Recombinant collagen type v and its encoding gene and application

Abstract

This invention relates to a recombinant type V collagen, its encoding gene, and its applications, belonging to the fields of genetic engineering and protein engineering technology. Based on the amino acid sequence of the α1 chain of human type V collagen, this invention utilizes bioinformatics and other methods to design the amino acid sequence, enabling the recombinant collagen to achieve higher expression efficiency and high stability while maintaining high biological activity. A novel collagen sequence containing 178 amino acids was designed. The nucleotide sequence of this recombinant collagen was optimized using E. coli codon preference to construct a high-expression recombinant strain. The recombinant collagen produced by fermentation of this strain contains no exogenous amino acids such as tags, making it a recombinant humanized type V collagen with good safety. Compared with commercially available type V collagen products, the recombinant type V collagen prepared using the above technical solution has better antioxidant activity and barrier repair capabilities, thus possessing good practical application value.

Description

Technical Field

[0001] This invention relates to a recombinant type V collagen, its encoding gene, and its applications, belonging to the fields of genetic engineering and protein engineering technology. Background Technology

[0002] Type V collagen (COLV) is a dermal collagen that regulates the formation of collagen fibers. It is relatively rare in the human body, accounting for only 2% to 5% of total collagen. It is mainly distributed in the dermis, tendons, ligaments, bones, blood vessels, and cornea. It plays a crucial role in increasing dermal collagen content, maintaining epidermal-dermal structural stability, promoting tissue repair, accelerating wound healing, and preventing scar formation. Type V collagen is a "regulator" of collagen fibers and also a "connector" between the dermal matrix and the basement membrane. Type V collagen is composed of at least three different homo or heterotrimers (α1, α2, and α3) consisting of three different polypeptide α chains (α1, α2, and α3): α1α1α1, α1α1α2, and α1α2α3.

[0003] Recombinant type V collagen, with its unique structure and functional properties, shows broad application prospects in fields such as medical aesthetics and biomedical materials. With continuous technological breakthroughs and market expansion, it is expected to become a significant growth driver in the collagen industry. Currently, there is limited research on recombinant type V collagen. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a recombinant type V collagen, its encoding gene, and its applications. Experiments have demonstrated that the type V collagen of this invention contains no exogenous amino acids such as tags, is a recombinant humanized collagen, and exhibits good safety; it possesses superior antioxidant and barrier repair bioactivity; and it boasts high protein yield and low purification costs, making it more suitable for large-scale industrial production.

[0005] To achieve the above-mentioned technical objectives, the technical solution of the present invention is as follows: A recombinant type V collagen, the amino acid sequence of which is shown in SEQ ID NO.1.

[0006] A gene encoding a recombinant type V collagen, which encodes the aforementioned recombinant type V collagen.

[0007] According to a preferred embodiment of the present invention, the nucleotide sequence of the encoding gene is shown in SEQ ID NO.2.

[0008] A recombinant expression vector containing the gene encoding the aforementioned recombinant type V collagen.

[0009] According to a preferred embodiment of the present invention, the plasmid vector of the recombinant expression vector is pET28a.

[0010] A recombinant expression strain containing the coding gene for the above-mentioned recombinant type V collagen or the above-mentioned recombinant expression vector.

[0011] According to a preferred embodiment of the present invention, the host bacterium of the recombinant expression strain is Escherichia coli.

[0012] More preferably, the Escherichia coli is Escherichia coli BL21(DE3).

[0013] Application of the above-mentioned recombinant expression strain in the preparation of recombinant type V collagen.

[0014] A method for preparing recombinant type V collagen by fermentation using the above-mentioned recombinant expression strain includes the following steps: inoculating the above-mentioned recombinant expression strain into a seed culture medium to obtain a seed solution; inoculating the seed solution into a fermentation culture medium at an inoculation rate of 2-5% by volume, and culturing until OD... 600 After being induced to express at a concentration of 0.6-0.8, a fermentation broth containing recombinant type V collagen was obtained.

[0015] According to a preferred embodiment of the present invention, the method further includes a purification step of recombinant type V collagen, specifically: the fermentation broth containing recombinant type V collagen is centrifuged to obtain bacterial sludge, and the bacterial broth after resuspension of bacterial sludge is homogenized, impurity protein removed, centrifuged, clarified by hollow fiber membrane, concentrated by ultrafiltration membrane, ion exchange chromatography, desalted and freeze-dried to obtain freeze-dried recombinant type V collagen.

[0016] More preferably, the protein removal is performed by acid precipitation, adjusting the pH of the bacterial solution to 2.9-3.1.

[0017] More preferably, the hollow fiber membrane has a molecular weight cutoff of 250 kDa.

[0018] More preferably, the ultrafiltration membrane has a molecular weight cutoff of 3 kDa.

[0019] More preferably, the ion exchange chromatography is cation exchange chromatography.

[0020] The above-mentioned recombinant type V collagen is used in the preparation of food, pharmaceuticals, cosmetics, health products or medical devices.

[0021] Beneficial effects: This invention, based on the amino acid sequence of the α1 chain of human collagen type V, utilizes bioinformatics and other methods to design the amino acid sequence, enabling recombinant collagen to achieve higher expression efficiency and stability while maintaining high biological activity. A novel collagen sequence containing 178 amino acids was designed. The nucleotide sequence of this recombinant collagen was optimized using E. coli codon preference to construct a high-expression recombinant strain. The recombinant collagen produced by fermentation of this strain contains no exogenous amino acids such as tags, making it a recombinant humanized type V collagen with good safety. Compared with commercially available type V collagen products, the recombinant type V collagen prepared using the above technical solution exhibits better antioxidant activity and barrier repair capabilities, thus possessing significant practical application value. Attached Figure Description

[0022] Figure 1 Recombinant expression strain E. coil SDS-PAGE analysis of BL21 / pET28a-C5A1 induced expression; lane M represents the standard protein with a molecular weight of 180 kDa; lane 1 represents the blank control bacteria. E. coil BL21 / pET28a cell lysis supernatant; lane 2 represents blank control bacteria. E. coil BL21 / pET28a cell lysis precipitation; lane 3 represents the recombinant expression strain. E. coil BL21 / pET28a-C5A1 cell lysis supernatant; lane 4 represents the recombinant expression strain. E. coil BL21 / pET28a-C5A1 cell disruption precipitation.

[0023] Figure 2 The bar chart shows the DPPH free radical scavenging rate of different collagens; where the comparative example represents the experimental group of commercially available recombinant collagen; C5A1 represents the experimental group of recombinant type V collagen prepared using Example 4 of this invention.

[0024] Figure 3 The bar chart shows the relative expression levels of FLG mRNA in HaCaT cells under the action of different collagens; where control group 1 represents the blank control group; control group 2 represents the commercially available recombinant collagen control group; C5A1 represents the recombinant type V collagen C5A1 group prepared in Example 4 of this invention; in the figure, compared with control group 1, P<0.0001 (****), which is considered to be significant. Detailed Implementation

[0025] The technical solution of the present invention will be further described below with reference to the embodiments. Unless otherwise specified, the experimental operations involved in the embodiments are all conventional experimental operations in the art.

[0026] The commercially available recombinant collagen involved in the examples is a common commercially available recombinant humanized type V collagen.

[0027] Example 1: Design of Recombinant Type V Collagen Sequence The amino acid sequence of the α1 chain protein of natural human type V collagen (UniProt ID: P20908) was obtained from the NCBI database. Through in-depth analysis of this sequence information, bioinformatics techniques were used to design sequences based on the hydrophilicity / hydrophobicity, charge distribution, and protein spatial structure of the amino acids. This enabled the recombinant collagen to achieve higher expression efficiency and stability while maintaining high biological activity. The recombinant collagen of this invention contains 178 amino acids, and the specific amino acid sequence is shown in SEQ ID NO.1. This sequence contains the main bioactive sites of human type V collagen, ensuring the realization of the biological function of the recombinant collagen. Furthermore, this sequence does not contain any exogenous amino acids such as tags, making it a recombinant humanized collagen with high biocompatibility and low risk of use.

[0028] Example 2: Recombinant Expression Strains E. coil Construction of BL21 / pET28a-C5A1 Based on the codon preference of *E. coli*, the coding gene for recombinant type V collagen shown in SEQ ID NO.1 of Example 1 was codon optimized. The optimized coding gene nucleotide sequence is shown in SEQ ID NO.2 and named C5A1. The nucleotide sequence of C5A1 was synthesized by Nanjing Genscript Biotech Co., Ltd., cloned into the *E. coli* expression vector pET28a, and transformed into... E. coil In the TOP10, the recombinant expression vector pET28a-C5A1 was obtained while ensuring that the reading frame did not shift. After DNA sequencing and alignment, the recombinant expression vector pET28a-C5A1 was successfully constructed.

[0029] The successfully validated recombinant expression vector pET28a-C5A1 was transformed into E. coli BL21(DE3) according to the E. coli manual. Transformants were screened using kanamycin antibiotics, and then verified by PCR to confirm the recombinant expression strain expressing recombinant type V collagen. E. coil BL21 / pET28a-C5A1 was successfully constructed.

[0030] Example 3: Recombinant Expression Strains E. coil Inducible expression of BL21 / pET28a-C5A1 Select recombinant expression strains E. coilA single colony of BL21 / pET28a-C5A1 was incubated overnight at 37°C and 200 rpm in LB liquid medium (1% NaCl, 0.5% yeast extract, 1% peptone, all by weight) containing 50 μg / mL kanamycin. Then, it was inoculated at a 2% (v / v) rate into TB medium (1.18% peptone, 2.36% yeast extract, 0.94% K₂HPO₄, 0.22% KH₂PO₄, 0.4% glycerol, all by weight) and incubated at 37°C and 200 rpm until OD₀. 600 The initial concentration was 0.6-0.8, followed by the addition of 0.5 mM IPTG (isopropyl-β-D-thiogalactopyranoside) to induce expression. The cells were cultured at 22°C for 20 h, collected by centrifugation, resuspended in Tris buffer (20 mM Tris, pH 7.5), and then sonicated. The supernatant and precipitate were analyzed by SDS-PAGE. The theoretical size of this recombinant type V collagen was 17.2 kDa. The SDS-PAGE results are shown below. Figure 1 As shown, the recombinant expression strain E. coil Cell lysis supernatant of BL21 / pET28a-C5A1 (lane 3) and blank control bacteria E. coil Compared to pET28a (lane 1), BL21 shows a distinct protein band at the theoretical size (indicated by the arrow). These results demonstrate that the recombinant type V collagen of this invention can be successfully induced to express.

[0031] Example 4: Purification and preparation of recombinant type V collagen recombinant expression strain E. coil BL21 / pET28a-C5A1 was fermented according to the method in Example 3. The fermentation broth after induction culture was centrifuged at 8000 rpm to obtain bacterial sludge. The bacterial sludge was then purified through the following steps to obtain pure recombinant type V collagen: ① Removal of impurities and proteins: The bacterial sludge was resuspended in Tris-HCl buffer solution at pH 7.5. After homogenization, the pH of the bacterial solution was adjusted to 3.0 with 1M hydrochloric acid solution. The bacterial solution was stirred continuously during the acid addition process to prevent local over-acidification and denaturation and precipitation of recombinant type V collagen. ② Centrifugation: Centrifugation removes acid-precipitated proteins. Adjust the pH of the supernatant to 7.0 with 1M sodium hydroxide solution. ③ Hollow fiber membrane clarification: The above centrifuged supernatant is filtered through a 250kDa hollow fiber membrane to remove bacteria and large molecular impurities; ④ Ultrafiltration membrane concentration: The feed solution is concentrated using a 3kDa ultrafiltration membrane pack, which can also remove small molecule impurities. ⑤ Ion exchange chromatography: Cation exchange chromatography is used. Recombinant type V collagen is positively charged. During chromatographic loading, the target protein is adsorbed on the column, while negatively charged impurities such as foreign proteins and endotoxins will flow through directly, thus removing impurities. After washing the column, the target protein on the column is eluted with 150mM NaCl solution, and the eluent is collected. ⑥ Ultrafiltration membrane desalination: The feed solution is desalted using a 3kDa ultrafiltration membrane until the conductivity of the sample solution is below 70μs / cm; ⑦ Freeze-drying: The desalted sample solution is freeze-dried under vacuum to obtain recombinant type V collagen freeze-dried product.

[0032] The recombinant type V collagen purified by the above steps has a purity greater than 97% and a yield greater than 60%.

[0033] Example 5: Antioxidant Experiment of Recombinant Type V Collagen Take 2.5 mg of the recombinant type V collagen lyophilized product from Example 4 and place it in a test tube. Add 2.5 mL of DPPH free radical solution (0.1 mmol / L) dissolved in 95% ethanol. Shake the mixture vigorously for 10 s and then let it react at room temperature for 30 min. After the reaction is complete, measure the absorbance of the reaction mixture at 517 nm. Use distilled water instead of the sample solution as a blank control and commercially available recombinant collagen as a comparative experiment.

[0034] DPPH free radical scavenging activity (%) = (OD 空白 -OD 样品 ) / OD 空白 ×100% Among them, OD 空白 The absorbance value of the blank control group is OD. 样品 The absorbance values ​​are for recombinant type V collagen groups or commercially available recombinant collagen groups.

[0035] The results are as follows Figure 2 As shown, the recombinant type V collagen C5A1 of the present invention has a significant DPPH free radical scavenging ability compared with the blank control, and its DPPH free radical scavenging ability is higher than that of commercially available recombinant collagen. Therefore, the recombinant type V collagen provided by the present invention has a significant antioxidant effect.

[0036] Example 7: Barrier Repair Experiment Filagrin (FLG) is an important component of the keratinized capsule, ensuring the integrity of the skin barrier. FLG deficiency leads to disordered lipid bilayer structure and delayed maturation, while also causing reduced keratinocyte density, increased intercellular permeability, and decreased photoprotective function, ultimately resulting in skin barrier damage.

[0037] Immortalized human keratinocytes (HaCaT cells, purchased from Shanghai Cell Bank) were collected, and a cell suspension was prepared using high-glucose DMEM cell culture medium. 2 mL of the cell suspension was added to each well of a 6-well plate, resulting in a cell count of 2.6 × 10⁶ cells. 5 / well. The experiment included a blank control group (control group 1), a commercially available recombinant collagen control group (control group 2), and the recombinant type V collagen C5A1 group prepared in Example 4 of this invention (experimental group), with 3 replicates for each group. The 6-well plate was incubated in a cell culture incubator (37℃, 5% CO2) for 24 h. When the cell confluence rate reached 50%~60%, the culture medium was discarded. 2 mL of high-glucose DMEM cell culture medium was added to each well of the blank control group; 2 mL of high-glucose DMEM cell culture medium containing the corresponding collagen was added to each of the other two groups. The concentration of collagen in the high-glucose DMEM cell culture medium was 1 mg / mL. After incubating the 6-well plates in an incubator (37℃, 5% CO2) for 24 hours, the cells in each well were washed twice with 2 mL of PBS buffer. Following the Total RNA Extraction Kit, 1 mL of RNAiso Plus was added, and the cells were lysed by pipetting. RNA extraction, reverse transcription, and quantitative real-time PCR were performed according to the kit instructions to detect the relative mRNA expression level of the barrier-related protein FLG. A 2... -△△CT The method is used for calculation.

[0038] Different collagen proteins promote the expression of FLG mRNA in HaCaT cells, such as Figure 3 As shown, compared with the blank control group (control group 1), both commercially available recombinant collagen (control group 2) and the recombinant type V collagen C5A1 prepared in Example 4 of this invention (experimental group) can significantly increase the relative expression level of FLG mRNA. Among them, the recombinant type V collagen prepared in this invention has a stronger ability to promote FLG mRNA expression than commercially available recombinant collagen. Therefore, the recombinant type V collagen of this invention has obvious skin barrier repair function.

Claims

1. A recombinant type V collagen, characterized in that, The amino acid sequence of the recombinant type V collagen is shown in SEQ ID NO.

1.

2. A gene encoding recombinant type V collagen, characterized in that, It encodes the recombinant type V collagen as described in claim 1; Preferably, the nucleotide sequence of the encoding gene is shown in SEQ ID NO.

2.

3. A recombinant expression vector, characterized in that, Contains the gene encoding the recombinant type V collagen as described in claim 2; Preferably, the plasmid vector of the recombinant expression vector is pET28a.

4. A recombinant expression strain, characterized in that, Contains the encoding gene of the recombinant type V collagen as described in claim 2 or the recombinant expression vector as described in claim 3.

5. The recombinant expression strain as described in claim 4, characterized in that, The host bacterium of the recombinant expression strain is Escherichia coli; More preferably, the Escherichia coli is Escherichia coli BL21(DE3).

6. The use of the recombinant expression strain according to claim 5 in the preparation of recombinant type V collagen.

7. A method for preparing recombinant type V collagen by fermentation using the recombinant expression strain according to claim 5, characterized in that, The steps include: inoculating the recombinant expression strain of claim 5 into a seed culture medium and culturing it to obtain a seed solution; Inoculate the seed culture into the fermentation medium at an inoculation rate of 2-5% by volume, and culture until OD... 600 After being induced to express at a concentration of 0.6-0.8, a fermentation broth containing recombinant type V collagen was obtained.

8. The method as described in claim 7, characterized in that, The method also includes a purification step for recombinant type V collagen, specifically: the fermentation broth containing recombinant type V collagen is centrifuged to obtain bacterial sludge, and the bacterial broth after resuspension of bacterial sludge is homogenized, impurity protein removed, centrifuged, clarified by hollow fiber membrane, concentrated by ultrafiltration membrane, ion exchange chromatography, desalted and freeze-dried to obtain freeze-dried recombinant type V collagen.

9. The method as described in claim 8, characterized in that, One or more of the following conditions must be met: i. The protein removal is performed by acid precipitation, adjusting the pH of the bacterial solution to 2.9-3.1; ii. The hollow fiber membrane has a molecular weight cutoff of 250 kDa; iii. The ultrafiltration membrane has a molecular weight cutoff of 3 kDa; iv. The ion exchange chromatography is cation exchange chromatography.

10. The use of the recombinant type V collagen according to claim 1 in the preparation of food, pharmaceuticals, cosmetics, health products or medical devices.

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