by IPTG.

8. The recombinant strain according to claim 7, characterized in that The concentration of the IPTG is 0.05-1 mM.

9. The recombinant strain according to claim 7, characterized in that The temperature for inducing expression is 15-20°C; The time for inducing expression is 12 to 72 hours.

10. Use of the recombinant humanized type IV collagen according to claim 1, the recombinant expression vector according to any one of claims 3 to 5, or the recombinant strain according to any one of claims 6 to 9 in the preparation of whitening products, repair products or anti-wrinkle products.

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