Recombinant human IV-type collagen as well as production method and application thereof

Abstract

The invention discloses a recombinant human IV type collagen as well as a production method and application thereof. The amino acid sequence of the recombinant human IV type collagen is shown as SEQ ID No: 1. The recombinant human IV-type collagen is a truncated protein of a human IV-type collagen alpha1 chain, special expression elements and means are not needed, the yield of the recombinant human IV-type collagen can reach up to 18g / L only through extracellular secretory expression of a commercial pichia pastoris expression system, the yield is obviously higher than the high yield level of the recombinant collagen considered in the technical field, and the recombinant human IV-type collagen can be used for preparing the recombinant human IV-type collagen. The total length of the alpha 1 chain of the human IV-type collagen or other truncated proteins is obviously higher than that of the human IV-type collagen. In addition, the safety and biological activity of the recombinant human IV-type collagen are superior to the full length of the alpha 1 chain of the human IV-type collagen.

Description

Technical Field

[0001] The invention belongs to the technical field of synthetic biology, and specifically relates to a high-yield genetically engineered recombinant human type IV collagen, a production method and use thereof. Background Art

[0002] Collagen is a biopolymer protein and the main component of animal connective tissue. It is also the most abundant and widely distributed functional protein in mammals, accounting for 25% to 30% of the total protein. There are many types of collagen, the most common types are type I, type II, type III, type V and type XI. Collagen has good biocompatibility, biodegradability and biological activity, so it is widely used in food, medicine, tissue engineering, cosmetics and other fields.

[0003] Currently, genetic engineering recombinant expression is one of the important methods for producing collagen. In particular, recombinant human collagen, whose amino acid composition is consistent with that of the human body, is considered to be the closest to the collagen naturally synthesized by the human body, with good biocompatibility and high safety. The commercial recombinant human collagen products developed are widely used in medical cosmetology, cosmetics, medical devices, health care products and other fields.

[0004] At present, the production of recombinant collagen mainly has the following problems: 1) easy degradation. Because recombinant collagen cannot form a stable triple helix structure like natural collagen under the existing expression system, it is easily degraded by the protease in the production system; 2) difficult to purify. Collagen is easy to degrade, resulting in the need to remove a large amount of degraded proteins in addition to host protein interference during the purification process of the prepared recombinant collagen, which increases the difficulty of purification and makes it difficult to prepare high-purity target proteins; 3) low yield. Due to the existence of degradation problems, the yield of recombinant collagen is low, far from people's expectations. It is generally believed that when using commercial expression hosts and expression vectors, the yield of recombinant collagen with a molecular weight of more than 70,000 reaches 10g / L, which is already a very high yield. Therefore, the types of mass-produced recombinant collagen are relatively small, which seriously restricts the further application of recombinant collagen.

[0005] As the market acceptance of recombinant collagen gradually increases, the market demand for recombinant collagen is gradually increasing. Therefore, recombinant collagen that can be mass-produced is urgently needed. Recombinant collagen that can be mass-produced must have the characteristics of high single-batch yield, good stability of expressed protein, and easy purification. Therefore, high-yield, good stability, and easy purification recombinant collagen is urgently needed in the current market.

[0006] Recombinant collagen can be obtained by constructing truncated proteins or truncated peptides of natural collagen. The uncertainty of the position and length of the truncated protein in natural collagen makes it possible to have tens of millions of recombinant collagen types in theory. However, there has been a lack of effective theoretical guidance on which of these recombinant collagens can have a higher secretion yield. The best results must be screened out through a large number of experimental verifications. This is the main reason why there are currently few types of recombinant collagen in mass production. Summary of the invention

[0007] In view of the technical problems existing in the above-mentioned prior art, the object of the present invention is to provide a recombinant human type IV collagen with high secretion yield, good safety and strong biological activity.

[0008] The present invention comprises:

[0009] 1. A recombinant human type IV collagen, the amino acid sequence of which is shown in SEQ ID No: 1.

[0010] SEQ ID No:1

[0011]

[0012]

[0013] 2. A nucleic acid encoding the recombinant human type IV collagen described in item 1. The nucleotide sequence of the nucleic acid may be, for example, as shown in SEQ ID No: 2.

[0014] 3. An expression vector comprising the nucleic acid described in item 2.

[0015] 4. A host cell introduced with the expression vector according to Item 3. The host cell is preferably a eukaryotic cell, for example, Pichia pastoris.

[0016] 5. A method for producing the recombinant human type IV collagen described in item 1, comprising culturing the host cell described in item 4 to secrete and express the recombinant human type IV collagen, and then isolating and purifying it.

[0017] 6. The method according to item 5, wherein the separation and purification is carried out by one or a combination of salting out, chromatography, affinity chromatography, acid-base precipitation, and membrane separation. Preferably, the combination of chromatography and membrane separation, or the combination of ion exchange chromatography and membrane separation is used.

[0018] 7. Use of the recombinant human type IV collagen according to item 1 in the preparation of cosmetics or medical devices.

[0019] 8. The use according to item 8, wherein the cosmetic or medical device is used to improve skin redness and repair sensitive skin.

[0020] 9. The use according to item 8, wherein the cosmetic or medical device is prepared in a form suitable for application to the skin, such as a facial mask, a facial cream, a spray, an essence water, etc.

[0021] The beneficial effects of the present invention are:

[0022] 1. High yield: In the technical field, it is generally believed that when using commercial expression hosts and expression vectors, the yield of recombinant collagen with a molecular weight of more than 70,000 reaches 10g / L, which is already quite high. The present invention provides a recombinant human type IV collagen composed of a new amino acid sequence. The recombinant human type IV collagen is a truncated protein of the α1 chain of human type IV collagen. No special expression elements and means are required. The yield of the recombinant human type IV collagen can be as high as 18g / L only through extracellular secretion expression of the commercial Pichia pastoris expression system, which is significantly higher than the high yield level of recombinant collagen considered in the technical field, and also significantly higher than the full-length α1 chain of human type IV collagen or other truncated proteins. The results of the extracellular protein SDS-PAGE test showed that the target protein accounted for more than 70%, and the purity could reach 80% after simple ultrafiltration decolorization.

[0023] 2. The cytotoxicity is significantly lower than that of the full-length human type IV collagen α1 chain (see Example 3).

[0024] 3. Its cell adhesion promoting activity and its effects on improving skin redness and repairing sensitive skin are significantly better than the full-length human type IV collagen α1 chain (see Examples 3 and 4). BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the SDS-PAGE protein electrophoresis diagram of the purified recombinant human type IV collagen-α1.

[0026] Figure 2 The graph shows the effect of different concentrations of recombinant human type IV collagen-α1 solution on cell viability.

[0027] Figure 3 The graph shows the effect of different concentrations of full-length human type IV collagen α1 chain solutions on cell viability.

[0028] Figure 4 Fluorescence microscopy images of cells before and after centrifugation.

[0029] Figure 5 The figure shows an example of how recombinant human type IV collagen-α1 dressing improves skin redness and repairs skin.

[0030] Figure 6 The figure shows an example of the improvement of skin redness and repair by full-length type IV collagen α1 chain dressing. DETAILED DESCRIPTION

[0031] Example 1 Preparation of recombinant human type IV collagen-α1 in a yeast expression system

[0032] 1. Experimental Methods

[0033] 1. Preparation of shuttle plasmid

[0034] According to the amino acid sequence shown in SEQ ID No: 1 (named as recombinant human type IV collagen-α1), the codon optimization of the yeast expression system was performed to obtain the type IV target gene sequence as shown in SEQ ID No: 2. The obtained target gene sequence was entrusted to Qingke Biotechnology Co., Ltd. for gene synthesis, and the synthesized gene was connected to the pPicZαA plasmid to obtain the pPicZαA-I-α1 plasmid.

[0035] SEQ ID No:2

[0036]

[0037] 2) Preparation of yeast expression strains

[0038] pPicZαA-I-α1 was linearized with Pme I and then transformed into Pichia pastoris X-33 competent cells. Bleomycin resistance was used as a selection marker to screen transformants to obtain a yeast expression strain.

[0039] 2. Induced expression of target protein

[0040] (1) Pick a single colony of the constructed yeast expression strain and add it to 5 ml of YPD liquid medium (1% yeast extract, 2% peptone and 2% glucose), and culture it at 30°C and 200 rpm for 48 h for activation;

[0041] (2) Transfer the inoculum to a 500 ml Erlenmeyer flask (containing 200 ml of YPD culture medium) at a 1% inoculum volume and culture at 30°C and 200 rpm for 24 h to serve as the seed for the tank.

[0042] (3) Prepare 3 L of BSM medium and add it to a 5 L fermenter, sterilize at 121° C. for 20 min, and after cooling to 30° C., adjust the pH to 5.0, and add the seeds prepared in (2) to the fermenter by flame inoculation for fermentation;

[0043] (4) Cultivate until OD 600 When OD was 70, 50% glycerol was added until 600 When the temperature reaches about 120, stop adding, and wait until the dissolved oxygen rebounds to 100%, then start adding methanol for induction;

[0044] (5) During the induction process, the dissolved oxygen was controlled to be not less than 30% and the pH was about 5.0. The induction was continued for 40 h, and the fermentation was terminated. The culture medium was centrifuged at 12,000 rpm for 2 min, and the supernatant was taken for detection of protein yield and purity using the BCA method and SDS-PAGE method.

[0045] 3. Collagen purification

[0046] The collected supernatant was passed through a 30KD ultrafiltration membrane, and then passed through a hollow fiber column and freeze-dried to obtain recombinant human type IV collagen-α1.

[0047] 2. Results

[0048] The BCA test result of the fermentation tank yield of recombinant human type IV collagen-α1 prepared by the above method is as high as 18g / L. The SDS-PAGE protein electrophoresis test result shows that the target protein accounts for more than 70%, and the protein purity after ultrafiltration through the hollow fiber column can reach more than 80%. The SDS-PAGE protein electrophoresis test results of the purified recombinant human type IV collagen-α1 are as follows Figure 1 shown.

[0049] Comparative Example 1 Preparation of other recombinant human type IV collagen (control protein) and full-length type IV collagen α1 chain in yeast expression system

[0050] The same design concept as recombinant human type IV collagen-α1 was used to design protein sequences with similar molecular weights, and the target gene was obtained after codon optimization. After the target gene was fully synthesized, the same shuttle plasmid and host bacteria were used to express the protein sequence. At the same time, the full-length type IV collagen α1 chain was expressed in the same way. The results of sequence design and expression are shown in the following table (Table 1).

[0051] Table 1 Different amino acid sequences and expression results

[0052]

[0053] From the results of Example 1 and Comparative Example 1, it can be seen that the yield of the recombinant human type IV collagen-α1 of the present invention is much higher than that of similarly designed sequences intercepted at different positions and different sizes, and is also much higher than that of the full-length type IV amino acid sequence. Therefore, the recombinant human type IV collagen-α1 of the present invention is easier to prepare on a large scale and has greater potential for mass production.

[0054] For a long time, there has been a lack of effective theoretical guidance on which recombinant collagen can have a higher secretion yield, and a large number of experiments must be used for screening and verification. Therefore, high-yield recombinant collagen is extremely rare. In the technical field, it is generally believed that when a commercial expression host and expression vector are used without adding special expression elements and expression means, the yield of recombinant collagen with a molecular weight of more than 70,000 reaches 10g / L, which is already quite high. The yield of the recombinant human type IV collagen of the present invention can be as high as 18g / L, which is significantly higher than the high yield level of recombinant collagen considered in the technical field.

[0055] Example 3 Evaluation of the biological activity of recombinant human type IV collagen-α1

[0056] 1. Cytotoxicity experiment HeLa cells grown to 70%-80% of the bottom area of ​​the culture flask were digested with 0.25% trypsin and made into a cell density of 1×10 5 / mL of cell suspension. Take 100μL of cell suspension and inoculate it in a 96-well culture plate and place it in an incubator at 37℃ and 5% CO2 saturated humidity. After 24h of cell culture, aspirate the complete culture medium. In the experimental group, different concentrations of recombinant human type IV collagen-α1 solution (0.001-0.5mg / ml, 4 replicates for each concentration) diluted with high-glucose DMEM medium and different concentrations of full-length human type IV collagen-α1 chain solution (0.001-0.5mg / ml, 4 replicates for each concentration) were added. The control group was cells cultured in DMEM medium, and the blank group was DMEM medium without cells. Continue to culture in an incubator at 37℃ and 5% CO2 saturated humidity for 24h. Add 10μL of CCK-8 reagent to each group, incubate in the cell culture incubator for 2h, and use an enzyme-linked immunosorbent assay to measure the absorbance value (OD value) of each well at a wavelength of 450nm. The cell survival rate was calculated according to the mean absorbance of each group using the following formula:

[0057] Experimental results: Figure 2 As shown, the cell viability of each concentration experimental group of the recombinant human type IV collagen-α1 solution was above 90%, indicating that the recombinant human type IV collagen-α1 of different concentrations prepared by the present invention had no cytotoxicity and good safety. Figure 3 As shown, the cell viability of the full-length human type IV collagen-α1 chain solution experimental group gradually decreased with the increase of concentration, especially when the concentration exceeded 0.1 mg / mL, the cell morphology began to change and the viability decreased significantly, indicating that the high-concentration full-length human type IV collagen-α1 chain solution has potential cytotoxicity.

[0058] 2. Cell Adhesion Assay NIH / 3T3 cells were cultured in a 37°C, 5% CO2 cell culture incubator, and the cell density and status were observed under an inverted microscope every day. When the cells grew to 80% to 90% of the culture flask, cell passage or cell inoculation was performed. Referring to the steps of Appendix B (Normative) Cell Adhesion Assay-Centrifugation Method of the Pharmaceutical Industry Standard of the People's Republic of China YY / T 1849-2022 "Recombinant Collagen", the cells were diluted to 5×10 using complete culture medium premixed with Hoechst 33342 fluorescent thermochromic agent (10%). 4 / mL, 100μL of cell suspension was added to a 96-well plate, covered with aluminum foil, and incubated at 37°C, 5% CO2 for 1h. The prepared D-PBS solution, recombinant human type IV collagen-α1 solution (0.05mg / mL) and full-length human type IV collagen-α1 chain solution (0.05mg / mL) were added to a 96-well culture plate, 4 wells were prepared for each sample coating, incubated in an incubator at 37°C, 5% CO2 for 2h, and the fluorescent tiled images were captured using a fluorescent inverted microscope. Subsequently, each well was filled with D-PBS, the bubbles were purged and covered with a sealing film, and inverted centrifuged at 350g for 5min. After centrifugation, the sealing film was discarded and the supernatant was removed from the well. After washing once with D-PBS, 100μL D-PBS was added and the fluorescent tiled images were captured again using a fluorescent inverted microscope. Finally, the number of fluorescently labeled cells was counted using ImageJ software, and data processing and analysis were performed.

[0059] Use an automated cell counting program to count the number of fluorescently labeled nuclei and determine the number of cells before and after centrifugation. Calculate the percentage of adhesion according to the formula:

[0060] V=Nt / Nc×100%

[0061] Where: V---adhesion percentage; Nt--cell number after centrifugation; Nc--cell number before centrifugation.

[0062] Using D-PBS as the blank control sample, recombinant human type IV collagen-α1 solution (0.05 mg / mL) and full-length human type IV collagen-α1 chain solution (0.05 mg / mL) were tested. By measuring the adhesion of cells under the same centrifugation conditions, the effects of the two type IV collagens on cell adhesion were compared and evaluated. The results are shown in the chart. Under the conditions of this experiment, the adhesion percentage (%) of the recombinant human type IV collagen-α1 solution was 47.546±3.045, which was significantly better than the adhesion percentage (%) of the full-length human type IV collagen-α1 chain solution of 26.518±2.089 (P<0.05). The specific experimental results are as follows Figure 4 , as shown in Table 2.

[0063] Table 2 Adhesion percentage of different experimental groups

[0064]

[0065] Example 4 Efficacy of recombinant collagen type IV protein-α1 on human skin

[0066] The recombinant human type IV collagen-α1 and full-length type IV collagen α1 chain protein prepared in Example 1 are respectively prepared into dressings, and 0.1% of recombinant collagen type IV protein, 0.1% of full-length type IV collagen α1 chain protein, 0.1% of glycerol, 0.1% of sodium hyaluronate, 0.02% of preservative, and the rest is purified water. The preparation steps are as follows: (1) Accurately weigh each component, dissolve in purified water, and stir and mix thoroughly; (2) Fill 28 mL into a medical aluminum foil bag containing a facial mask non-woven fabric substrate, irradiate and sterilize, and seal to obtain a collagen patch product.

[0067] Methods: 35 Asian healthy adult female subjects aged 25 to 45 years old used recombinant collagen type IV protein-α1 dressing samples and full-length type IV collagen α1 chain dressing samples on their faces continuously under normal circumstances for 7 days each. The redness of the facial skin of the subjects was randomly tested on the 0th, 3rd, and 7th days of use. The average values ​​of the Visia-CR redness a value test results of the 35 subjects were calculated. After statistical software analysis, the differences between the recombinant collagen type IV protein-α1 dressing samples and the full-length IV collagen α1 chain dressing samples on the 3rd and 7th days of use and before use were compared.

[0068] Table 3: Visia-CR red zone redness a value detection results:

[0069]

[0070] *The data in the table are mean ± standard error

[0071] Table 4: Visia-CR red zone redness a value detection results:

[0072]

[0073] *Statistical methods: Rank sum test and t-test were used for analysis, with the test level α=0.05.

[0074] *Significance marking method: (“ns” indicates no statistical difference, p≥0.05; p<0.05 indicates a significant difference (“*” indicates 0.01≤p<0.05; “**” indicates 0.001≤p<0.01; “***” indicates p<0.001).

[0075] *Number of people using the product = 30.

[0076] Test results: As shown in Tables 3 and 4, after using the recombinant collagen type IV protein-α1 dressing sample, the redness a value of the skin red area on the 3rd day of use was not significantly different from that before use, while on the 7th day of use, the redness a value of the skin red area was significantly improved, and the difference was significant.

[0077] Reference Figure 5 , Figure 6 As shown in the imaging, the skin redness of the recombinant collagen type IV protein-α1 dressing sample was significantly reduced on the 3rd and 7th days after use compared with before use, with a significant difference, especially on the 7th day.

[0078] Test conclusion: The recombinant collagen type IV protein-α1 provided by the present invention has a significant effect of improving skin redness and repairing sensitive skin, and its effect is significantly better than the full-length recombinant collagen type IV protein α1 chain.

Claims

1. A recombinant human type IV collagen, the amino acid sequence of which is shown in SEQ ID No:

1.

2. A nucleic acid encoding the recombinant human type IV collagen according to claim 1; the nucleotide sequence of the nucleic acid may be, for example, as shown in SEQ ID No:

2.

3. An expression vector comprising the nucleic acid of claim 2.

4. A host cell into which the expression vector according to claim 3 has been introduced; the host cell is preferably a eukaryotic cell, such as Pichia pastoris.

5. A method for producing the recombinant human type IV collagen according to claim 1, comprising culturing the host cell according to claim 4 to secrete and express the recombinant human type IV collagen, and then separating and purifying it.

6. The method according to claim 5, wherein: The separation and purification adopts one or a combination of salting out, chromatography, affinity chromatography, acid-base precipitation, and membrane separation; preferably, a combination of chromatography and membrane separation, or a combination of ion exchange chromatography and membrane separation.

7. Use of the recombinant human type IV collagen according to claim 1 in the preparation of cosmetics, external medicines or medical devices.

8. The use according to claim 8, wherein The cosmetics, external medicines or medical devices are used to improve skin redness and repair sensitive skin.

9. The use according to claim 8, wherein The cosmetics, external medicines or medical devices are prepared in a form suitable for application to the skin, such as facial masks, facial creams, sprays, essence water, etc.

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