Recombinant IV type collagen for adjuvant therapy of xerodermia

Abstract

The invention discloses a recombinant IV type collagen capable of being used for adjuvant therapy of xerodermia. The amino acid sequence of the recombinant IV type collagen is as shown in SEQ ID No. 1. The novel recombinant IV type collagen provided by the invention has the advantages that the yield is high; in addition, synthesis of lipid-related enzymes is adjusted, synthesis and secretion of lipid in sebaceous gland cells are adjusted, completeness of skin barriers is maintained, keratinocyte gaps are filled, and hydrophobic barriers are formed; the biological activity or the effect related to moisturizing and skin dryness relieving such as moisture loss prevention is obviously superior to that of commercialized human IV type collagen or other truncated proteins of the human IV type collagen alpha1 chain.

Description

Technical Field

[0001] The present invention belongs to the technical field of synthetic biology, and particularly relates to a novel genetically engineered recombinant type IV collagen, which has good moisturizing efficacy and can be used to alleviate xerosis cutis. Background Art

[0002] Xerosis cutis is a common skin problem with very complex causes. For example, in autumn and winter, the air is less humid, the weather is dry and windy, and the human body loses too much water, making the skin prone to dryness. In addition, as people age, the skin's oil secretion decreases, leading to dry skin. Moreover, malnutrition and certain diseases such as diabetes, hypothyroidism, renal insufficiency, ichthyosis, psoriasis, atopic dermatitis, etc. may also cause xerosis cutis. When xerosis cutis is severe, symptoms of seborrheic dermatitis such as erythema, papules, and desquamation may occur. This inflammation not only causes itching and pain but also may further damage the skin barrier function, making the skin more vulnerable to external stimuli.

[0003] Collagen is a biological macromolecular protein and the main component of animal connective tissue. Due to the risks of animal - source virus infection and immune rejection, it is difficult to be widely used in medical devices and tissue engineering products. With the large - scale application of genetic engineering technology, genetically engineered recombinant expression of collagen has successfully solved the drawbacks of animal collagen in the form of expressing foreign proteins using model expression hosts. It has good biocompatibility, biodegradability, and biological activity, and can be prepared on a large scale. Summary of the Invention

[0004] The purpose of the present invention is to provide a recombinant type IV collagen with high yield, good biological activity related to moisturizing efficacy, and can be used to alleviate xerosis cutis.

[0005] The present invention includes:

[0006] 1. A recombinant type IV collagen, whose amino acid sequence is as shown in SEQ ID No.:1.

[0007] SEQ ID No.:1

[0008]

[0009] 2. A nucleic acid molecule encoding the recombinant type IV collagen described in item 1. The nucleotide sequence of the nucleic acid molecule can be as shown in SEQ ID No:4.

[0010] SEQ ID No:4

[0011]

[0012] 3. An expression vector comprising the nucleic acid of item 2.

[0013] 4. A host cell into which the expression vector of item 3 has been introduced. The host cell is preferably a eukaryotic cell, such as Pichia pastoris.

[0014] 5. A method for producing the recombinant type IV collagen of item 1, which comprises culturing the host cell of item 4 to secrete and express the recombinant type IV collagen, and then performing separation and purification.

[0015] 6. According to the method of item 5, wherein the separation and purification employs one or a combination of several selected from 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.

[0016] 7. Use of the recombinant type IV collagen of item 1 in the preparation of cosmetics, topical drugs, or medical devices for moisturizing.

[0017] 8. According to the use of item 7, wherein the cosmetics, topical drugs, or medical devices are prepared in a form suitable for application to the skin.

[0018] 9. Use of the recombinant type IV collagen of item 1 in the preparation of topical drugs or medical devices for adjuvant treatment of xerosis cutis.

[0019] 10. According to the use of item 9, wherein the topical drugs or medical devices are prepared in a form suitable for application to the skin.

[0020] 11. Use of the recombinant type IV collagen of item 1 in the preparation of cosmetics, medical devices, or topical drugs for regulating skin lipid metabolism, repairing the skin barrier, moisturizing, alleviating skin dryness, and / or adjuvant treatment of xerosis cutis.

[0021] 12. According to the use of item 11, wherein the cosmetics, medical devices, or topical drugs are prepared in a form suitable for application to the skin.

[0022] Advantages of the Invention

[0023] The novel recombinant type IV collagen provided by the present invention does not require a special expression system, and its secretion yield in a conventional commercial Pichia pastoris expression system can be as high as about 14.2 g / L. The extracellularly secreted protein is basically not degraded during the fermentation process, and a purity of more than 85% can be achieved through simple ultrafiltration and decolorization. It can be easily mass-produced industrially, and its yield is significantly higher than that of other truncated proteins of human type IV collagen α1 chain and also higher than that of the full-length human type IV collagen. Moreover, this recombinant type IV collagen is significantly superior to commercial human type IV collagen (Merck C6745) or other truncated proteins of human type IV collagen α1 chain not only in terms of yield, but also in terms of biological activities or effects such as regulating the synthesis of lipid-related enzymes, regulating the synthesis and secretion of lipids in sebaceous gland cells, maintaining the integrity of the skin barrier, filling the gaps between keratinocytes, forming a hydrophobic barrier, and preventing water loss. Description of the Drawings

[0024] Figure 1 It is the purified electrophoresis pattern of different sequence fragments of recombinant type IV collagen.

[0025] Figure 2 It is a figure showing the effect of the recombinant type IV collagen solution on cell moisturizing effect.

[0026] Figure 3 It is a figure showing the effect of the recombinant type IV collagen solution on the expression of AQP3 gene.

[0027] Figure 4 It is a figure showing the effect of the recombinant type IV collagen dressing on the content of PPARα.

[0028] Figure 5 It is a figure showing the effect of the recombinant type IV collagen dressing on the content of PPARγ.

[0029] Figure 6 It is a figure showing the effect of the recombinant type IV collagen dressing on the content of FABP5.

[0030] Compared with the normal control group: **P < 0.01, compared with the model group: #P < 0.05, ##P < 0.01. Detailed Description of the Invention

[0031] Example 1 Preparation of Recombinant Type IV Collagen in Yeast Expression System

[0032] I. Experimental Method

[0033] 1. Preparation of Shuttle Plasmid

[0034] According to the amino acid sequence shown in SEQ ID No.1 (also known as recombinant type IV-α1 chain collagen), codon optimization was carried out for the yeast expression system to obtain the target gene sequence of type IV. The obtained target gene sequence was entrusted to Tsingke Biotechnology Co., Ltd. for gene synthesis. After synthesis, the gene was ligated into the pPicZαA plasmid to obtain the pPicZαA-IV plasmid.

[0035] 2) Preparation of yeast expression strain

[0036] After linearizing pPicZαA-IV with Pme I, it was transformed into the competent cells of Pichia pastoris X-33. Using bleomycin resistance as the screening marker, transformants were screened to obtain the yeast expression strain.

[0037] 2. Induced expression of target protein

[0038] (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). Incubate at 30 °C and 200 rpm for 48 h for activation;

[0039] (2) Transfer with an inoculation amount of 1% to a 500 ml Erlenmeyer flask (containing 200 ml of YPD culture medium), and incubate at 30 °C and 200 rpm for 24 h as the seed for the fermenter;

[0040] (3) Prepare 3 L of BSM medium and add it to a 5 L fermenter. Sterilize at 121 °C for 20 min. After cooling to 30 °C, adjust the pH to 5.0. Add the seed for the fermenter prepared in (2) to the fermenter in the form of flame inoculation for fermentation culture;

[0041] (4) When the OD 600 reaches 70, start supplementing 50% glycerol until the OD 600 reaches about 120, stop supplementing. Wait for the dissolved oxygen to rebound to 100%, and start feeding methanol for induction;

[0042] (5) During the induction process, control the dissolved oxygen not less than 30% and the pH around 5.0. Induce for 40 h to end the fermentation. Take the culture solution and centrifuge at 12000 rpm for 2 min. Take the supernatant and detect the protein yield and purity by the BCA method and SDS-PAGE method.

[0043] 3. Collagen purification

[0044] The bacterial cells were sonicated and then centrifuged. The precipitate was washed with TritonX-100 washing solution, centrifuged again to remove the supernatant. 6M guanidine hydrochloride was added to the precipitate and the pH was adjusted to 8. After the precipitate was fully dissolved, the solution was passed through nickel affinity chromatography resin, and eluted successively with imidazole solutions with concentration gradients of 20mM, 50mM, 100mM, 200mM, and 500mM. After the eluate was concentrated using a protein concentrator column, the concentrated solution was collected and dialyzed overnight (dialysis bag, RC membrane, 14KD, flat width 44mm (Sangon Biotech F600112)). Subsequently, the protein concentration was detected using a BCA kit, and mixed with a mannitol aqueous solution at a ratio of 0.5 mL of a 5% (v / v) mannitol aqueous solution per 5 mg of protein. Then, it was pre-frozen at -40°C for 10 min using a freeze dryer, and finally freeze-dried at -35°C to obtain the recombinant collagen freeze-dried powder.

[0045] 4. Preparation and Identification

[0046] The BCA test result of the recombinant type IV-α1 chain collagen fermentation tank production prepared by the above method was as high as 14.2 g / L. The test result of electrophoresis by SDS-PAGE method showed that the target protein accounted for more than 70%. The protein purity could reach more than 85% after ultrafiltration through a hollow fiber column. Using the same design concept and preparation method as the recombinant collagen type IV-α1 chain, protein sequences with similar molecular weights were designed respectively. After codon optimization, the target genes were obtained and the protein sequences were expressed. The results of sequence design and expression are shown in the following table (Table 1). The protein electrophoresis purification results (IV-1, IV-α1, IV-2) are as Figure 1 shown.

[0047] Table 1 Different Amino Acid Sequences and Expression Results

[0048]

[0049] Example 2 Effect of Recombinant Type IV Collagen Solution on Cell Viability in a Dry Injury Model

[0050] The logarithmically growing HaCat cells were seeded at a density of 1×10 5Inoculate at a density of / mL into a 96-well plate and culture for 24 h. Observe the cell survival rate when the drying time is 10 - 50 min to determine the optimal drying time. The experimental results show that within the range of air-drying time from 20 to 60 min, the cell survival rate decreases significantly, but when it exceeds 20 min, the cell survival rate drops sharply and is lower than 50%. Subsequently, the following groups are set: normal group (without air-drying), model group (add basal medium after air-drying for 20 min), experimental group (add recombinant type IV different fragment collagen solutions and full-length type IV collagen solution at corresponding concentrations after drying for 20 min). The total volume added to each group is 100 μL, and continue to culture for 24 h. After the culture ends, discard the supernatant, add MTT solution, incubate for 2.5 h, and measure the absorbance (A) at a wavelength of 570 nm after dissolving with 150 μL DMSO.

[0051] Analyze the absorbance values. Compared with the normal value, the cell survival rate of the model group is 68.32% (P < 0.01), indicating that the dry injury model is successfully established. Compared with the model group, treating HaCaT cells with recombinant type IV collagen solutions at different concentrations reduces the cell dry mortality to a certain extent, showing a concentration dependence. When the concentration is 0.20%, the cell survival rate is 89.94% (P < 0.05), and the difference is statistically significant, with the most significant effect, indicating that the recombinant type IV-α1 chain collagen solution has good moisturizing efficacy ( Figure 2 ).

[0052] Example 3 Effect of Recombinant Type IV Collagen Solution on Related Barrier Moisturizing Factors

[0053] (1) Establish a sodium dodecyl sulfate-induced immortalized human epidermal cell (HaCat) model

[0054] Detect the content of AQP3 gene in cells after intervention with recombinant collagen type IV and evaluate its moisturizing efficacy. Inoculate cells into a 6-well plate at an inoculation density of 2×10 5 cells / well and incubate overnight in an incubator (37 °C, 5% CO2). When the cell confluence rate in the 6-well plate reaches 40% - 60%, conduct grouping. Except for the blank control group, add 30 μg / mL sodium dodecyl sulfate solution to the other wells. After reacting for 24 h, the experimental groups are recombinant type IV different fragment collagen solutions and full-length type IV collagen solution at corresponding concentrations respectively, the positive control group adds 50 μM WY14643 solution, and the blank control group and the model group add the same volume of cell culture medium respectively and react for 24 h.

[0055] (2) Detect aquaporin AQP3 in cells by RT-qPCR

[0056] mRNA extraction and reverse transcription: After 24 h, the culture medium was discarded, and total mRNA was extracted by the Trizol method. The RNA concentration was measured using a ultra-micro spectrophotometer. Samples with a concentration greater than 20 ng / μL and an A260 / A280 value greater than 1.8 were qualified. cDNA was synthesized using the RevertAidTM First Strand cDNA Synthesis Kit.

[0057] Gene expression detection: SYBR Green qPCR Mix, cDNA template, primers (sequences are shown in Table 1), and RNase-free dd H2O were dissolved at 4°C, and the Real-Time PCR reaction system was prepared on ice. The PCR reaction was carried out using a two-step method, and the reaction program was set as follows: the first step: pre-denaturation at 95°C for 300 s; the second step: denaturation at 95°C for 20 s, annealing at 55°C for 20 s, and extension at 72°C for 20 s, for 40 reaction cycles.

[0058] (3) Experimental results

[0059] Sodium dodecyl sulfate was used to stimulate HaCat. Figure 3 As shown, compared with the normal group, the gene expression level of AQP3 in the model group was significantly down-regulated, indicating successful modeling. When the concentration of the recombinant type IV-α1 chain collagen solution was 0.20%, the gene expression level of AQP3 in HaCat cells was significantly up-regulated, and there was a statistical difference compared with the model group (p < 0.001), indicating that the recombinant type IV-α1 chain collagen has good moisturizing efficacy.

[0060] Table 2: Primer sequence table

[0061]

[0062] Example 4 Effect of recombinant type IV collagen on transepidermal water loss (TEWL)

[0063] The recombinant type IV collagen with different amino acid fragments prepared in Example 1 was made into dressings, and various types of recombinant type IV collagen (or full-length type IV collagen), trehalose dihydrate, theaflavin, fucoidan oligosaccharides, carbomer, xanthan gum, butanediol, glycerol, triethanolamine, and purified water were mixed in sequence; the specific preparation steps refer to the method disclosed in the authorized Chinese patent application CN116688212 B.

[0064] Thirty-five Asian adult female subjects with dry skin aged 35 to 55 years old continuously used various types of recombinant collagen type IV dressing samples on their faces for 7 days each in autumn and winter. The transdermal water loss of the facial skin of the female subjects was randomly measured on the 0th, 3rd, and 7th days of use. After statistical software analysis, the differences between the 3rd and 7th days of using the recombinant collagen type IV protein dressing sample and before use were compared. Results: As shown in Table 1, after the female subjects used the recombinant collagen type IV protein dressing sample, the transdermal water loss rate of the skin decreased on the 3rd day of use compared with before use, and the transdermal water loss situation of the skin improved significantly on the 7th day of use, with an improvement rate of 31.82%. It can be seen that the recombinant collagen type IV protein provided by the present invention has a significant moisturizing effect.

[0065] Table 3: Detection results of transdermal water loss of skin

[0066]

[0067] Table 4: Detection results of transdermal water loss of skin

[0068]

[0069] * Statistical method: The rank sum test and t-test methods were used for analysis, and the test level α = 0.05.

[0070] * Significance marking method: ("n.s" 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).

[0071] * Number of people using the product = 35.

[0072] Example 5 Effect of recombinant type IV collagen on related inflammatory factors

[0073] (1) Establish a mouse model of skin xerosis induced by AEW

[0074] Sixty C57BL / 6 mice were randomly divided into a normal control group and an acetone-ether-water (AEW) group, with 7 mice in each group. They were depilated. In the AEW group, the skin on the neck and back was sequentially smeared with acetone / ether (1:1) and distilled water, while the control group was smeared with an equal amount of distilled water for 7 consecutive days. On the 1st, 3rd, 5th, and 7th days of modeling, the changes in the smeared skin were observed; on the 1st, 3rd, 5th, and 7th days of modeling, the skin of the smeared area in the control group was normal; on the 1st day of modeling in the AEW group, the smeared skin was normal, on the 3rd day, there was a phenomenon of reduced epidermal water content, on the 5th and 7th days, epidermal exfoliation and scales appeared on the mouse epidermis, and it was more severe on the 7th day than on the 5th day. In addition, the mice had itching, indicating that the model was successfully constructed. The AEW group was randomly divided again into a model group, a recombinant type IV collagen group with different sequence fragments, etc.; both the blank control group and the model group were smeared with a collagen-free basal dressing every day, and the other groups were smeared with the corresponding recombinant type IV collagen dressings with different concentrations (the dressing was the same as in Example 4) for 7 consecutive days. The mice were sacrificed, and skin tissue RNA samples were extracted, and the contents of PPAR and FABP5 genes were detected by q-PCR (the primers were provided by Beijing Tsingke Biotechnology Co., Ltd.);

[0075] Table 5: Primer ID Table

[0076] Gene Primer ID PPARα 19013 PPARγ 19016 FABP5 16592 Actin 11461

[0077] (2) Experimental Results

[0078] AEW stimulates the mouse skin and induces a dry skin model in mice. In dry skin, lipid synthesis is reduced. PPARα is involved in lipid metabolism and anti-inflammatory responses, and the barrier function is damaged, resulting in a decrease in PPARα expression; PPARγ plays a key role in epidermal differentiation and lipid synthesis, and FABP5 is involved in fatty acid transport and metabolism. Abnormal epidermal differentiation and lipid metabolism disorders will lead to a decrease in the expression of PPARγ and FABP5. Figure 4 、 5 As shown in Figures 6, compared with the normal group, the contents of PPARα, PPARγ, and FABP5 in the model group were significantly down-regulated, indicating that the model was successfully constructed. When the concentration of the recombinant type IV-α1 chain collagen solution was 0.20%, the contents of PPARα, PPARγ, and FABP5 in the mouse skin were significantly up-regulated, and there were statistical differences compared with the model group (p < 0.01, p < 0.001), indicating that the recombinant type IV-α1 chain collagen has a good effect on promoting lipid synthesis. The effectiveness of the skin barrier depends on the content and composition of lipids in the stratum corneum. These lipids can fill the gaps between keratinocytes, form a hydrophobic barrier, and prevent water loss.

[0079] Sequence Information:

[0080]

[0081]

Claims

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

1. 2 . A nucleic acid encoding the recombinant type IV collagen according to claim 1 . An expression vector comprising the nucleic acid according to 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 type IV collagen according to claim 1, comprising culturing the host cell according to claim 4 to secrete and express the recombinant type IV collagen, and then isolating and purifying the recombinant type IV collagen.

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 type IV collagen according to claim 1 in the preparation of moisturizing cosmetics, external medicines or medical devices.

8. The use according to claim 7, wherein The cosmetic, external medicine or medical device is prepared in a form suitable for application to the skin.

9. Use of the recombinant type IV collagen according to claim 1 in the preparation of external medicines or medical devices for auxiliary treatment of xeroderma.

10. The use according to claim 9, wherein The external medicine or medical device is prepared in a form suitable for application to the skin.

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