High-temperature-resistant and high-hydrophilic recombinant collagen, recombinant bacterium and application of high-temperature-resistant and high-hydrophilic recombinant collagen

Abstract

The invention relates to a high-temperature-resistant and high-hydrophilic recombinant collagen, a recombinant bacterium and application of the high-temperature-resistant and high-hydrophilic recombinant collagen and belongs to the technical field of genetic engineering, the amino acid sequence of the recombinant collagen is shown as SEQ ID NO: 2, and the nucleotide sequence of the recombinant collagen is shown as SEQ ID NO: 1. The invention further provides engineering bacteria containing the gene and application of the recombinant collagen. The recombinant collagen is not prone to denaturation under the high-temperature condition, has the good hydrophilic performance, meanwhile has the good biological activity and stability, and has the functions of promoting collagen regeneration and enhancing the skin barrier.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a high-temperature resistant and highly hydrophilic recombinant collagen, recombinant bacteria, and their applications. Background Technology

[0002] Collagen is a large protein molecule widely found in mammals, accounting for about one-third of the body's total protein. In the human body, collagen is mainly found in skin, bones, cartilage, ligaments, and blood vessels. It is a major structural protein that constitutes connective tissue, providing support, connection, nutrition, and protection, playing a central role in maintaining tissue integrity. Thanks to its good biocompatibility, excellent biodegradability, and water and air permeability, collagen is now widely used in biomedicine, pharmaceuticals, cosmetics, and the food industry, among other fields.

[0003] Currently, collagen preparation methods fall into two main categories: animal tissue extraction and recombinant genetic engineering. Animal tissue extraction is relatively mature, primarily using raw materials such as pigskin, cowhide, beef tendon, or fish scales. However, it carries risks related to pathogens, potential immunogenicity, and ethical concerns. Furthermore, obtaining collagen with identical molecular size and structure presents numerous technological challenges. Recombinant genetic engineering expresses collagen in E. coli, yeast, plant cells, or mammalian cells using genetic engineering techniques. While this addresses the safety concerns of animal-derived collagen, its widespread application is limited by low protein expression levels, complex fermentation and purification processes, and high production costs.

[0004] Furthermore, animal-derived collagen typically requires cryogenic storage, is prone to denaturation, and is easily degraded by enzymes in vivo. While some recombinant collagens have eliminated potential MMP cleavage sites, they still suffer from common problems such as insufficient mechanical strength, poor thermal stability, and poor low-temperature solubility, severely limiting their application and efficacy in cosmetics and biomedicine. Developing recombinant collagen that is non-degradable, heat-resistant, and highly hydrophilic not only avoids the risks associated with animal-derived collagen but also enables room-temperature storage and low-temperature applications, significantly reducing transportation, storage, and usage costs. This is an effective method to expand its application scenarios. Therefore, recombinant expression of heat-resistant and highly hydrophilic collagen through genetic engineering technology has significant market value and research significance. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a high-temperature resistant and highly hydrophilic recombinant collagen, its preparation method, and its applications. This recombinant collagen is not easily denatured under high-temperature conditions and possesses good hydrophilicity, along with excellent biological activity and stability.

[0006] This invention is achieved through the following technical solution: A heat-resistant and highly hydrophilic recombinant collagen, the amino acid sequence of which is shown in SEQ ID NO: 26.

[0007] The present invention also provides a gene encoding the recombinant collagen, the nucleotide sequence of which is shown in SEQ ID NO:25.

[0008] The present invention also provides a recombinant vector containing the genes described above, wherein the original expression vector is pPIC9K.

[0009] The present invention also provides a genetically engineered bacterium, comprising a host cell and a target gene transferred into the host cell, wherein the target gene comprises a recombinant collagen gene with a nucleotide sequence as shown in SEQ ID NO: 25.

[0010] Furthermore, the host cell is Pichia pastoris GS115; the target gene is inserted into the original expression vector and subjected to restriction endonuclease restriction. Sac After I enzyme digestion, the cells were transformed into GS115 competent cells, and high-copy transformants were selected by G418.

[0011] The present invention also provides a method for preparing the above-mentioned heat-resistant and highly hydrophilic recombinant collagen, comprising culturing host cells to secrete the recombinant collagen into a culture medium, and separating and purifying the target protein.

[0012] Furthermore, the separation and purification employs one or a combination of several of the following: ammonium sulfate precipitation, alcohol precipitation, activated carbon decolorization, membrane separation, and gel size exclusion chromatography.

[0013] The present invention also provides applications of the aforementioned heat-resistant and highly hydrophilic recombinant collagen, including using the aforementioned collagen to promote the proliferation of keratinocytes (HaCaT) and fibroblasts (HSF), or using the aforementioned collagen to regulate the expression of genes for various keratinocytes (KRT1, KRT5, KRT10, KRT14), epidermal protein (IVL-2), lobe-like protein (LOR1), and filaggrin (FLG) in HaCaT cells, and using the aforementioned collagen to promote the expression of genes COL1A1 and COL3A1 in HSF cells.

[0014] The present invention also provides the application of the collagen in the preparation of products that promote collagen regeneration and enhance skin barrier function.

[0015] A composition for promoting collagen regeneration and enhancing skin barrier function, the composition comprising the collagen.

[0016] The beneficial effects of this invention compared to the prior art are as follows: (1) The recombinant collagen has significant high temperature resistance; it retains the integrity of its structure and sequence even after being sterilized by moist heat at 121°C for 30 min. (2) The recombinant collagen has high hydrophilicity, and the apparent molecular weight on SDS-PAGE is 2.3 times the actual molecular weight; (3) The results of the five toxicological assessments of the recombinant collagen showed that it has good safety; (4) The recombinant collagen can significantly increase the expression of keratin (KRT1, KRT5, KRT10, KRT14), epidermal protein (IVL-2), lipoprotein (LOR1) and filaggrin (FLG) genes in HaCaT cells, and also significantly promotes the expression of COL1A1 and COL3A1 genes in HSF cells, indicating that it can promote collagen regeneration and enhance skin barrier function. Attached Figure Description

[0017] Figure 1 Schematic diagram of recombinant plasmid; Figure 2 This is an SDS-PAGE electrophoresis image of purified recombinant collagen; lanes 1 and 2 are 2 mg / mL, and lanes 3 and 4 are 5 mg / mL. Figure 3 This is a graph showing the analysis of recombinant collagen samples after high-temperature treatment by HPLC; where S represents the high-temperature treated sample and CK represents the untreated control sample. Figure 4 The sample is analyzed by LC-MS / MS. Figure 5 Images showing the morphology and growth status of collagen cells at different concentrations and in the control group under a microscope; Figure 6 Example diagram of injection site arrangement for New Zealand rabbits; where 1--head end; 2--0.2 mL polar extract injection site; 3--0.2 mL non-polar extract injection site; 4--0.2 mL polar solvent control solution injection site; 5--0.2 mL non-polar solvent control solution injection site; 6--tail end; Figure 7 Example diagram of intradermal injection sites in guinea pigs; where point a: emulsifier prepared by mixing Freund's complete adjuvant and a selected solvent in equal volume proportions; point b: the prepared extract; point c: the emulsion obtained by mixing the extract in equal volume proportions with an emulsifier prepared by Freund's complete adjuvant and a solvent (50%). The negative control group was treated in the same manner. Figure 8 Bar charts showing the effects of different concentrations of collagen on HaCaT cell proliferation after 24 h, 48 h, and 72 h of culture; where A is the 24 h figure, B is the 48 h figure, and C is the 72 h figure. Figure 9 Bar charts showing the effects of different concentrations of collagen on HSF cell proliferation after 24 h, 48 h, and 72 h of culture; where A is the 24 h chart, B is the 48 h chart, and C is the 72 h chart. Figure 10 The effects of 500 μg / mL and 750 μg / mL collagen treatment for 48 h on gene expression in HaCaT cells are shown in the figure. A represents the relative expression levels of KRT1, B of KRT5, C of KRT10, D of KRT14, E of IL-1β, F of TNF-α, G of IVL-2, H of FLG, and I of LOR1. The expression levels of each gene were normalized using GAPDH as an internal control. Figure 11 The effects of 500 μg / mL and 750 μg / mL collagen treatment for 48 h on gene expression in HSF cells are shown in the figure. In the figure, A is the relative expression level of TIMP1, B is the relative expression level of COL1A1, and C is the relative expression level of COL3A1. The expression levels of each gene were normalized using GAPDH as an internal reference. Detailed Implementation

[0018] The present invention will be further described below with reference to specific embodiments. The following examples are only specific embodiments of the present invention.

[0019] The materials used in the following examples are all commercially available, and the experimental procedures employed conventional molecular biology methods and biochemical analyses. The experimental methods involved are as follows: Example 1 Construction of recombinant collagen engineered strains (1) Construction of recombinant plasmid: Based on the core sequence of human type III collagen (hCOL3A1) (GeneBank No.: AGL34959.1), this invention carried out a systematic and rational design to obtain recombinant collagen with both heat resistance and high hydrophilicity. Unlike conventional sequence substitution, this design systematically removes the highly hydrophobic GXY triplet that is prone to aggregation, degradation or decreased solubility, removes cysteine ​​Cys(C), and preferably mutates the sequence to the highly hydrophilic triplets GSP, GQP, GEP, and GSQ, with S and Q distributed every 1-7 amino acid residues. At the same time, to enhance its biological activity and cell recognition ability, GER and RGD integrin recognition sites are introduced at the C-terminus of the collagen, finally obtaining the recombinant collagen amino acid sequence with both heat resistance and high hydrophilicity as shown in SEQ ID NO: 26. Subsequently, using Pichia pastoris as the target expression host, the gene encoding the optimized amino acid sequence was codon-optimized. The resulting gene sequence is shown in SEQ ID NO:25. This sequence was then chemically synthesized by General Biosystems (Anhui) Co., Ltd., and integrated into the pPIC9K expression vector to obtain the recombinant plasmid: pPIC9KCOL3α1 (as shown in SEQ ID NO:25). Figure 1 (As shown).

[0020] (2) Construction of engineered strains: using restriction endonucleases Sac I digested pPIC9KCOL3α1 with enzymes (37℃, 2 h), and then transformed the linearized recombinant vector into Pichia pastoris GS115 competent cells by electroporation. The transformed cells were then plated onto MD solid culture plates and incubated at 30℃ for 48-72 h. The transformants were then picked and photocopied into YPD solid medium containing 4 mg / mLG418 and incubated at 30℃ for 48 h to obtain high-copy engineered strains tolerant to high concentrations of G418.

[0021] MD solid culture medium formula (g / L): 13.4 g YNB (amino-free yeast nitrogen source), 20 g glucose, 20 g agar powder (YNB and glucose are sterilized separately).

[0022] YPD solid culture medium formula (g / L): 20 g peptone, 10 g yeast extract, 20 g glucose, 20 g agar powder.

[0023] Example 2: Shake-flask fermentation of recombinant collagen Using BMGY as the growth medium (100 mL), the culture was carried out on a shaker at 30℃ and 200-220 rpm until OD. 600 The range is 2 to 6.

[0024] Take 5 mL of bacterial suspension from BMGY medium and inoculate it into 100 mL of BMMY medium. Continue culturing at 28℃ and 200-220 rpm. Add anhydrous methanol to the culture medium every 24 h to a final concentration of 1% to induce the expression of the target collagen. Stop culturing after 72 h of induction.

[0025] BMGY culture medium formulation (g / L): 20 g peptone, 10 g yeast extract, 700 mL ultrapure water, 100 mL 1 M potassium phosphate buffer (pH 6.0), sterilized at 121℃ for 20 min, and after cooling, add 100 mL 10×YNB, 100 mL 10% glycerol, and 2 mL 500×Biotin, which have been sterilized by filtration through a 0.22 μm membrane.

[0026] BMMY medium formulation (g / L): 20 g peptone, 10 g yeast extract, 790 mL ultrapure water, 100 mL 1 M potassium phosphate buffer (pH 6.0), sterilized at 121℃ for 20 min, and after cooling, add 100 mL 10×YNB, 2 mL 500×Biotin, and 10 mL methanol that have been sterilized by filtration through a 0.22 μm filter membrane.

[0027] Example 3 Purification of recombinant collagen (1) Sample pretreatment: Centrifuge at 4℃ and 8,000×g for 30 min and collect the fermentation supernatant; (2) Salting out protein: Ammonium sulfate was slowly added to the fermentation supernatant at a rate of 56.8 g / 100 mL and stirred continuously until completely dissolved. After dissolution, stirring was continued for 30 min, followed by centrifugation at 4°C and 10,000×g for 30 min, and the supernatant was discarded.

[0028] (3) Precipitation dissolution: Dissolve the above centrifuged solid with 10% of the sample volume of pure water and stir at 4°C for 15-20 min until the precipitate is completely dissolved.

[0029] (4) Decolorization: Add granular activated carbon (200 mesh) to the sample for decolorization treatment, and then use a decarbonizer to obtain a clear filtrate.

[0030] (5) Ultrafiltration concentration: Turn on the ultrafiltration machine, turn on the cooling, maintain the temperature at 4-8℃, and use a 10 kDa filter membrane to ultrafilter the feed solution. Wash with pure water and concentrate to a total protein concentration of about 50 g / L.

[0031] (6) Gel filtration chromatography: A G25 column was used. The equilibration and elution solutions were 10 mM Tris-HCl buffer (pH 7.4) containing 50 mM NaCl. The elution process was monitored at 245 nm using a UV detector, and the fraction corresponding to the main peak was collected.

[0032] (7) Secondary ultrafiltration desalination: Turn on the cooling and maintain the temperature at 4-8℃. Turn on the ultrafiltration machine to perform ultrafiltration of the feed liquid. Wash with pure water until the conductivity of the solution is lower than 25 μs / cm.

[0033] (8) Freeze-drying: The protein solution was filtered through a 0.22 μm sterile filter and then freeze-dried.

[0034] Protein electrophoresis (SDS-PAGE) of purified collagen as follows Figure 2 As shown, these are samples of different concentrations, with lanes 1 and 2 containing 2 mg / mL, and lanes 3 and 4 containing 5 mg / mL. The sample loading volume for each lane is 10 μL.

[0035] Example 4: High-Temperature Resistance Test of Recombinant Collagen The purified collagen sample was placed in an autoclave and sterilized at 121℃ for 30 min. An untreated sample stored at 4℃ was used as a negative control. After the high-temperature treatment, the sample was cooled to an appropriate temperature. The heat-treated and untreated samples were analyzed by HPLC to verify the high-temperature resistance of the recombinant collagen. The results are as follows: Figure 3 As shown. Simultaneously, peptide coverage analysis based on liquid chromatography-mass spectrometry (LC-MS / MS) was used to perform sequence analysis on the high-temperature treated samples: first, the samples were subjected to SDS-PAGE, then the corresponding gel strips were cut and destained, the destained gel particles were digested with trypsin, and then the treated samples were analyzed by LC-MS / MS to obtain the raw file of the mass spectrometry results. Figure 4 The data was analyzed and matched using PEAKSStudio 10.6 software to obtain the identification results. The results showed that the sequence coverage of the collagen sample after high-temperature treatment was 100% compared with the theoretical sequence (SEQ ID NO: 26).

[0036] Example 5: In vitro cytotoxicity test of high-temperature resistant and highly hydrophilic collagen (1) Experimental procedure: L929 mouse fibroblasts were cultured in a 5% CO2, saturated humidity, 37℃ constant temperature incubator. The culture medium was MEM medium (containing 10% fetal bovine serum and 1% penicillin). Observe under an inverted microscope. After the cells have filled the culture flask, they were digested with 0.25% trypsin. When the cells began to round and shrink, 10% fetal bovine serum medium was added to stop the digestion. The cells were then gently pipetted with a sterile pipette to disperse them into single cells for later use.

[0037] After enzymatic digestion (trypsin), the cultured L929 cells were removed from the cell culture flask and the density was adjusted to 1×10⁻⁶. 5 Cells were seeded at a density of 100 μL / well in 96-well cell culture plates and cultured in a cell culture incubator (37℃, 5% CO2) until a semi-confluent monolayer was formed. Each well was observed under an inverted microscope to ensure relatively equal cell growth. After 24 h of incubation, the original culture medium was aspirated from the cell culture plates. For each group of 6 replicate wells (excluding peripheral wells), 100 μL of different concentrations of the test sample extract (100%, 50%, 25%, and 12.5%, located in columns 3-6 of the culture plate) were added to each group. Positive controls (column 9), negative controls (column 7), and blank controls (two groups, columns 2 and 11) were also added. The plates were then incubated for 24 h. After culture, the growth characteristics of the control group and the cells in each concentration group were observed under a microscope. After observation, carefully remove the culture medium, add 50 μL of MTT (1 mg / mL) solution to each well, incubate at 37℃ for 2 h, discard the MTT solution, add 100 μL of isopropanol solution to each well, vortex to mix, place in a microplate reader, and measure the absorbance at a wavelength of 570 nm (reference wavelength 650 nm).

[0038] (2) Result evaluation: Qualitative evaluation: After 24 h of culture, the cell morphology was qualitatively evaluated by microscopic observation according to Table 1. A grade greater than 2 was considered to have cytotoxic effects. Table 1 Qualitative classification of cytotoxic morphology of extracts ; Quantitative evaluation: Calculate cell viability using the following formula; ; Where: OD 570e : Average optical density of the extract of the test sample; OD 570b : Average optical density of the blank control group.

[0039] If cell viability drops to less than 70% of the control group, the test sample has potential cytotoxicity. The cell viability of the 50% extract group should be the same as or higher than that of the 100% extract group; otherwise, the test should be repeated.

[0040] (3) Test Results: Qualitative Evaluation: Under a microscope, discrete particles were observed in the cytoplasm of the 100%, 50%, 25%, and 12.5% ​​concentration groups of the test sample extract, indicating a cytotoxicity morphological grade of 0. The cell morphology and growth of each concentration group and the control group were observed under a microscope, and the results are shown in […]. Figure 5 .

[0041] Quantitative evaluation: Based on the test results, the test data of the test sample are analyzed. Please refer to Table 2 for specific test results. Table 2. Cytotoxicity test data for the test sample group and each control group. .

[0042] (4) Conclusion: Under the experimental conditions, the qualitative evaluation results showed that the cytotoxicity morphology grade of the 100% concentration group of the test sample extract was 0, indicating no cytotoxic reaction. The quantitative evaluation results showed that the cell viability rates of the 100%, 50%, 25%, and 12.5% ​​concentration groups of the test sample extract were 87.9%, 98.8%, 100.9%, and 104.6%, respectively, and the cell viability rate of the 100% concentration group of the test sample extract was greater than 70%.

[0043] Example 6 Intradermal Reaction Test (1) Experimental steps: Animal preparation: Weigh the animals 4 to 18 hours before the experiment and remove enough hair from both sides of the spine on the back of the animals in preparation for the injection of the test solution.

[0044] Each New Zealand rabbit was assigned 20 injection sites, with 5 injection sites per group (e.g., ...). Figure 6 ). Hair removal area on one side of the spine of each New Zealand rabbit ( Figure 6 Inject 0.2 mL of polar extract into each of the five sites in region 2, and inject 0.2 mL of non-polar extract into each of the five sites in region 3. The smallest needle size should be selected for intradermal injection based on the viscosity of the test material.

[0045] Inject either a polar solvent control solution or a non-polar solvent control solution into the opposite side of the spine of each New Zealand rabbit, following the same procedure as above. (e.g.) Figure 5 ) Animals were observed and their condition was recorded immediately after injection, at (24+2) h, (48+2) h, and (72+2) h.

[0046] The tissue response of erythema and edema at each injection site during each observation period was scored according to the scoring system given in Table 3, and the test results were recorded. Table 3 Endothelial Response Scoring System .

[0047] (2) Result Evaluation: After scoring at (72+2) h, the scores for all erythema and edema at (24+2) h, (48+2) h, and (72+2) h for each animal test sample or solvent control were added together and then divided by 15 [3 (scoring time points) × 5 (injection points of test sample or solvent control)] to calculate the score for each animal test sample or solvent control. The scores of the three animals were added together and divided by 3 to obtain the total average score for each test sample and the corresponding solvent control. The final score of the test sample was obtained by subtracting the score of the solvent control from the score of the test sample. If the final score of the test sample was not greater than 1.0, it met the requirements of the test. If, during any observation period, the average reaction of the test sample was suspected to be greater than that of the solvent control, three more rabbits should be retested. If the final score of the test sample was not greater than 1.0, it met the requirements of the test.

[0048] (3) Experimental results: The skin reactions of animals at each injection site were observed at (24+2) h, (48+2) h and (72+2) h. The specific scores are shown in Table 4. Table 4. Scoring results of skin irritation response in each group of animals .

[0049] (4) Conclusion: Under the experimental conditions, the difference between the average scores of the polar (or non-polar) extract of the test sample and the solvent control was 0. The final scores of both the polar and non-polar extracts of the test sample were less than 1.0, indicating no potential skin irritation.

[0050] Example 7 Guinea Pig Skin Sensitization Test - Maximum Dose Method (1) Experimental steps: Animal preparation: skinning, disinfection, and preparation of the test and observation sites.

[0051] Animal grouping: Animals were randomly divided into two groups: 10 animals each for the polar and non-polar test sample groups and 5 animals each for the polar and non-polar negative control groups.

[0052] Intradermal induction: according to Figure 7 As shown, three symmetrical points are marked on both sides of the midline of the hair removal area on the back of the neck, and 0.1 mL of the following solution is injected intradermally at each point.

[0053] Local induction: Intradermal induction for 6 days. The test area of ​​the test group was pretreated with 10% sodium dodecyl sulfate and massaged into the skin. 24 h later, a 2.0 cm × 4.0 cm absorbent gauze soaked in the extract was applied topically to the induction injection site of the guinea pigs and secured with a closed bandage. The bandage and patch were removed after 48 h. The control group was treated in the same manner.

[0054] Triggering contact: On day 13 of local induction, the abdominal hair of guinea pigs in the test group was removed. 24 hours later, the patch was soaked in an emulsion of the test extract and Freund's complete adjuvant and applied topically to the untested areas during the induction phase. It was secured with a closed bandage. The bandage and patch were removed 24 hours later. The control group was treated in the same way.

[0055] Animal observation: 24 h and 48 h after removing the patch, observe the skin condition at the stimulation site of the test group and the control group. According to the Magnusson and Kligman grading criteria as shown in Table 5 below, the skin erythema and edema reactions were graded and recorded. Table 5 Magnusson and Kligman grading criteria .

[0056] (2) Evaluation of results: If the negative control group animals have a grade of less than 1, while the experimental group animals have a grade of 1 or greater, it generally indicates sensitization. If the negative control group animals have a grade of 1 or greater, and the experimental group animals have a reaction that exceeds the most severe reaction in the control group, it is considered sensitization.

[0057] (3) Experimental results: Skin reactions at the stimulation sites of animals in each group were observed and recorded 24 h and 48 h after stimulation and removal of the patch. The specific grading is shown in Table 6. The skin reaction grade of animals in the test group and the negative control group was less than 1; Table 6. Magnusson and Kligman grading records for each group of skin. .

[0058] (4) Conclusion: Under the experimental conditions, the skin reaction grade of the polar (or non-polar) extract of the test sample was less than 1, and there was no skin sensitization reaction of the polar and non-polar extracts of the test sample.

[0059] Example 8 Acute systemic toxicity test (1) Experimental procedure: Animal grouping: Animals were randomly grouped into two groups: 6 animals each in the polar and non-polar test sample groups and 6 animals each in the polar and non-polar control groups, with half males and half females in each group.

[0060] Before the experiment, the mice were marked and weighed. The polar test extract and polar control extract were administered by intravenous injection, while the non-polar test extract and non-polar control extract were administered by intraperitoneal injection. The injection dose was 50 mL / kg.

[0061] Immediately after injection, the clinical response of mice was observed, and the general condition, toxic manifestations, and number of dead animals in the test group and control group were observed and recorded at 4 h, 24 h, 48 h, and 72 h after injection. The weight of the animals was also recorded at 24 h, 48 h, and 72 h after injection. Common clinical symptoms and observation items are shown in Table 7. Table 7 Common Clinical Symptoms and Observation Items .

[0062] (2) Result evaluation: If the biological response of the animals exposed to the test sample is no greater than that of the control group animals during the observation period, then the test sample meets the test requirements. If two or more animals in the test group die, or two or more animals convulse or lie prone, or three or more animals lose more than 10% of their body weight, then the test sample does not meet the test requirements. If the animals in the test group only show mild biological response, and no more than one animal shows general biological response symptoms or dies, the test should be repeated with 10 animals as the test group.

[0063] If, during repeated experiments, all 10 animals exposed to the test sample show no scientifically significant biological response greater than that of the control group animals during the observation phase, then the test sample meets the experimental requirements.

[0064] (3) Experimental results: Under the experimental conditions, no clinical toxicity was observed in either the test group or the control group. The specific results are shown in Table 8. There was no significant difference in the changes in animal body weight between the test group and the control group. The specific results are shown in Table 9. Table 8 Clinical observation results of animals in each group ;

[0065] Table 9. Body weight and number of deaths of experimental animals in each group .

[0066] (4) Conclusion: Under the experimental conditions, no animal deaths or obvious clinical symptoms were observed after intravenous and intraperitoneal injection of polar or nonpolar test extracts. No acute systemic toxicity reactions were observed with the polar or nonpolar test extracts.

[0067] Example 9: Rabbit Heat Source Experiment (1) Experimental procedure: Rabbits were not fed for at least one hour before the experiment and were placed in a spacious and suitable environment. Their body temperature was measured twice, with an interval of 30 minutes. The difference between the two body temperatures should not exceed 0.2℃. The average of the two body temperatures was taken as the rabbit's average body temperature. After the average body temperature was measured, the test sample extract, preheated to about 38℃, was slowly injected into the ear vein at a dose of 10 mL / g. After injection, the body temperature was measured every 30 minutes for a total of 6 times. The highest measured body temperature minus the average body temperature was taken as the elevated body temperature of the rabbit in this experiment.

[0068] (2) Result evaluation: Result shall be deemed to meet the following conditions: In the initial test, the body temperature of all three rabbits was less than 0.6℃, and the total body temperature increase of the three rabbits was less than 1.3℃. Alternatively, in the retest, no more than one rabbit had a body temperature increase of 0.6℃ or higher, and the total body temperature increase of the eight rabbits in the combined initial and retest was not higher than 3.5℃.

[0069] If any of the following conditions are met, the application will be deemed non-compliant: In the initial trial, more than one of the three rabbits had a body temperature increase of 0.6℃ or higher. Or, in the retest, more than one of the five rabbits had a body temperature increase of 0.6℃ or higher. Or, the combined body temperature increase of the eight rabbits in the initial and retest trials exceeded 3.5℃. When the temperature of a rabbit rises to a negative value, it is considered as 0℃.

[0070] (3) Experimental results: Under the conditions of this experiment, the average body temperature of the three rabbits and the body temperature after injection are shown in Table 10; Table 10. Rabbit body temperature results .

[0071] (4) Conclusion: Under the experimental conditions, after the test sample extract was injected into the rabbits, the body temperature of each rabbit increased by less than 0.6℃, and the total body temperature increase of the three rabbits was less than 1.3℃. The pyrogen test of this test sample met the requirements.

[0072] Example 10: Effects of recombinant collagen at different concentrations on the proliferation of HaCaT and HSF cells (1) Experimental method: HaCaT cells were revived at 37℃ for 1 min in DMEM medium containing 10% fetal bovine serum and 1% triple antibodies. They were cultured in an incubator at 37℃ and 5% CO2, and passaged once every 48 h at a passage ratio of 1:2.

[0073] HSF cells were revived at 37°C for 1 min in DMEM medium containing 10% fetal bovine serum and 1% triple antibodies. Cells were cultured in a 37°C, 5% CO2 incubator, and passaged once every 48 h at a passage ratio of 1:2.

[0074] Collect cells in the exponential growth phase, count them, and then divide HaCaT and HSF cells into groups of 2 × 10⁻⁶. 3 / hole, 3×10 3 The cells were seeded in 96-well plates with 100 μL of complete DMEM medium per well. After 24 h of culture, collagen was added for treatment. After 24 h, 48 h, and 72 h, 10 μL of CCK8 reagent was added to each well to detect cell proliferation.

[0075] (2) Experimental results: such as Figure 8 , Figure 9 As shown, after treatment for 24 h, 48 h and 72 h, concentrations of 500 μg / mL and 750 μg / mL promoted the proliferation activity of HaCaT and HSF cells.

[0076] Example 11: Effects of recombinant collagen on KRT1, KRT5, KRT10, KRT14, IL1-β, TNF-α, IVL-2, LOR1, and FLG genes in HaCaT cells. (1) Experimental methods: Collect cells in the exponential growth phase, seed HaCaT cells in 6-well plates, and add samples according to the sample concentrations selected in the table above; after culturing at 37℃ for 24 h and 48 h, extract cellular RNA using the TRIzol method; use a kit to reverse transcribe RNA to obtain cDNA; qPCR was used to detect the gene expression of keratin (KRT1, KRT5, KRT10, KRT14), interleukin (IL1-b), tumor necrosis factor (TNF-α), epithelial protein (IVL-1), lipoprotein (LOR1), and filaggrin (FLG) in each group; Table 11 qPCR primer sequences ; Table 12 qPCR cycling parameters .

[0077] (2) Experimental results: HaCaT cells were cultured for 48 h in collagen-containing media containing 500 μg / mL and 750 μg / mL, respectively. The expression levels of KRT1, KRT5, KRT10, KRT14, IL1-b, TNF-a, IVL-2, LOR1, and FLG genes were detected. The results are as follows: Figure 10As shown, this collagen can significantly increase the expression levels of keratin (KRT1, KRT5, KRT10, KRT14), epidermal protein (IVL-2), laminarin (LOR1), and filaggrin (FLG) genes in various groups of HaCaT cells.

[0078] Example 12 Effects of recombinant collagen on COL1A1, COL3A1, and TIMP1 genes in HSF cells (1) Experimental methods: Collect cells in the exponential growth phase, seed HaCaT cells in 6-well plates, and add samples according to the sample concentrations selected in the table above; after culturing at 37℃ for 24 h and 48 h, extract cellular RNA using the TRIzol method; use a kit to reverse transcribe RNA to obtain cDNA; qPCR method to detect the gene expression of collagen (COL1A1, COL3A1) and matrix metalloproteinase inhibitor (TIMP1) in each group; Table 13 qPCR primer sequences ; Table 14 qPCR cycling parameters .

[0079] (2) Experimental results: HSF cells were cultured for 48 h using collagen medium containing 500 μg / mL and 750 μg / mL, respectively. The expression levels of COL1A1, COL3A1, and TIMP1 genes were then detected. The results showed that... Figure 11 As shown, a concentration of 500 μg / mL of this collagen can significantly increase the expression levels of COL1A1 and COL3A1 genes in HSF cells.

[0080] Although the present invention has been disclosed above with reference to preferred embodiments, any person skilled in the art may make various modifications and adjustments without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be defined by the claims.

[0081] The nucleotide sequence of the recombinant collagen gene is SEQ ID NO: 25. Amino acid sequence of recombinant collagen SEQ ID NO: 26 GPPGEPGNPGSPGNQGQPGNKGQPGSQGNPGKNGQPGSPGNPGQPGNEGSPGEPGSNGPQGQPGSQGQPGQPGQNGQPGSPGEPGSNGPQGQPGSQGNPGKNGQPGSPGSQGQPGSPGEPGSNGPQGSPGNQGSPGQPGNPGQPGEQGKPGNQGPAGEPGNPGSPGNQGQPGNKGQPGSQGNPGKNGQPGSPGNPGQPGNEGSPGEPGSNGPQGQPGSQGQPGQPGQNGQPGSPGEPGSNGPQGQPGSQGNPGKNGQPGSPGSQGQPGSPGEPGSNGPQGSPGNQGSPGQPGNPGQPGNQGQPGNKGQPGSQGNPGKNGQPGSPGNPGQPGNEGSPGEPGSNGPQGQPGSQGQPGQPGQNGQPGSPGEPGSNGPQGQPGSQGNPGKNGQPGSPGSQGQPGSPGEPGSNGPQGSPGNQGSPGQPGNPGQPGEQGKPGNQGPAGEPGNPGSPGNQGQPGNKGQPGSQGNPGKNGQPGSPGNPGQPGNEGSPGEPGSNGPQGQPGSQGQPGQPGQNGQPGSPGEPGSNGPQGQPGSQGNPGKNGQPGSPGSQGQPGSPGEPGSNGPQGSPGNQGSPGQPGNPGQPGEQGKPGNQGPAGERRGD。

Claims

1. A high-temperature resistant and highly hydrophilic recombinant collagen, characterized in that, The amino acid sequence of the recombinant collagen is shown in SEQ ID NO:

26.

2. The gene encoding the recombinant collagen of claim 1, characterized in that, Its nucleotide sequence is shown in SEQ ID NO:

25.

3. A recombinant vector comprising the gene as described in claim 2, characterized in that, The original expression vector for the recombinant vector is pPIC9K.

4. A genetically engineered bacterium, characterized in that, The engineered bacteria include a host cell and a target gene transferred into the host cell. The target gene includes a recombinant collagen gene with a nucleotide sequence as shown in SEQ ID NO:

25.

5. The genetically engineered bacterium according to claim 4, characterized in that, The host cell was Pichia pastoris GS115.

6. A method for preparing the high-temperature resistant and highly hydrophilic recombinant collagen of claim 1, characterized in that, The method includes culturing host cells to secrete the recombinant collagen into a culture medium, and separating and purifying the target protein.

7. The method according to claim 5, characterized in that, The separation and purification process employs one or a combination of several of the following: ammonium sulfate salting-out, alcohol precipitation, activated carbon decolorization, membrane separation, and gel size exclusion chromatography.

8. The application of the high-temperature resistant and highly hydrophilic recombinant collagen according to claim 1, characterized in that, The applications include using the collagen to promote the proliferation of keratinocytes and fibroblasts, or using the collagen to regulate the expression of keratin, epidermal protein, lipofuscin, and filaggrin genes in HaCaT cells, and using the collagen to promote the growth of HSF cells. COL1A1、COL3A1 Gene expression.

9. The use of the collagen according to claim 1 in the preparation of products that promote collagen regeneration and enhance skin barrier function.

10. A composition for promoting collagen regeneration and enhancing skin barrier function, characterized in that, The composition comprises the recombinant collagen of claim 1.

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