CN117886923B - Recombinant humanized collagen and encoding gene and application thereof - Google Patents
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Abstract
The invention relates to recombinant humanized collagen and a coding gene and application thereof, belonging to the technical fields of genetic engineering and protein engineering. The invention is based on the amino acid sequence of human collagen, designs the amino acid sequence by means of bioinformatics and the like, realizes higher expression efficiency and high stability on the premise of maintaining high bioactivity of the recombinant collagen, designs and obtains a brand new collagen sequence containing 658 amino acids, has large protein molecular weight, and can better play the biological function of the human collagen. The nucleotide sequence of the recombinant collagen is optimized by the codon preference of escherichia coli, a high-expression recombinant strain is constructed, and the recombinant collagen produced by fermenting the recombinant strain has no exogenous amino acid such as any tag and the like, is recombinant humanized collagen, and has good safety. The invention also provides a low-cost and high-efficiency protein purification process which is suitable for large-scale production.
Description
Technical Field
The invention relates to recombinant humanized collagen and a coding gene and application thereof, belonging to the technical fields of genetic engineering and protein engineering.
Background
Collagen is an important structural protein, and is one of the most abundant proteins in many organisms. The main function of the collagen is to provide structural support for the body and maintain the stability and elasticity of the tissues. In tissues and organs, collagen is mainly present in tissues such as bones, joints, muscles, skin, blood vessels, cornea, and the like. In addition, collagen can be used as a signal molecule to regulate proliferation, differentiation and migration of cells, and has important effects on maintaining normal physiological functions and overall health of organisms. 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 present stage, large research institutions mainly carry out recombination work around human type III collagen. Some research institutions select a short segment of human collagen fragment as a repeating unit, and splice the segments together to express long-segment recombinant humanized collagen (such as CN102443057A, CN 106554410A), and the recombinant humanized collagen cannot well replicate the sequence of the human collagen and cannot fully exert the biological function of the human collagen; some research institutions can do some optimization work on the basis of the human collagen sequence, namely recombinant collagen (such as CN114262368A, CN 113735964A), but the recombinant collagen has low safety and is easy to cause adverse reactions such as allergy; in the prior art, purification tags are added on protein amino acid sequences to obtain products with high purity, and the purification cost of the scheme is high (such as CN 111004319A).
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a recombinant humanized collagen and a coding gene and application thereof. Experiments prove that the collagen produced by the invention has large molecular weight and can better exert the biological functions of the humanized collagen; no foreign amino acid such as any label, is recombinant humanized collagen, and has good safety; the preparation has better antioxidant activity and barrier repair biological activity; and the protein yield is high, the purification cost is low, and the method is more suitable for industrialized mass production.
In order to achieve the technical purpose, the technical scheme of the invention is as follows:
a recombinant humanized collagen has an amino acid sequence shown in SEQ ID NO. 1.
A recombinant humanized collagen encoding gene, which encodes the recombinant humanized collagen.
According to the invention, the nucleotide sequence of the coding gene is shown as SEQ ID NO. 2.
A recombinant expression vector containing the coding gene of the recombinant humanized 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 humanized 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 humanized collagen.
A method for preparing recombinant humanized collagen by utilizing the recombinant expression strain to perform high-density fermentation comprises the following steps: after the recombinant expression strain is fermented and cultured in a fermentation tank until the OD 600 is 79-81, the recombinant humanized collagen is induced to be expressed.
A method for purifying recombinant humanized collagen, comprising the steps of: and centrifuging the fermentation liquor after the recombinant expression strain is induced and cultured to obtain bacterial mud, homogenizing bacterial liquor after bacterial mud is resuspended, removing impurity proteins, centrifuging, clarifying by a hollow fiber membrane, concentrating by an ultrafiltration membrane and performing ion exchange chromatography to obtain recombinant humanized collagen purified stock solution.
According to the invention, the impurity-removed protein is removed by an acid precipitation method, and the pH of the bacterial liquid is adjusted to 2.7-2.9.
According to a preferred embodiment of the invention, the hollow fiber membrane has a molecular weight cut-off of 250kDa.
According to a preferred embodiment of the invention, the ultrafiltration membrane has a molecular weight cut-off of 10kDa.
Preferably, according to the present invention, the ion exchange chromatography is anion exchange chromatography.
The application of the recombinant humanized collagen in preparing food, medicine, cosmetics, health care products or appliance products.
The beneficial effects are that:
The invention is based on the amino acid sequence of human collagen, designs the amino acid sequence by means of bioinformatics and the like, realizes higher expression efficiency and high stability on the premise of maintaining high bioactivity of the recombinant collagen, designs and obtains a brand new collagen sequence containing 658 amino acids, has large protein molecular weight, and can better play the biological function of the human collagen. The nucleotide sequence of the recombinant collagen is optimized by the codon preference of escherichia coli, a high-expression recombinant strain is constructed, and the recombinant collagen produced by fermenting the recombinant strain has no exogenous amino acid such as any tag and the like, is recombinant humanized collagen, and has good safety. The invention also provides a low-cost and high-efficiency protein purification process which is suitable for large-scale production. Compared with the collagen product sold in the market, the recombinant humanized collagen prepared by the technical scheme has better antioxidant activity and barrier repair capability, so that the recombinant humanized collagen has good practical application value.
Drawings
FIG. 1 is a SDS-PAGE analysis of recombinant E. coliBL21/pET28a-Col after induced expression; wherein lane M represents a standard protein having a molecular weight of 180 kDa; lane 1 represents the supernatant of the control bacterium E. coliBL21/pET28a cell disruption; lane 2 represents recombinant E. coliBL21/pET28a-Col cell supernatant.
FIG. 2 is an SDS-PAGE protein electrophoresis of recombinant E. coliBL21/pET28a-Col for 5-L fermenter high density culture induction phase timing sampling; wherein lane M represents a standard protein having a molecular weight of 180 kDa; lane 1 represents pre-induction; lane 2 represents induction for 5h; lane 3 represents induction for 10h; lane 4 represents induction for 12h; lane 5 represents induction for 15h.
FIG. 3 is a bar graph of DPPH radical scavenging for different collagens; wherein, the comparative example represents a commercial recombinant collagen comparative experiment group; col represents the experimental group of recombinant humanized collagen prepared using example 5 of the present invention.
FIG. 4 is a bar graph showing the relative expression of FLG mRNA in HaCaT cells under the action of different collagens; wherein, the control group 1 represents a blank control group; control group 2 represents a commercially available recombinant collagen control group; col represents the recombinant humanized collagen Col set prepared in example 5 of the present invention; 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.
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 percentages referred to in the examples are mass percentages unless otherwise indicated.
Example 1: recombinant humanized collagen sequence design
The NCBI database is utilized to obtain the amino acid sequence (GenBank: KAI 2526099.1) of the natural human III type collagen alpha 1 chain protein, the deep mining analysis of the sequence information is utilized, and the means such as bioinformatics and the like are utilized to carry out sequence design on the hydrophilicity and hydrophobicity, charge distribution and protein space structure of the amino acid, so that the recombinant collagen can realize higher expression efficiency and high stability on the premise of maintaining high bioactivity. The recombinant collagen of the invention has 658 amino acids in total, the specific amino acid sequence is shown as SEQ ID NO.1, the sequence contains the main bioactive site of the natural human III type collagen alpha 1 chain, the realization of the biological function of the recombinant collagen is ensured, the sequence does not contain any exogenous amino acid such as labels, and the recombinant collagen is recombinant humanized collagen, and has high biocompatibility and low use risk.
Example 2: construction of recombinant E. coliBL21/pET28a-Col
The coding gene of the recombinant humanized 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 Col. The nucleotide sequence of Col entrusts the large gene to synthesize, clone onto E.coli expression vector pET28a, convert into E. coliTOP10, obtain recombinant expression vector pET28a-Col under the precondition of ensuring that reading frame is not shifted, and construct recombinant expression vector pET28a-Col successfully by DNA sequencing comparison.
The verified recombinant expression vector pET28a-Col 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. coliBL21/pET28a-Col expressing recombinant humanized collagen are successfully constructed.
Example 3: induction expression of recombinant bacteria E. coliBL21/pET28a-Col
A single colony of recombinant E. coliBL/pET 28a-Col was picked up, cultured overnight at 37℃in LB liquid medium (1% NaCl,0.5% yeast extract, 1% peptone) containing 50. Mu.g/mL kanamycin, inoculated in 2% inoculum size into TB medium (1.18% peptone, 2.36% yeast extract, 0.94% K 2HPO4,0.22% KH2PO4, 0.4% glycerol) at 37℃at 200rpm until OD 600 was 0.7, then added with 0.5mM IPTG (isopropyl-. Beta. -D-thiogalactoside) to induce expression, cultured at 25℃for 16h, centrifuged to collect the cells, resuspended in Tris buffer (20 mM Tris, pH 7.5), and SDS-PAGE was performed on the cell supernatant and the cell pellet, the theoretical size of the recombinant humanized collagen was 60.08kDa, SDS-PAGE results were shown in FIG. 1, and the recombinant E coliBL/pET 28 a-pET 28a was significantly compared with the theoretical supernatant (FIG. 28a small-35) of the control (FIG. 2A-pE) in a small lane, indicated by the arrow mark of FIG. 1). The above results indicate that the recombinant humanized collagen of the present invention can be successfully induced to express.
Example 4: recombinant E. coliBL21/pET28a-Col 5-L tank high-density culture
Recombinant E. coliBL21/pET28a-Col was subjected to 5-L tank high-density culture. The bacterial liquid in the glycerol tube was inoculated into 60mL seed medium (1% peptone, 1% sodium chloride, 0.5% yeast powder) containing 50. Mu.g/mL kanamycin at an inoculum size of 1%o, and cultured at 37℃for about 15 hours at 180 rpm. The whole seed liquid after cultivation is transferred into a 5-L fermentation tank containing 3L fermentation medium (3% yeast powder, 1.5% glycerol, 3.25% ammonium sulfate, 0.2% sodium chloride, 1.0% disodium hydrogen phosphate dodecahydrate, 0.31% potassium dihydrogen phosphate, 0.17% magnesium sulfate heptahydrate, 0.02% defoamer, 0.43% trace element, pH adjusted to 7.0 with sodium hydroxide, 500mL trace element formulation: boric acid 1.625g, manganese sulfate monohydrate 0.275g, cupric chloride dihydrate 0.3325g, zinc sulfate monohydrate 0.1375g, cobalt chloride 0.211g, sodium molybdate 0.1735 g). The initial fermentation parameters were set as follows: stirring at 150rpm, fermenting at 37deg.C, ventilating at 4L/min, regulating dissolved oxygen, stirring to 20%, and automatically adding ammonia water to control pH to 7.0. After the dissolved oxygen is raised to 35%, feeding (formula of feeding culture medium: 3% ammonium sulfate, 0.22% magnesium sulfate heptahydrate, 30% glycerol, 0.5% trace elements), initial feeding speed of 0.8 mL/min, regulating dissolved oxygen to be 20% by increasing ventilation, adding 0.5mM IPTG after OD 600 is 80, regulating temperature to 25 ℃, controlling dissolved oxygen to be about 30%, and induction time of 10-20h. Samples were taken at regular intervals after induction time and the fermentation broth was subjected to SDS-PAGE detection.
As shown in FIG. 2, the SDS-PAGE detection result shows that the yield of the recombinant humanized collagen in the fermentation broth gradually increases along with the induction time, and the gray values of the standard protein and the recombinant humanized collagen are judged by BioAnaly biological analysis software analysis, and when the recombinant bacterium E. coliBL21/pET28a-Col is subjected to 5-L tank high-density fermentation induction for 15 hours, the yield of the recombinant humanized collagen is 8.3 g/L, so that the high expression efficiency of the recombinant humanized collagen is realized.
Example 5: purification preparation of recombinant humanized collagen
Carrying out high-density fermentation on recombinant bacteria E. coliBL21/pET28a-Col in a 5-L tank, centrifuging fermentation liquor after induced culture for 15h at 8000rpm to obtain bacterial sludge, and purifying the bacterial sludge to obtain a recombinant humanized collagen pure product through the following purification steps:
① Impurity removal protein: the pH of the bacterial liquid after bacterial mud re-suspension homogenization is adjusted to 2.8 by low-concentration hydrochloric acid, and the bacterial liquid is continuously stirred in the acid adding process, so that partial peracid in the process is prevented, and the recombinant humanized collagen is denatured and precipitated;
② And (3) centrifuging: centrifuging to remove acid precipitated impurity protein, and regulating pH of the supernatant to 7.0 with low concentration sodium hydroxide;
③ Clarifying by a hollow fiber membrane: filtering the centrifugal supernatant by a 250kDa hollow fiber membrane, sterilizing and removing macromolecular impurities;
④ Concentrating by ultrafiltration membrane: concentrating the feed liquid by using a10 kDa ultrafiltration membrane bag, and removing small molecular impurities;
⑤ Ion exchange chromatography: anion exchange chromatography is adopted, recombinant humanized collagen is positively charged, and the flow-through liquid is directly collected during chromatography sample loading, impurities such as negatively charged impurity proteins and endotoxin can be adsorbed on a column, so that the aim of removing the impurities is achieved, and the obtained flow-through liquid is the purified stock solution.
The purity of the protein purified by the steps is more than 97%, and the yield is more than 60%.
Example 6: antioxidant experiment of recombinant humanized collagen
An appropriate amount of the purified stock solution of recombinant humanized collagen of example 5 was placed in a test tube, 2.5mL of DPPH (1, 1-diphenyl-2-trinitrophenylhydrazine) free radical solution (0.1 mmoL/L) dissolved in 95% ethanol was added, the mixture was vigorously shaken for 10s, then placed at room temperature for reaction for 30min, after the reaction was completed, absorbance of the reaction mixture was measured at 517nm, distilled water was used as a blank control instead of the sample solution, and a comparative experiment was performed with commercially available recombinant collagen (comparative example, which was purchased from Hebei Nake biosciences, model 3CH-FD 05).
DPPH radical scavenging activity= (OD Blank space -OD Sample of ）/OD Blank space ,
Wherein OD Blank space is the absorbance of the blank control group, and OD Sample of is the absorbance of the recombinant humanized collagen or a commercially available recombinant collagen group.
As shown in FIG. 3, the recombinant humanized collagen Col of the present invention has an obvious DPPH radical scavenging ability compared with the blank, and the DPPH radical scavenging rate is higher than that of the commercially available recombinant collagen, so that the recombinant humanized collagen provided by the present invention has an obvious antioxidant effect.
Example 7: 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, purchased from the Shanghai cell bank of the national academy of sciences) were collected and cell suspensions were prepared with high-sugar DMEM cell culture broth, 2mL of cell suspension was added per well in 6-well plates, and the number of cells was 2.6x10 5/well. A blank control group (control group 1), a commercially available recombinant collagen control group (control group 2), and a recombinant humanized collagen Col group (experimental group) prepared in example 5 of the present invention were experimentally set, and 3 duplicate wells were set in each group. Placing the 6-hole plate in a cell incubator (37 ℃ C., 5% CO 2) for incubation for 24 hours, 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 a blank control group; the other two groups are respectively added with 2mL of high-sugar DMEM cell culture solution containing corresponding collagen, wherein the concentration of the collagen in the high-sugar DMEM cell culture solution is 1mg/mL. After incubating the 6-well plate in an incubator (37 ℃ C., 5% CO 2) for 24 hours, the cells were washed twice with 2mL of PBS buffer per well, 1mL RNAiso Plus was added according to the Total RNA extraction kit (Nanjinofuzan FastPure Cell/Tissue Total RNA Isolation Kit V2 kit (RC 112-01)), the lysed cells were blown down and collected, RNA extraction, reverse transcription and fluorescent quantitative PCR detection experiments were performed according to the kit instructions, and the relative expression level of mRNA of the barrier-associated protein FLG was detected and calculated by the method of 2 -△△CT.
As shown in FIG. 4, compared with the blank control group (control group 1), the mRNA expression capacity of FLG in HaCaT cells is promoted by different collagens, and compared with the blank control group (control group 2), the mRNA relative expression capacity of FLG can be obviously improved by the commercially available recombinant collagen (control group 2) and the recombinant humanized collagen Col (experimental group) prepared by the embodiment 5 of the invention, wherein the mRNA expression capacity of FLG is promoted by the prepared recombinant humanized collagen, which is stronger than that of the commercially available recombinant collagen, so that the recombinant humanized collagen has an obvious skin barrier repair function.
Claims (13)
1. The recombinant humanized collagen is characterized in that the amino acid sequence of the recombinant humanized collagen is shown as SEQ ID NO. 1.
2. A gene encoding recombinant humanized collagen, wherein the gene encodes the recombinant humanized collagen of claim 1.
3. The recombinant humanized collagen encoding gene according to claim 2, wherein the nucleotide sequence of the encoding gene is shown in SEQ ID NO. 2.
4. A recombinant expression vector comprising the recombinant humanized collagen encoding gene according to claim 2.
5. The recombinant expression vector of claim 4, wherein the plasmid vector of the recombinant expression vector is pET28a.
6. A recombinant expression strain comprising the recombinant humanized collagen encoding gene of claim 2 or the recombinant expression vector of claim 4.
7. The recombinant expression strain of claim 6, wherein the host strain of the recombinant expression strain is escherichia coli.
8. The recombinant expression strain of claim 7, wherein the E.coli is E.coli BL21 (DE 3).
9. Use of the recombinant expression strain of claim 6 for the preparation of recombinant humanized collagen according to claim 1.
10. A method for preparing recombinant humanized collagen by high-density fermentation using the recombinant expression strain of claim 6, comprising the steps of: after fermenting and culturing the recombinant expression strain of claim 6 in a fermenter until the OD 600 is 79-81, recombinant humanized collagen is induced to be expressed.
11. A method for purifying recombinant humanized collagen, comprising the steps of: centrifuging the fermentation broth after the recombinant expression strain is induced and cultured to obtain bacterial sludge, homogenizing the bacterial liquid after bacterial sludge is resuspended, removing impurity proteins, centrifuging, clarifying by a hollow fiber membrane, concentrating by an ultrafiltration membrane, and performing ion exchange chromatography to obtain the recombinant humanized collagen purification stock solution.
12. The method of purifying recombinant humanized collagen according to claim 11, wherein one or more of the following conditions are satisfied:
i. The impurity-removing protein is removed by adopting an acid precipitation method, and the pH value of the bacterial liquid is adjusted to 2.7-2.9;
the molecular weight cut-off of the hollow fiber membrane is 250kDa;
The ultrafiltration membrane has a molecular weight cut-off of 10kDa;
the ion exchange chromatography is anion exchange chromatography.
13. The use of the recombinant humanized collagen according to claim 1 for the preparation of food, cosmetics, appliance products or antioxidant drugs.
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| CN113150173A (en) * | 2021-05-24 | 2021-07-23 | 钟小强 | Recombinant human collagen peptide and preparation method and application thereof |
| CN116640206A (en) * | 2023-07-19 | 2023-08-25 | 山东福瑞达生物股份有限公司 | Recombinant humanized III type collagen and preparation method and application thereof |
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| CN113150173A (en) * | 2021-05-24 | 2021-07-23 | 钟小强 | Recombinant human collagen peptide and preparation method and application thereof |
| WO2023221787A1 (en) * | 2022-05-19 | 2023-11-23 | 华北制药集团新药研究开发有限责任公司 | Pichia pastoris engineering strain for recombinant type i collagen, construction method therefor and use thereof |
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