Method for fermenting recombinant humanized collagen by using escherichia coli and application of recombinant humanized collagen

Abstract

The invention discloses a method for fermenting recombinant humanized collagen by using escherichia coli and application of the recombinant humanized collagen, and belongs to the field of collagen. The amino acid sequence of the prepared recombinant humanized III-type collagen is shown as SEQ ID NO.4-7, the recombinant humanized III-type collagen is formed by connecting a plurality of repetitive units of natural collagen in series and has the advantages of being good in biocompatibility and high in safety, in addition, the nucleotide sequence for coding the recombinant collagen is designed according to the codon optimization theory, and the recombinant humanized III-type collagen has the advantages of being good in biocompatibility and high in safety. And carrying out fermentation expression on escherichia coli to obtain the recombinant humanized III-type collagen. The humanized III-type collagen can significantly improve the cell adhesion activity and proliferation activity, and can be widely applied to the fields of food, cosmetics, health care products, medical instruments and the like.

Description

Technical Field

[0001] The present invention relates to the field of collagen, and in particular to a method for fermenting recombinant humanized collagen using Escherichia coli and an application thereof. Background Art

[0002] Collagen is one of the most important and abundant proteins in mammals. It is generally white, transparent, and unbranched fibrils. It is the basic support for skin and bones, accounting for 25% to 35% of the total protein. It is mainly distributed in human skin, blood vessels, bones, tendons, teeth, and cartilage. It is the main matrix and scaffold of these tissues, protecting and connecting various tissues, and plays an important physiological function in the body. Therefore, collagen can be widely used in industries such as medicine and cosmetics.

[0003] Human type III collagen is a transmembrane non-fibroblastic collagen. It is a homogeneous trimer composed of three identical α1 (XVII) chains and is divided into three major structural domains: intracellular, transmembrane, and extracellular. Human type III collagen is a component of hemidesmosomes in cells and plays an important role in the function of epithelial cells-basement membranes. It can regulate the adhesion, separation, and development and differentiation of epithelial cells, and plays an important role in the differentiation and regeneration of keratinocytes. The content of human type III collagen in the human body is extremely low, and the content of type III collagen in animals is also extremely rare. The extraction is also very difficult and cannot be mass-produced. At the same time, animal-derived collagen products inevitably have immunogenicity and potential biosafety risks such as viruses and epidemics, and are not widely used. Limited by legal and ethical constraints, human type III collagen can only be used in scientific research. This has led to the fact that there are no commercialized type III collagen products from humans or other animals on the market, which further limits the research and application of human type III collagen. The main way to solve such problems now is to complete collagen through biotechnology such as genetic engineering.

[0004] The application of recombinant collagen requires large-scale, high-density, high-expression fermentation production and purification. Escherichia coli is an ideal expression host. However, as a eukaryotic organism, when a large amount of heterologous proteins are expressed and secreted, that is, when the intracellular biological resources are occupied in large quantities, Escherichia coli will inevitably regulate the expression of heterologous proteins accordingly. Its outstanding performance is actually the degradation of the expressed recombinant protein. Recombinant collagen is constructed by genetic engineering technology to express engineered bacteria containing the full-length gene or partial fragment gene of collagen, and then the engineered bacteria are used to synthesize collagen, and finally a specific purification process is used to obtain high-purity recombinant collagen. The use of genetic engineering technology to construct genetically engineered strains is of great significance for the preparation of high-quality human-like collagen. Summary of the invention

[0005] The technical problem to be solved by the present invention is to utilize genetic engineering technology to recombinantly express a new recombinant humanized type III collagen, specifically a method for fermenting recombinant humanized collagen using Escherichia coli and its application.

[0006] Based on the above, the technical solution of the present invention is achieved as follows: The present invention provides a recombinant humanized type III collagen, the amino acid sequence of the collagen is shown as SEQ ID NO.4-7.

[0007] The present invention also provides a nucleic acid molecule encoding the recombinant humanized type III collagen.

[0008] Furthermore, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.8-11.

[0009] The invention also provides a recombinant expression vector comprising the nucleic acid molecule.

[0010] The present invention also provides a host cell comprising the recombinant expression vector or expressing the collagen.

[0011] Furthermore, the host cell is Escherichia coli.

[0012] It should be noted that host cell refers to any cell type, which is susceptible to transformation, transfection, transduction, etc. of nucleic acid constructs or expression vectors containing polynucleotides of the present application. Host cell can be any cell useful in the production of recombinant humanized collagen of the present application. In order to produce recombinant collagen, nucleic acid encoding recombinant collagen can be separated and inserted into one or more vectors to further clone and / or express in host cells. Conventional techniques (e.g., by using oligonucleotide probes that can specifically bind to genes encoding recombinant collagen) can be easily separated and sequenced. The host cell refers to a cell into which exogenous nucleic acid has been introduced, including the offspring of such cells. Host cells include transformants and transformed cells, which include primary transformed cells and offspring derived therefrom, regardless of the number of passages. Offspring may not be completely identical to parental cells in nucleic acid content, but may contain mutations. It is well known that the method for introducing a vector into a host cell is, for example, using electrotransformation to introduce a vector into a host cell, and the method can also be transfection, microinjection technology, gene gun technology, liposome-mediated method, etc. The host cell is a prokaryotic cell or a eukaryotic cell. The host cell is selected from any one of Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, and Bacillus subtilis. Preferably, the prokaryotic cell is Escherichia coli; preferably, the eukaryotic cell is Pichia pastoris.

[0013] The present invention also provides a method for producing the recombinant humanized type III collagen, which is characterized by comprising the following steps: (1) culturing the host cell in a culture medium; (2) Isolating the recombinant humanized type III collagen from the host cells.

[0014] The present invention also provides a composition, which includes the recombinant humanized type III collagen, or the recombinant humanized type III collagen encoded by the nucleic acid molecule, or the recombinant humanized type III collagen expressed by the recombinant expression vector, or the recombinant humanized type III collagen produced by the host cell, or the recombinant humanized type III collagen prepared by the production method.

[0015] The present invention also provides a product comprising the composition, which is a pharmaceutical composition, a medical device or a cosmetic.

[0016] Furthermore, the product is one or more of biological dressings, human bionic materials, plastic surgery materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue injection filling materials, ophthalmic materials, obstetrics and gynecology biomaterials, nerve repair and regeneration materials, liver tissue materials and vascular repair and regeneration materials, 3D printed artificial organ biomaterials, cosmetic raw materials, pharmaceutical excipients and food additives.

[0017] Furthermore, the part of the cosmetic is not particularly limited, and can be the face, hands, legs, torso, etc. The form of the composition is not particularly limited, as long as the intended function can be achieved. For example, the composition is a solid, liquid or gel composition.

[0018] The present invention also provides the use of the composition in preparing pharmaceutical compositions, medical devices or cosmetics.

[0019] The present invention provides use of recombinant humanized type III collagen in preparing a medicine for promoting cell adhesion, promoting cell proliferation and / or promoting wound healing.

[0020] Compared with the prior art, the present invention has the following beneficial effects: The recombinant humanized type III collagen of the present invention is formed by a plurality of repeating units connected in series, and the repeating units are derived from the amino acid sequence of natural type III collagen, so it has the advantages of good biocompatibility and high safety. The recombinant humanized type III collagen of the present invention has good effects of promoting cell adhesion, promoting cell proliferation and / or promoting wound healing, and can be widely used in the fields of food, cosmetics, health care products and medical and medical products. The preparation method of the recombinant humanized type III collagen of the present invention is simple and easy to operate, and is suitable for industrial large-scale production, so it has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 : Evaluation of cytotoxicity of recombinant humanized type III collagen.

[0022] Figure 2 : Cell proliferation promoting activity of recombinant humanized type III collagen.

[0023] Figure 3 : Cell adhesion promoting activity of recombinant humanized type III collagen. DETAILED DESCRIPTION

[0024] The present invention is described below by way of examples to make the technical solution of the present invention easier to understand and grasp, but the present invention is not limited thereto. The experimental methods described in the following examples are conventional methods unless otherwise specified; the medicinal materials and reagents described can be obtained from commercial channels unless otherwise specified; and the performance of products from different sources does not have a significant impact.

[0025] For those skilled in the art, the features, benefits and advantages of the present invention will become apparent by reading the contents disclosed in this specification.

[0026] Unless otherwise indicated, all percentages, fractions and ratios are calculated based on the total weight of the composition of the present invention. The term "mass content" herein may be represented by the symbol "%".

[0027] As used herein, "medical device" means instruments, equipment, apparatus, in vitro diagnostic reagents and calibrators, materials, and other similar or related items that are intended for direct or indirect use on the human body.

[0028] As used herein, "isolation" refers to separating the target polypeptide from the cultured host cells, for example, the host cells are broken and the target polypeptide is purified. In the case where the purified target polypeptide carries a purification tag, such as a Trx or His tag, "isolation" also includes enzymatic removal of the Trx or His tag.

[0029] As used herein, "includes," "comprising," "including," "containing," "having," or other variations are intended to encompass non-exclusive inclusions, and no distinction is made between these terms. The term "comprising" means that other steps and ingredients that do not affect the end result may be added. The term "comprising" also includes the terms "consisting of" and "consisting essentially of." The compositions and methods / processes of the present invention may comprise, consist of, and consist essentially of the essential elements and limitations described herein, as well as any additional or optional ingredients, components, steps, or limitations described herein.

[0030] Example 1 Design of amino acid sequence of recombinant humanized type III collagen In the present invention, the human natural type III collagen COL-III sequence is selected for screening and optimization. The sequence of human type III collagen is the NCBI reference sequence: KAI4037253.1 (SEQ ID No. 12), see https: / / www.ncbi.nlm.nih.gov / protein / KAI4037253.1. The details are as follows: MMSFVQKGSWLLLALLHPTIILAQQEAVEGGCSLGQSYADRDVWKPEPCQICVCDSGSVLCDDIICDDQELDCPNPEIPFGECCAVCPQPPTAPTRPPN GQGPQGPKGDPGPPGIPGRNGDPGIPGQPGSPGSPGPPGIC ESCPTGPQNYSPQYDSYDVKSGVAVGGLAGYPGPAGPPGPPGPPGTSGHPGSP GSPGYQGPPGEPGQAGPSGPPGPPGAIGP SGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHR GFDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSDGQPGPPGPPGTAGFPGSPGAKGEVGPAGSPGSNGAPGQRGEPGPQGHAGAQGPPGPPGINGSPGGKGEMGPAGIPGAPGLMG ARGPPGPAGANGAPGLRGGAGEPGKNGAKGEPGPRGERGEAGIPGVPGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQ GESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPP GKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPGPGLAGAPGLRGGGAGPPGPEGGKGAAGPPGPPGAAGTPGLQGMPGERGGLGSPGPKGDKGEPGGPGADGVPGKDGPRGPTGPIGPPGPAGQPGDKGEGGAPGLPGIAG PRGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGEGGPPGVAGPPGKDGTSGHPGPIGPPGPRGNRGERGSEGSPGHPGQPGPPGPPGAPGPCCGGVGAAA IAGIGGEKAGGFAPYYGDEPMDFKINTDEIMTSLKSVNGQIESLISPDGSRKNPARNCRDLKFCHPELKSGEYWVDPNQGCKLDAIKVFCNMETGETCISANPLNVPRK HWWTDSSAEKKHVWFGESMDGGFQFSYGNPELPEDVLDVQLAFLRLLSSRASQNITYHCKNSIAYMDQASGNVKKALKLMGSNEGEFKAEGNSKFTYTVLEDGCTKHTGE WSKTVFEYRTRKAVRLPIVDIAPYDIGGPDQEFGVDVGPVCFL (SEQ ID No. 12).

[0031] The bold underlined part in the above sequence is the amino acid sequence selected by the present invention. The applicant has found through extensive research that the above sequence has strong water solubility, high recombinant expression yield, simple purification process, and better biological activity than commercial collagen or other sequences in SEQ ID No.12.

[0032] The present invention is based in part on the following discovery: a polypeptide comprising at least 41 consecutive amino acid residues in SEQ ID No. 12 can have better biomaterial properties than commercial human collagen, so the sequences of several specific amino acid regions were tested: COL-HR-1: GQGPQGPKGDPGPPGIPGRNGDPGIPGQPGSPGSPGPPGIC (SEQ ID NO. 1).

[0033] COL-HR-2: GSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHR (SEQ ID NO. 2).

[0034] COL-HR-3: GESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGER (SEQ ID NO. 3).

[0035] The recombinant humanized type III collagen COL-HR-111 of the present invention is a three-repeat sequence of COL-HR-1 (SEQ ID No. 1), including 123 amino acids. The amino acid sequence of COL-HR-111 is as follows: GQGPQGPKGDPGPPGIPGRNGDPGIPGQPGSPGSPGPPGICGQGPQGPKGDPGPPGIPGRNGDPGIPGQPGSPGSPGPPGICGQGPQGPKGDPGPPGIPGRNGDPGIPGQPGSPGSPGPPGIC (SEQ ID NO. 4).

[0036] The DNA sequence of COL-HR-111 is as follows: GGCCAGGGCCCGCAGGGCCCGAAAGGCGATCCGGGCCCGCCGGGCATTCCGGGCCGCAACGGCGATCCGGGCATTCCGGGCCAGGGGCAGCCCGGGCAGCCCGGGCCCGCCGGGCATTTGCGGCCAGGGCCCGCAGGGCCCGAAAGGCGATCCGGGCCCGCCGGGCATTCCGG GCCGCAACGGCGATCCGGGCATTCCGGGCCAGCCGGGCAGCCCGGGCAGCCCGGGCCCGCCGGGCATTTGCGGCCAGGGCCCGCAGGGCCCGAAAGGCGATCCGGGCCCGCCGGGCATTCCGGGCCGCAACGGCGATCCGGGCATTCCGGGCCAGCCGGGCAGCCCGGGCAGCCCGGGCCCGCCGGGCATTTGC.

[0037] After codon optimization of the above DNA sequence, the GC content changed from 80.49% before optimization to 69.38% after optimization. The sequence is as follows: GGTCAGGGTCCACAAGGCCCGAAAGGCGATCCTGGTCCGCCAGGCATTCCGGGCCGTAACGGTGATCCGGGCATCCCTGGCCAGCCGGGCTCTCCGGGTTCCCTGGTCCGCCGGGTATTTGTGGCCAGGGTCCTCAGGGCCCAAAAGGTGATCCTGGTCCG. CCTGGTATCCCGGGCCGCAACGGTGATCCTGGCATCCCGGGCCAGCCGGGTTCCCCGGGTTCCCCGGGCCCGCCAGGCATCTGCGGTCAGGGTCCACAGGGCCCAAAGGGTGACCCGGGTCCACCGGGTATTCCGGGTCGCAATGGTGATCCGGGTATTCCGGGCCAGCCAGGTTCTCCTGGCTCTCCGGGTCCGCCGGGTATCTGC (SEQ ID NO.8).

[0038] The recombinant humanized type III collagen COL-HR-222 of the present invention is a three-repeat sequence of COL-HR-2 (SEQ ID No. 2), including 316 amino acids. The amino acid sequence of COL-HR-222 is as follows: GSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGES GRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHR (SEQID NO.5).

[0039] The DNA sequence of COL-HR-222 is as follows: GGCAGCCCGGGCTATCAGGGCCCGCCGGGCGAACCGGGCCAGGCGGGCCCGAGCGGCCCGCCGGGCCCGCCGGGCGCGATTGGCCCGAGCGGCCCGGCGGGCAAAGATGGCGAAAGCGGCCGCCCGGGCCGCCCGGGCGAACGCGGCCTGCCGGGCCCGCCGGGCATTAAAGGCCCGGCGGGCATTCCGGGCTTTCCGGGCATGAAAGGCCATCGCGGCAGCCCGGGCTATCAGGGCCCGCCGGGCGAACCGGGCCAGGCGGGCCCGAGCGGCCCGCCGGGCCCGCCGGGCGCGATTGGCCCGAGCGGCCCGGCGGGCAAAGATGGCGAAAGCGGCCGCCCGGGCCGCCCGGGCGAACGCGGCCTGCCGGGCCCGCCGGGCATTAAAGGCCCGGCGGGCATTCCGGGCTTTCCGGGCATGAAAGGCCATCGCGGCAGCCCGGGCTATCAGGGCCCGCCGGGCGAACCGGGCCAGGCGGGCCCGAGCGGCCCGCCGGGCCCGCCGGGCGCGATTGGCCCGAGCGGCCCGGCGGGCAAAGATGGCGAAAGCGGCCGCCCGGGCCGCCCGGGCGAACGCGGCCTGCCGGGCCCGCCGGGCATTAAAGGCCCGGCGGGCATTCCGGGCTTTCCGGGCATGAAAGGCCATCGC。

[0040] After codon optimization, the GC content of the above DNA sequence changed from 80.09% before optimization to 69.91% after optimization. The sequence is as follows: (SEQ ID NO.9).

[0041] The recombinant humanized type III collagen COL-HR-333 of the present invention is a three-repeat sequence of COL-HR-3 (SEQ ID No. 3), including 378 amino acids. The amino acid sequence of COL-HR-333 is as follows: GESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGER(SEQ ID NO.6)。

[0042]

[0043]

[0044] The recombinant humanized type III collagen COL-HR-123 of the present invention is a connection sequence of COL-HR-1, COL-HR-2 and COL-HR-3, including 239 amino acids. The amino acid sequence of COL-HR-123 is as follows: GQGPQGPKGDPGPPGIPGRNGDPGIPGQPGSPGSPGPPGICGSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGESGRPGP PGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGER (SEQ ID NO.7).

[0045] The DNA sequence of COL-HR-123 is as follows: GGCCAGGGCCCGCAGGGCCCGAAAGGCGATCCGGGCCCGCCGGGCATTCCGGGCCGCAACGGCGATCCGGGCATTCCGGGCCAGCCGGGCAGCCCGGGCAGCCCGGGCCCGCCGGGCATTTGCGGCAGCCCGGGCTATCAGGGCCCGCCGGGCGAACCGGGCCAGGCGGGCCCGAGCGGCCCGCCGGGCCCGCCGGGCGCGATTGGCCCGAGCGGCCCGGCGGGCAAAGATGGCGAAAGCGGCCGCCCGGGCCGCCCGGGCGAACGCGGCCTGCCGGGCCCGCCGGGCATTAAAGGCCCGGCGGGCATTCCGGGCTTTCCGGGCATGAAAGGCCATCGCGGCGAAAGCGGCCGCCCGGGCCCGCCGGGCCCGAGCGGCCCGCGCGGCCAGCCGGGCGTGATGGGCTTTCCGGGCCCGAAAGGCAACGATGGCGCGCCGGGCAAAAACGGCGAACGCGGCGGCCCGGGCGGCCCGGGCCCGCAGGGCCCGCCGGGCAAAAACGGCGAAACCGGCCCGCAGGGCCCGCCGGGCCCGACCGGCCCGGGCGGCGATAAAGGCGATACCGGCCCGCCGGGCCCGCAGGGCCTGCAGGGCCTGCCGGGCACCGGCGGCCCGCCGGGCGAAAACGGCAAACCGGGCGAACCGGGCCCGAAAGGCGATGCGGGCGCGCCGGGCGCGCCGGGCGGCAAAGGCGATGCGGGCGCGCCGGGCGAACGC。

[0046] After codon optimization, the GC content of the above DNA sequence changed from 80.89% before optimization to 69.87% after optimization. The sequence is as follows: (SEQ ID NO.11).

[0047] The above collagen DNA sequences SEQ ID NOs. 8-11 are all codons of recombinant genes optimized based on codon preference and expression capacity in an E. coli expression host using the Exp Optimizer codon optimization tool.

[0048]

[0049] Example 2 Construction and expression of recombinant humanized type III collagen expression vector The gene sequence of collagen is shown in SEQ ID NO.8-11, and was submitted to Sangon Biotech (Shanghai) Co., Ltd. for full gene synthesis.

[0050] The same method as in Example 2 of the patent CN118324899B authorized by the applicant was used to construct vectors and express and purify target collagens COL-HR-111, COL-HR-222, COL-HR-333 and COL-HR-123. The specific steps are as follows: (1) The DNA fragments of SEQ ID NOs. 8-11 were cloned and amplified by PCR, and the target genes were recovered by agarose gel electrophoresis. Using the EcoRI and NotI restriction sites added to the primers, the corresponding restriction endonucleases were added to cut the gene fragment into sticky ends, and the fragments were incubated with the pPIC9K expression vector (purchased from Thermo Fisher Scientific) treated with the corresponding restriction enzymes. T4 DNA ligase (TaKaRa, catalog number 2011A) was added, and the fragments were connected at 4°C for 12 hours, and transformed into Escherichia coli DH5α (purchased from Sangon Biotech (Shanghai) Co., Ltd.). White spot clones were selected using ampicillin sodium-LB plates, plasmids were extracted using the alkaline lysis method of molecular cloning, and positive clones were identified as correct using restriction enzyme digestion and PCR methods. Finally, sequencing confirmed that the recombinant plasmids contained the correct gene sequence reading frame, and the pPIC9K-COL-HR-111, pPIC9K-COL-HR-222, pPIC9K-COL-HR-333 and pPIC9K-COL-HR-123 recombinant expression vectors were successfully constructed, and sequenced by Sangon Biotech (Shanghai) Co., Ltd.

[0051] (2) The correct pPIC9K-COL-HR-111, pPIC9K-COL-HR-222, pPIC9K-COL-HR-333 and pPIC9K-COL-HR-123 expression vectors were transformed into Escherichia coli BL21 (DE3) (purchased from Sangon Biotech (Shanghai) Co., Ltd.), and single-plaque clones were selected using ampicillin sodium-LB plates. They were cultured overnight in 5 ml LB medium, transferred at a ratio of 1:100, and cultured in a shake flask at 37°C to an OD600 of 0.5. IPTG stock solution was added at a ratio of 1:5000, and cultured at 20°C for 8 h. The cells were collected by centrifugation and stored at -20°C or immediately proceeded to the next purification step.

[0052] (3) Wash the mixed bacterial precipitate with PBS buffer, resuspend about 500 ml of bacterial solution in 20-40 ml volume, use lysozyme and Triton X-100 to help lyse the bacteria, sonicate in an ice-water mixture for 60 min, centrifuge at 12000 rpm / min for 20 min, and collect the supernatant. At this time, the solution contains a large amount of the target recombinant humanized type III collagen COL-HR-111, COL-HR-222, COL-HR-333 and COL-HR-123.

[0053] (4) Ammonium sulfate precipitation and graded salting out were used for preliminary purification. After exploring the saturation of ammonium sulfate, the optimal saturation of ammonium sulfate salting out was 20%. The nickel ion affinity column (Ni-NTA His-Bind Resin) was washed with PBS buffer, and then the column material and the above-mentioned desalted and purified solution were mixed and incubated, and gently shaken at room temperature or on ice for 30 minutes, and then loaded onto the column, and the impurities were washed with a PBS solution containing 15mM imidazole, and only pure type III collagen remained on the column. An appropriate amount of Prescission Protease with a His tag was added to the column, and gently shaken at room temperature or on ice for 2 hours to release the type III collagen from the column, and eluted with PBS solution to obtain high-purity recombinant humanized type III collagen COL-HR-111 of SEQ ID NO.4, recombinant humanized type III collagen COL-HR-222 of SEQ ID NO.5, recombinant humanized type III collagen COL-HR-333 of SEQ ID NO.6, and recombinant humanized type III collagen COL-HR-123 of SEQ ID NO.8, respectively, and freeze-dried for use.

[0054] Example 3 Evaluation of cytotoxicity of recombinant humanized type III collagen HeLa cells grown to 80% of the bottom area of ​​the culture flask were taken, digested with 0.25% trypsin, and made into a cell density of 1×10 5 / mL of cell suspension. Take 100μL of cell suspension and inoculate it in a 96-well culture plate and culture it in an incubator at 37℃ and 5% CO2 saturated humidity. After 24h of cell culture, aspirate the complete culture medium. Add a recombinant humanized type III collagen solution diluted with high-glucose DMEM medium to the experimental group at a concentration of 0.05mg / ml (3 replicates for each concentration), the control group is cells cultured in DMEM medium, and the blank group is DMEM medium without cells. Continue to culture in an incubator at 37℃ and 5% CO2 saturated humidity for 24h. Add 10μL of CCK-8 reagent to each group, incubate in the cell culture incubator for 2h, and use an enzyme-linked immunosorbent assay to measure the absorbance value (OD value) of each well at a wavelength of 450nm. Calculate the cell survival rate according to the mean absorbance of each group according to the following formula: ; In this example, the positive control group was a commercially available recombinant type III collagen sample solution, which was added into the cell culture medium at 0.05 mg / ml, with 3 parallel samples in each group.

[0055] The experimental results are as follows Figure 1 As shown, the cell viability of each experimental group was above 100%, indicating that the recombinant humanized type III collagen COL-HR-111, COL-HR-222, COL-HR-333 and COL-HR-123 prepared by the present invention had no cytotoxicity and good safety.

[0056] Example 4 Evaluation of the cell proliferation promoting activity of recombinant humanized type III collagen BALB / c 3T3 cells (mouse embryonic fibroblasts) in good logarithmic growth phase (purchased from Shanghai Cell Bank, Chinese Academy of Sciences) were selected and inoculated into 96-well cell culture plates. After digestion with 0.25% trypsin, the cells were centrifuged and resuspended in DMEM medium containing 10% fetal bovine serum and counted. BALB / c 3T3 cells were inoculated into 96-well culture plates, with the number of cells per well being approximately 5×10 3 , and set up a blank control without cells, and cultured overnight at 37°C, 5% CO2. Recombinant humanized type III collagen COL-HR-111, COL-HR-222, COL-HR-333 and COL-HR-123 prepared in Example 2 were added at a final concentration of 0.05 mg / ml and 0.1 mg / ml, respectively, with 3 replicate wells in each group, and cultured at 37°C, 5% CO2 for 48 hours. 4 hours before the end of the culture, 5 g / L of MTT solution was added, and the culture was terminated after another 4 hours of culture; the liquid in the well was discarded, washed once with PBS, DMSO was added to dissolve the crystals, and the absorbance value of each well was measured at a wavelength of 570 nm.

[0057] Cell proliferation rate = (absorbance value of the assay well - absorbance value of the blank control group) / (absorbance value of the cell control group - absorbance value of the blank control group) × 100%.

[0058] In this example, the positive control group was a commercially available recombinant type III collagen sample solution, with 3 parallel samples in each group.

[0059] like Figure 2 As shown, the experimental results show that under the conditions of concentrations of 0.05 mg / ml and 0.1 mg / ml, the collagen in the control group and the recombinant humanized type III collagen of the present application both promote the proliferation of BALB / c 3T3 cells, and the effect is better than that of commercially available recombinant type III collagen.

[0060] Example 5 Evaluation of the cell adhesion promoting activity of recombinant humanized type III collagen The recombinant humanized type III collagen COL-HR-111, COL-HR-222, COL-HR-333 and COL-HR-123 prepared in Example 2 were dissolved in PBS and added at a final concentration of 0.05 mg / ml and 0.1 mg / ml, respectively. 50 μL of the recombinant humanized type III collagen solution was added to each well of a 96-well cell culture plate and placed at 37°C for 2 h. PBS was added to the control wells. BALB / 3T3 cells were digested with trypsin, counted, and 5×10 4 Cells were cultured in a 37°C, 5% CO2 incubator for 2 hours. PBS was washed three times to remove non-adherent cells, and then 200 μL DMEM culture medium was added; 10 μL CCK-8 reagent was added to each well, and the cells were incubated in a 37°C, 5% CO2 cell incubator for 2 hours before removal. The absorbance values ​​of the 96-well plate at 450nm and 630nm were read with an ELISA reader, and the absorbance was measured at 450nm with 630nm as the reference wavelength, and the measurement results were recorded.

[0061] Cell adhesion promotion rate = (450nm absorbance of the experimental group - 450nm absorbance of the negative control group) / 450nm absorbance of the negative control group × 100%.

[0062] In this example, the positive control group was a commercially available recombinant type III collagen sample solution, which was added into the cell culture medium at 0.10 mg / ml, with 3 parallel samples in each group.

[0063] The experimental results are as follows Figure 3 As shown, the experimental results show that under the conditions of concentrations of 0.05 mg / ml and 0.1 mg / ml, the collagen in the control group and the recombinant humanized type III collagen of the present application both promote the adhesion of BALB / c 3T3 cells, and the effect is better than that of commercially available recombinant type III collagen.

[0064] Based on the above embodiments, the recombinant humanized type III collagen expressed by the present invention has uniform molecular weight, good solubility, good cell adhesion and proliferation properties, and can be applied in various fields, such as food, cosmetics, health products or pharmaceutical and medical products.

[0065] The above describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and the specification only describe the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention. The scope of protection claimed by the present invention is defined by the attached claims and their equivalents.

Claims

1. A recombinant humanized type III collagen, characterized in that: The amino acid sequences of the collagen are shown in SEQ ID NO.4-7 respectively.

2. A nucleic acid molecule encoding the recombinant humanized type III collagen according to claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that The nucleotide sequences of the nucleic acid molecules are shown in SEQ ID NOs. 8-11 respectively.

4. A recombinant expression vector comprising the nucleic acid molecule according to any one of claims 2 to 3.

5. A host cell comprising the recombinant expression vector according to claim 4 or expressing the collagen according to claim 1.

6. The host cell according to claim 5, characterized in that The host cell is Escherichia coli.

7. The method for producing recombinant humanized type III collagen according to claim 1, characterized in that: The method comprises the following steps: (1) culturing the host cell according to claim 5 in a culture medium; (2) isolating the recombinant humanized type III collagen according to any one of claim 1 from the host cell.

8. A composition, characterized in that The composition comprises the recombinant humanized type III collagen according to any one of claims 1, or the recombinant humanized type III collagen encoded by the nucleic acid molecule according to any one of claims 2-3, or the recombinant humanized type III collagen expressed by the recombinant expression vector according to claim 4, or the recombinant humanized type III collagen produced by the host cell according to any one of claims 5-6, or the recombinant humanized type III collagen prepared by the production method according to claim 7.

9. A product comprising the composition according to claim 8, characterized in that The product is a pharmaceutical composition, a medical device or a cosmetic.

10. Use of the composition according to any one of claims 8 to 9 in the preparation of a medicament for promoting cell adhesion, promoting cell proliferation and / or promoting wound healing.

Images