CN118324930A - Recombinant humanized lactoferrin and preparation method thereof - Google Patents
Recombinant humanized lactoferrin and preparation method thereof Download PDFInfo
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- CN118324930A CN118324930A CN202410762512.5A CN202410762512A CN118324930A CN 118324930 A CN118324930 A CN 118324930A CN 202410762512 A CN202410762512 A CN 202410762512A CN 118324930 A CN118324930 A CN 118324930A
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- lactoferrin
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- CSSYQJWUGATIHM-IKGCZBKSSA-N l-phenylalanyl-l-lysyl-l-cysteinyl-l-arginyl-l-arginyl-l-tryptophyl-l-glutaminyl-l-tryptophyl-l-arginyl-l-methionyl-l-lysyl-l-lysyl-l-leucylglycyl-l-alanyl-l-prolyl-l-seryl-l-isoleucyl-l-threonyl-l-cysteinyl-l-valyl-l-arginyl-l-arginyl-l-alanyl-l-phenylal Chemical compound C([C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 CSSYQJWUGATIHM-IKGCZBKSSA-N 0.000 title claims abstract description 83
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Classifications
-
- C—CHEMISTRY; METALLURGY
- 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
- C07K14/79—Transferrins, e.g. lactoferrins, ovotransferrins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
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- Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a recombinant humanized lactoferrin and a preparation method thereof. The recombinant humanized lactoferrin sequence comprises a derivative peptide that mimics the antibacterial alpha-helical region of the LfcinH N terminus of human lactoferrin and retains the key active amino acid residues LFCINH CYS, trp23, arg28, lys29, and Arg31, the LfcinH derivative peptide having hydrophilic amino acid side chain modifications; or fusion of a human FLG 2C-terminal active fragment, said FLG 2C-terminal active fragment comprising the B-type repeat 14 (2247-2321) or the C-term sequence (2322-2391) of FLG 2. The invention utilizes the bioengineering technology to construct engineering bacteria containing the structural characteristic protein fragments, and obtains the high-efficiency expression of the series of recombinant humanized lactoferrin. The recombinant humanized lactoferrin produced by the invention has biological activity of promoting skin wound repair and inhibiting inflammatory reaction, and is expected to be developed into active raw materials to be applied to the fields of biological medicine, cosmetics, large health products and the like.
Description
Technical Field
The invention relates to the field of bioengineering, in particular to recombinant humanized lactoferrin and a preparation method thereof.
Background
Sensitive skin problems have become a prominent health issue in today's society. The core problem of sensitive skin is that the skin barrier function is declined and the immune system is unbalanced, so that the skin is easy to be stimulated, red swelling, itching and other symptoms are generated, and inflammatory response is caused by serious skin. The skin barrier deterioration is mainly characterized by loose stratum corneum structure and increased physical permeability, and further causes physiological lipid loss among keratinocytes, reduction of natural moisturizing factors, deep skin water loss, pH rise, and easy invasion of harmful substances such as external environment irritants and bacteria into the skin, so that irritant dermatitis and allergic dermatitis are caused. Solving the sensitive skin problem, strengthening the skin barrier function from the source and restoring the normal activity of immune cells.
Lactoferrin (Lactoferrin, lf) is a non-heme iron-binding glycoprotein secreted by mammalian mucosal epithelial cells and studies have reported that Lf released from neutrophils plays a key role in immune and inflammatory responses. Human lactoferrin (LfcinH) is a multifunctional short peptide consisting of 49N-terminal residues, obtained by hydrolysis of Lf by acid pepsin. Lfcin is highly basic and contains multiple positively charged Arg and Lys residues, the Trp residues in the sequence being important for bacteriostatic activity. The activity of Lfcin depends on the hydrophobicity, cationicity, secondary structure, amino acid composition, etc. of the protein. Thus, altering these properties provides an effective strategy for optimizing the activity of lfmin. A human-like lactoferrin peptide as disclosed in CN112500475A is designed and expressed as a series of LfcinH structural analogues, and the functions of inhibiting the growth of various pathogenic bacteria and regulating skin micro-ecology are verified.
Filaggrin (FLG) is a key structural protein in the epidermis. FLG can promote the differentiation of epidermal cells, and then a special barrier structure of the stratum corneum is constructed, and the barrier is not only helpful for preventing the loss of in-vivo water, but also can effectively resist the invasion of external pathogens, so that the basic defense function of the skin is maintained. Human silk fibroin-2 (FLG 2) is a 248 kDa s100 fusion protein that is insoluble in water, and is present in the stratum granulosum and stratum corneum. The N-terminal portion of FLG2 (including the A-type repeat) shares identity with the S100 member keratin, while the C-terminal portion (including the B-type repeat) shares similarity with the filaggrin. The B-type repeat of FLG2 may have keratin bundle activity similar to that of the silk fibroin subunit. Recombinant FLG 2C-term has been reported to terminate the growth of pathogenic bacteria on the skin surface by interfering with bacterial replication, thus potentially contributing to the antimicrobial defensive barrier of the skin.
Disclosure of Invention
The invention aims to optimize and develop new functional recombinant lactoferrin by adopting a molecular design strategy based on LfcinH active sites, key structures and amino acid sequences. The invention provides recombinant humanized lactoferrin with skin wound repair promoting and inflammatory reaction inhibiting activities, and a method for producing the recombinant humanized lactoferrin.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a recombinant humanized lactoferrin comprising:
(1) A derivative peptide mimicking the antibacterial alpha-helical region at the terminus of human lactoferrin LfcinH N;
Or (2) a derivative peptide which mimics the antibacterial alpha-helical region at the terminus of human lactoferrin LfcinH N and fused to the FLG 2C-terminal active fragment, the derivative peptide and FLG 2C-terminal active fragment being linked by a linker peptide.
Preferably, in the recombinant humanized lactoferrin, the antibacterial alpha-helical region at the end of the humanized lactoferrin LfcinH N retains LFCINH CYS, trp23, arg28, lys29 and Arg31 active amino acid residues, and the amino acid sequence of the derivative peptide of the antibacterial alpha-helical region at the end of the humanized lactoferrin LfcinH N is shown in SEQ ID No. 1.
Preferably, in the recombinant humanized lactoferrin, the derivative peptide of the antibacterial alpha-helical region at the end of the human-derived lactoferrin LfcinH N is subjected to hydrophilic amino acid side chain modification.
Preferably, in the recombinant humanized lactoferrin, the C-terminal active fragment of FLG2 comprises a B repeat sequence B-14, 2244-2321 or C-term sequence 2322-2391 of FLG2, and the amino acid sequence is shown as SEQ ID NO.2 or SEQ ID NO.3 respectively.
Preferably, in the recombinant humanized lactoferrin, the amino acid sequence of the recombinant humanized lactoferrin is further linked with a histidine tag which is favorable for identifying and purifying the target protein, and the linking site is amino-terminal or carboxyl-terminal.
Preferably, in the recombinant humanized lactoferrin, the amino acid sequence of the recombinant humanized lactoferrin is shown as SEQ ID NO. 4.
The nucleotide sequence of the nucleic acid for encoding the recombinant humanized lactoferrin is shown as SEQ ID NO. 5.
A method of expressing a recombinant humanized lactoferrin as above, comprising the steps of:
(1) Optimizing a nucleotide sequence for encoding the recombinant humanized lactoferrin according to codon preference of host engineering bacteria and synthesizing genes;
(2) Respectively connecting the gene sequences obtained in the step (1) into an expression vector pET20b to construct a recombinant expression plasmid pET20b-rhLF;
(3) The recombinant plasmid polyclonal copy is obtained in the step (2) and is guided into escherichia coli BL21 or BL21 STAR PLYSS to construct recombinant engineering bacteria;
(4) Performing induction expression screening and expression optimization on the recombinant engineering bacteria constructed in the step (3); the recombinant humanized lactoferrin is isolated and purified by column chromatography and molecular sieve combination or dialysis.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention simulates and applies LfcinH N end antibacterial alpha-helical region derived peptide and carries out hydrophilic amino acid side chain modification, and combines with human silk fibroin 2 (FLG 2) C end antibacterial activity fragment to carry out innovative fusion expression. The B-14 and C-term sequences have 100% homology with the corresponding human silk fibroin sequences.
(2) The LfcinH derived peptide contained in the recombinant humanized lactoferrin of the present invention retains the key amino acid residues of Cys20, trp23, arg28, lys29 and Arg31 in the alpha-helical region, and the contained FLG 2C-terminal sequence has 100% homology with the corresponding human filaggrin sequence.
(3) The invention utilizes bioengineering technology to obtain high-efficiency expression of series recombinant humanized lactoferrin, and the purification process provided by the invention can obtain target protein with purity higher than 95%.
(4) The recombinant humanized lactoferrin has the activity of promoting cell adhesion and migration and reducing inflammation-related cell generation, shows that the recombinant humanized lactoferrin has biological functions in the aspects of promoting skin wound repair and inhibiting inflammatory reaction, and is expected to be developed into an active raw material to be applied to the fields of biological medicines, cosmetics, large health products (oral administration) and the like.
Drawings
FIG. 1 is a map of pET20b-rhLF expression plasmid constructed according to the invention.
FIG. 2 is a SDS-PAGE identification of recombinant humanized lactoferrin rhLF expression.
FIG. 3 is an optimized expression profile of recombinant humanized lactoferrin rhLF; wherein A is an SDS-PAGE detection graph of using concentrations of different inducers; b is SDS-PAGE detection graph of different induction temperatures.
FIG. 4 is a SDS-PAGE detection of pilot expression levels of recombinant humanized lactoferrin rhLF.
FIG. 5 is a SDS-PAGE detection of purified product of recombinant humanized lactoferrin rhLF; lanes 1-9 are whole bacteria, marker, broken bacterial supernatant, broken bacterial pellet, flow-through, renaturation, 100 mmol/L elution, 200 mmol/L elution and 400 mmol/L imidazole elution, respectively.
FIG. 6 is a Western Blot identification of recombinant humanized lactoferrin rhLF; lane 1: marker, lane 2: recombinant humanized lactoferrin rhLF.
FIG. 7 is a graph of the analysis of the cell adhesion promoting activity of recombinant humanized lactoferrin rhLF.
FIG. 8 is a graph of the analysis of the cell migration promoting activity of recombinant humanized lactoferrin rhLF.
FIG. 9 is an assay for the inhibitory activity of recombinant humanized lactoferrin rhLF on zebra fish macrophage activation; * P < 0.05, P < 0.001 vs. Control ns indicates no significant difference.
FIG. 10 is a graph of the analysis of the inhibitory activity of recombinant humanized lactoferrin rhLF on zebra fish neutrophil migration; * P < 0.01, P < 0.001 vs control group.
Detailed Description
In order that the invention may be more readily understood, the invention will be further described with reference to the following examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. Any modifications and substitutions of the details and form of the technical solution of the present invention without departing from the spirit and scope of the present invention will be apparent to those skilled in the art.
Example 1: construction and expression identification of recombinant humanized lactoferrin expression vector
(1) Construction of recombinant humanized lactoferrin rhLF expression plasmid: the amino acid sequence of the recombinant humanized lactoferrin rhLF is shown as SEQ ID NO.4, and the nucleotide sequence of the recombinant humanized lactoferrin rhLF is shown as SEQ ID NO. 5. Synthesizing a nucleotide sequence shown in SEQ ID NO.5 by a gene, introducing NdeI at an amino terminal and introducing XhoI enzyme cutting site sequences at a carboxyl terminal; double-enzyme cutting the gene synthesis product and the pET20b vector by NdeI and XhoI, and then recombining by using ligase; transformation of pET20b-rhLF ligation into E.coli DH 5. Alpha. Competent cells (available from Beijing qingke biosciences 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) Preparing and expressing identification of recombinant expression engineering strains: the sequenced pET20b-rhLF recombinant plasmid was transformed into BL21 (DE 3) and BL21 STAR PLYSS (DE 3) competent cells (available from Beijing Optimago technologies Co., ltd.), positive clones were screened on LB resistant plates containing ampicillin (100. Mu.g/mL) and colony PCR verified. Then test tube expression identification is carried out, and high expression strains are screened, and the specific operation is as follows: 4 positive colonies are picked up and inoculated into a culture medium test tube containing 5mL LB (100 mu g/mL ampicillin), 220 rpm is cultivated at 37 ℃ until OD 600 reaches 0.4-0.8, 100 mmol/L isopropyl thiocysteine (IPTG) is added to a final concentration of 1.0 mmol/L, 4h is induced to be expressed, and thalli are collected. The strain is resuspended in lysis buffer for complete lysis, and then added with loading buffer and heated in boiling water bath for 10min, and the expression level is detected by SDS-PAGE.
SDS-PAGE detection results are shown in figure 2, recombinant humanized lactoferrin rhLF is successfully expressed in two engineering bacteria BL21 (DE 3) and BL21 STAR PLYSS (DE 3), and the apparent molecular weight of the recombinant humanized lactoferrin rhLF is consistent with the theoretical molecular weight.
Example 2: expression condition optimization of recombinant humanized lactoferrin
The results of example 1 show that the expression level of recombinant humanized lactoferrin rhLF in BL21 (DE 3) is higher than BL21 STAR PLYSS (DE 3), so BL21 (DE 3) is selected as the preferred expression engineering bacterium. The present example aims at optimizing the expression parameters of the selected recombinant expression engineering bacteria, i.e., the inducer concentration and the inducer expression temperature, to enhance the yield of recombinant proteins. Glycerol seed solution was inoculated in 5mL LB medium containing ampicillin (100 μg/mL) at 37 ℃ 220 rpm overnight, transferred to new 5mL medium at five thousandths after culturing, and continued to OD 600 =0.4. 100 mmol/L IPTG was added to a final concentration of 0.5 mmol/L and 1.0 mmol/L respectively and induced at 37℃for 4h, and the preferred IPTG use concentration was determined by SDS-PAGE analysis. The induction expression temperature is optimized and selected according to induction of 6 h at 30 ℃ and induction of 4h at 37 ℃.
As a result, as shown in FIG. 3, recombinant humanized lactoferrin rhLF had no significant difference in expression level at IPTG use concentrations of 0.5 mmol/L and 1.0 mmol/L, and the preferred induction expression temperature was 37 ℃.
Example 3: pilot fermentation of recombinant humanized lactoferrin 20L
Inoculating glycerol seed bacteria into a 30 mL LB culture medium according to 1 per mill, culturing at 37 ℃ and 220: 220 rpm/min for 14-18 h, inoculating the culture into a 250 mL modified culture medium according to 1 percent, continuously culturing for 6: 6 h, and transferring to a 50: 50L bioreactor. After inoculation, glucose is gradually added into the fermentation tank by a peristaltic pump as a carbon source and microelements such as magnesium sulfate. The stirring speed and the aeration rate are respectively 200 rpm and 5L/min, and the dissolved oxygen content is kept above 30%. After about 3.5 h, feeding until the bacterial weight is more than or equal to 40g/L, increasing the dissolved oxygen to 80%, adding IPTG for induction, monitoring and sampling for SDS-PAGE detection every hour, stopping fermentation after induction for 4 hours, and centrifugally collecting bacterial cells at 4 ℃ under the conditions of 30,000 g and 10min.
As shown in FIG. 4, the expression level of recombinant humanized lactoferrin rhLF is proportional to the induced fermentation time, the gray scale analysis of the target protein band after fermentation of 4h is up to 40%, and the cell density is up to 57 g/L.
Example 4: purification of recombinant humanized lactoferrin
In the embodiment, the recombinant humanized lactoferrin is separated and purified by adopting a nickel column affinity chromatography and molecular sieve combination technology. Firstly, a stable coordination bond is formed between a target protein and Ni ⁺ ions through a histidine tag of the target protein in a protein denaturation and on-column renaturation mode, so that the target protein is adsorbed on a column, wherein a denaturation buffer solution is a 0.2M PBS buffer solution containing 8M urea, and a renaturation buffer solution is a 0.2M PBS buffer solution containing 4M urea; the target protein was then eluted with a gradient concentration of imidazole in 0.2M PBS buffer followed by desalting purification using G25 column. The immune response of the target protein was identified using Western Blot (Anti His, purchased from Biotechnology (Shanghai) Inc., mouse Anti His monoclonal antibody).
The purification results are shown in FIG. 5, and the recombinant humanized lactoferrin rhLF was completely eluted with 400mM imidazole, with a gray scale analysis of over 95%. The results show that recombinant humanized lactoferrin rhLF with high purity can be obtained by the purification method of the example. Western Blot identification is shown in FIG. 6, and the immunoblots of recombinant humanized lactoferrin rhLF are single bands and consistent with the molecular weight in SDS-PAGE.
Example 5: cell adhesion promoting Activity assay of recombinant humanized lactoferrin
DMEM medium (GIBCO, cat# 12800017) was added to 24 well plates at 400. Mu.L/well, respectively, with 0.025. Mu. Mol/L and 0.25. Mu. Mol/L recombinant humanized lactoferrin rhLF as the basal medium; embedding overnight at 4deg.C; inoculating 4x10 4 human immortalized epidermal cells (HaCaT) with good growth state every hole, and culturing in a 5% CO 2 incubator at 37deg.C for normal culture 3 h; cells were fixed with 4% paraformaldehyde 20 min, stained with 1% crystal violet 20 min, washed 2-3 times with PBS, and examined by microscopy.
The results are shown in fig. 7, in which the number of cell adhesion increases significantly in rhLF-administered groups relative to blank groups, and the dose dependence is shown under the administration conditions, indicating that the recombinant humanized lactoferrin of the present invention has activity in promoting HaCaT cell adhesion.
Example 6: analysis of cell migration promoting Activity of recombinant humanized lactoferrin
HaCaT with good growth state was added to 6-well plate at 2×10 4/well, and cultured in a 5% CO 2 incubator at 37℃until cells were confluent. The 200. Mu.L tip was streaked and the floating cells were washed with PBS. 2 mL DMEM basal medium was added to each well, and 0.025. Mu. Mol/L and 0.25. Mu. Mol/L recombinant humanized lactoferrin rhLF were added to each of the dosing groups, respectively, with the blank group as basal medium. Culturing in a 5% CO 2 incubator at 37deg.C, sampling and photographing at 0h, 24h and 48 h, respectively.
The results are shown in fig. 8, in which the scratch healing area of rhLF-administered group is significantly increased relative to the blank group and the administration conditions are dose-dependent, indicating that the recombinant humanized lactoferrin of the present invention has HaCaT cell migration promoting activity.
Example 7: macrophage activation test of zebra fish
An increased number of activated macrophages is an important marker of inflammation. Thus, measuring the relative intensity of macrophage activation allows for assessment of the efficacy of a test sample in inhibiting macrophage activation. This example uses macrophage fluorescence-labeled transgenic zebra fish Tg (mpeg 1: EGFP) in an AB-series background. Male and female fish were treated with 2:1, mixing and culturing, collecting zebra fish embryo, culturing at 28+ -1deg.C with density of not more than 1 fish embryo in 200 μl fish embryo culture solution to fertilize 2 d; zebra fish embryos are randomly selected and transferred into 6 well plates at 10 tails/well, wherein the blank group is 6 mL embryo culture broth, the model group is added with 2.5 μg/L phorbol ester (PMA), the positive group is added with 2.5 μg/L PMA and 3.96 mg/L dexamethasone, and the dosing groups are added with 0.25 μmol/L, 0.5 μmol/L and 1.0 μmol/L recombinant humanized lactoferrin rhLF and 2.5 μg/L PMA, respectively; the 6-well plate was placed in an incubator at 28.+ -. 1 ℃ for 24.+ -. 1h. Each zebra fish embryo macrophage was labeled using Image J software and macrophages were counted for each group of fish.
The results are shown in FIG. 9, and compared with the model group, the recombinant humanized lactoferrin rhLF can obviously reduce the quantity of macrophages under the administration condition of 0.05 mu mol/L and 0.1 mu mol/L, which shows that the recombinant humanized lactoferrin rhLF has the activity of inhibiting the activation of the macrophages.
Example 8: zebra fish neutrophil migration experiment
Neutrophils are produced after the skin is damaged by the outside or invaded by bacteria and actively remove infectious agents, thus inhibiting inflammatory response. In this example, the AB zebra fish was used as a model, and the male fish and the female fish were used as 2:1, mixing and culturing, collecting zebra fish embryo, culturing at 28+ -1deg.C with density of not more than 1 fish embryo in 200 μl fish embryo culture solution to fertilize 3 d; zebra fish embryos are randomly selected and transferred to a 6-well plate according to 24 tails/well, wherein a blank group is 5mL fish embryo culture solution, a model group is added with 10 mu mol/L anhydrous copper sulfate to stimulate inflammation, a positive group is added with 10 mu mol/L anhydrous copper sulfate and 10 mu mol/L dexamethasone, and a dosing group is respectively added with 0.25 mu mol/L, 0.5 mu mol/L and 1.0 mu mol/L recombinant humanized lactoferrin rhLF and 10 mu mol/L anhydrous copper sulfate; placing a 6-hole plate in a constant temperature cabinet at 28+/-1 ℃ for culturing 40-45 min, fixing for 1h by paraformaldehyde, washing by a PBST solution for 3 times, treating by 50% ethanol for 3 min times, dyeing by a Sudan black dyeing solution for 1h, washing by 70% ethanol for 3 times, washing by the PBST solution for 3 times, treating by 70% ethanol for 5min, and placing in a position of 50% glycerol after PBST treatment for 1 min. The number of neutrophils in the three-quarter tail lateral line region from the anus was counted per fish embryo with the fish embryo placed in the lateral position under a split microscope.
The results are shown in figure 10, in which the number of neutrophils was significantly reduced at all three groups of dosing concentrations of recombinant humanized lactoferrin rhLF compared to the model group, from low to high at 20.76%, 90.00% and 91.45% for the neutrophils, respectively. The recombinant humanized lactoferrin rhLF has obvious activity of inhibiting inflammatory reaction.
Claims (9)
1. A recombinant humanized lactoferrin, comprising:
(1) A derivative peptide mimicking the antibacterial alpha-helical region at the terminus of human lactoferrin LfcinH N;
Or (2) a derivative peptide which mimics the antibacterial alpha-helical region at the terminus of human lactoferrin LfcinH N and fused to the FLG 2C-terminal active fragment, the derivative peptide and FLG 2C-terminal active fragment being linked by a linker peptide.
2. The recombinant humanized lactoferrin of claim 1, wherein the antibacterial α -helical region at the terminus of human lactoferrin LfcinH N retains the active amino acid residues LFCINH CYS, trp23, arg28, lys29 and Arg31 and the amino acid sequence of the peptide derived from the antibacterial α -helical region mimicking the terminus of human lactoferrin LfcinH N is shown in SEQ ID No. 1.
3. The recombinant humanized lactoferrin of claim 1, wherein the derivative peptide that mimics the antibacterial alpha-helical region of human lactoferrin LfcinH N is subjected to hydrophilic amino acid side chain modification.
4. The recombinant humanized lactoferrin of claim 1, wherein the FLG 2C-terminal active fragment comprises the B-repeat of FLG 2B-14, 2244-2321 or C-term 2322-2391, the amino acid sequence of which is set forth in SEQ ID No.2 or SEQ ID No.3, respectively.
5. The recombinant humanized lactoferrin of claim 1, wherein the amino acid sequence of the recombinant humanized lactoferrin is further linked to a histidine tag that facilitates the identification and purification of the protein of interest, and the site of linkage is amino-or carboxy-terminal.
6. The recombinant humanized lactoferrin of claim 1, wherein the amino acid sequence of said recombinant humanized lactoferrin is set forth in SEQ ID No. 4.
7. A nucleic acid encoding the recombinant humanized lactoferrin of claim 6, the nucleotide sequence of which is set forth in SEQ ID No. 5.
8. A method of expressing recombinant humanized lactoferrin according to claim 1, comprising the steps of:
(1) Optimizing a nucleotide sequence for encoding the recombinant humanized lactoferrin according to codon preference of host engineering bacteria and synthesizing genes;
(2) Respectively connecting the gene sequences obtained in the step (1) into an expression vector pET20b to construct a recombinant expression plasmid pET20b-rhLF;
(3) The recombinant plasmid polyclonal copy is obtained in the step (2) and is guided into escherichia coli BL21 or BL21 STAR PLYSS to construct recombinant engineering bacteria;
(4) Performing induction expression screening and expression optimization on the recombinant engineering bacteria constructed in the step (3); the recombinant humanized lactoferrin is isolated and purified by column chromatography and molecular sieve combination or dialysis.
9. Use of the recombinant humanized lactoferrin in the manufacture of a medicament or cosmetic or health product for promoting skin wound repair or inhibiting skin inflammation according to claim 1.
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| CN116574172A (en) * | 2023-06-07 | 2023-08-11 | 广州暨南大学医药生物技术研究开发中心有限公司 | Recombinant humanized type I collagen and preparation method thereof |
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