CN116874590A - Recombinant III type collagen and preparation method thereof - Google Patents
Recombinant III type collagen and preparation method thereof Download PDFInfo
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
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Abstract
The application discloses recombinant III type collagen and a preparation method thereof, and relates to the technical field of genetic engineering. The amino acid sequence of the recombinant III type collagen is shown as SEQ ID NO. 3. The recombinant III type collagen is obtained by taking a nucleotide sequence of human III type collagen as a template for design and optimization, and has good biocompatibility, low toxicity and low immunogenicity compared with natural III type collagen. According to the experimental result of cell proliferation promotion, the recombinant III type collagen provided by the application can obviously promote proliferation and migration of fibroblasts, and has wide application prospects in industries such as biological medicine, cosmetics, skin care products, food and the like.
Description
Technical Field
The application relates to the technical field of genetic engineering, in particular to recombinant III-type collagen and a preparation method thereof.
Background
Collagen is the most abundant protein in mammals, accounting for about 25% of the total protein content in humans. Collagen, which is an adhesive substance of connective tissue, plays an important role in maintaining normal physiological functions of cells, tissues, and organs, has been demonstrated to be directly or indirectly involved in promotion of cell adhesion and differentiation, which are associated with embryonic development and regeneration control.
Collagen is a major structural protein in the extracellular matrix and connective tissue, the basic structure of which consists of three helical subunits: two α1 (I) chains and one α2 (I) chain. At least 28 different types of collagen are currently known, all of which contain three-ply helical segments of similar structure, each differing by breaking the segments of the three-ply helical structure and folding into other types of three-dimensional structures. Among them, type I, type II and type III collagens occupy 80% of the collagen content in the body.
Type III collagen plays an important role in the regulation of the organization structure and function of the extracellular matrix, and is involved in many physiological and pathological processes, including structural support, cell adhesion and differentiation, inflammation-related pathological processes, and association with certain diseases. Type III collagen, an extracellular matrix protein synthesized by cells as procollagen, is found as a major structural component of hollow organs such as large blood vessels, uterus and intestines. Other functions of type III collagen include interactions with platelets in the coagulation cascade, which is also an important signaling molecule in wound healing.
Collagen has biodegradability, good biocompatibility and low immunogenicity; can promote proliferation and adhesion of cells; in addition, the collagen can form collagen fibers, has certain mechanical properties, is a good biological material, and can be widely used. The collagen is mainly used as collagen-based biological materials in medicine, such as heart valves, vascular repair, soluble collagen periodontal repair, burn repair, collagen hemostatic, gelatin, artificial skin, immobilized enzyme carriers, capsules, collagen membranes and the like.
At present, most of collagen used in the medicine industry, the food industry and the cosmetic industry is obtained by extracting pigskin, fish skin and cowhells by an acid, alkali and enzyme method. Collagen extracted by this method has many drawbacks that are difficult to overcome: (1) has virus hidden trouble; (2) Has certain antigenicity, and can lead to immune rejection reaction when entering the organism; (3) the extraction method is seriously polluted; (4) raw materials are limited in source.
The recombinant collagen is an expression engineering bacterium which is constructed by utilizing a genetic engineering technology and contains a full-length gene or a partial fragment gene of the collagen, the collagen is synthesized by the engineering bacterium, and finally, the high-purity recombinant collagen is obtained by adopting a specific purification process. The recombinant collagen produced by adopting the genetic engineering technology has higher controllability, consistency and high purity, can be produced in a large scale and has better biocompatibility; has wide application prospect, including the fields of tissue engineering, drug delivery systems, biomedical research and the like.
Disclosure of Invention
The technical problem to be solved by the application is to use a genetic engineering technology to recombine and express a new III type collagen and a preparation method of the recombined III type collagen.
In order to solve the problems, the application provides the following technical scheme:
in a first aspect, the present application provides a recombinant type III collagen, wherein the amino acid sequence of the recombinant type III collagen is shown as SEQ ID No.3, or has an amino acid sequence with 1 or more amino acids added, substituted, deleted, and the like compared to the amino acid sequence shown as SEQ ID No.3, which retains a similar effect.
The technical scheme is that the amino acid sequence of the recombinant III type collagen comprises 2-10 repeated SEQ ID NO:3.
further, the application provides a nucleic acid molecule which codes for the recombinant type III collagen.
The further technical scheme is that the sequence of the nucleic acid molecule is shown as SEQ ID NO. 4.
The application also provides a vector comprising the nucleic acid molecule.
The application also provides a host cell comprising said collagen or said nucleic acid molecule or said vector.
By host cell is meant any cell type that is susceptible to transformation, transfection, transduction, etc. by a nucleic acid construct or expression vector comprising a polynucleotide of the application. "host cell" encompasses any progeny of a parent cell that is not exactly identical to the parent cell due to mutations that occur during replication. The host cell may be any cell useful in the production of recombinant humanized collagen of the present application. To produce recombinant collagen, nucleic acid encoding recombinant collagen may be isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional techniques (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding recombinant collagen). The host cell refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include transformants and transformed cells, including primary transformed cells and progeny derived therefrom, regardless of the number of passages. The offspring may not be identical in nucleic acid content to the parent cell, but may contain mutations. Methods for introducing vectors into host cells are well known, for example, using electrotransformation to introduce vectors into host cells, and may also be transfection, microinjection techniques, gene gun techniques, liposome-mediated methods, and the like. 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.
The application also provides a method for preparing the recombinant type III collagen, which comprises the following steps:
and expressing the recombinant III type collagen by using the host cell, and then separating and purifying to obtain the recombinant III type collagen.
The application also provides the application of the recombinant type III collagen according to the first aspect, or the recombinant type III collagen encoded by the nucleic acid molecule, or the recombinant type III collagen produced by the host cell in preparing food, cosmetics or pharmaceutical products.
The application also provides a composition comprising the recombinant type III collagen according to the first aspect, or the recombinant type III collagen encoded by the nucleic acid molecule, or the recombinant type III collagen produced by the host cell.
The application also provides application of the composition in preparing food, cosmetics or medical products.
Compared with the prior art, the application has the following technical effects:
the application uses the nucleotide sequence of human III type collagen as a template, utilizes the genetic engineering technology to construct a new recombinant III type collagen, synthesizes the recombinant III type collagen by genetic engineering bacteria, and obtains high-purity recombinant collagen through a purification process. According to the experimental result of cell proliferation promotion, the recombinant III type collagen provided by the application can obviously promote the proliferation of fibroblasts, and has wide application prospects in the industries of biological medicines, cosmetics, skin care products, foods and the like.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows SDS-PAGE electrophoresis of recombinant type III collagen HC8 prepared in the embodiment of the application at different induction expression times; in the figure, band 1, protein Marker standard, band 2, uninduced expressed bacteria, band 3: inducing the expression bacteria for 1h, inducing the expression bacteria for 2h in a band 4, and inducing the expression bacteria for 4h in a band 5.
FIG. 2 shows the SDS-PAGE of recombinant type III collagen purified according to the example of the present application; in the figure, band 1 is a protein Marker standard, and band 2 is purified recombinant type III collagen HC8.
FIG. 3 shows the results of the proliferation rate of recombinant type III collagen HC8 according to the present application.
Detailed Description
The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, in which like reference numerals represent like components. It will be apparent that the embodiments described below are only some, but not all, embodiments of the application. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
It should be understood that the terms "comprises" and "comprising," when used in this specification and the appended claims, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.
It is also to be understood that the terminology used in the description of the embodiments of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of embodiments of the application. As used in the specification of the embodiments of the application and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.
The main materials related to the embodiment of the application are as follows: plasmid pET-30a (Merck), host bacterium E.coli BL21 (DE 3) (Merck), protein Marker were purchased from Thermo Fisher Scientific company, ni Sepharose TM 6Fast Flow was purchased from Bozuron (Shanghai) Biotechnology Co., ltd, and CCK-8 kit was purchased from Biyun Biotechnology Co., ltd.
Buffer used for Ni-NTA affinity chromatography, equilibration buffer 20mmol/L Tris-HCl,0.25mol/LNaCl,10mmol/L imidazole (pH 8.0), elution buffer A20 mmol/L Tris-HCl,0.25mol/LNaCl,40mmol/L imidazole (pH 8.0), elution buffer B20 mmol/L Tris-HCl,0.25mol/LNaCl,300mmol/L imidazole (pH 8.0).
Example 1: recombinant III type collagen expression vector construction
The application takes the nucleotide sequence of human III type collagen as a (NM_ 000090.3) template, analyzes the composition and structure of the sequence, screens out a specific peptide on the human III type collagen by analyzing the stability of the peptide and the receptor binding capacity on cells, and the like, wherein the amino acid sequence of the peptide is SEQ ID NO:1. the nucleotide sequence of the peptide fragment is shown as SEQ ID NO:2.
the peptide segment is used as a basic structural unit for optimization design to obtain recombinant human III type collagen, which is marked as HC8 and consists of 503 amino acids, and the amino acid sequence is shown as SEQ ID NO:3. The nucleotide sequence of the recombinant human III type collagen HC8 is shown as SEQ ID NO: 4.
And optimizing the designed nucleotide sequence of the recombinant III type collagen HC8 according to the codon preference of the escherichia coli, adopting total gene synthesis of the DNA sequence of the recombinant III type collagen, and constructing the DNA sequence on a pET-30a expression vector, wherein the recombinant plasmid vector is named pET-30a-HC8.
The DNA sequence of the recombinant III type collagen subjected to codon optimization is shown as SEQ ID NO: shown at 5.
Example 2: expression and purification of recombinant type III collagen HC8
(1) Preparation of recombinant type III collagen HC8 expressing strain:
(1) competent cells of E.coli BL21 (DE 3) were prepared.
(2) The expression vector pET-30a-HC8 was transformed into competent cells of E.coli BL21 (DE 3) at 42℃for 90s by heat shock.
(2) Recombinant type III collagen HC8 induced expression and solubility analysis:
inoculating the expression strain pET-30a-HC8 obtained in the step (1) into 100mL of LB medium containing 50 mug/mL kanamycin content, culturing at 37 ℃ and 200rpm, adding IPTG when OD600 = 0.8, carrying out induction expression for 4h at 37 ℃, and centrifuging at 8000g and 4 ℃ for 10min to collect thalli. The cells were resuspended in PBS buffer, homogenized and disrupted at high pressure (1000 bar), centrifuged at 18000g at 4℃for 60min, and the supernatant and pellet were respectively subjected to subsequent SDS-PAGE (5% gel concentrate, 12% gel isolate) and Westernblot analysis.
(3) Recombinant III type collagen HC8 shake flask fermentation and purification
Inoculating recombinant type III collagen HC8 expression strain obtained in the step (1) into 2L LB culture medium with kanamycin content of 50 mug/mL respectively, and carrying out shake flask fermentation and induction according to the expression conditions. Centrifuging at 4 ℃ for 6000 Xg for 10min to collect thalli, and precipitating the thalli according to the volume ratio of 1:12 ratio resuspended in Ni-NTA affinity chromatography equilibration buffer and the cells were broken up homogeneously at high pressure (1000 bar). Supernatant was collected by centrifugation at 25000 Xg for 60min at 4 ℃.
The bacterial cell disruption and centrifugation supernatant is purified by Ni-NTA affinity chromatography, and the specific steps are as follows: loading the supernatant onto a Ni-NTA affinity chromatographic column with a bed volume of 10mL, a flow rate of 5mL/min, washing back to a base line with an equilibrium buffer solution, a flow rate of 5mL/min, eluting the protein impurity with an elution buffer solution A, and eluting the target protein with an elution buffer solution B; purified recombinant type III collagen HC8 is subjected to SDS-PAGE electrophoresis to identify the purity of the purified collagen. The results are shown in FIGS. 1-2.
Example 3: evaluation of recombinant type III collagen HC8 proliferation promoting Activity
Taking BALB/c 3T3 cells (mouse embryo fibroblasts) in logarithmic growth phase (purchased from Shanghai cell bank of China academy of sciences) and inoculating into 96-well cell culture plate with an inoculating density of 1×10 5 100 mu L of each well is placed in each volume of the solutionIn a carbon dioxide cell incubator at 37 ℃ and 5% CO 2 Culturing for 24h conventionally. The culture was continued for 12h with a serum-free medium. Then 100. Mu.L of serum-free culture solution or 100. Mu.L of recombinant type III collagen HC8 sample solution was added thereto, and the culture was continued for 24 hours. Each solution was 400. Mu.g/mL and the solution was sterilized by filtration through a 0.22 μm filter. After further culturing for 24 hours, the culture broth was discarded, 100. Mu.L of CCK-8 (available from Shanghai Biotechnology Co., ltd.) diluted 10-fold with serum-free culture broth was added to each well, and the mixture was placed in a cell incubator at 37℃with 5% CO 2 And taking out after incubation for 2 hours.
Reading the absorbance values of the 96-well plate at 450nm and 630nm by using an enzyme-labeled instrument, measuring absorbance at 450nm by taking 630nm as a reference wavelength, recording the measurement result, and obtaining the relative proliferation rate according to the following formula:
relative proliferation% = (sample OD-control)/(control OD-medium OD).
In the test, a blank control group is added with an equivalent serum-free culture solution, a control group is a commercially available recombinant type III collagen sample solution, an experimental group is added with 100 mu L of the recombinant type III collagen sample solution, and 3 parallel samples are taken in each group.
As shown in fig. 3, the experimental results indicate that: under the condition of the concentration of 1 mug/mL, the mobility of the collagen of the control group and the recombinant type III collagen HC8 relatively promote the fibroblasts is 12.95% and 26.64%, respectively, and compared with the control group, the recombinant type III collagen HC8 provided by the application can obviously promote the proliferation of the fibroblasts.
In the foregoing embodiments, the descriptions of the embodiments are focused on, and for those portions of one embodiment that are not described in detail, reference may be made to the related descriptions of other embodiments.
While the application has been described with reference to certain preferred embodiments, it will be understood by those skilled in the art that various changes and substitutions of equivalents may be made and equivalents will be apparent to those skilled in the art without departing from the scope of the application. Therefore, the protection scope of the application is subject to the protection scope of the claims.
Claims (10)
1. The recombinant type III collagen is characterized in that the amino acid sequence of the recombinant type III collagen is shown as SEQ ID NO.3 or has an amino acid sequence with 1 or more amino acids added, substituted and deleted compared with the amino acid sequence shown as SEQ ID NO. 3.
2. A nucleic acid molecule encoding the recombinant type III collagen of claim 1.
3. The nucleic acid molecule of claim 2, wherein the sequence of the nucleic acid molecule is set forth in SEQ ID No. 4.
4. A vector comprising the nucleic acid molecule of claim 2 or 3.
5. A host cell comprising the collagen of claim 1 or the nucleic acid molecule of claim 2 or 3 or the vector of claim 4.
6. The host cell of claim 5, wherein the host cell is a prokaryotic cell or a eukaryotic cell.
7. A method of preparing the recombinant type III collagen of claim 1, comprising the steps of:
expressing the recombinant type III collagen using the host cell of any one of claims 5-6, followed by isolation and purification.
8. Use of the recombinant type III collagen according to claim 1, or the recombinant type III collagen encoded by the nucleic acid molecule of claim 2 or 3, or the recombinant type III collagen produced by the host cell of any one of claims 5-6, in the preparation of a food, cosmetic or pharmaceutical product.
9. A composition comprising the recombinant type III collagen according to claim 1, or the recombinant type III collagen encoded by the nucleic acid molecule of claim 2 or 3, or the recombinant type III collagen produced by the host cell of any one of claims 5-6.
10. Use of a composition according to claim 9 for the preparation of a food, cosmetic or pharmaceutical product.
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| CN116284339A (en) * | 2022-12-29 | 2023-06-23 | 深圳柏垠生物科技有限公司 | Recombinant III type collagen, nucleic acid, expression vector, strain and application thereof |
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