CN116574172A - Recombinant humanized type I collagen and preparation method thereof - Google Patents
Recombinant humanized type I collagen and preparation method thereof Download PDFInfo
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Classifications
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
- A61K8/65—Collagen; Gelatin; Keratin; Derivatives or degradation products thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61Q19/00—Preparations for care of the skin
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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Abstract
The invention discloses a recombinant humanized type I collagen and a preparation method thereof. The recombinant humanized type I collagen consists of or comprises an amino acid sequence shown in (aa) n, wherein aa is a sequence shown in SEQ ID NO.1 or an optimized sequence with 80% of homology with the sequence; n is an integer greater than or equal to 1, and each aa is the same or different and is spliced in series by taking aa as a motif. The protein has temperature sensitive property: the water solubility is reduced when the temperature is lower than 4 ℃, the fibers are automatically assembled and separated out, and the fibers are re-dissolved when the temperature returns to the room temperature. Based on the characteristics, the preparation method adopts an escherichia coli expression system and a temperature control inducible recombinant expression vector, has strong promoter and strong terminator elements connected in series, and ensures the high-level expression of the recombinant humanized type I collagen; the recombinant humanized type I collagen has good skin wound repair promoting activity and cell adhesion and migration promoting activity, and can be developed into active raw materials to be applied to the fields of biological medicines, cosmetics and the like.
Description
Technical Field
The invention relates to the field of bioengineering, in particular to recombinant humanized type I collagen and a preparation method thereof.
Background
Collagen (Collagen) is distributed in connective tissue in human and animal bodies, accounting for about 25-33% of total protein in the bodies, has important functions for maintaining normal physiological functions and injury repair of cells, tissues and organs, and is widely applied to industries of medical materials, medicines, chemical engineering, health care products, cosmetics and the like. Most of the collagen sold in the market at present is extracted from animal sources by an acid, alkali and enzymolysis method, and the human collagen has the defects of hidden virus trouble, heterologous rejection, low absorption and utilization rate and the like.
Recombinant humanized type I Collagen (Human-like Collagen) is an active biological protein produced by high-density fermentation and purification processes by utilizing bioengineering and fermentation engineering. The recombinant humanized type I collagen has the structure and function similar to that of natural human collagen, and has gene affinity with human skin cell homoroot. Compared with the traditional animal-derived collagen, the human-like collagen has the characteristics of no toxic hidden trouble, excellent biological compatibility and efficacy and low immunogenicity, and effectively avoids the virus hidden trouble and immune rejection of the animal-derived collagen. On the other hand, the human-like collagen overcomes the defects of inaccurate clinical curative effect and unstable quality of the end product caused by animal age difference and species difference of animal-derived collagen.
Disclosure of Invention
The invention aims to provide recombinant humanized type I collagen and a method for producing the recombinant humanized type I collagen. Therefore, the invention provides a recombinant humanized type I collagen (rhC), a nucleotide sequence and an amino acid sequence for encoding the recombinant humanized type I collagen, a recombinant expression plasmid for encoding the recombinant humanized type I collagen, engineering bacteria and a process for producing the recombinant humanized type I collagen. The invention utilizes the temperature control vector to construct the recombinant plasmid for expressing the recombinant humanized type I collagen, and can realize the high-efficiency expression of the recombinant humanized type I collagen in escherichia coli based on the temperature control recombinant expression plasmid. The recombinant humanized type I collagen produced by the invention has good biological activities of promoting skin wound repair, cell adhesion promotion or migration and the like.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a recombinant humanized type I collagen consisting of or comprising an amino acid sequence shown as (aa) n, wherein aa is at least 85% amino acid sequence homology to human collagen; aa is a sequence shown in SEQ ID NO.1 or an optimized sequence with 80% of homology with the sequence, wherein the optimized sequence refers to amino acid substitution, insertion or knockout based on the sequence shown in SEQ ID NO. 1; wherein n is an integer greater than or equal to 1; wherein each aa is the same or different and uses aa as a motif, and n tandem splices are performed.
Preferably, in the recombinant humanized type I collagen, aa is an amino acid sequence shown in SEQ ID No. 1; when n=8, the amino acid sequence of the recombinant humanized type I collagen is shown as SEQ ID No. 2.
Preferably, in the recombinant humanized type I collagen, the recombinant humanized type I collagen has histidine tags attached to the amino-and/or carboxyl-termini thereof for identifying and purifying a target protein, and the attachment site is the amino-and/or carboxyl-termini, and the histidine tags contain at least 6 histidine residues.
The nucleotide sequence of the recombinant humanized type I collagen is shown as SEQ ID NO.3 when n=8.
The recombinant expression vector containing the nucleotide sequence of the recombinant humanized type I collagen is a temperature control inducible or inducible type. The inducible expression vector is pET3c, and the temperature control inducible vector is pBV-220. The saidThe temperature control inducible vector pBV-220 has strong promoter and strong terminator elements connected in series, and can ensure the high-level transcription and translation of the recombinant humanized I-type collagen. The temperature control inducible vector pBV-220 has lambda inhibitor, but can regulate and express target genes under the condition of thermal induction. The gene sequence for encoding the recombinant humanized type I collagen can be obtained by artificial synthesis or PCR amplification or enzymatic digestion. The temperature control inducible vector pBV-220 is obtained by selecting a designed primer containing an enzyme cutting site for PCR amplification. The temperature control inducible recombinant expression vector has strong promoter and strong terminator elements connected in series, and can ensure high-level expression of recombinant humanized I-type collagen 。
Recombinant engineering bacteria containing the recombinant expression vector, wherein the engineering bacteria are selected from bacteria or fungi.
The recombinant humanized type I collagen has temperature sensitive property: the water solubility is reduced at a temperature below 4 ℃, the fibers are self-assembled and separated out, and the fibers can be redissolved when the temperature returns to room temperature. Based on this feature, the present invention employs two expression methods:
the first expression method is to adopt an inducible pET3c-rhC escherichia coli expression system, and comprises the following steps:
(1) The humanized type I collagen amino acid sequence is subjected to tandem connection with n equal to or greater than 1 by referring to the amino acid sequence shown in SEQ ID NO. 1;
(2) Optimizing a DNA sequence for encoding the humanized type I collagen according to the codon preference of the escherichia coli and synthesizing genes;
(3) Connecting the gene sequence synthesized in the step (2) into a pET3c linearization vector subjected to restriction enzyme digestion to construct a recombinant expression plasmid pET3c-rhC;
(4) Introducing the recombinant plasmid pET3c-rhC constructed in the step (3) into a competent cell of escherichia coli DH5 alpha, and further extracting polyclonal copy of the recombinant plasmid pET3c-rhC;
(5) The recombinant plasmid polyclonal copy obtained in the step (4) is introduced into competent cells of escherichia coli BL21, and engineering bacteria capable of expressing and producing recombinant humanized type 1 collagen are constructed;
(6) Carrying out induction expression on the engineering bacteria constructed in the step (5); then separating and purifying the target protein by utilizing Ni column affinity chromatography and molecular sieve combination;
(7) The purified humanized type 1 collagen is filtered by a microporous filter membrane.
The second expression method is to use a temperature control induction type expression system, and comprises the following steps:
(1) Construction of an expression vector: the recombinant expression vector pET3c-rhC is used as a template, a human-like collagen gene fragment is obtained through PCR amplification or double-enzyme digestion, a temperature control inducible vector pBV220 sequence is amplified through a primer, the rhC gene fragment is connected with the temperature control inducible vector pBV220, and competent cells of escherichia coli DH5 alpha are transformed, so that recombinant plasmids pBV220-rhC are obtained;
(2) Expression identification: the recombinant plasmid polyclonal copy obtained in the step (1) is introduced into competent cells of escherichia coli BL21 to construct expression engineering bacteria pBV220-rhC-BL21 containing recombinant plasmid pBV220-rhC;
(3) Pilot fermentation: inoculating the recombinant engineering bacteria to a culture medium for culture to obtain seed liquid, inoculating the seed liquid to a fermentation tank, adopting an inducer or inducing at 42 ℃ for high-density fermentation, and centrifuging after fermentation to obtain fermentation bacterial sludge;
(4) Protein purification: homogenizing and crushing the bacterial sludge, centrifugally separating supernatant and precipitating, separating and purifying the recombinant humanized type I collagen by adopting a Ni affinity chromatography column and a molecular sieve, and filtering the purified recombinant humanized type I collagen by a 0.22 mu m microporous filter membrane.
The recombinant humanized type I collagen is applied to the preparation of medicaments or cosmetics for promoting the repair of skin wound surfaces, promoting cell adhesion or migration.
Compared with the prior art, the invention has the following beneficial effects:
(1) The recombinant humanized type I collagen has temperature-sensitive property, water solubility is reduced when the temperature is lower than 4 ℃, fibers are separated out through self-assembly, and the fibers are redissolved when the temperature returns to the room temperature.
(2) Compared with an inducible expression vector pET3c-rhC, the temperature control inducible expression vector pBV220-rhC is used, so that the density of fermentation thalli and the yield of target protein are obviously improved.
(3) The invention adopts a temperature control induction type expression vector, and provides a temperature control process flow for regulating and controlling the expression of target proteins through high-density fermentation. The fermentation process is regulated and controlled through the temperature control process, the target protein expression is induced, the pollution and the cost improvement caused by adding the inducer can be avoided through the thermal induction, the temperature control process flow is simple and convenient to operate, the production yield is greatly improved, and the method is suitable for industrial production. The temperature control inducible recombinant expression vector is fermented by a temperature control process, the weight of the obtained bacterial mud is 1.8 times that of the inducible expression vector, and the yield of the prepared recombinant humanized type I collagen is improved by 2.36 times compared with that of the inducible expression vector.
(4) The recombinant humanized type I collagen prepared by the method has good skin wound repair promoting activity and cell adhesion and migration promoting activity, and can be developed into an active raw material to be applied to the fields of biological medicines, cosmetics and the like.
Drawings
FIG. 1 is a diagram of pET3c-rhC and pBV220-rhC expression plasmids constructed in the present invention;
FIG. 2 shows SDS-PAGE results of recombinant humanized type I collagen expression of example 1, having an apparent molecular weight of about 25.9kDa;
FIG. 3 shows the Western Blot detection results of recombinant humanized type I collagen expression in example 1;
FIG. 4 shows the SDS-PAGE results of recombinant humanized type I collagen expressed in different vectors of pET3c-rhC and pBV220-rhC at a pilot level;
FIG. 5 shows the SDS-PAGE results of pilot level recombinant humanized type I collagen expressed in different vectors of pET3c-rhC and pBV220-rhC;
FIG. 6 shows the yield of different engineering bacteria fermentation bacteria of the pilot level pET3c-rhC-BL21 and pBV220-rhC-BL21;
FIG. 7 shows the SDS-PAGE detection of purified recombinant humanized type I collagen;
FIG. 8 is an HPLC chromatogram of purified recombinant humanized type I collagen;
FIG. 9 is an electron microscope scanning view of recombinant humanized type I collagen;
FIG. 10 shows the results of cell adhesion activity assay for human-like collagen;
FIG. 11 shows the results of measurement of the cell migration activity of human-like collagen;
FIG. 12 shows the results of detection of skin minimally invasive repair activity of human-like collagen.
Detailed Description
The following specific examples are provided to further illustrate the present invention, and it will be understood by those skilled in the art that the present invention is not limited to these specific examples.
EXAMPLE 1 construction of recombinant humanized type I collagen expression vector and expression identification (1) construction of pET3c-rhC E.coli expression System
The amino acid sequence of the recombinant humanized type I collagen is shown as SEQ ID NO.2, and the nucleotide sequence for encoding the sequence shown as SEQ ID NO.2 is shown as SEQ ID NO. 3. In order to facilitate the expression, identification and purification of the target protein, hexahistidine tags are respectively introduced at the amino terminal and the carboxyl terminal of the target protein, the amino acid sequence of the recombined and fused humanized I-type collagen is shown as SEQ ID NO.4, and the nucleotide sequence is shown as SEQ ID NO. 5. The nucleotide sequence shown in SEQ ID NO.5 is synthesized by the gene, nde I is introduced at the amino terminal, and BamH I enzyme cutting site sequence is introduced at the carboxyl terminal. The gene synthesis product and pET-3c vector were digested with NdeI and BamHI, and recombined with ligase. The pET3c-rhC ligation was transformed into E.coli DH 5. Alpha. Competent cells (available from Beijing Optimago Biotech Co., ltd.). Positive clones were screened on LB-resistant plates containing ampicillin (100. Mu.g/mL) and subjected to colony PCR amplification and sequencing verification (Shenzhen Dai Gene technologies Co.). (2) Construction of temperature control inducible expression system pBV220-rhC
Based on recombinant expression vector pET3c-rhC, the rhC gene fragment is obtained through double restriction enzyme digestion of restriction enzymes NdeI and BamHI, in order to make the rhC gene fragment be connected into temperature control inducible vector pBV220, the invention uses upstream and downstream primers containing NdeI and BamHI respectively to amplify the full-length sequence of temperature control inducible vector pBV220 and connect the rhC gene fragment into temperature control inducible vector pBV220 through homologous recombination, and simultaneously hexahistidine tags are respectively introduced at the amino terminal and carboxyl terminal of the rhC gene fragment to construct recombinant expression plasmid pBV220-rhC. The amino acid sequence of the recombinant humanized type I collagen is shown as SEQ ID NO.2, and the nucleotide sequence of the sequence shown as SEQ ID NO.2 is shown as SEQ ID NO. 3. The amino acid sequence after the histidine tag is introduced is shown as SEQ ID NO. 4. The pBV220-rhC ligation product was transformed into E.coli DH 5. Alpha. Competent cells (purchased from Beijing Optimago technologies Co., ltd.). Positive clones were screened on LB-resistant plates containing ampicillin (100. Mu.g/mL) and subjected to colony PCR amplification and sequencing verification (Shenzhen Dai Gene technologies Co.). (3) Preparation of expression Strain and expression identification
The pET3c-rhC and pBV220-rhC recombinant plasmids after sequencing verification were transformed into BL21 (DE 3) competent cells (purchased from Beijing qing Biotechnology Co., ltd.) respectively, positive clones were screened on LB-resistant plates containing ampicillin (100. Mu.g/mL) and subjected to colony PCR verification. Then test tube expression identification is carried out, and high expression strains are screened, and the specific operation is as follows: selecting positive colony, inoculating the colony into a culture medium test tube containing 5mL LB (100 mug/mL ampicillin), and culturing at 37 ℃ until OD600 reaches 0.6-0.8; culturing pET3c-rhC recombinant expression engineering bacteria for 3-4 h under the induction of IPTG of one thousand at 37 ℃; the pBV220-rhC recombinant expression engineering bacteria are placed in a preheated shaking table at 42 ℃ for expression culture for 3-4 hours, and the thalli are collected. The strain is resuspended in lysis buffer for full lysis, and then added with loading buffer and heated in boiling water bath for 10min, and the expression quantity is detected by SDS-PAGE. Because the target protein contains a hexahistidine tag, western Blot (Anti His, antibodies are available from Shanghai Biotechnology Co., ltd., mouse Anti-His monoclonal antibody) can be used to detect the immunoreactivity of rhC.
The SDS-PAGE detection result is shown in figure 2, the recombinant expression engineering bacteria of the pET3c-rhC and the pBV220-rhC successfully express rhC protein, the expression level of the pBV220-rhC group is higher than that of the pET3c-rhC group, the apparent molecular weight is about 25.9kDa, and the amino acid sequence is shown in SEQ ID NO. 4. Western Blot detection was performed using anti-6 XHis Tag antibodies, and the results are shown in FIG. 3, in which the immunoblots were single and the size was consistent with the apparent molecular weight in SDS-PAGE, indicating successful expression of recombinant humanized type I collagen in the present invention.
EXAMPLE 2 comparison of the expression levels of different vectors
(1) Bench scale level comparison
Expression of pBV220-rhC in vitro: the pBV220-rhC-BL21 strain obtained in example 1 was inoculated into 5mL of LB (100. Mu.g/mL ampicillin) medium and cultured at 37℃for 14-16 hours, and transferred to 5mL of a new LB medium in percentage, followed by continuous culture to OD 600 After reaching 0.6 to 0.8, placing the strain in a shaking table at 42 ℃ for induced expression culture for 4 hours, taking 0.5mL of bacterial liquid, centrifugally collecting bacterial bodies, and detecting the expression quantity by SDS-PAGE;
expression of pET3c-rhC in vitro: pET3c-rhC-BL21 strain is inoculated in 5mL LB (100 mug/mL ampicillin) culture medium and cultured for 14-16h at 37 ℃, and is transferred to 5mL new LB culture medium according to percentage for continuous culture until OD 600 And adding IPTG to 0.6-0.8 in a thousandth, then placing the mixture in a shaking table at 37 ℃ to induce expression and culture for 4 hours, taking 0.5mL of bacterial liquid, centrifuging and collecting bacterial bodies, and detecting the expression quantity by SDS-PAGE.
As shown in FIG. 4, the human-like collagen can be successfully expressed by the pET-3c vector and the pBV-220 vector, and the sizes of the human-like collagen and the pBV-220 vector are consistent with the apparent molecular weight. By comparison, the expression level of the human-like collagen in the invention through the temperature control vector pBV220-rhC is obviously higher than that of the recombinant vector pET3c-rhC.
(2) Pilot level comparison
15L pilot fermentation pBV220-rhC engineering bacteria: inoculating glycerol seed strain into 50mL LB culture medium, culturing at 37deg.C and 220rpm/min to OD 600 Up to 0.6-0.8, the culture was transferred to a modified medium, cultured for a further 6h and transferred to a 15L bioreactor. The initial liquid loading amount was 7L, and sterile glucose and 0.65g/L sterile magnesium sulfate were added at an initial concentration of 5g/L, the stirring speed and aeration rate were 200rpm and 5L/min, respectively, and the dissolved oxygen concentration was maintained at 30% or more of air. When the wet weight of the cells reached about 130g/L, the temperature was adjusted to 42 ℃. During the induction, glucose was fed at a constant low flow rate (0.6 g/L/h)The pH is stabilized at 7.2-7.4. After 4h of induction, the cells were collected by centrifugation at 4℃at 30,000g for 10 min.
15L pilot fermentation pET3c-rhC engineering bacteria: inoculating glycerol seed strain into 50mL LB culture medium, culturing at 37deg.C and 220rpm/min to OD 600 Up to 0.6-0.8, the culture was transferred to a modified medium, cultured for a further 6h and transferred to a 15L bioreactor. The initial liquid loading amount is 7L, and sterile glucose with the initial concentration of 5g/L and sterile magnesium sulfate with the initial concentration of 0.65g/L are added, the stirring speed and the aeration rate are respectively 200rpm and 5L/min, and the dissolved oxygen amount is kept above 30 percent. After about 3.5h, feeding until the bacterial weight is more than or equal to 40g/L, raising the dissolved oxygen to 80%, adding 7ml of 1M/L IPTG, and centrifugally collecting bacterial bodies at 4 ℃ and 30,000g for 10min after induction for 4 h.
As shown in FIG. 5 and FIG. 6, the yield of the pET3c-rhC-BL21 engineering bacteria is about 64.24g/L, and the yield of the pBV220-rhC-BL21 engineering bacteria is about 115.82g/L, which is obviously higher than the yield of the pBV220-rhC-BL21 engineering bacteria and is 1.79 times higher than the yield of the pBV220-rhC-BL21 engineering bacteria. The expression quantity of the target protein of the pET3c-rhC-BL21 engineering bacteria is about 535.83mg/L, while the expression quantity of the target protein in the pBV220-rhC-BL21 engineering bacteria is about 1801.67mg/L, which is obviously higher than the expression quantity of the target protein and is 3.36 times higher than the expression quantity of the target protein in the target protein. The pilot test comparison result shows that compared with the recombinant expression vector pET3c-rhC, the recombinant temperature control inducible expression vector pBV220-rhC provided by the invention can obviously improve the thallus fermentation density and the target protein yield, and realize the large-scale production of recombinant humanized I-type collagen.
EXAMPLE 3 purification of recombinant humanized type I collagen
The soluble fraction of recombinant humanized type I collagen was separated and purified using Ni column affinity chromatography and molecular sieve combination, washed with PBS buffer containing 200mM imidazole, the contaminated protein was removed from the Ni-NTA resin, then rhC was eluted in a single pass, the main effluent peak was eluted using PBS buffer containing 500mM imidazole, and then desalted using G25 column. The rhC-like human collagen purified product was filtered through a 0.22 μm filter, and 10. Mu.L (Sepax Bio-C18 column, LC-2040C high performance liquid chromatograph) was introduced to analyze the purity of the purified rhC.
The purification results are shown in figures 7 and 8, SDS-PAGE detection result of rhC purification products has single band, and HPLC detection result has single peak, which shows that the purification method is reliable and effective, and high-purity recombinant humanized type I collagen can be obtained by the purification method.
EXAMPLE 4 recombinant humanized type I collagen scanning and viewing
And (3) attaching a conductive adhesive tape to a sample stage, taking a small amount of the freeze-dried recombinant humanized type I collagen sample, adhering the small amount of the recombinant humanized type I collagen sample to the conductive adhesive tape, performing metal spraying treatment, and placing the recombinant humanized type I collagen sample under a scanning electron microscope for observation.
As a result, as shown in FIG. 9, the recombinant humanized type I collagen prepared by the method of the present invention was reduced in water solubility at a temperature lower than 4 ℃ (left FIG. 2), and self-assembled to precipitate fibers (right panel), and re-dissolved when the temperature was returned to room temperature (left panel 1).
EXAMPLE 5 cell adhesion Activity of recombinant humanized type I collagen
Human-like collagen concentration was determined using BCA protein quantification kit (available from zemoeimer technologies limited) and samples were diluted using DMEM medium (GIBCO, cat No. 12800017). 400 mu L of recombinant humanized type I collagen sample is added into a 24-well plate, a blank control is DMEM medium, a positive control is commercial collagen, and the mixture is treated overnight at 4 ℃; every hole of the next day 4 х 10 4 Hacat cells with good growth state are placed at 37 ℃ and 5% CO 2 Culturing in an incubator for 3 hours; cells were fixed with 4% paraformaldehyde for 20min, stained with 1% crystal violet for 20min, washed with PBS and examined by microscopy.
As shown in FIG. 10, the recombinant humanized type I collagen obtained by the invention stimulates the acceleration adhesion matrix of keratinocytes at the concentration of 2.5nmol/mL, and has better cell adhesion activity compared with the positive control.
EXAMPLE 6 cell migration Activity of recombinant humanized type I collagen
Hacat cells with good growth state are pressed by 2x10 4 Adding the mixture into 6-well plate, placing at 37deg.C and 5% CO 2 The incubator cultures until the cells confluent to the bottom. The 200. Mu.L gun head was streaked and the floating cells were washed with PBS. 2mL of recombinant humanized type I collagen sample was added to each well, the blank was DMEM medium, and the positive control was commercial collagen. Placing at 37deg.C and 5% CO 2 The incubator continues to culture, and samples are taken at 0h and 48h, respectively.
In vitro cell migration experiments simulate the process of in vivo cell migration to a certain extent, and directly reflect the interaction between cells and extracellular matrix and the interaction between cells under the influence of the matrix. Cell migration activity is an indicator of more effective characterization of collagen biological activity, and faster migration indicates better collagen biological activity. As shown in FIG. 11, the human-like collagen obtained by the invention has better cell migration promoting activity.
Example 7 Effect of recombinant humanized type I collagen on wound repair on rat skin
SD rats (6-8 weeks old, purchased from the medical laboratory animal center in Guangdong province) were anesthetized by intraperitoneal injection with 1% sodium pentobarbital solution (Sigma, germany, 3 mL/kg). After depilating the back skin for 24 hours, the skin was abraded with sand paper to slight bleeding and scalded with a sterilizing weight at 70 ℃ for 15s, creating four circular (12 mm diameter) abraded and scalded wounds. Rats were randomly divided into 3 groups after wound formation, natural healing (Normal), blank matrix (Vehicles) and rhC, and the photographs were recorded as day 0, and wound healing status was recorded after drug administration treatment on days 1, 3, 5, 7, 10 and 14, respectively.
The evaluation effect of the recombinant humanized type I collagen on wound repair is shown in FIG. 12. After the wound surface is subjected to drug administration treatment, the wound surface healing speed of the rhC group is obviously higher than that of the Normal group and the Vehicles group, the wound surface of the rhC group is completely healed on the 10 th day, and the other two groups are not completely healed. The result shows that the recombinant humanized type I collagen produced by the invention has good wound repair effect.
Claims (10)
1. A recombinant humanized type I collagen characterized by consisting of or comprising an amino acid sequence shown as (aa) n, wherein aa is at least 85% amino acid sequence homology to human collagen; aa is a sequence shown in SEQ ID NO.1 or an optimized sequence with 80% of homology with the sequence, wherein the optimized sequence refers to amino acid substitution, insertion or knockout based on the sequence shown in SEQ ID NO. 1; wherein n is an integer greater than or equal to 1; wherein each aa is the same or different and n tandem splices are made with aa as a motif.
2. The recombinant humanized type I collagen according to claim 1, wherein aa is the amino acid sequence shown in SEQ ID No. 1; when n=8, the amino acid sequence of the recombinant humanized type I collagen is shown as SEQ ID No. 2.
3. Recombinant humanized type I collagen according to claim 1 or 2, characterized in that it has attached at its amino-and carboxy-terminus a histidine tag for the identification and purification of the protein of interest, the attachment site being amino-and/or carboxy-terminus, said histidine tag containing at least 6 histidine residues.
4. A nucleotide sequence encoding the recombinant humanized type I collagen of any one of claims 1-3.
5. The nucleotide sequence of recombinant humanized type I collagen according to claim 4, wherein when n=8, the nucleotide sequence is set forth in SEQ ID No. 3.
6. A recombinant expression vector comprising the nucleotide sequence of recombinant humanized type I collagen of claim 5, wherein the expression vector is a temperature-controlled inducible or inducible dosage form.
7. The recombinant expression vector of claim 6, wherein the temperature-controlled inducible vector is pBV220-rhC.
8. The method for expressing recombinant humanized type I collagen according to claim 1, wherein an inducible pET3c-rhC escherichia coli expression system is used, comprising the steps of:
(1) The humanized type I collagen amino acid sequence is subjected to tandem connection with n equal to or greater than 1 by referring to the amino acid sequence shown in SEQ ID NO. 1;
(2) Optimizing a DNA sequence for encoding the humanized type I collagen according to the codon preference of the escherichia coli and synthesizing genes;
(3) Connecting the gene sequence synthesized in the step (2) into a pET3c linearization vector subjected to restriction enzyme digestion to construct a recombinant expression plasmid pET3c-rhC;
(4) Introducing the recombinant plasmid pET3c-rhC constructed in the step (3) into a competent cell of escherichia coli DH5 alpha, and further extracting polyclonal copy of the recombinant plasmid pET3c-rhC;
(5) The recombinant plasmid polyclonal copy obtained in the step (4) is introduced into competent cells of escherichia coli BL21, and engineering bacteria capable of expressing and producing recombinant humanized type 1 collagen are constructed;
(6) Carrying out induction expression on the engineering bacteria constructed in the step (5); then separating and purifying the target protein by utilizing Ni column affinity chromatography and molecular sieve combination;
(7) The purified humanized type 1 collagen is filtered by a microporous filter membrane.
9. The method for expressing recombinant humanized type I collagen according to claim 1, wherein a temperature-controlled inducible expression system is used, comprising the steps of:
(1) Construction of an expression vector: the recombinant expression vector pET3c-rhC is used as a template, a human-like collagen gene fragment is obtained through PCR amplification or double-enzyme digestion, a temperature control inducible vector pBV220 sequence is amplified through a primer, the rhC gene fragment is connected with the temperature control inducible vector pBV220, and competent cells of escherichia coli DH5 alpha are transformed, so that recombinant plasmids pBV220-rhC are obtained;
(2) Expression identification: the recombinant plasmid polyclonal copy obtained in the step (1) is introduced into competent cells of escherichia coli BL21 to construct expression engineering bacteria pBV220-rhC-BL21 containing recombinant plasmid pBV220-rhC;
(3) Pilot fermentation: inoculating the recombinant engineering bacteria to a culture medium for culture to obtain seed liquid, inoculating the seed liquid to a fermentation tank, adopting an inducer or inducing at 42 ℃ for high-density fermentation, and centrifuging after fermentation to obtain fermentation bacterial sludge;
(4) Protein purification: homogenizing and crushing the obtained bacterial sludge, centrifugally separating supernatant and precipitating, separating and purifying recombinant humanized type I collagen by adopting a Ni affinity chromatographic column and a molecular sieve in combination, and filtering the purified recombinant humanized type I collagen by a microporous filter membrane.
10. Use of the recombinant humanized type I collagen according to claim 1 for the preparation of a medicament or cosmetic for promoting skin wound repair, cell adhesion or migration.
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