Heat-resistant stable recombinant humanized V-type collagen and preparation method thereof

Abstract

The embodiment of the invention discloses heat-resistant stable recombinant humanized V-type collagen and a preparation method thereof, relates to the technical field of recombinant protein, and aims to solve the technical problem that existing collagen is difficult to meet application requirements. The preparation method of the heat-resistant and stable recombinant humanized V-type collagen comprises the following steps: respectively obtaining recombinant collagen with gene sequences as shown in SEQ ID No.2, SEQ ID No.4 and SEQ ID No.6, and respectively inserting the recombinant collagen into pPIC9K plasmids to obtain corresponding recombinant plasmids; the recombinant plasmids comprise a recombinant plasmid pPIC9K-ColV alpha 1-1, a recombinant plasmid pPIC9K-ColV alpha 1-2 and a recombinant plasmid pPIC9K-ColV alpha 1-4, and the recombinant plasmid pPIC9K-ColV alpha 1-1, the recombinant plasmid pPIC9K-ColV alpha 1-2 and the The recombinant plasmid is sequentially subjected to electrotransformation treatment, screening treatment, identification treatment and purification treatment, and the recombinant V-type collagen alpha1 is obtained; the recombinant V type collagen alpha1 comprises a recombinant V type collagen alpha1-1, a recombinant V type collagen alpha1-2 and a recombinant V type collagen alpha1-4.

Description

Technical Field

[0001] This application relates to the field of recombinant protein technology, specifically to a heat-resistant and stable recombinant humanized type V collagen and its preparation method. Background Technology

[0002] Collagen is a family of proteins widely distributed in animal connective tissues, playing a vital role in maintaining the normal physiological functions of cells, tissues, and organs, as well as in damage repair. Composed of polysaccharide-protein molecules, collagen is mainly found in animal skin, bones, teeth, tendons, ligaments, and blood vessels, and is a major protein component of connective tissues. Its function is to maintain the structure and morphology of the skin and various organs. Type I and Type III collagen are widely used in skincare, while Type V collagen is a key component in maintaining skin elasticity. It works synergistically with Type I and Type III collagen, significantly affecting skin smoothness, firmness, and repair capabilities. Current extraction methods for Type V collagen from animal tissues result in poor water solubility and purity, making it difficult to meet the application requirements in the biomedical field. Furthermore, naturally extracted collagen carries the risk of infection and rejection reactions from zoonotic viruses, greatly limiting its application in pharmaceuticals, biomedical materials, or drug carriers. Recombinant type V collagen obtained through existing biosynthetic methods generally faces the industry challenge of poor thermal stability, making it difficult to maintain stable storage in liquid formulations such as lotions, creams, and ointments for extended periods, thus limiting its widespread application in skincare products and skin dressings. Summary of the Invention

[0003] The main objective of this application is to provide a heat-resistant and stable recombinant humanized type V collagen and its preparation method, aiming to solve the technical problem that existing collagen cannot meet application requirements.

[0004] To achieve the above objectives, the technical solutions adopted in the embodiments of this application are as follows: In a first aspect, embodiments of this application provide a method for preparing heat-resistant and stable recombinant humanized type V collagen, comprising the following steps: Recombinant collagen sequences as shown in SEQ ID No. 2, SEQ ID No. 4, and SEQ ID No. 6 were obtained, and the recombinant collagen sequences were inserted into the pPIC9K plasmid to obtain the corresponding recombinant plasmids; the recombinant plasmids include recombinant plasmids pPIC9K-ColⅤα1-1, pPIC9K-ColⅤα1-2, and pPIC9K-ColⅤα1-4; The recombinant plasmid was subjected to electroporation, screening, identification and purification processes in sequence to obtain recombinant type V collagen α1; the recombinant type V collagen α1 includes recombinant type V collagen α1-1, recombinant type V collagen α1-2 and recombinant type V collagen α1-4.

[0005] As some optional embodiments of this application, the recombinant collagen with the gene sequence shown in SEQ ID No. 2 is obtained based on the target amino acid sequence shown in SEQ ID No. 1; The recombinant type V collagen with the gene sequence shown in SEQ ID No. 4 was obtained based on the target amino acid sequence shown in SEQ ID No. 3; The recombinant type V collagen with the gene sequence shown in SEQ ID No. 6 was obtained based on the target amino acid sequence shown in SEQ ID No. 5.

[0006] As some optional embodiments of this application, the target amino acid sequence shown in SEQ ID No. 1 is gene sequence 643 to 730 in the gene sequence of recombinant expression of ColⅤα1 chain, which contains two RGD domains; The target amino acid sequence shown in SEQ ID No. 3 is obtained by repeating the target amino acid sequence shown in SEQ ID No. 1 twice in tandem; The target amino acid sequence shown in SEQ ID No. 5 is obtained by repeating the target amino acid sequence shown in SEQ ID No. 1 four times in series.

[0007] As some optional embodiments of this application, the electroconversion process includes the following steps: The recombinant plasmid was linearized with restriction endonuclease SacⅠ, then electroporated into GS115 competent cells, plated on MD solid plates, incubated at room temperature for 10 min, and then inverted in a 30℃ incubator for 2 to 5 days until single colonies appeared.

[0008] As some optional embodiments of this application, the screening process includes the following steps: The single colonies were inoculated onto YPD solid plates containing 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL and 4 mg / mL G418, respectively; Single colonies that could grow simultaneously on YPD solid plates containing 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL and 4 mg / mL G418 were screened and identified as high-copy recombinant strains.

[0009] As some optional embodiments of this application, the identification process includes the following steps: Four high-copy recombinant strains were selected and inoculated into BMGY medium, and cultured at 30 °C and 220 rpm for 24 h until OD500. 600 Values ​​range from 2 to 6; According to actual measured OD 600 Adjust the volume of the BMGY culture medium, collect the cells at 3000 rpm for 10 min for resuspending, and adjust the initial OD value. 600 The value is 2.0; The culture was continued at 30 ℃ and 220 rpm. 0.5% methanol was added to the culture medium every 24 h. After 72 h of methanol induction, bacterial culture samples were taken and the expression supernatant was collected by centrifugation. The expression of proteins in the supernatant of the recombinant strain was analyzed by SDS-PAGE electrophoresis to obtain the target protein.

[0010] As some optional embodiments of this application, the purification process includes the following steps: The target protein was subjected to a strong anion exchange medium. The chromatography column was equilibrated with phosphate buffer until the conductivity and A280 absorbance remained constant. The sample loading flow rate was set to 5 mL / min. The UV A280 absorbance was detected. When the absorbance rose, the sample loading began. After the sample loading is completed, the chromatography medium is equilibrated with phosphate buffer until the UV A280 absorbance and conductivity values ​​drop to their minimum and no longer change, at which point the equilibration is complete. The protein was then eluted with a phosphate buffer containing NaCl and collected to obtain recombinant type V collagen.

[0011] Secondly, embodiments of this application also provide a heat-resistant and stable recombinant humanized type V collagen, prepared by the method described above; the heat-resistant and stable recombinant humanized type V collagen includes recombinant type V collagen α1-1, recombinant type V collagen α1-2 and recombinant type V collagen α1-4.

[0012] As one of the optional embodiments of this application, the Hacat cell adhesion-promoting activity of recombinant type V collagen α1-1 is 1.10 × 10⁻⁶. 4 U / mg; The recombinant type V collagen α1-2 showed a Hacat cell adhesion-promoting activity of 1.27 × 10⁻⁶. 5 U / mg; The recombinant type V collagen α1-4 exhibited Hacat cell adhesion-promoting activity of 9.58 × 10⁻⁶. 5 U / mg.

[0013] As some optional embodiments of this application, the recombinant type V collagen α1-1 has a NIH 3T3 cell proliferation-promoting activity of 1.43 × 10⁻⁶. 4 U / mg; Recombinant type V collagen α1-2 showed a proliferation-promoting activity of 2.34 × 10⁻⁶ NIH 3T3 cells. 4 U / mg; Recombinant type V collagen α1-4 showed a proliferation-promoting activity of 9.38 × 10⁻⁶ NIH 3T3 cells. 4 .

[0014] Compared with the prior art, the method for preparing heat-resistant and stable recombinant humanized type V collagen provided in this application includes the following steps: obtaining recombinant collagen with gene sequences as shown in SEQ ID No. 2, SEQ ID No. 4 and SEQ ID No. 6 respectively, and inserting the recombinant collagen into the pPIC9K plasmid to obtain the corresponding recombinant plasmid; the recombinant plasmid includes recombinant plasmid pPIC9K-ColⅤα1-1, recombinant plasmid pPIC9K-ColⅤα1-2 and recombinant plasmid pPIC9K-ColⅤα1-4; subjecting the recombinant plasmid to electroporation, screening, identification and purification treatment in sequence to obtain recombinant type V collagen α1; the recombinant type V collagen α1 includes recombinant type V collagen α1-1, recombinant type V collagen α1-2 and recombinant type V collagen α1-4. The recombinant type V collagen α1-1 prepared by the above steps exhibited Hacat cell adhesion-promoting activity of 1.10 × 10⁻⁶. 4 U / mg, the proliferation-promoting activity of NIH 3T3 cells was 1.43×10. 4 U / mg; the Hacat cell adhesion-promoting activity of recombinant type V collagen α1-2 was 1.27 × 10⁻⁶ U / mg. 5 U / mg, the proliferation-promoting activity of NIH 3T3 cells was 2.34×10 4 U / mg; the Hacat cell adhesion-promoting activity of recombinant type V collagen α1-4 was 9.58 × 10⁻⁶ U / mg. 5 U / mg, the proliferation-promoting activity of NIH 3T3 cells was 9.38×10. 4 . Attached Figure Description

[0015] Figure 1 This is a graph showing the results of identifying the expression level of the target protein ColⅤ α1-1 involved in the embodiments of this application; Figure 2 This is a graph showing the expression levels of the target proteins ColⅤ α1-2 and ColⅤ α1-4 involved in the embodiments of this application. Figure 3The images show the purification results of the target proteins ColⅤ α1-1, ColⅤ α1-2, and ColⅤ α1-4 involved in the embodiments of this application. Figure 4 The figure shows the toxicity test results based on mouse macrophage RAW264.7 involved in the embodiments of this application; Figure 5 This is a diagram showing the results of recombinant type V collagen inhibiting TNFα secretion according to the embodiments of this application; Figure 6 This is a diagram showing the results of recombinant type V collagen inhibiting IL-6 secretion in the embodiments of this application; Figure 7 This is a diagram illustrating the effect of recombinant type V collagen on promoting HaCaT cell migration, as described in the embodiments of this application. Detailed Implementation

[0016] It should be understood that the specific embodiments described herein are merely illustrative of this application and are not intended to limit this application.

[0017] With the development of genetic engineering and synthetic biology technologies, researchers at home and abroad are using conventional techniques to industrialize the mass production of recombinant humanized collagen to meet current biomedical needs.

[0018] Type V collagen is a fibrous collagen found in the lungs, bones, and fetal membranes, co-distributed with type I collagen. Other fibroblastic collagens include type I, type II, type III, and type XI collagen. These fibroblasts possess a typical quarter-interlaced array of fibrils, ranging in diameter from 25 nm to 400 nm, and are used to assemble supramolecular aggregates with highly oriented superstructures. Type V collagen is located in the extracellular matrix, is present in low amounts, and interacts with matrix collagen and structural proteins to maintain tissue structural integrity. Low levels of type V collagen can lead to loss of corneal transparency and Ehlers-Danlos syndrome.

[0019] Therefore, it is crucial to biosynthesize a heat-resistant and stable recombinant humanized type V collagen.

[0020] Based on this, this application proposes a heat-resistant and stable recombinant humanized type V collagen and its preparation method, namely: The method for preparing the heat-resistant and stable recombinant humanized type V collagen includes the following steps: Step S10: Obtain the recombinant collagen sequences shown in SEQ ID No. 2, SEQ ID No. 4, and SEQ ID No. 6 respectively, and insert the recombinant collagen into the pPIC9K plasmid to obtain the corresponding recombinant plasmids; the recombinant plasmids include recombinant plasmid pPIC9K-ColⅤα1-1, recombinant plasmid pPIC9K-ColⅤα1-2, and recombinant plasmid pPIC9K-ColⅤα1-4; Step S20: The recombinant plasmid is subjected to electroporation, screening, identification and purification processes in sequence to obtain recombinant type V collagen α1; the recombinant type V collagen α1 includes recombinant type V collagen α1-1, recombinant type V collagen α1-2 and recombinant type V collagen α1-4.

[0021] It should be noted that the recombinant collagen with the gene sequence shown in SEQ ID No. 2 is obtained based on the target amino acid sequence shown in SEQ ID No. 1; the target amino acid sequence shown in SEQ ID No. 1 is gene sequence 643 to 730 in the recombinant gene sequence expressing the ColⅤα1 chain, which contains two RGD domains; The recombinant collagen with the gene sequence shown in SEQ ID No. 4 is obtained based on the target amino acid sequence shown in SEQ ID No. 3; the target amino acid sequence shown in SEQ ID No. 3 is obtained by repeating the target amino acid sequence shown in SEQ ID No. 1 twice in tandem. The recombinant collagen with the gene sequence shown in SEQ ID No. 6 is obtained based on the target amino acid sequence shown in SEQ ID No. 5; the target amino acid sequence shown in SEQ ID No. 5 is obtained by repeating the target amino acid sequence shown in SEQ ID No. 1 four times in tandem.

[0022] It should be noted that the electroconversion treatment in step S20 includes the following steps: The recombinant plasmid was linearized with restriction endonuclease SacⅠ, then electroporated into GS115 competent cells, plated on MD solid plates, incubated at room temperature for 10 min, and then inverted in a 30℃ incubator for 2 to 5 days until single colonies appeared.

[0023] It should be noted that the screening process described in step S20 includes the following steps: The single colonies were inoculated onto YPD solid plates containing 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL and 4 mg / mL G418, respectively; Single colonies that could grow simultaneously on YPD solid plates containing 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL and 4 mg / mL G418 were screened and identified as high-copy recombinant strains.

[0024] It should be noted that the identification process described in step S20 includes the following steps: Four single colonies of the high-copy recombinant strain were picked and inoculated into BMGY medium, and cultured at 30 ℃ and 220 rpm for 24 h until OD. 600 Values ​​range from 2 to 6; based on measured OD values. 600 Adjust the volume of the BMGY culture medium, collect the cells at 3000 rpm for 10 min, and resuspend the cells in an equal volume of BMMY medium to adjust the initial OD. 600 The value was 2.0; the culture was continued at 30 ℃ and 220 rpm, and 0.5% methanol was added to the culture medium every 24 h. After 72 h of methanol induction, bacterial culture samples were taken and the expression supernatant was collected by centrifugation. The protein expression in the supernatant of the recombinant strain was analyzed by SDS-PAGE electrophoresis to obtain the target protein.

[0025] It should be noted that the purification process described in step S20 includes the following steps: The target protein was equilibrated with a strong anion exchange medium using phosphate buffer until the conductivity and A280 absorbance remained constant. The sample loading flow rate was set to 5 mL / min, and the UV A280 absorbance was measured. When the absorbance increased, sample loading began. After loading, the chromatography medium was equilibrated again with phosphate buffer until the conductivity and A280 absorbance reached their minimum and stopped changing. The equilibration was then complete. The protein was eluted with a phosphate buffer containing NaCl and the corresponding protein was collected to obtain recombinant type V collagen. The recombinant humanized type V collagen includes recombinant type V collagen α1-1, recombinant type V collagen α1-2, and recombinant type V collagen α1-4.

[0026] The recombinant type V collagen α1-1 prepared by the above steps exhibited Hacat cell adhesion-promoting activity of 1.10 × 10⁻⁶. 4 U / mg, the proliferation-promoting activity of NIH 3T3 cells was 1.43×10. 4 U / mg; the Hacat cell adhesion-promoting activity of recombinant type V collagen α1-2 was 1.27 × 10⁻⁶ U / mg. 5 U / mg, the proliferation-promoting activity of NIH 3T3 cells was 2.34×10 4 U / mg; the Hacat cell adhesion-promoting activity of recombinant type V collagen α1-4 was 9.58 × 10⁻⁶ U / mg. 5 U / mg, the proliferation-promoting activity of NIH 3T3 cells was 9.38×10. 4 .

[0027] The following detailed description, with reference to specific embodiments, further illustrates the heat-resistant and stable recombinant humanized type V collagen and its preparation method described in this application: Example 1 Construction, identification and preparation of engineered bacteria 1.1 Gene Sequence Selection Studies have shown that the type V α1 chain (Col Vα1) intervenes in the release of inflammatory factors and enhances cell adhesion through the guanine nucleotide exchange factor (GFF) / integrin signaling pathway, which is beneficial to tissue repair. The integrin receptor on the cell surface is RGD, and the type V α1 chain (GenBank: KAI4009059.1) contains two RGD domains (located at positions 645-647 and 662-665, respectively).

[0028] The gene sequence used for recombinant expression of the ColⅤα1 chain was selected from positions 643 to 730. This sequence contains two RGD domains, and the amino acid sequence is SEQ ID No. 1. Codon optimization was performed on this sequence for the Pichia pastoris expression system to obtain the recombinant collagen type V α1-1 gene sequence, as shown in SEQ ID No. 2. The obtained target gene sequence was then synthesized by Nanjing Genscript Biotech Co., Ltd. The synthesized gene was inserted into the pPIC9K plasmid to obtain the recombinant plasmid pPIC9K-ColⅤ α1-1.

[0029] The amino acid sequence SEQ ID No.1 was repeated twice in tandem to obtain the amino acid sequence as shown in SEQ ID No.3. Based on the above method, the recombinant collagen shown in SEQ ID No.4 was obtained and inserted into the pPIC9K plasmid to obtain the recombinant plasmid pPIC9K-ColⅤ α1-2.

[0030] Similarly, the amino acid sequence SEQ ID No.1 was repeated 4 times in tandem, resulting in a total of 352 amino acids, which is SEQ ID No.5. Following the method described above, the recombinant collagen shown in SEQ ID No.6 was obtained and inserted into the pPIC9K plasmid to obtain the recombinant plasmid pPIC9K-ColⅤ α1-4.

[0031] Among them, SEQ ID No. 1-SEQ ID No. 6 are shown below: SEQ ID No. 1: GHRGDPGPSGPPGPPGDDGERGDDGEVGPRGLPGEPGPRGLLGPKGPPGPPGPPGVTGMDGQPGPKGNVGPQGEPGPPGQQGNPGAQG。

[0032] SEQ ID No.2: GGTCACAGGGGGGACCCTGGCCCTTCGGGACCGCCAGGGCCACCCGGCGACGATGGTGAACGTGGAGATGATGGTGAGGTGGGGCCCCGGGGACTCCCCGGGGAACCGGGACCTAGAGGACTGCTTGGACCAAAGGGTCCGCCCGGCCCGCCTGGTCCGCCAGGGGTAACAGGGATGGACGGGCAGCCAGGACCAAAAGGCAACGTCGGGCCCCAAGGCGAGCCTGGCCCGCCTGGACAGCAAGGTAATCCCGGCGCTCAAGGT。

[0033] SEQ ID No.3: GHRGDPGPSGPPGPPGDDGERGDDGEVGPRGLPGEPGPRGLLGPKGPPGPPGPPGVTGMDGQPGPKGNVGPQGEPGPPGQQGNPGAQGGHRGDPGPSGPPGPPGDDGERGDDGEVGPRGLPGEPGPRGLLGPKGPPGPPGPPGVTGMDGQPGPKGNVGPQGEPGPPGQQGNPGAQG。

[0034] SEQ ID No.4: GGACATAGAGGCGACCCCGGTCCTAGCGGCCCGCCGGGCCCACCCGGCGACGACGGGGAGCGTGGAGATGACGGAGAAGTAGGCCCACGCGGCCTACCAGGAGAACCTGGTCCCAGGGGCCTGTTAGGTCCGAAGGGGCCTCCTGGGCCTCCCGGCCCCCCTGGAGTCACGGGAATGGATGGTCAGCCCGGTCCGAAGGGAAACGTCGGGCCTCAGGGAGAGCCAGGACCACCGGGACAACAAGGCAACCCCGGTGCCCAAGGGGGACACCGAGGGGATCCAGGACCGAGTGGACCGCCAGGGCCTCCGGGTGATGACGGTGAACGCGGTGATGATGGTGAGGTGGGACCTCGTGGGCTTCCGGGCGAACCCGGCCCCCGGGGGCTCTTGGGTCCTAAAGGTCCCCCTGGTCCCCCGGGTCCACCGGGTGTGACAGGGATGGACGGACAGCCGGGGCCAAAAGGGAATGTTGGGCCACAAGGCGAGCCAGGCCCTCCAGGGCAACAGGGCAATCCCGGCGCGCAGGGG。

[0035] SEQ ID No.5: GHRGDPGPSGPPGPPGDDGERGDDGEVGPRGLPGEPGPRGLLGPKGPPGPPGPPGVTGMDGQPGPKGNVGPQGEPGPPGQQGNPGAQGGHRGDPGPSGPPGPPGDDGERGDDGEVGPRGLPGEPGPRGLLGPKGPPGPPGPPGVTGMDGQPGPKGNVGPQGEPGPPGQQGNPGAQGGHRGDPGPSGPPGPPGDDGERGDDGEVGPRGLPGEPGPRGLLGPKGPPGPPGPPGVTGMDGQPGPKGNVGPQGEPGPPGQQGNPGAQGGHRGDPGPSGPPGPPGDDGERGDDGEVGPRGLPGEPGPRGLLGPKGPPGPPGPPGVTGMDGQPGPKGNVGPQGEPGPPGQQGNPGAQG。

[0036] SEQ ID No.6:

[0037] 1.2 Preparation of yeast expression strains Step 1): Recombinant plasmids pPIC9K-ColⅤ α1-1, pPIC9K-ColⅤ α1-2, and pPIC9K-ColⅤ α1-4 were linearized with restriction endonuclease SacⅠ (enzyme digestion system: 15 μg recombinant plasmid, 10 μL Quick Cut Sac Ⅰ, 5 μL 10× Quick Cut buffer, and ddH2O to a final volume of 50 μL), and then digested in a water bath at 37℃ for 5 h.

[0038] Step 2): Preparation of GS115 competent cells After streaking GS115 strain onto a plate, a single colony was picked and inoculated into 5 mL of YPD medium. The culture was incubated overnight at 30 °C with shaking at 225 r / min. The next day, the colony was transferred to 50 mL of YPD liquid medium at a 1:100 inoculation ratio and incubated at 30 °C with shaking at 225 r / min until OD (out of control) was reached. 600 When the pH value is 1.2-1.5, transfer the bacterial culture to a sterile 50 mL centrifuge tube and incubate on ice for 10 min. Centrifuge at 4 ℃, 5000 rpm for 5 min, discard the supernatant, collect the bacterial cells, resuspend the bacterial cells in 50 mL of pre-chilled sterile ultrapure water, centrifuge at 4 ℃, 5000 rpm for 5 min, discard the supernatant, and resuspend the bacterial cells in 25 mL of pre-chilled sterile ultrapure water. Centrifuge at 4 ℃, 5000 rpm for 5 min, discard the supernatant, and resuspend the bacterial cells in 25 mL of pre-chilled sterile 1 M sorbitol. Centrifuge at 4 ℃, 5000 rpm for 5 min, discard the supernatant, and resuspend the bacterial cells in 10 mL of pre-chilled sterile 1 M sorbitol. Centrifuge at 4 ℃, 5000 rpm for 5 min, discard the supernatant, resuspend the cells in 200 μL of pre-chilled sterile 1 M sorbitol, gently vortex to mix, and place on ice for later use.

[0039] It should be noted that the "pre-cooling" mentioned in the technical solution described in this application refers to pre-cooling to about 4°C.

[0040] Step 3): Electroporate the recombinant plasmid obtained in Step 1.1 into GS115 competent cells, spread it on MD solid plates, let it stand at room temperature for 10 min, and then incubate it upside down in a 30℃ incubator for 2-5 days until single colonies appear.

[0041] Single colonies from the MD plates were inoculated using sterile white pipette tips onto YPD solid plates containing G418 at concentrations of 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, and 4 mg / mL. If a transformant could grow simultaneously on YPD solid plates containing G418 at concentrations of 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, and 4 mg / mL, it indicated that the transformant contained multiple copies of the target gene. This screening step yielded a high-copy recombinant yeast strain capable of efficiently expressing the gene.

[0042] Experiments have shown that GS115 / pPIC9K-ColⅤ α1-1, GS115 / pPIC9KColⅤ α1-2, and GS115 / pPIC9K-ColⅤ α1-4 can all grow on plates with high concentrations of G418 (0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, and 4 mg / mL), indicating that they are all high-copy recombinant strains.

[0043] Step 4): Four single colonies from each of the high-copy recombinant strains GS115 / pPIC9K-ColⅤ α1-1, GS115 / pPIC9KColⅤ α1-2, and GS115 / pPIC9K-ColⅤ α1-4 were picked and inoculated into BMGY medium. The cultures were incubated at 30 °C and 220 rpm for 24 h until OD (Organic Difference) was reached. 600 Values ​​range from 2 to 6. Based on measured OD values. 600 Adjust the volume of the BMGY culture medium, collect the cells at 3000 rpm for 10 min, and resuspend the cells in an equal volume of BMMY medium to adjust the initial OD. 600 The value was 2.0. The culture was continued at 30 ℃ and 220 rpm, with 0.5% methanol added to the medium every 24 h. After 72 h of methanol induction, bacterial samples were collected, and the expression supernatant was collected by centrifugation. SDS-PAGE electrophoresis was used to analyze the protein expression in the supernatant of the recombinant strain. The expressed target proteins were named ColⅤα1-1 (molecular weight 8.18 kD), ColⅤα1-2 (molecular weight 16.30 kD), and ColⅤα1-4 (molecular weight 33.20 kD), respectively. The expression level identification results are shown below. Figure 1 and Figure 2 .

[0044] Step 5): Using a strong anion exchange medium (Q Purose 6 FF from Dongfulong Qianchun Biotechnology, loaded into a GE Akta chromatography system), equilibrate the column with phosphate buffer (20 Mm, pH 7.0) until the conductivity and A280 absorbance remain constant. Set the sample loading flow rate to 5 mL / min and monitor the UV A280 absorbance. When the absorbance rises, begin sample loading. After loading, equilibrate the chromatography medium again with phosphate buffer until the conductivity and A280 absorbance reach their minimum and no longer change, indicating the end of equilibration. Elute with phosphate buffer containing NaCl (500 mM) and collect the corresponding proteins to obtain recombinant type V collagen α1-1, recombinant type V collagen α1-2, and recombinant type V collagen α1-4 (e.g., ...). Figure 3 ).

[0045] Experimental Example 1 The Hacat cell adhesion-promoting activities of recombinant type V collagen α1-1, recombinant type V collagen α1-2, and recombinant type V collagen α1-4 described in Example 1 were tested respectively: Dilute to 0.5 μg / mL with 20 mM pH 7.4 PBS solution and perform serial dilutions (2-fold) in 96-well plates, for a total of 10 dilutions, 50 μL per well. Each sample is tested in triplicate. 20 mM PBS is used as a negative control, and a commercially available recombinant Col-V protein control is used as a positive control. Incubate overnight at 4°C. After incubation, discard the liquid in the plate, add 100 μL of 30 g / L BSA to each well for blocking, and incubate at 37°C for 1 h. Remove the plate, discard the liquid, and add 1.0 × 10⁶ Hacat immortalized human skin cells in logarithmic growth phase to each well. 5 / mL, 100μL per well, incubated at 37℃ for 5h. After incubation, the cell plates were washed three times with 20mM PBS, and the cell adhesion was observed under a microscope. Five points were selected under a 200x microscope (excluding the edge) to count the number of adherent cells. The titer was obtained by fitting a four-parameter curve based on the counting results.

[0046] The results are shown in Table 1. It can be seen that the recombinant type V collagen prepared in Example 1 of this application has a significantly higher effect on promoting Hacat cell adhesion than the commercial recombinant Col-V protein control. Furthermore, the cell adhesion activity increases with the increase of the n cycle number, with the highest activity when n is 4.

[0047] Table 1. Hacat cell adhesion-promoting activity of recombinant type V collagen

[0048] Experimental Example 2 The proliferation-promoting activities of recombinant type V collagen α1-1, recombinant type V collagen α1-2, and recombinant type V collagen α1-4 described in Example 1 on NIH 3T3 cells were tested respectively. Referring to Appendix 3528 of the 2020 edition (Part III) of the Chinese Pharmacopoeia, based on the stimulatory effect of recombinant type V collagen on the growth of mouse embryonic fibroblasts (NIH 3T3 cells), and considering the differences in the biological activities of different type V collagens in the growth status of NIH 3T3 cells, the biological activity of recombinant type V collagen in promoting cell proliferation in vitro was tested.

[0049] 2.1 Test Materials: Complete cell culture medium: 1640 culture medium (containing penicillin and streptomycin) with 10% fetal bovine serum added, stored at 4°C; Serum-free culture medium: 1640 culture medium (containing penicillin and streptomycin), stored at 4°C; Digestive fluid: 0.25% trypsin; Thiazol blue (MTT) solution: Weigh 0.10 g of MTT powder, add 20 mL of PBS to dissolve it, filter through a 0.22 μm filter membrane for sterilization, and store at 4°C protected from light; NIH 3T3 cells (purchased from Wuhan Pronosai); Samples: Recombinant type V collagen α1-1, recombinant type V collagen α1-2 and recombinant type V collagen α1-4 from Example 1, and commercial recombinant Col-V protein control.

[0050] 2.2 Specific implementation methods: The NIH 3T3 cell line was cultured in complete cell culture medium at 37°C and 5% carbon dioxide, with a cell concentration controlled at 5.0 × 10⁻⁶ cells / year. 5 / mL, used for biological activity assay 24-36h after subculturing; discard the culture medium in the culture flask, digest and collect the cells, and prepare 5.0×10⁶ cells / mL solution with complete cell culture medium. 5 Cell suspension of 100 μL / mL was seeded into 96-well cell culture plates and cultured at 37°C with 5% CO2. After 24 h, the solution was replaced with 20 mM PBS buffer and cultured at 37°C with 5% CO2 for another 24 h. The PBS buffer was discarded, and 100 μL of the test solution was added to each well. The plates were then cultured at 37°C with 5% CO2 for 64-72 h. 20 μL of MTT solution was added to each well and the plates were cultured at 37°C with 5% CO2 for 5 h. After discarding the liquid in the culture plates, 100 μL of DMSO was added to each well, mixed well, and the absorbance was measured at 570 nm using a microplate reader with 630 nm as the reference wavelength. The results were recorded.

[0051] 2.3 Data Processing: The experimental data were processed using a computer program or a four-parameter regression calculation method. The results are shown in Table 2 below. It can be seen that the recombinant type V collagen prepared in Example 1 of this application has a significantly higher effect on promoting the proliferation of NIH 3T3 cells than the commercial recombinant Col-V protein control. Furthermore, the cell proliferation activity increases with the increase of the n cycle number, with the highest activity when n is 4.

[0052] Table 2. Cell proliferation-promoting activity of recombinant type V collagen.

[0053] Experimental Example 3: Recombinant Type V Collagen Promotes Hacat Cell Migration Activity The specific method is as follows: 3.1 Using a black marker, draw horizontal lines evenly on the back of a 6-well cell culture plate, aligning with a ruler, with each line approximately every 0.5cm to 1cm, passing through the wells. 3.2 Collect Hacat cells in logarithmic growth phase and adjust the cell density to 5 × 10⁻⁶. 5 Add 2 mL / well to the above 6-well cell plate and incubate at 37°C with 5% CO2 for 24 h. Once the cells have grown into a monolayer, use a pipette tip to make two horizontal lines perpendicular to the back of the plate, using a ruler as a guide. Wash the cells three times with PBS to remove the suspended cells that were drawn. Then add 1.8 mL of serum-free DMEM medium to the well.

[0054] 3.3 Add recombinant type V collagen to the wells and use commercial recombinant Col-V protein as a positive control. The concentration of all proteins was 50 μg / mL (at which concentration there is no cytotoxicity). PBS was used as a blank control.

[0055] 3.4 The 6-well cell culture plates containing the samples to be tested were placed in a 37℃, 5% CO2 incubator for 24 hours. Photos were taken at hour 0 of culture, recording the location of each well. At hour 24 of culture, observations and photos were taken at the fixed locations. ImageJ software was used to process the cell migration images to obtain the initial scratch area and the area of ​​the cell-free blank region, and the migration rate was calculated. The migration rate was calculated as: (Area of ​​cell-free blank region / Initial scratch area) × 100%.

[0056] Results of cell control wells and individual sample wells at 0h and 18h Figure 7 As shown. The effect of each treatment group on promoting the migration of HaCaT keratinocytes was also calculated.

[0057] Compared with the blank control and the commercial recombinant Col-V protein control, the recombinant type V collagen of this application showed a significantly higher effect on promoting HaCat cell migration, and the cell migration-promoting activity increased with the increase of n cycles.

[0058] Experiment Example 4: Soothing Efficacy Test – In Vitro Determination of TNF-α and IL-6 Levels 4.1 Toxicity test based on mouse macrophage RAW264.7 Refer to Biological Evaluation of Medical Devices Part 5: In Vitro Cytotoxicity Tests (GB / T 16886.5-2017). Cytotoxicity is assessed by observing cell morphology and measuring the MTT assay, and by quantifying the number of live and dead cells in the sample and measuring cell metabolic activity.

[0059] Experimental materials: Complete cell culture medium: DMEM culture medium (containing double antibiotics) with 10% fetal bovine serum added, stored at 4°C.

[0060] Serum-free culture medium: DMEM culture medium (containing double antibiotics), stored at 4℃.

[0061] Digestive fluid: 0.25% trypsin.

[0062] PBS buffer: Weigh 8.0g sodium chloride, 0.20g potassium chloride, 1.44g disodium hydrogen phosphate, and 0.24g potassium dihydrogen phosphate, dissolve in water and bring the volume to 1000mL, then autoclave at 121℃ for 15 minutes.

[0063] MTT (thiazolyl blue) solution: Weigh 0.10 g of MTT powder, add 20 mL of PBS to dissolve, and filter through a 0.22 μm filter membrane for sterilization. Store at 4°C protected from light.

[0064] Mouse macrophages RAW264.7: purchased from the Cell Culture Center of the Chinese Academy of Medical Sciences.

[0065] Samples: Recombinant type V collagen α1-1, recombinant type V collagen α1-2 and recombinant type V collagen α1-4, and commercial recombinant Col-V protein control.

[0066] Specific implementation method: RAW264.7 macrophage cell line was cultured in complete culture medium at 37℃ and 5% CO2, with the cell concentration controlled at 1.0×10⁻⁶. 5 Cells / mL, after passage, discard the culture medium in the culture flask 24 hours later. After digestion, collect cells with complete culture medium and prepare 5.0 × 10⁶ cells / mL solutions. 5 / mL cell suspension. 100 μL of cell suspension was seeded into each well of a 96-well plate and cultured for 24 h. After the cells formed a monolayer, the culture medium was discarded, and 100 μL of different concentrations of experimental sample diluent (100%, 50%, 25%, 12.5%, 6.25%, 3.13%, 1.56%, 0.78%), blank control (culture medium), and negative control (PBS) were added to each well, with 6 replicates per group. The plates were incubated at 37°C with 5% CO2. After 24 hours, the 96-well plates were removed, and cell morphology was observed under a microscope. The liquid was then aspirated, and 50 μL of MTT solution was added to each well. The plates were incubated in a CO2 incubator for 2 h, and the MTT solution was discarded. 100 μL of LDMSO solution was added to each well. The plates were shaken, and the absorbance was measured at 570 nm (reference wavelength 650 nm) using a microplate reader. Cell viability was calculated. Compared with the cell control group, when the cell viability was lower than that of the blank control group, the sample was cytotoxic. Conversely, there is no cytotoxicity.

[0067] The statistical method is as follows: Mean ± Standard Deviation ±s); Survival rate (%) = (Experimental group OD 570 Mean / Blank Control Group OD 570 (mean) * 100%.

[0068] Result: As Figure 4 As shown, compared with the NC group, the relative cell viability was >90%, and there was no significant difference (P>0.05). Cell morphology showed no significant change compared to the NC group. The highest dose group, i.e., 10 μg / mL, with an average cell viability exceeding 90%, was selected.

[0069] 4.2 Enzyme-linked immunosorbent assay (ELISA) for the detection of TNF-α and IL-6 levels Studies have shown that lipopolysaccharide (LPS) can induce a strong inflammatory response in RAW264.7 macrophages, manifested by upregulation of the expression levels of inflammatory factors TNF-α and IL-6 and increased cytokine secretion. By establishing an LPS-induced pro-inflammatory differentiation model of RAW264.7 macrophages, the anti-inflammatory effects of the samples in vitro were investigated.

[0070] Experimental materials: Cell lines, culture media and others: Same as 4.1.

[0071] Main reagents: Lipopolysaccharide (Sigma), dexamethasone (Sinopharm), TNF-α and IL-6 ELISA detection kit (purchased from R&D Company).

[0072] Specific implementation method: RAW264.7 cells passaged for 24 hours were digested, resuspended, and diluted to 1×10⁻⁶. 5100 μL of cell suspension was added to each well of a 96-well plate and incubated at 37°C with 5% CO2 for 24 h. Then, all culture medium was discarded, and subsequent culture medium used was FBS-free DMEM basal medium. 0.9 mL of DMEM culture medium containing different components was added to different groups (see Table 3 for specific groupings), with three replicates per group, and incubated at 37°C with 5% CO2 for 6 h. After pretreatment, the blank control group was added with 0.1 mL of DMEM culture medium, and the LPS model group and each drug-treated group were added with 0.1 mL of DMEM culture medium containing 10 μg / mL LPS, and incubated at 37°C with 5% CO2 for 24 h. Cell culture supernatant was collected 24 h after stimulation. The levels of cytokines TNF-α and IL-6 were detected using an ELISA kit. Specific procedures were performed according to the manufacturer's instructions.

[0073] Data processing and analysis: All experimental results were repeated three times and expressed as mean ± standard deviation. One-way ANOVA (SNK test) and multiple comparisons were performed using SPSS 22.0 for anti-inflammatory activity data. The inhibition rate of TNF-α and IL-6 was calculated as follows: Inhibition rate (%) = (1 - T / C) × 100%, where: T: average content of TNF-α and IL-6 in the test substances; C: average content of TNF-α and IL-6 in the negative controls.

[0074] Table 3. Grouping of RAW264.7 cell inflammation model experiment

[0075] Test results: According to Table 4 of the test results Figure 5 and Figure 6 Compared with the blank control, the negative control showed significantly upregulated expression of TNFα and IL-6, indicating the validity of the experimental system. Compared with the negative control (LPS), the positive control (dexamethasone) showed an inhibition rate of 31.61% for TNFα and 29.67% for IL-6. The results of TNFα and IL-6 level detection and inhibition rates are shown in Table 4. The experimental results indicate that recombinant type V collagen can significantly inhibit the secretion of TNFα and IL-6, and the anti-inflammatory effect increases with the number of cycles.

[0076] Table 4. Statistical table of inhibition rates of TNFα and IL-6 levels.

[0077] Experimental Example 5: The thermal stability of the recombinant humanized type V collagen prepared in Example 1 was tested.

[0078] The recombinant humanized type V collagen prepared in Example 1 was diluted to 1 mg / mL with 20 mM pH 7.0 PBS solution and dispensed into 5 mL vials (2 mL per vial). These vials were placed in a 60°C constant temperature and humidity incubator for accelerated degradation experiments. Samples were collected at 0 h, 7 d, 14 d, 28 d, and 60 d to detect the effects on BALB / c 3T3 mouse embryo fibroblast adhesion activity and HaCaT keratinocyte migration and adhesion at each time point. The detection methods were the same as in Examples 1, 2, and 3. The results of the thermostability test on HaCaT keratinocyte adhesion activity are shown in Table 5, the results on the thermostability test on BALB / c 3T3 mouse embryo fibroblast adhesion activity are shown in Table 6, and the results on the thermostability test on HaCaT keratinocyte migration activity are shown in Table 7.

[0079] Table 5. Thermal stability test results show the activity of HaCaT keratinocytes in promoting adhesion.

[0080] Table 6. Thermal stability test results on the proliferation activity of BALB / c 3T3 mouse embryonic fibroblasts.

[0081] Table 7. Thermal stability test promotes HaCaT keratinocyte migration rate.

[0082] As shown in Table 5, in the accelerated degradation experiment at 60℃, the recombinant humanized type V collagen α1-1, recombinant humanized type V collagen α1-2, and recombinant humanized type V collagen α1-4 showed almost no decrease in their HaCaT keratinocyte adhesion activity after heating for 7 and 14 days. However, after heating for 28 and 60 days, the HaCaT keratinocyte adhesion activity remained above 80% of its initial value. Commercially available human type V collagen lost its activity after heating at 60℃ for 7 days.

[0083] As shown in Table 6, in the accelerated degradation experiment at 60℃, recombinant humanized type V collagen α1-1, recombinant humanized type V collagen α1-2, and recombinant humanized type V collagen α1-4 maintained over 90% of their proliferative activity in promoting BALB / c 3T3 mouse embryonic fibroblasts after heating for 7 and 14 days, and still maintained over 80% of their proliferative activity after heating for 28 and 60 days. Commercially available human type V collagen lost its activity after heating at 60℃ for 7 days.

[0084] As shown in Table 7, in the accelerated degradation experiment at 60℃, the recombinant humanized type V collagen α1-1, recombinant humanized type V collagen α1-2, and recombinant humanized type V collagen α1-4 maintained over 90% of their effect in promoting HaCaT keratinocyte migration after heating for 7 and 14 days, and over 80% of their cell migration-promoting activity after heating for 28 and 60 days. Commercially available human type V collagen lost its activity after heating at 60℃ for 7 days.

[0085] Therefore, it can be seen that the selected type V collagen sequence, when synthesized using genetic engineering technology, produces recombinant humanized type V collagen with long-lasting heat resistance.

[0086] The above description is only a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for preparing heat-resistant and stable recombinant humanized type V collagen, characterized in that, Includes the following steps: Recombinant collagen sequences as shown in SEQ ID No. 2, SEQ ID No. 4, and SEQ ID No. 6 were obtained, and the recombinant collagen sequences were inserted into the pPIC9K plasmid to obtain the corresponding recombinant plasmids; the recombinant plasmids include recombinant plasmids pPIC9K-ColⅤα1-1, pPIC9K-ColⅤα1-2, and pPIC9K-ColⅤα1-4; The recombinant plasmid was subjected to electroporation, screening, identification and purification processes in sequence to obtain recombinant type V collagen α1; the recombinant type V collagen α1 includes recombinant type V collagen α1-1, recombinant type V collagen α1-2 and recombinant type V collagen α1-4.

2. The method for preparing heat-resistant and stable recombinant humanized type V collagen according to claim 1, characterized in that, The recombinant collagen with the gene sequence shown in SEQ ID No. 2 is obtained based on the target amino acid sequence shown in SEQ ID No. 1; The recombinant type V collagen with the gene sequence shown in SEQ ID No. 4 was obtained based on the target amino acid sequence shown in SEQ ID No. 3; The recombinant type V collagen with the gene sequence shown in SEQ ID No. 6 was obtained based on the target amino acid sequence shown in SEQ ID No.

5.

3. The method for preparing heat-resistant and stable recombinant humanized type V collagen according to claim 2, characterized in that, The target amino acid sequence shown in SEQ ID No. 1 is gene sequence 643 to 730 of the recombinant gene expressing the ColⅤα1 chain, containing two RGD domains; The target amino acid sequence shown in SEQ ID No. 3 is obtained by repeating the target amino acid sequence shown in SEQ ID No. 1 twice in tandem; The target amino acid sequence shown in SEQ ID No. 5 is obtained by repeating the target amino acid sequence shown in SEQ ID No. 1 four times in series.

4. The method for preparing heat-resistant and stable recombinant humanized type V collagen according to any one of claims 1-3, characterized in that, The electroconversion process includes the following steps: The recombinant plasmid was linearized with restriction endonuclease SacⅠ, then electroporated into GS115 competent cells, plated on MD solid plates, incubated at room temperature for 10 min, and then inverted in a 30℃ incubator for 2 to 5 days until single colonies appeared.

5. The method for preparing heat-resistant and stable recombinant humanized type V collagen according to claim 4, characterized in that, The screening process includes the following steps: The single colonies were inoculated onto YPD solid plates containing 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL and 4 mg / mL G418, respectively; Single colonies that could grow simultaneously on YPD solid plates containing 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL and 4 mg / mL G418 were screened and identified as high-copy recombinant strains.

6. The method for preparing heat-resistant and stable recombinant humanized type V collagen according to claim 5, characterized in that, The identification process includes the following steps: Four high-copy recombinant strains were selected and inoculated into BMGY medium, and cultured at 30 °C and 220 rpm for 24 h until OD500. 600 Values ​​range from 2 to 6; According to actual measured OD 600 Adjust the volume of the BMGY culture medium, collect the cells at 3000 rpm for 10 min for resuspending, and adjust the initial OD value. 600 The value is 2.0; The culture was continued at 30 ℃ and 220 rpm. 0.5% methanol was added to the culture medium every 24 h. After 72 h of methanol induction, bacterial culture samples were taken and the expression supernatant was collected by centrifugation. The expression of proteins in the supernatant of the recombinant strain was analyzed by SDS-PAGE electrophoresis to obtain the target protein.

7. The method for preparing heat-resistant and stable recombinant humanized type V collagen according to claim 6, characterized in that, The purification process includes the following steps: The target protein was subjected to a strong anion exchange medium. The chromatography column was equilibrated with phosphate buffer until the conductivity and A280 absorbance remained constant. The sample loading flow rate was set to 5 mL / min. The UV A280 absorbance was detected. When the absorbance rose, the sample loading began. After the sample loading is completed, the chromatography medium is equilibrated with phosphate buffer until the UV A280 absorbance and conductivity values ​​drop to their minimum and no longer change, at which point the equilibration is complete. The protein was then eluted with a phosphate buffer containing NaCl and collected to obtain recombinant type V collagen.

8. A heat-resistant and stable recombinant humanized type V collagen, characterized in that, The recombinant humanized type V collagen is prepared by the method described in any one of claims 1-7; the heat-resistant and stable recombinant humanized type V collagen includes recombinant type V collagen α1-1, recombinant type V collagen α1-2 and recombinant type V collagen α1-4.

9. The heat-resistant and stable recombinant humanized type V collagen according to claim 8, characterized in that, The recombinant type V collagen α1-1 showed a Hacat cell adhesion-promoting activity of 1.10 × 10⁻⁶. 4 U / mg; The recombinant type V collagen α1-2 showed a Hacat cell adhesion-promoting activity of 1.27 × 10⁻⁶. 5 U / mg; The recombinant type V collagen α1-4 exhibited Hacat cell adhesion-promoting activity of 9.58 × 10⁻⁶. 5 U / mg.

10. The heat-resistant and stable recombinant humanized type V collagen according to claim 8, characterized in that, Recombinant type V collagen α1-1 showed a proliferation-promoting activity of 1.43 × 10⁻⁶ NIH 3T3 cells. 4 U / mg; Recombinant type V collagen α1-2 showed a proliferation-promoting activity of 2.34 × 10⁻⁶ NIH 3T3 cells. 4 U / mg; Recombinant type V collagen α1-4 showed a proliferation-promoting activity of 9.38 × 10⁻⁶ NIH 3T3 cells. 4 U / mg.

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