Recombinant humanized type-A type-III collagen, preparation method thereof and recombinant bacteria

Abstract

The invention discloses a recombinant humanized type-A type-III collagen, a preparation method thereof and a recombinant bacterium, and relates to the technical field of bioengineering. According to the preparation method, peptide fragments with 100% identical sequences of III-type collagen from human are integrated into host bacteria to obtain recombinant bacteria for efficiently synthesizing recombinant humanized A-type III-type collagen, and the recombinant humanized collagen produced by utilizing the recombinant bacteria does not contain any gene sequence of non-human collagen sources, and has the advantages of high efficiency, high yield, high yield and the like. The collagen has the characteristics of small molecular weight, high yield, high purity, water solubility and the like, and can be widely applied to the fields of skin care products, medical cosmetology, skin repair dressings and the like, so that the recombinant humanized A type III collagen, the preparation method thereof and the recombinant bacteria provided by the invention have better industrial application prospects.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to a recombinant humanized type A and type III collagen, a preparation method and application thereof, a recombinant bacterium, and a method for constructing the recombinant bacterium. Background Art

[0002] Collagen is a crucial protein that forms the structure of human tissues and is widely distributed in animal connective tissue. It plays a vital role in maintaining the normal physiological functions and repairing injuries of cells, tissues, and organs. Recombinant humanized collagen, due to its excellent physical and biological properties, has a wide range of applications in medicine, tissue engineering, food, and cosmetics.

[0003] Collagen is a biopolymer and the primary component of animal connective tissue, providing support, protection, and connection. Currently, there are over 29 known types of collagen, the most widely distributed of which are types I, II, III, IV, and V. Type I collagen is the most abundant, accounting for approximately 80-90% of the total collagen content. Type I collagen is primarily found in adult skin, tendons, and bone tissue; type II collagen is primarily found in cartilage, the vitreous humor of the eye, and intervertebral discs; and type III collagen is primarily found in infant skin, the lining of blood vessels, and the intestines.

[0004] Type III collagen, compared to type I collagen, has a fine, loose, mesh-like structure and is distributed at the epidermal-dermal junction, providing skin with elasticity and stress resistance. Type III collagen accounts for approximately 80% of the skin's production during infancy. However, as we age, this level of collagen is lost, resulting in a dull, matte appearance. Therefore, type III collagen is also known as baby collagen.

[0005] Currently, collagen is generally derived from two sources: animal extraction and biosynthesis. Animal-derived collagen is complex, has poor product stability, and carries the risk of viral transmission. Recombinant collagen derived from biosynthesis offers low cost, high purity, and enhanced biosafety, with a market growth rate approximately twice that of animal-derived collagen. However, existing biosynthesis methods suffer from low purity and limited yield.

[0006] On March 15, 2021, the "Guiding Principles for the Naming of Recombinant Collagen Biomaterials" (hereinafter referred to as the "Principles") were issued to address the phenomenon of non-standard terminology in the industry. The "Principles" classify recombinant collagen into three categories: recombinant human collagen, recombinant humanized collagen, and recombinant collagen-like proteins. Recombinant humanized collagen is further divided into Type A and Type B. Type A refers to materials that do not contain non-human collagen amino acid sequences; Type B refers to materials that have non-human collagen amino acid sequences such as linker amino acids and tag amino acids added to the combination of functional human collagen fragments. Because Type A is a completely human collagen sequence, allergic reactions after use are greatly reduced.

[0007] Recombinant type III collagen is prepared by DNA recombination technology and contains a full-length or partial amino acid sequence fragment encoded by a specific type of collagen gene, or a combination of functional collagen fragments. However, it is not easy to accurately screen the amino acid sequence of the target functional region in the complete gene of a specific type of collagen. It is necessary to analyze the full-chain gene sequence of the protein and screen the amino acids in the core functional region to ensure that the screened functional region has the desired effect.

[0008] There are two types of recombinant type III collagen. One is conventional recombinant type III collagen, which is an amino acid sequence or fragment thereof encoded by a designed and modified specific gene, prepared by DNA recombination technology. Although its production is high, its amino acid sequence has low homology with the human collagen sequence, low activity, and a risk of immunogenicity. The other type is recombinant humanized type III collagen, which is the full-length or partial amino acid sequence of human collagen. Its production is lower, but it has high homology with the human collagen amino acid sequence, high activity, and no immunogenicity. However, there is still no solution to effectively increase the production of humanized type III collagen.

[0009] Therefore, there is an urgent need to provide a method for preparing recombinant humanized type A and type III collagen with high yield to meet the rapidly growing market demand for high-purity humanized collagen in the fields of medical aesthetics, medical devices, etc. Summary of the Invention

[0010] In response to the problems existing in the prior art, the purpose of the present invention is to provide a recombinant humanized type A and type III collagen protein, its preparation method, application, recombinant bacteria and its construction method. This method integrates a peptide segment with 100% identical sequence to type III collagen from humans (Homo sapiens) into the host bacteria to obtain a recombinant bacteria that efficiently synthesizes recombinant humanized type A and type III collagen protein. The recombinant humanized collagen protein produced by the recombinant bacteria does not contain any gene sequence derived from non-human collagen. This collagen protein has the characteristics of small molecular weight, high yield, high purity and easy solubility in water, and can be widely used in skin care products, medical beauty and skin repair dressings and other fields.

[0011] To solve the above problems, the present invention adopts the following technical solutions.

[0012] A method for preparing recombinant humanized type A and type III collagen, the preparation steps are as follows: S1: Construction of recombinant bacteria: The target gene as shown in SEQ ID NO. 2 is ligated to an expression vector by enzyme digestion to obtain a recombinant vector. The recombinant vector is amplified and transformed into an expression host and positive clones are performed to obtain recombinant bacteria; S2: The recombinant bacteria obtained in step S1 are induced with methanol and high-expressing strains are screened by detecting protein expression. After screening, the strains are inoculated into BSM medium for fermentation to obtain a fermentation broth, and the bacteria are collected by centrifugation. The bacteria are disrupted and centrifuged to obtain a supernatant, which is the crude protein solution; S3: Purify and dialyze the crude protein solution to obtain recombinant humanized type A and type III collagen solution.

[0013] Furthermore, in step S1, the enzymes used for enzyme digestion are EcoRI and NotI; the expression vector is pPICZa A, the amplification host is Escherichia coli; the expression host is Pichia pastoris; preferably Pichia pastoris X33; the positive cloning operation is: the expression host transformed with the recombinant vector is spread on a YPD plate supplemented with bleomycin and cultured at 28°C for 2-3 days.

[0014] Furthermore, in step S2, the fermentation temperature is 30° C., the fermentation pH is 5.0, the fermentation time is 70 h, the centrifugation time is 30 min, and the centrifugal speed is 10,000 rpm.

[0015] Furthermore, in step S3, purification includes: passing the crude protein solution through a desalting column and a cation exchange chromatography column in sequence; the desalting column is HiPre™ 26 / 10 Desalting; the cation exchange chromatography column is SP-FF, the dialysate of the dialysis comprises 10 mM Tris and 5 mM NaCl, and the pH of the dialysate is 8.0.

[0016] The present invention also provides a recombinant bacterium expressing recombinant humanized type A and type III collagen, comprising a recombinant plasmid pPICZa A-colla III and an expression host; the recombinant plasmid comprises a target gene and an expression vector; the nucleotide sequence of the target gene is shown in SEQ ID NO. 2, the protein product has a molecular weight of 12 kDa, and the expression vector is pPICZa A.

[0017] Furthermore, the expression host is Pichia pastoris, preferably Pichia pastoris X33.

[0018] The present invention also provides a method for constructing a recombinant bacterium that expresses recombinant humanized type A and type III collagen. The construction method comprises the following steps: connecting the target gene to an expression vector to obtain a recombinant plasmid, and then transforming the recombinant plasmid into an expression host to obtain the recombinant bacterium.

[0019] Furthermore, the nucleotide sequence of the target gene is shown in SEQ ID NO. 2, the expression vector is pPICZa A, the recombinant plasmid is pPICZa A-colla III, and the expression host is Pichia pastoris, preferably Pichia pastoris X33.

[0020] Furthermore, the construction method of the recombinant bacteria is specifically as follows: S1: Use EcoRI and NotI enzymes to digest the target gene and connect it to pPICZa A to obtain the recombinant plasmid pPICZa A-colla III; S2: The pPICZa A-colla Ⅲ obtained in step S1 was transformed into E. coli for amplification, and the plasmid was screened and extracted; then it was linearized; S3: The pPICZa A-colla Ⅲ linearized in step S2 is transferred into the expression host for positive cloning. The cloned bacteria are the recombinant bacteria.

[0021] Furthermore, in step S2, the culture medium used for amplification is an LB plate with zeocin (the host in step S2 is Escherichia coli, and LB plate screening is used), the culture temperature is 37 degrees, and the culture is overnight; the linearization enzyme is Sac I enzyme; in step S3, the culture dish for positive clones is a YPD plate with zeocin (bleomycin), the culture temperature for positive clones is 28 degrees, and the cloning time is 2-3 days.

[0022] Furthermore, the Escherichia coli is preferably Escherichia coli DH5α.

[0023] The present invention also provides a recombinant humanized type A and type III collagen, which is prepared by the above-mentioned method for preparing recombinant humanized type A and type III collagen or expressed by the above-mentioned recombinant bacteria expressing recombinant humanized type A and type III collagen.

[0024] Furthermore, the amino acid sequence of the recombinant humanized type A and type III collagen is shown in SEQ ID NO.1.

[0025] The present invention also provides an application of recombinant humanized type A and type III collagen, which is used in preparing external excipients or biomaterials for hydrating, moisturizing or skin repairing.

[0026] The present invention also provides a use of recombinant humanized type A and type III collagen in the preparation of hydrating or moisturizing skin care products.

[0027] The present invention also provides a polypeptide, the amino acid sequence of which is shown in SEQ ID NO.1.

[0028] The present invention also provides a DNA molecule encoding the above polypeptide or the above recombinant humanized type A and type III collagen, the base sequence of which is shown in SEQ ID NO.2.

[0029] Specifically, the present invention is achieved as follows: In the first aspect, the present invention provides a recombinant bacterium that efficiently synthesizes recombinant humanized type A and type III collagen, which includes a recombinant plasmid and an expression host; the recombinant plasmid contains a target gene and an expression vector; the target gene is a gene with a sequence 100% identical to human type III collagen.

[0030] In order to obtain a higher yield of recombinant humanized type A and type III collagen, the inventors used the Kyte-Doolittle hydrophilicity index to screen multiple highly hydrophilic regions; and confirmed the collagen triple helical domain through InterPro or Pfam to obtain candidate molecules. The candidate molecules were then sent to Shanghai Sangon Biotechnology Co., Ltd. to synthesize the coding DNA of the candidate molecules and then transferred into cells for expression. Multiple candidate fragments with hydrating and moisturizing functions were screened out, and the coding DNA of the candidate fragments was loaded into recombinant bacteria to screen out functional fragments that can be highly expressed in the recombinant bacteria.

[0031] Specifically, the amino acid sequence of the functional fragment obtained by screening is shown in SEQ ID NO. 1: PGADGQPGAKGEPGDAGAKGDAGPPGPAGPAGPPGPIGNVGAPGAKGARGSAGPPGATGFPGAAGRVGPPGPSGNAGPPGPPGPAGKEGGKGPRGETGPAGRPGEVGPPGPPGPAGEKGSPGADGPAGAPGTPGPQGIAGQRGV.

[0032] Since different species and cells have codon biases, in order to enable host cells to utilize their codon preferences to obtain stable and highly expressed recombinant humanized type A and type III collagen, the present invention reversely designed the gene sequence of the functional fragment gene obtained by the above screening using the online design tool Jcat, and codon-optimized it according to the expression requirements of the host Pichia pastoris. In some embodiments, the optimized nucleotide sequence of the recombinant humanized type A and type III collagen is as shown in SEQ ID NO. 2: CCTGGTGCCGATGGACAACCTGGAGCAAAGGGTGAACCTG GAGACGCTGGTGCTAAAGGTGATGCTGGTCCACCTGGTCCAGCAGGTCCAGCTGGTCCACCTGGTCCTATCGGTAACGTGGGAGCCCCCG GTGCTAAAGGCGCACGTGGTTCTGCTGGACCCCCTGGAGCTACTGGATTTCCCGGTGCCGCAGGTAGAGTTGGACCACCTGGACCTTCTGGAAACGCCGGACCTCCTGGTCCACCAGGTCCTGCCGGTAAAGAGGGAGGA AAGGGCCCAAGAGGAGAAACGGGTCCTGCTGGCAGACCCGGAGAAGTTGGTCCTCCAGGACCACCAGGTCCAGCCGGAGAGAAAGGTTCCCCCGGAGCTGATGGTCCAGCTGGAGCACCTGGTACTCCCGGTCCCCAGGG TATAGCTGGTCAGCGTGGTGTT.

[0033] In some embodiments, the expression host is Pichia pastoris, preferably Pichia pastoris X33.

[0034] In some embodiments, the expression vector is pPICZa A.

[0035] In a second aspect, the present invention provides a method for constructing the above-mentioned recombinant bacteria, which comprises: connecting the target gene to an expression vector to obtain a recombinant plasmid, and then transforming the recombinant plasmid into an expression host to obtain the recombinant bacteria.

[0036] In some embodiments, the recombinant plasmid is pPICZa A-colla III, and the expression host is Pichia pastoris, preferably Pichia pastoris X33.

[0037] In a third aspect, the present invention provides the use of the above-mentioned recombinant bacteria in recombinant humanized type A and type III collagen.

[0038] In a fourth aspect, the present invention also provides a method for producing recombinant humanized type A and type III collagen, comprising constructing the above-mentioned recombinant bacteria, then inoculating the recombinant bacteria into BSM culture medium for fermentation to obtain fermentation broth, collecting the bacteria by centrifugation, disrupting the cells of the bacteria, and centrifuging to obtain a supernatant, which is the crude protein solution.

[0039] In some embodiments, the fermentation conditions of the recombinant bacteria in BSM medium are: pH=5.0, and the fermentation time is 70 hours.

[0040] In some embodiments, the method further comprises purifying and dialysis the crude protein solution.

[0041] In some embodiments, purification comprises: passing the crude protein solution through a desalting column and a cation exchange chromatography column in sequence.

[0042] In some embodiments, the desalting column is a HiPre™ 26 / 10 Desalting column.

[0043] In some embodiments, the cation exchange chromatography column is SP-FF.

[0044] In some embodiments, the dialysate for dialysis comprises 10 mM Tris and 5 mM NaCl, and the pH of the dialysate is 8.0.

[0045] Compared with the prior art, the advantages of the present invention are: The present invention integrates a peptide segment with 100% identical sequence to type III collagen from humans (Homo sapiens) into a host bacterium to obtain a recombinant bacterium that efficiently synthesizes recombinant humanized type A and type III collagen. The recombinant humanized collagen produced by this recombinant bacterium does not contain any gene sequences from non-human collagen sources. This collagen has the characteristics of small molecular weight, high yield, high purity, and easy solubility in water. It can be widely used in skin care products, medical cosmetology, skin repair dressings and other fields. Therefore, the recombinant humanized type A and type III collagen provided by the present invention and the preparation method thereof have good industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 The plasmid pPICZa A-colla Ⅲ is used for synthesizing and expressing recombinant humanized type A and type III collagen by recombinant Pichia pastoris in Example 1; Figure 2 This is a DNA identification diagram for the construction of the plasmid pPICZa A-colla III molecule in Example 1; Figure 3 This is an SDS-PAGE image of the protein expressed by the recombinant Pichia pastoris pPICZa A-colla III in Example 2; Figure 4 This is the SDS-PAGE pattern of the cation exchange chromatography column purification in Example 2; Figure 5 The results of thermal stability study of the purified collagen in Example 2 are as follows; Figure 6 This is the water solubility test result of the purified collagen in Example 2. DETAILED DESCRIPTION

[0047] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0048] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0049] The Escherichia coli DH5α used in the following examples was purchased from Sangon Biotech (Shanghai) Co., Ltd.; Pichia pastoris X33 was purchased from Miaoling Plasmid Platform.

[0050] The culture media used in the following examples: Yeast extract peptone dextrose (YPD) medium was purchased from Sangon Biotech (Shanghai) Co., Ltd.; BMGY medium was purchased from Sangon Biotech (Shanghai) Co., Ltd.; BMMY medium was purchased from Sangon Biotech (Shanghai) Co., Ltd.; BSM medium was purchased from Sangon Biotech (Shanghai) Co., Ltd.; and SOC medium was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0051] The restriction endonucleases and ligases involved in the following examples were purchased from New England Bioland (New England Biotechnology (Beijing) Co., Ltd.).

[0052] The features and performance of the present invention are further described in detail below with reference to the embodiments. Example

[0053] This example is a method for constructing a recombinant bacterium for producing recombinant humanized type A and type III collagen, and the steps are as follows: (1) The synthetic gene (the gene fragment shown in SEQ ID NO. 2) was ligated to the vector pPICZa A by digestion with EcoRI and NotI (using T4 DNA ligase at 16°C overnight). The ligation product was named pPICZa A-collaⅢ, and then sequenced to verify the correctness of the connection. The results of double enzyme digestion of the complete vector were then sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for gene sequencing verification. Sequence comparison confirmed that the construction was successful. (2) The successfully constructed pPICZa A-colla Ⅲ was transformed into Escherichia coli DH5α (after ice bath for 30 minutes, heat shock at 42℃ for 45 seconds, ice bath again for 2 minutes, 800μL SOC medium was added and recovered at 37℃ for 1 hour), pPICZa A-colla Ⅲ was amplified, and then it was spread on LB plates containing 25μg / mL Zeocin (bleomycin), and cultured overnight at 37℃ for screening. Positive transformants appeared as white single colonies; a conventional plasmid extraction kit (such as SanPrep column extraction) was used to extract plasmids from positive transformants according to standard procedures (the operating procedures are routine operations and are not described in detail), and then the extracted plasmids were verified by electrophoresis (electrophoresis results are shown in Figure 2). Figure 2 As shown), the electrophoresis operation is conventional; (3) Use Sac I to digest pPICZa A-colla III (37°C, 2 h) to linearize it, and perform electrophoresis to verify that the linearization is complete; (4) The linearized pPICZa A-colla Ⅲ was electroporated into Pichia pastoris X33 (the electroporation operation here is a routine operation and will not be described in detail). It was spread on a YPD plate supplemented with 300 μg / mL zeocin and cultured at 28°C for 2-3 days to obtain positive clones and obtain recombinant bacteria.

[0054] Pick a single colony with a sterile toothpick or inoculating loop and inoculate it into YPD + Zeocin liquid medium for further verification. Then add 20% glycerol and freeze at -80°C.

[0055] in Figure 1 Schematic diagram of the construction of the recombinant plasmid pPICZa A-colla Ⅲ; Figure 2 This is a diagram verifying the construction results of the recombinant plasmid pPICZa A-colla Ⅲ. Figure 2 In the figure, M is a DNA marker; 1, 2, 3, and 4 are complete plasmids, i.e., the target vector; 5, 6, 7, and 8 are the results of double enzyme digestion of the complete vector. This result indicates that the constructed vector is the same size as the target vector. The electrophoresis bands show that the constructed vector is the same size as the target vector, and the double enzyme digestion results are also as expected. Example

[0056] This example is a method for producing and purifying recombinant humanized type A and type III collagen, and the steps are as follows: (1) The synthetic gene (the gene fragment shown in SEQ ID NO. 2) was ligated to the vector pPICZa A by digestion with EcoRI and NotI (using T4 DNA ligase at 16°C overnight). The ligation product was named pPICZa A-collaⅢ, and then sequenced to verify the correctness of the connection. The results of double enzyme digestion of the complete vector were then sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for gene sequencing verification. Sequence comparison confirmed that the construction was successful. (2) The successfully constructed pPICZa A-colla Ⅲ was transformed into Escherichia coli DH5α (after ice bath for 30 minutes, heat shock at 42℃ for 45 seconds, ice bath again for 2 minutes, 800μL SOC medium was added and recovered at 37℃ for 1 hour), pPICZa A-colla Ⅲ was amplified, and then it was spread on LB plates containing 25μg / mL Zeocin (bleomycin), and cultured overnight at 37℃ for screening. Positive transformants appeared as white single colonies; a conventional plasmid extraction kit (such as SanPrep column extraction) was used to extract plasmids from positive transformants according to standard procedures (the operating procedures are routine operations and are not described in detail), and then the extracted plasmids were verified by electrophoresis (electrophoresis results are shown in Figure 2). Figure 2 As shown), the electrophoresis operation is conventional; (3) Use Sac I to digest pPICZa A-colla III (37°C, 2 h) to linearize it, and perform electrophoresis to verify that the linearization is complete; (4) The linearized pPICZa A-colla Ⅲ was electroporated into Pichia pastoris X33, plated on a YPD plate supplemented with 300 μg / mL zeocin, and cultured at 28°C for 2–3 days to obtain positive clones and obtain recombinant bacteria. (5) The colonies selected from the YPD plate containing 300 μg / mL zeocin were inoculated into BMGY medium for fermentation (30°C). After culturing for 24 h, the culture was transferred to BMMY medium and 0.5% methanol was added to induce expression. (6) After 5 days of methanol induction, the supernatant was collected and the protein expression level was detected by SDS-PAGE, and the protein with the thickest protein band (e.g. Figure 3 The bacteria corresponding to the protein band indicated by the blue arrow in the middle were screened out to find the strain with the highest expression level (numbered: A1); (7) The strain A1 with the highest expression level was subjected to pilot fermentation in BSM medium (pH controlled at 5.0, 30°C, 70 h) (8) After fermentation, the fermentation liquid was centrifuged at 10000 r·min -1 After centrifugation for 30 min, the supernatant was collected, which was the crude protein solution; (9) The purification method is ion exchange column purification. The crude protein solution is purified by HiPre™ 26 / 10 Desalting and SP-FF respectively to obtain collagen solution; (10) The collagen solution obtained after dialysis (4°C, 12-16 h) using a dialysate consisting of 10 mM Tris (tris(hydroxymethylaminomethane)) and 5 mM NaCl (pH = 8.0) is the recombinant humanized type A and type III collagen solution.

[0057] The obtained recombinant humanized type A and type III collagen was sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for LC-MS / MS analysis. Sequence comparison showed that the obtained recombinant humanized type A and type III collagen was consistent with the amino acid sequence of SEQ ID NO.1, and no gene sequence of non-human collagen origin was detected.

[0058] Figure 3 The figure shows the SDS-PAGE of the protein expressed by recombinant Pichia pastoris X33 / pPICZa A-colla Ⅲ. NC is the blank control. M represents a DNA marker. 1 to 10 represent the expression patterns of 10 screened seeds. The blue arrow indicates the seed with the highest relative expression level, namely the colony numbered A1 (the seed refers to the colony screened from a YPD plate containing 300 μg / mL zeocin). The figure shows that the target protein is expressed as a single band at 12 kDa.

[0059] Figure 4 This is an SDS-PAGE profile of recombinant humanized type A and type III collagen purified using an ion exchange column. M represents a DNA marker; S represents fermentation supernatant; FT represents flow-through; E0 represents buffer A eluent; P1 represents 50 mM NaCl eluent; P2 represents 100 mM NaCl eluent; P3 represents 200 mM imidazole eluent; P4 represents 300 mM imidazole eluent; and P5 represents 500 mM imidazole eluent. The 65 kDa band is the brightest in the protein marker, facilitating comparison with the target protein. The target protein is primarily present in peak P3, with contaminants in peaks P1 and P2.

[0060] Test Example 1: The purified recombinant humanized collagen solution in Example 2 was subjected to a thermal stability test, and the test method was as follows: The purified protein solution was placed in a refrigerator at 4 degrees Celsius, and samples were taken at different times and run on SDS-PAGE to detect whether the collagen solution was degraded.

[0061] Figure 5The figure shows the thermal stability study results of the purified collagen; M is a protein marker, 1 is the test result at day 0, 2, 3, and 4 are the test results at days 3, 8, and 14, respectively. The absence of degradation bands in the figure indicates that the collagen has good thermal stability in a 4-degree refrigerator, that is, the recombinant humanized collagen has good thermal stability, and the purified collagen will not degrade when placed at 4 degrees for 3, 8, and 14 days.

[0062] Test Example 2: The purified recombinant humanized collagen solution in Example 2 was tested for water solubility using the following method: 150 mg of freeze-dried recombinant humanized collagen powder was weighed and dissolved in 10 mL of pure water to observe the solubility.

[0063] like Figure 6 As shown, 150 mg of freeze-dried powder was dissolved in 10 mL of pure water, and the powder was completely dissolved, indicating that the recombinant humanized collagen had good water solubility.

[0064] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A method for preparing recombinant humanized type A and type III collagen, characterized by: The preparation steps are as follows: S1: Construction of recombinant bacteria: The target gene as shown in SEQ ID NO. 2 is ligated to an expression vector by enzyme digestion to obtain a recombinant vector. The recombinant vector is amplified and transformed into an expression host and positive clones are performed to obtain recombinant bacteria; S2: The recombinant bacteria obtained in step S1 are induced with methanol and high-expressing strains are screened by detecting protein expression. After screening, the strains are inoculated into BSM medium for fermentation to obtain a fermentation broth, and the bacteria are collected by centrifugation. The bacteria are disrupted and centrifuged to obtain a supernatant, which is the crude protein solution; S3: Purify and dialyze the crude protein solution to obtain recombinant humanized type A and type III collagen solution.

2. The method for preparing recombinant humanized type A and type III collagen according to claim 1, characterized in that: In step S1, the enzymes used for enzyme digestion are EcoRI and NotI; the expression vector is pPICZa A, the amplification host is Escherichia coli; the expression host is Pichia pastoris; the positive cloning operation is: the expression host transformed with the recombinant vector is spread on a YPD plate supplemented with bleomycin and cultured at 28°C for 2-3 days.

3. The method for preparing recombinant humanized type A and type III collagen according to claim 1, wherein: In step S2, the fermentation temperature is 30° C., the fermentation pH is 5.0, the fermentation time is 70 h, the centrifugation time is 30 min, and the centrifugal speed is 10,000 rpm.

4. The method for preparing recombinant humanized type A and type III collagen according to claim 1, wherein: In step S3, purification includes: passing the crude protein solution through a desalting column and a cation exchange chromatography column in sequence; the desalting column is HiPre™ 26 / 10 Desalting; the cation exchange chromatography column is SP-FF, the dialysate of the dialysis comprises 10 mM Tris and 5 mM NaCl, and the pH of the dialysate is 8.

0.

5. A recombinant bacterium expressing recombinant humanized type A and type III collagen, characterized by: It includes a recombinant plasmid pPICZaA-colla III and an expression host; the recombinant plasmid contains a target gene and an expression vector; the nucleotide sequence of the target gene is shown in SEQ ID NO. 2, the protein product has a molecular weight of 12 kDa, and the expression vector is pPICZa A.

6. The recombinant bacterium expressing recombinant humanized type A and type III collagen according to claim 5, characterized in that: The expression host is Pichia pastoris.

7. The method for constructing a recombinant bacterium expressing recombinant humanized type A and type III collagen according to any one of claims 5 to 6, characterized in that: The construction method is: connecting the target gene to an expression vector to obtain a recombinant plasmid, and then transforming the recombinant plasmid into an expression host to obtain the recombinant bacteria.

8. The method for constructing a recombinant bacterium expressing recombinant humanized type A and type III collagen according to claim 7, characterized in that: The nucleotide sequence of the target gene is shown in SEQ ID NO. 2, the expression vector is pPICZa A, the recombinant plasmid is pPICZa A-colla III, and the expression host is Pichia pastoris.

9. A recombinant humanized type A and type III collagen, characterized by: The protein is prepared by the method for preparing a recombinant humanized type A and type III collagen according to any one of claims 1 to 4, or expressed by a recombinant bacterium expressing a recombinant humanized type A and type III collagen according to any one of claims 5 to 6.

10. The recombinant humanized type A and type III collagen according to claim 9, characterized in that: The amino acid sequence of the recombinant humanized type A and type III collagen is shown in SEQ ID NO.

1.

11. The use of recombinant humanized type A and type III collagen according to claim 10, characterized in that: Application in the preparation of external excipients or biomaterials for hydration, moisturizing or skin repair.

12. Use of the recombinant humanized type A and type III collagen according to claim 10 in the preparation of hydrating or moisturizing skin care products.

13. A polypeptide, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

1.

14. A DNA molecule encoding a polypeptide according to claim 13 or a recombinant humanized type A and type III collagen according to claim 10, characterized in that: Its base sequence is shown in SEQ ID NO.2.

Images