CN116987175A - Recombinant collagen, recombinant expression strain and application - Google Patents
Recombinant collagen, recombinant expression strain and application Download PDFInfo
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- 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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- 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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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
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Abstract
The invention relates to recombinant collagen, recombinant expression strain and application thereof, belonging to the technical field of protein engineering and genetic engineering. The invention discloses a method for constructing a fusion recombinant collagen mutant and an engineering strain of a high-yield fusion recombinant collagen mutant, wherein the fusion recombinant collagen mutant is improved by 13 times compared with the soluble expression of recombinant collagen before optimization by splicing a brand-new human collagen sequence formed by screening sequences with high activity and high stability of cell binding sites, optimizing the nucleotide sequence through escherichia coli codon preference and carrying out site-directed mutagenesis on fusion promotion tags TrxA and hydrophobic amino acid fragments to obtain specific cell binding sites.
Description
Technical Field
The invention relates to recombinant collagen, recombinant expression strain and application thereof, belonging to the technical field of protein engineering and genetic engineering.
Background
Collagen is the most abundant protein in mammals, accounting for 25% -35% of the protein content in the bodies, is widely distributed in various tissues and organs of human bodies, such as skin, bones, cornea, blood vessels and the like, and especially contains a large amount of collagen in skin tissues. Collagen, which is a connective tissue adhesive substance, plays an important role in maintaining normal physiological functions of cells, tissues and organs. The recombinant collagen has the advantages of low toxicity, low antigenicity, low immunity, capability of guiding cell regeneration, good biocompatibility and the like, and is widely applied to industries such as biological medicine, cosmetics, food and the like.
In the prior art, the most important method for producing collagen is to treat animal-derived tissues by an acid-base method. In order to improve the biological safety, purity and activity of collagen, the application of collagen in the fields of cosmetics, medical instruments and the like is widened, and the genetic engineering technology is widely used. Some research institutions and biological companies at home and abroad are sequentially put into research and development of recombinant collagen, wherein an escherichia coli expression system is widely applied, but most of the prior art adopts a short segment of human collagen fragments as a repeating unit, and the short segment of human collagen fragments are spliced together to express long-segment recombinant collagen, so that the sequence of the human collagen cannot be well replicated, and the protein is expressed in the form of inclusion bodies, so that the expression quantity is not high.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides recombinant collagen, recombinant expression strain and application. Experiments prove that the recombinant collagen produced by the invention has better barrier repair and biological activities of promoting the synthesis of type I collagen, and the like, has high protein yield, and is more suitable for industrial mass production.
The technical scheme of the invention is as follows:
the amino acid sequence of the recombinant collagen is shown as SEQ ID NO.1 or SEQ ID NO. 3; wherein, the recombinant collagen with the amino acid sequence of SEQ ID NO.3 is obtained by mutating amino acids 19-24 of the recombinant collagen with the amino acid sequence of SEQ ID NO.1 into GLKGEN.
A fusion recombinant collagen is formed by connecting a dissolution promoting tag TrxA to the N-terminal of the recombinant collagen.
A recombinant collagen encoding gene, which encodes the recombinant collagen.
According to the invention, the nucleotide sequence of the coding gene is shown as SEQ ID NO.2 or SEQ ID NO. 4; wherein the nucleotide sequence of SEQ ID NO.2 encodes the amino acid sequence of SEQ ID NO.1, and the nucleotide sequence of SEQ ID NO.4 encodes the amino acid sequence of SEQ ID NO. 3.
A recombinant expression vector containing the coding gene of the recombinant collagen.
According to a preferred embodiment of the present invention, the plasmid vector of the recombinant expression vector is pET28a.
A recombinant expression strain containing the coding gene of the recombinant collagen or the recombinant expression vector.
According to a preferred embodiment of the present invention, the host bacterium of the recombinant expression strain is E.coli.
Further preferably, the E.coli is E.coli BL21 (DE 3).
The recombinant expression strain is applied to the preparation of recombinant collagen.
A method for preparing recombinant collagen by using the recombinant expression strain comprises the following steps: culturing the recombinant expression strain to OD 600 And the content is 0.6 to 0.8, recombinant collagen is obtained by induced expression and separation and purification.
The application of the recombinant collagen or the fusion recombinant collagen in preparing foods, medicines, cosmetics, health products or medical products.
Coli (Escherichia coli) BL21/TrxA-dcol M The strain is preserved in China Center for Type Culture Collection (CCTCC) for 7 months and 18 days in 2023, wherein the preservation unit address is university of Wuhan, hubei province, and the preservation number is CCTCC No. M20231326.
The beneficial effects are that:
the invention discloses a novel human collagen sequence which is spliced by a sequence with high activity and high stability and a cell binding site, wherein the sequence contains 264 amino acids, the protein size is 24.4kDa, the nucleotide sequence is optimized by the codon preference of escherichia coli, and a fusion recombinant collagen mutant and an engineering strain of a high-yield fusion recombinant collagen mutant are constructed by a method of replacing a fusion promotion tag TrxA and a hydrophobic amino acid fragment with the cell binding site. Compared with the commercial collagen products and the recombinant humanized III type collagen of patent application number 202310882902.1, the fusion recombinant collagen and the fusion recombinant collagen mutant prepared by the technical scheme have better barrier repair and bioactivity effects of promoting I type collagen synthesis and the like, so that the fusion recombinant collagen and the fusion recombinant collagen mutant have good practical application value.
Drawings
FIG. 1 shows SDS-PAGE (A) and Western-Blot (B) detection patterns of recombinant E.coil BL21/pET28a-dcol after induced expression; wherein lane M represents a standard protein having a molecular weight of 180 kDa; lane 1 represents the control E.coil BL21/pET28a cell-destroying supernatant; lane 2 represents recombinant E.coil BL21/pET28a-dcol cell supernatant; lane 3 represents recombinant E.coil BL21/pET28a-dcol cell disruption pellet.
FIG. 2 is a SDS-PAGE detection graph of fusion recombinant strain E.coil BL21/pET28a-TrxA-dcol after induced expression; wherein lane M represents a standard protein having a molecular weight of 180 kDa; lane 1 represents the control E.coil BL21/pET28a cell-destroying supernatant; lane 2 represents recombinant E.coil BL21/pET28a-dcol cell supernatant; lane 3 represents the cell-disrupting supernatant of the fusion recombinant E.coil BL21/pET28 a-TrxA-dcol; lane 4 represents the fusion recombinant E.coil BL21/pET28a-TrxA-dcol cell disruption pellet.
FIG. 3 is a graph of amino acid hydrophobicity scores before and after mutation of amino acid sequences of recombinant collagen; wherein, FIG. A represents the amino acid sequence hydrophobicity score of SEQ ID NO. 1; panel B represents the hydrophobicity score of the amino acid sequence of SEQ ID NO.3 after mutation.
FIG. 4 shows a mutant recombinant strain E.coil BL21/TrxA-dcol M SDS-PAGE detection after induced expression; wherein lane M represents a standard protein having a molecular weight of 180 kDa; lane 1 represents the control E.coil BL21/pET28a cell-destroying supernatant; lane 2 represents the supernatant of the fusion recombinant E.coil BL21/pET28a-TrxA-dcol cell; lane 3 represents mutant recombinant E.coil BL21/TrxA-dcol M Breaking cell supernatant; lane 4 represents mutant recombinant E.coil BL21/TrxA-dcol M And (5) cell disruption and precipitation.
FIG. 5 is a graph showing the comparison of the results of different collagens for promoting the mRNA expression of FLG in HaCaT cells; in the figure, P <0.01 (×0.0001 (×0) and P <0.0001 (×0) are considered to have significant differences compared to control group 1.
FIG. 6 is a graph showing comparison of results of different collagens in promoting COL-I secretion of HSF cells; wherein P <0.05 (, P <0.001 (, x) compared to comparative example 1, is considered to have a significant difference.
Detailed Description
The technical scheme of the present invention will be described in further detail with reference to examples and drawings, but the scope of the present invention is not limited thereto. The medicines and reagents related to the examples are common commercial products unless specified; the experimental procedures referred to in the examples, unless otherwise specified, are conventional in the art.
The microorganism deposit information referred to in the examples:
coli (Escherichia coli) BL21/TrxA-dcol M Has been preserved in China center for type culture Collection (CCTCC for short) for 7.18.2023, the collection is in the field of ChinaThe site is university of Wuhan in Wuhan, hubei province, and the preservation number is CCTCC No. M20231326.
The formulation of the medium referred to in the examples:
LB liquid medium: 10g/L NaCl, 5g/L yeast extract, 10g/L peptone;
TB medium: 11.8g/L peptone, 23.6g/L yeast extract, 9.4g/L K 2 HPO 4 ，2.2g/L KH 2 PO 4 4ml/L glycerol.
The commercially available recombinant collagen used in the examples was purchased from Hebei Nake biosciences, inc., model 3CH-FD05, protein size 70kDa.
Example 1: splicing construction of recombinant collagen
The NCBI database was used to obtain the amino acid sequence of the native human type III collagen alpha 1 chain protein (SEQ ID NO: NP-000081), and the biological function of collagen was mainly related to the cell binding site in the amino acid sequence. The recombinant collagen of the invention has 264 amino acids in total, the specific amino acid sequence is shown as SEQ ID NO.1, and is formed by splicing and assembling three sections of sequences with high activity in a natural human III type collagen alpha 1 chain, and the sequences contain RGD, GLKGEN and other cell binding sites, thereby ensuring the realization of the biological functions of the recombinant collagen.
Example 2: construction of recombinant E.coil BL21/pET28a-dcol
The coding gene of the recombinant collagen shown in SEQ ID NO.1 in example 1 is subjected to codon optimization according to the codon preference of the escherichia coli, and the nucleotide sequence of the optimized coding gene is shown as SEQ ID NO.2 and is named dcol. The nucleotide sequence of dcol is entrusted to the Nanjing Jinsri biotechnology Co.Ltd for synthesis, cloned to an escherichia coli expression vector pET28a, converted into E.coil TOP10, and subjected to DNA sequencing comparison to obtain a recombinant expression vector pET28a-dcol on the premise of ensuring that the reading frame is not shifted, and the recombinant expression vector pET28a-dcol is successfully constructed.
The verified recombinant expression vector pET28a-dcol is transformed into escherichia coli BL21 (DE 3) according to an escherichia coli operation manual, kanamycin antibiotics are utilized to screen recombinant expression transformants, and PCR verification proves that recombinant bacteria E.coil BL21/pET28a-dcol expressing recombinant collagen are successfully constructed.
Example 3: induction expression of recombinant bacterium E.coil BL21/pET28a-dcol
Selecting recombinant strain E.coil BL21/pET28a-dcol single colony, culturing in LB liquid medium containing 50 μg/mL kanamycin at 37deg.C and 200rpm overnight, inoculating into TB medium with 2% inoculum size, culturing at 37deg.C and 200rpm to OD 600 After 0.7, 0.5mM IPTG (isopropyl-beta-D-thiogalactoside) with the final concentration is added for induction expression, the culture is carried out for 16 hours at 25 ℃, the thalli are collected by centrifugation, the thalli are resuspended by Tris buffer (20 mM Tris, pH 7.5), then the thalli are subjected to ultrasonic disruption, the broken supernatant and the broken sediment are subjected to SDS-PAGE detection, the theoretical size of the recombinant collagen is 24.4kDa, the SDS-PAGE result is shown as A diagram in figure 1, and the broken supernatant and the broken sediment of the recombinant bacterium E.cobL 21/pET28a-dcol have a weak protein band at the theoretical size, and the protein band is the target recombinant collagen through Western-Blot verification (shown as B diagram in figure 1). The above results indicate that the recombinant collagen of the present invention can be successfully induced to express.
Example 4: construction of fusion recombinant bacterium E.coil BL21/pET28a-TrxA-dcol
In order to improve the soluble expression level of the recombinant collagen, a dissolution-promoting tag, namely thioredoxin TrxA, is fused to the N-terminal of the recombinant collagen. Taking the escherichia coli K12 MG1655 genome as a template, designing a primer, performing PCR amplification to obtain a coding gene of TrxA, introducing an NcoI restriction enzyme site into an N-terminal coding region of the TrxA, and introducing an NdeI restriction enzyme site into a C-terminal coding region of the TrxA;
wherein, the primer sequence is as follows:
TrxA-F：5’-CCATGGATGAGCGATAAAATTATT-3’，
TrxA-R：5’-CATATGGGCCAGGTTAGCGTCGAG-3’。
the PCR amplified product is connected to the linear recombinant expression vector pET28a-dcol cut by the same endonuclease after being cut by the endonuclease NcoI and NdeI, and is converted into E.coil TOP10, the fusion recombinant expression vector pET28a-TrxA-dcol is obtained on the premise of ensuring that the reading frame is not shifted, and the fusion recombinant expression vector is successfully constructed through DNA sequencing comparison.
And (3) converting the verified fusion recombinant expression vector pET28a-TrxA-dcol into escherichia coli BL21 (DE 3) according to an escherichia coli operation manual, screening recombinant expression transformants by using kanamycin antibiotics, and then verifying by PCR that the fusion recombinant bacterium E.coil BL21/pET28a-TrxA-dcol expressing fusion recombinant collagen is successfully constructed.
Example 5: induced expression of fusion recombinant bacterium E.coil BL21/pET28a-TrxA-dcol
The recombinant bacterium E.coil BL21/pET28a-TrxA-dcol was induced and expressed according to the method of example 3, the theoretical size of the recombinant bacterium E.coil BL21/pET28a-TrxA-dcol was 42.1kDa, and as shown in FIG. 2, the expression level of the recombinant bacterium E.coil BL21/pET28a-TrxA-dcol in the cell supernatant was significantly higher than that of the recombinant bacterium E.coil BL21/pET28a-dcol, and the soluble protein expression level of the recombinant bacterium E.coil BL21/pET28a-TrxA-dcol (lane 3) was improved by 6.5 times compared with that of the recombinant bacterium E.coil BL21/pET28a-dcol (lane 2) after normalized analysis by BioAnaly biological analysis software. Further separating and purifying to obtain the fusion recombinant collagen TrxA-dcol.
Example 6: recombinant collagen mutant and mutant recombinant bacterium E.coil BL21/TrxA-dcol M Construction of (3)
Lane 4 in fig. 2 is a sample of a cell disruption precipitate of the recombinant bacterium e.coil BL21/pET28a-TrxA-dcol, and it can be seen from the figure that the recombinant bacterium e.coil BL21/pET28a-TrxA-dcol is fermented to generate a large amount of insoluble target protein, and in order to further improve the soluble expression of the recombinant collagen, the amino acid sequence (SEQ ID No. 1) of the recombinant collagen is analyzed by using protein hydrophobicity analysis software, as shown in a graph a of fig. 3, the vertical axis represents hydrophobicity, the greater represents hydrophobicity, and if negative, the greater represents hydrophilicity, the 19 th to 24 th amino acids in the amino acid sequence of the recombinant collagen have higher hydrophobicity scores, so that the 19 th to 24 th amino acids are mutated into a cell binding site GLKGEN, and the amino acid sequence after mutation has hydrophobicity analysis result as shown in a graph B of fig. 3, and the amino acid sequence of the obtained recombinant collagen mutant is shown in a graph B of fig. 3.
The fusion recombinant expression vector pET28a-TrxA-dcol is used as a template, a mutation primer and a one-step cloning kit PCR of the novzan are utilized to construct a mutation vector, and the nucleotide sequence of the obtained recombinant collagen mutant coding gene is shown as SEQ ID NO. 4. Sequencing and verifying to obtain a mutation vector pET28a-TrxA-dcol M 。
Wherein the sequence of the mutation primer is as follows:
mutation-F: 5'-GGCCTGAAAGGTGAGAACGGCTTCCCGGGTATG-3' the number of the individual pieces of the plastic,
mutation-R: 5'-GTTCTCACCTTTCAGGCCTTTGATACCCGGTGG-3'.
The successful mutation vector pET28a-TrxA-dcol is verified M Transforming into Escherichia coli BL21 (DE 3) according to Escherichia coli operation manual, screening recombinant expression transformant with kanamycin antibiotic, and PCR verification to obtain mutant recombinant strain E.coil BL21/TrxA-dcol for expressing recombinant collagen mutant M The construction was successful.
Coli (Escherichia coli) BL21/TrxA-dcol M The strain is preserved in China Center for Type Culture Collection (CCTCC) for 7 months and 18 days in 2023, wherein the preservation unit address is university of Wuhan, hubei province, and the preservation number is CCTCC No. M20231326.
Example 7: mutant recombinant E.coil BL21/TrxA-dcol M Is expressed by induction of (a)
Mutant recombinant E.coil BL21/TrxA-dcol was obtained in the same manner as in example 3 M Induced expression, SDS-PAGE verification, and the result is shown in figure 4, the mutant recombinant bacterium E.coil BL21/TrxA-dcol M The target protein content in the cell disruption sediment of (2) is obviously reduced, and the protein band is known to mutate recombinant bacterium E.coil BL21/TrxA-dcol after normalized analysis by BioAnaly biological analysis software M The expression level of the soluble protein (lane 3) was increased 2-fold compared with that of the recombinant bacterium E.coil BL21/pET28a-TrxA-dcol (lane 2). Further separating and purifying to obtain fusion recombinant collagen mutant TrxA-dcol M 。
Example 8: barrier repair experiments
Filaggrin (FLG) is one of the important components of the keratinized envelope, ensuring the integrity of the skin barrier. FLG deficiency can lead to disorder of lipid bilayer structure, delayed maturation, reduced cell tightness of the stratum corneum, enhanced intercellular permeability and reduced photoprotection, ultimately leading to impaired skin barrier.
Human immortalized keratinocytes (HaCaT cells) were collected and cell suspensions were prepared using high sugar DMEM cell culture broth, 2mL of cell suspension was added to each well in a 6-well plate, and the number of cells was 2.6X10 5 /well. Experiment setting blank control group (control group 1), commercially available recombinant collagen control group (control group 2), recombinant humanized III type collagen control group (control group 3) in patent application No. 202310882902.1, fusion recombinant collagen TrxA-dcol group prepared in example 5 of the invention (experiment group 1) and fusion recombinant collagen mutant TrxA-dcol prepared in example 7 of the invention M Groups (experimental group 2) were each provided with 3 duplicate wells. The 6-well plate was placed in a cell incubator (5% CO) 2 Incubating for 24 hours at 37 ℃, discarding the culture solution when the cell fusion rate reaches 50% -60%, and adding 2mL of high-sugar DMEM cell culture solution into each hole of the blank control group; the other groups were each added with 2mL of high sugar DMEM cell culture solution containing the corresponding collagen, wherein the concentration of the collagen in the high sugar DMEM cell culture solution was 1mg/mL. 6-well plates were placed in an incubator (37 ℃, 5% CO) 2 ) After incubation for 24h, washing the cells twice with 2mL of PBS buffer solution in each well, adding 1mL of RNAiso Plus according to the Total RNA extraction kit, blowing and lysing the cells, collecting samples, carrying out RNA extraction, reverse transcription and fluorescent quantitative PCR detection experiments according to the kit specification, detecting the relative expression level of mRNA of the barrier-associated protein FLG, and adopting 2 -△△CT The method performs the calculation.
The ability of different collagens to promote expression of FLG mRNA in HaCaT cells is shown in FIG. 5, and compared with the blank control (control 1), commercially available recombinant collagen (control 2), recombinant humanized type III collagen (control 3) of patent application No. 202310882902.1, and fusion recombinant collagen TrxA-dcol prepared in example 5 of the present invention (experimentGroup 1) fusion recombinant collagen mutant TrxA-dcol prepared in example 7 of the present invention M The relative expression quantity of the mRNA of the FLG can be obviously improved, and the mRNA expression capacity of the FLG promoted by four kinds of collagen is sequentially from strong to weak, namely fusion recombinant collagen mutant TrxA-dcol M The fusion recombinant collagen TrxA-dcol > recombinant humanized III type collagen > commercial recombinant collagen, and the mutant TrxA-dcol of the mutated fusion recombinant collagen M Is obviously superior to fusion recombinant collagen TrxA-dcol before mutation.
Example 9: anti-wrinkle experiment
Collagen is one of the important components of the dermis, and provides support for the skin, giving it its elasticity and texture. Wherein, the collagen type I (COL-I) accounts for 80-85% of the collagen component of skin, promotes the increase of the content of COL-I, and can achieve the effect of resisting the generation of wrinkles to a certain extent.
Collecting human skin fibroblast (HSF cell) with good growth state, preparing cell suspension with high sugar DMEM cell culture solution, adding 2mL cell suspension into each well of 6-well plate, and counting cell number of 2.6X10 5 /well. Experiment setting blank control group (control group 1), commercially available recombinant collagen control group (control group 2), recombinant humanized III type collagen control group (control group 3) in patent application No. 202310882902.1, fusion recombinant collagen TrxA-dcol group prepared in example 5 of the invention (experiment group 1) and fusion recombinant collagen mutant TrxA-dcol prepared in example 7 of the invention M Groups (experimental group 2) were each provided with 3 duplicate wells. The 6-well plate was placed in a cell incubator (5% CO) 2 Incubating for 24 hours at 37 ℃ and ensuring that the cell fusion rate reaches 60-70%. The culture solution is discarded, and 2mL of low-sugar DMEM cell culture solution is added into each hole of a blank control group; the other groups were each added with 2mL of low-sugar DMEM cell culture broth containing the corresponding collagen, wherein the concentration of collagen in the low-sugar DMEM cell culture broth was 1mg/mL. After the completion of the administration, the 6-well plate was placed in an incubator (5% CO) 2 Culturing for 24h at 37 ℃), collecting HSF cell culture solution, centrifuging at 12000rpm for 10min, collecting cell precipitate, and detecting the content of type I collagen according to ELISA kit instructions.
The results of the ability of different collagens to promote COL-I secretion of HSF cells are shown in FIG. 6, and compared with the blank control (control 1), commercially available recombinant collagen (control 2), recombinant humanized type III collagen (control 3) in patent application No. 202310882902.1, fusion recombinant collagen TrxA-dcol prepared in example 5 of the present invention (experimental 1), and fusion recombinant collagen mutant TrxA-dcol prepared in example 7 of the present invention M Can obviously improve the secretion amount of COL-I, and the COL-I secretion promotion capability of four kinds of collagen is fusion recombinant collagen mutant TrxA-dcol from strong to weak in turn M The fusion recombinant collagen TrxA-dcol > recombinant humanized III type collagen > commercial recombinant collagen, and the mutant TrxA-dcol of the mutated fusion recombinant collagen M Is obviously superior to fusion recombinant collagen TrxA-dcol before mutation.
Claims (10)
1. The recombinant collagen is characterized in that the amino acid sequence of the recombinant collagen is shown as SEQ ID NO.1 or SEQ ID NO. 3; wherein, the recombinant collagen with the amino acid sequence of SEQ ID NO.3 is obtained by mutating amino acids 19-24 of the recombinant collagen with the amino acid sequence of SEQ ID NO.1 into GLKGEN.
2. A fusion recombinant collagen, wherein the fusion recombinant collagen is prepared by connecting a dissolution promoting tag TrxA at the N-terminal of the recombinant collagen according to claim 1.
3. A recombinant collagen encoding gene, which encodes the recombinant collagen of claim 1.
4. The recombinant collagen encoding gene according to claim 3, wherein the nucleotide sequence of the encoding gene is shown in SEQ ID NO.2 or SEQ ID NO. 4; wherein the nucleotide sequence of SEQ ID NO.2 encodes the amino acid sequence of SEQ ID NO.1, and the nucleotide sequence of SEQ ID NO.4 encodes the amino acid sequence of SEQ ID NO. 3.
5. A recombinant expression vector comprising the recombinant collagen encoding gene of claim 3 or 4;
preferably, the plasmid vector of the recombinant expression vector is pET28a.
6. A recombinant expression strain comprising the recombinant collagen encoding gene of claim 3 or 4 or the recombinant expression vector of claim 5;
preferably, the host bacterium of the recombinant expression strain is escherichia coli;
further preferably, the E.coli is E.coli BL21 (DE 3).
7. Use of the recombinant expression strain of claim 6 for the preparation of recombinant collagen.
8. A method for preparing recombinant collagen using the recombinant expression strain of claim 6, comprising the steps of: culturing the recombinant expression strain of claim 6 to OD 600 And the content is 0.6 to 0.8, recombinant collagen is obtained by induced expression and separation and purification.
9. Use of the recombinant collagen according to claim 1 or the fusion recombinant collagen according to claim 2 in the preparation of a food, pharmaceutical, cosmetic, health care or pharmaceutical product.
10. Coli (Escherichia coli) BL21/TrxA-dcol M The strain is preserved in China center for type culture Collection (China) for 7 months and 18 days in 2023, wherein the preservation unit address is university of Wuhan in Wuhan, hubei province, and the preservation number is CCTCC No. M20231326.
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| CN117510650A (en) * | 2023-11-08 | 2024-02-06 | 广西福莱明生物制药有限公司 | Protein for pelvic floor dysfunction and composition thereof |
| CN118359703A (en) * | 2024-06-19 | 2024-07-19 | 山东福瑞达生物股份有限公司 | Recombinant transdermal collagen and preparation method and application thereof |
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| CN117510650A (en) * | 2023-11-08 | 2024-02-06 | 广西福莱明生物制药有限公司 | Protein for pelvic floor dysfunction and composition thereof |
| CN117510650B (en) * | 2023-11-08 | 2024-05-24 | 广西福莱明生物制药有限公司 | Protein for pelvic floor dysfunction and composition thereof |
| CN118359703A (en) * | 2024-06-19 | 2024-07-19 | 山东福瑞达生物股份有限公司 | Recombinant transdermal collagen and preparation method and application thereof |
| CN118359703B (en) * | 2024-06-19 | 2024-08-16 | 山东福瑞达生物股份有限公司 | Recombinant transdermal collagen and preparation method and application thereof |
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