CN114854744A - Recombinant anti-miR-129-5p, preparation method thereof and application thereof in resisting skin aging - Google Patents
Recombinant anti-miR-129-5p, preparation method thereof and application thereof in resisting skin aging Download PDFInfo
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Abstract
The invention belongs to the technical field of molecular biology and medicine, and discloses a recombinant anti-miR-129-5p, a preparation method thereof and application thereof in resisting skin aging, wherein the sequence of the recombinant gene is SEQ ID NO: 1, or is a peptide corresponding to SEQ ID NO: 1 with a sequence similarity of more than 90%. The invention also discloses a production method and application of the recombinant anti-miR-129-5 p. Compared with chemically synthesized anti-miR-129-5p, the anti-miR-129-5p with biological activity expressed by the tRNA stent has the advantages of fast production, high yield, low price, good functionality and high safety. The recombinant anti-miR-129-5p prepared by the invention can inhibit the expression of aging marker genes, promote the expression of collagen and play a role in resisting skin aging by improving the activity of skin fibroblasts.
Description
Technical Field
The invention belongs to the technical field of molecular biology and medicine, and particularly relates to recombinant anti-miR-129-5p, a preparation method thereof and application thereof in resisting skin aging.
Background
At present, the skin of a human body is positioned on the outermost layer of the human body and is directly influenced by various factors such as the external environment and the like. In aged skin, epidermal renewal is slowed, barrier function is diminished, keratinocyte viability is reduced, and repair capacity after epidermal injury is diminished, wherein the diminished barrier function results in dry skin, desquamation, wrinkles, etc. Dermal senescence is characterized in that the number of fibroblasts in the dermal layer is gradually reduced, the thickness of the dermal layer is reduced, the capacity of synthesizing collagen and elastin is reduced, the decomposition is increased, the I, III type collagen ratio is inverted, the collagen is gradually thickened, abnormal cross-linking occurs, the activities of a plurality of metabolism-related enzymes are reduced, the content of hyaluronic acid and dermatan sulfate in extracellular matrixes is obviously reduced, and the total content of glycosaminoglycan is reduced. Fibroblasts, which are the most important cellular components in the dermis, are gradually aged in their proliferative capacity and function, which is one of the important causes of aging changes in the skin.
MicroRNA (miRNA) is single-stranded non-coding small-molecule RNA, has the length of about 20-24 nucleotides, and is combined on the 3' -UTR of mRNA through base pairing or directly degrades the mRNA so as to regulate the expression of genes. The gene is closely related to skin aging, and with the increase of age, the repair ability of cells to DNA is reduced, causing apoptosis, and the gene related to cell activity is inhibited. Therefore, miRNA is involved in many important processes related to aging, and is widely considered as a key regulator of aging. Researches show that the up-regulation of miR-129-5p expression in dermal tissues can reduce the activity of fibroblasts and inhibit the synthesis of collagen, so that the expression can participate in the aging process of the dermal tissues.
Currently, the RNA reagents used for research mainly adopt chemical synthesis, but the chemically synthesized RNA is not only expensive and low in yield, but also may have more artificial gene modifications to improve stability, and thus RNA folding, biological activity and safety are affected.
Through the above analysis, the problems and defects of the prior art are as follows: the RNA reagents currently used for research are expensive and low in yield, and may have more artificial gene modifications for improving stability, so that RNA folding, biological activity and safety are affected.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a preparation method and application of recombinant anti-miR-129, and particularly designs a recombinant anti-miR-129-5p and a preparation method and application thereof in resisting skin aging.
The invention is realized by a recombinant gene, and the sequence of the recombinant gene is SEQ ID NO: 1, or a sequence identical to SEQ ID NO: 1 with a sequence similarity of more than 90%.
The invention also aims to provide a preparation method of the recombinant anti-miR-129 applying the recombinant gene, wherein the preparation method of the recombinant gene comprises the following steps:
designing an anti-miR-129-5p primer according to a genebank database and constructing a vector; designing anti-miR-129-5p, embedding the designed small interfering RNA anti-miR-129-5p into a tRNA stent, and performing recombinant expression in escherichia coli.
Further, the sequence of the small interfering RNA anti-miR-129-5p is SEQ ID NO: 2.
further, the chimeric sequence is a chimeric sequence identical to SEQ ID NO: 2 sequences with more than 90% sequence similarity.
Further, the sequence of the tRNA scaffold is a sequence with similarity of more than 90% with a human serine tRNA sequence, and the human serine tRNA sequence is SEQ ID NO: 3.
further, the precursor sequence of the small interfering RNA anti-miR-129-5p is an hsa-miR-34a precursor sequence of which the mature sequence part is replaced by an anti-miR-129-5p sequence, and the precursor sequence of the small interfering RNA anti-miR-129-5p is SEQ ID NO: 4.
further, the preparation method of the recombinant anti-miR-129 specifically comprises the following steps:
designing an hsa-miR-34a precursor primer for synthesizing a chimeric anti-miR-129-5p sequence;
secondly, inserting a precursor sequence of the small interfering RNA anti-miR-129-5p into the pBSMrnaSeph plasmid by utilizing a restriction enzyme cutting site of the pBSMrnaSeph plasmid at a tRNA anticodon ring to construct an expression vector;
step three, transforming the expression vector of the chimeric target sequence into competent escherichia coli;
step four, after escherichia coli is cultured and amplified, total RNA in the bacteria is extracted, and target recombinant anti-miR-129-5p is separated and purified through FPLC.
The invention also aims to provide application of the recombinant anti-miR-129 in preparation of anti-aging cosmetics.
Further, the dosage forms of the anti-aging cosmetic comprise creams, emulsions, lotions and mistura.
In combination with the above technical solutions and the technical problems to be solved, please analyze the advantages and positive effects of the technical solutions to be protected in the present invention from the following aspects:
first, aiming at the technical problems existing in the prior art and the difficulty in solving the problems, the technical problems to be solved by the technical scheme of the present invention are closely combined with results, data and the like in the research and development process, and some creative technical effects are brought after the problems are solved. The specific description is as follows:
the human serine tRNA chimeric has-miR-34a precursor and the human serine tRNA is human source tRNA, so that the development prospect of the recombinant small RNA produced by using the human serine tRNA chimeric has better prospect, and because human cells contain serine tRNA but do not contain bacterially derived methionyl tRNA, the toxicity and immunogenicity of the human serine tRNA used as a scaffold are probably lower. The invention shortens the length of the expressed recombinant tRNA, thereby reducing the cytotoxicity and improving the expression quantity. The sequence in the has-miR-34a precursor is modified, complementary pairing is improved, and the stability of recombinant anti-miR-129-5p is improved; the recombinant anti-miR-129-5p obtained through bioengineering has good biological activity, promotes the proliferation rate of skin fibroblasts, can obviously improve aged skin fibroblasts and promotes the expression of type I collagen.
Through Real-time PCR detection, the recombinant anti-miR-129-5p has correlation with the cell activity of dermal fibroblasts, and has correlation with the expression of aging marker genes and collagen genes in aging skin fibroblasts. Therefore, the recombinant anti-miR-129-5p can be used for developing cosmetics for improving exogenous aging of skin. Specifically, the method comprises the steps of carrying out in-vitro culture on HKFs of skin fibroblasts derived from normal people, and treating the skin fibroblasts by using the recombinant anti-miR-129-5p to be detected; after the culture is finished, the cells are harvested, Real-time PCR detection is carried out on the product recombinant anti-miR-129-5p, the collagen expression effect of skin fibroblasts is remarkably promoted, and the collagen expression product can be used for developing cosmetics for improving skin exogenous aging.
The invention uses tRNA as a bracket, and a chimeric anti-miR-129-5p sequence expresses recombinant anti-miR-129-5p in escherichia coli. The recombinant anti-miR-129-5p produced by the invention can be processed and matured in skin fibroblasts and successfully expressed, simultaneously improves the activity of the skin fibroblasts, can effectively reduce the expression of senescence genes in the senescence skin fibroblasts, can promote the expression of type I collagen, and further has an anti-aging effect in the skin.
Secondly, considering the technical scheme as a whole or from the perspective of products, the technical effect and advantages of the technical scheme to be protected by the invention are specifically described as follows:
the invention adopts optimized tRNA as a bracket, embeds anti-miR-129-5p sequence, and recombines and expresses anti-miR-129-5p in enterobacter. Experimental detection shows that the recombinant anti-miR-129-5p can promote the proliferation of human skin fibroblasts, the recombinant anti-miR-129-5p can reduce the expression of aging-related genes in aging skin fibroblasts and promote collagen synthesis, the mask containing the recombinant anti-miR-129-5p can repair a sensitive area of a face of a test person, fade color spots, improve fine lines and dry wrinkles, brighten the skin color and repair damaged skin, and the safety is good, so that the recombinant anti-miR-129-5p can be used for an anti-skin aging cosmetic. The recombinant anti-miR-129-5p prepared and produced by the biological engineering technology has the advantages of high yield, low cost, good activity and the like.
The recombinant anti-miR-129-5p biosynthesized by using the tRNA stent has the advantages of natural modification, high activity, good safety, large-scale production, low cost and the like. The biosynthetic recombinant small RNA can be applied to biological and medical research and small nucleic acid drug development. Compared with the prior art, the invention promotes the proliferation of skin fibroblasts and the expression of type I collagen, improves the expression of genes related to aging skin fibroblasts, realizes the synergistic interaction among the components, and can effectively delay skin aging because the prepared product has the effects of resisting wrinkles and improving skin barriers.
Compared with the prior art, the invention also has the following advantages:
(1) compared with chemically synthesized anti-miR-129-5p, the anti-miR-129-5p with biological activity expressed by using a novel tRNA (Optimal non-coding RNA screening, OnRS) bracket has the advantages of fast production, high yield, low price, good functionality and high safety.
(2) The human serine tRNA chimeric has-miR-34a precursor and the human serine tRNA is human source tRNA, and the development prospect of the recombinant small RNA produced by using the human serine tRNA chimeric has better prospect, because human cells contain serine tRNA, but no methionine tRNA derived from bacteria, the human serine tRNA used as the scaffold has lower toxicity and immunogenicity. The invention shortens the length of the expressed recombinant tRNA, thereby reducing the cytotoxicity and improving the expression quantity. The invention modifies the sequence in the precursor of has-miR-34a, improves complementary pairing and improves the stability of the recombinant miR-140.
(3) The recombinant anti-miR-129-5p designed and prepared by the invention can inhibit the expression of aging marker genes, promote the expression of collagen and play a role in resisting skin aging by improving the activity of skin fibroblasts.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments of the present invention will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a schematic diagram of an expression result of recombinant anti-miR-129-5p in Escherichia coli detected by denaturing polyacrylamide gel electrophoresis according to an embodiment of the present invention.
FIG. 2 is an FPLC chromatogram for purifying recombinant anti-miR-129-5p by using Bio-Rad NGCTM Chromatography System provided in the present invention.
FIG. 3 is a schematic diagram of the purity of the fractions collected by the purification of recombinant anti-miR-129-5p by means of denaturing polyacrylamide gel electrophoresis and identification of Bio-Rad NGCTM Chromatography System according to the embodiment of the present invention.
FIG. 4 is a schematic diagram of the effect of the CCK-8 method on the identification of the toxicity of the transfected recombinant anti-miR-129-5p on skin fibroblasts.
FIG. 5 is a schematic diagram of the effect of the CCK-8 method on the proliferation of skin fibroblasts of the transfected recombinant anti-miR-129-5 p.
FIG. 6 is a schematic diagram of the effect of transfection of recombinant anti-miR-129-5p on the expression of senescence marker genes (p21, p53, IL6) in senescent skin fibroblasts by Real-time PCR detection provided by the embodiment of the invention.
FIG. 7 is a schematic diagram of the effect of transfection of recombinant anti-miR-129-5p on type I collagen gene expression in aging skin fibroblasts detected by Real-time PCR provided by the embodiment of the invention.
Fig. 8A and 8B are schematic diagrams for detecting the influence of the facial mask on the rate of fine lines and the rate of dry lines of a tester by using a small nucleic acid anti-aging facial mask containing recombinant anti-miR-129-5p according to an embodiment of the invention.
Fig. 9A and 9B are schematic diagrams for detecting the influence of the facial mask on the improvement of facial red region inflammation and related fade-out rate of a tester by using a small nucleic acid anti-aging facial mask containing recombinant anti-miR-129-5p according to an embodiment of the invention.
Fig. 10A and fig. 10B are schematic diagrams for detecting the influence of the facial mask on the brown spot index and the related fade-out rate of a tester by using a small nucleic acid anti-aging facial mask containing recombinant anti-miR-129-5p, provided by an embodiment of the invention.
FIG. 11 is a flow chart of a preparation method of recombinant anti-miR-129 provided by the embodiment of the invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Aiming at the problems in the prior art, the invention provides a recombinant anti-miR-129-5p, a preparation method thereof and application thereof in resisting skin aging, and the invention is described in detail below with reference to the accompanying drawings.
First, an embodiment is explained. This section is an explanatory embodiment expanding on the claims so as to fully understand how the present invention is embodied by those skilled in the art.
As shown in fig. 11, the preparation method of the recombinant anti-miR-129 provided by the embodiment of the present invention includes:
s101, designing and synthesizing hsa-miR-34a precursor primer of a chimeric anti-miR-129-5p sequence, and inserting the primer into pBSMrnaSeph plasmid to construct an expression vector;
s102, transforming the expression vector of the chimeric target sequence into competent escherichia coli;
s103, after escherichia coli is cultured and amplified, total RNA in the bacteria is extracted, and target recombinant anti-miR-129-5p is separated and purified through FPLC.
Example 1: constructing a recombinant anti-miR-129-5p plasmid by using a pBSKrnaSeph/has-miR-34a expression vector, and expressing recombinant anti-miR-129-5 p;
a primer is designed according to an effective sequence (SEQ ID NO: 1) of the recombinant anti-miR-129-5p and a sequence on a pBSKrnaSeph/has-miR-34a expression vector, is named as miR-34 a/recombinant anti-miR-129-5p, and homologous sequences on two sides of an insertion site of a 1-15nt vector are added to two ends of the primer.
(2) Synthesis of the insert
The two primers in the table 2 are used as templates, and a precursor sequence (SEQ ID NO: 3) of the recombinant anti-miR-129-5p is inserted into a pBSMrnaSeph plasmid by utilizing a restriction enzyme cutting site of the pBSMrnaSeph plasmid in a tRNA (SEQ ID NO: 2) anticodon loop to construct an expression vector; the reaction system is shown in Table 1, and the reaction process is shown in Table 2.
TABLE 1 polymerase in vitro amplification chain reaction System (50. mu.L)
TABLE 2 polymerase in vitro amplification Strand reaction Process
(3) Double enzyme digestion of pBSKrnaSeph/has-mir-34a vector
The vector was digested with Eag I-HFTM, Sac II restriction enzymes at 37 ℃ in the reaction system shown in Table 3.
TABLE 350. mu.L double enzyme digestion System
In the embodiment of the invention, the precursor sequence of the small interfering RNA anti-miR-129-5p is an hsa-miR-34a precursor sequence of which the mature sequence part is replaced by an anti-miR-129-5p sequence, and the precursor sequence of the small interfering RNA anti-miR-129-5p is SEQ ID NO: 4. comprises the following steps: ggccagctgtgagtgtttcttgcaagcccagaccgcaaaaagutgtgagcaatagtaaggaagacuuuuugcgucuugggcuugauagaagtgctgcacgttgttggccc are provided.
(4) Recovery and purification of enzyme digestion plasmid and PCR fragment
The PCR product and the digested plasmid were identified by agarose Gel electrophoresis, and recovered and purified using an OMEGA Gel Extraction Kit (OMEGA). Observing the DNA separation result after agarose gel electrophoresis by using 365nm ultraviolet light in a gel imaging system, carefully cutting off the gel with the required DNA zone by using a blade, cutting off less gel as much as possible, and placing the gel into a 1.5mL EP centrifuge tube; weighing the mass of the gel; adding Binding Buffer solution into a centrifugal tube filled with agarose gel according to the volume ratio of 1:1, placing the mixture into a water bath at 60-65 ℃ for 7min, and shaking and mixing once every two to three minutes until the gel is completely melted; putting the solution obtained by melting in the step 3 in a room temperature for cooling, then transferring the solution into a DNA Mini Column centrifuge, and putting the Column centrifuge into a Collection Tube of 2ml Collection Tube; centrifuging at 10000rpm for 1 min. The volume of the solution centrifuged each time is at most 700 mu L, the solution can be centrifuged for multiple times until the solution is completely centrifuged, filtrate in the collecting pipe is discarded, and the collecting pipe is recycled; add 700. mu.L of the absolute ethanol added SPW Wash Buffer to the spin column. Centrifuging at room temperature for 1min at 10000rpm in a centrifuge; discarding the filtrate, centrifuging the column at 13000rpm for 2min at room temperature to completely remove ethanol from the column; the column was placed in a fresh clean centrifuge tube. Suspending and dropping 30-100 mu L of eluation Buffer eluent into the center of the centrifugal column, and standing for 2min to completely dissolve the DNA in the eluent. Centrifuging at 13000rpm for 1min at room temperature, and recovering the eluate at the bottom of the tube. A small amount of the eluate was subjected to DNA gel electrophoresis to determine whether the product was the desired product, and stored at-20 ℃.
(5) Ligation of the insert to the vector
For recovering fragments from glue
Ligation was performed by Ligation using the Ligation-Free Cloning System, and the reaction System is shown in Table 4.
TABLE 4 seamless ligation reaction System (20. mu.L)
After mixing, putting on ice and incubating for 30 minutes; transforming escherichia coli HST08 competent bacteria; ampicillin resistance screening was performed on the cloned colonies.
(6) Bacterium liquid PCR and DNA sequencing identification tRNA (ribonucleic acid) support recombinant anti-miR-129-5p-5p expression vector
Single colonies were picked and cultured in 1mL of LB medium containing ampicillin for about 3 h. 20 mu L of the bacterial liquid is taken, 180 mu L of water is added, and the mixture is placed at 95 ℃ for 10min to serve as a template. And carrying out PCR identification on the bacterial liquid by using a sequencing primer M13Fow-GTAAAACGACGGCCAGT and Rev-CAGGAAACAGCTATGAC. The reaction system and reaction conditions are shown in tables 3 and 4. Another 100. mu.L of the bacterial solution was used for DNA sequencing and identification with the same primers. The nucleotide sequence of the sequencing primer M13 is SEQ ID NO: 5.
(7) expression of tRNA (tRNA) scaffold recombinant anti-miR-129-5p
After 200ng of tRNA stent recombinant anti-miR-129-5p expression plasmid is transformed into HST08 competent bacteria, 5mL of 2XYT culture medium is added, and shake culture is carried out at 37 ℃ and 200rpm overnight. The bacterial liquid is centrifuged for 2min at 10000g, and then the precipitate is collected. Adding 180 mu L of 10mM magnesium acetate-Tris-HCl solution into the precipitate for resuspension, adding 200 mu L of saturated phenol, and shaking at room temperature for 20-60 min. After centrifugation at 10000g for 10min, the aqueous phase was collected and 0.1 volume of 5M NaCl was added to precipitate the macromolecular impurities. Adding 2 times volume of anhydrous ethanol into the supernatant, centrifuging at 10000g for 10min, and removing the supernatant. Absorbing residual ethanol with absorbent paper, adding DEPC water to dissolve RNA after the RNA is dried, measuring the concentration, and storing in a refrigerator at-80 ℃.
(8) Modified polyacrylamide gel electrophoresis identification
Mu.g of RNA sample was mixed with 2 XRNA loading buffer and added to the denatured gel sample wells. And (3) after electrophoresis at 120-150V for 40-60 min, putting the mixture into a solution containing 0.5 mu g/mL ethidium bromide, slightly shaking for 20-30 min, observing the mixture under a gel imaging system, and taking a picture for storage.
FIG. 1 shows that the modified polyacrylamide gel electrophoresis is used for detecting the expression of tRNA stent recombinant anti-miR-129-5p in Escherichia coli. In the figure, M represents RNA marker; 1 represents HST08 E.coli transformed by tRNA scaffold recombinant anti-miR-129-5p expression plasmid. Compared with the bacterial RNA of the untransformed recombinant anti-miR-129-5p expression plasmid, the transformed bacteria have an additional band between 150 nt and 300 nt. The result shows that the recombinant anti-miR-129-5p expression plasmid can highly express the recombinant anti-miR-129-5 p.
(9) FPLC purified recombinant anti-miR-129-5p
Recombinant anti-miR-129-5p was purified on an ion exchange Column (ENrichTMQ 10X 100Column) using a Bio-Rad NGCTM Chromatography System. Mobile phase A: 10mM NaH 2 PO 4 Solution, pH 7.0. Mobile phase B: 10mM NaH2PO4 solution, 1M NaCl solution, pH 7.0. The flow rate was 2.0 mL/min. The column was washed alternately with DEPC water, mobile phase A, and mobile phase B for about 1 h. Each timeWash 5 column volumes. Total RNA was isolated by running the following program: 0-8.9 min (0% B), 8.9-13.7 min (55% B), 13.7-53.7 min (55-75% B), 53.7-73.7 min (75-85% B), 73.7-83.7 min (100% B), 83.7-93.7 min (0% B). The RNA was detected by absorbance at 260nm, and the peak corresponding to the recombinant RNA was collected. The purity was verified by denaturing polyacrylamide gel electrophoresis.
FIG. 2 is a FPLC chromatogram of the invention for purifying recombinant anti-miR-129-5p by using Bio-Rad NGCTM Chromatography System, wherein the target RNA anti-miR-129-5p is well separated from other endogenous RNAs in the total bacterial RNA, and the expression of the target RNA is more than 15% in the total bacterial RNA as shown by peak area integration.
FIG. 3 shows the purity of the fraction collected by purifying recombinant anti-miR-129-5p by using denaturing polyacrylamide gel electrophoresis to identify Bio-Rad NGCTM Chromatography System in the invention. The result shows that after FPLC purification, the recombinant anti-miR-129-5p with the purity of more than 95% can be obtained.
(10) FPLC fraction collection and concentration for desalination
The purity of the collected fractions was verified by denaturing polyacrylamide gel electrophoresis. The mixed components were subjected to 2-fold volume of absolute ethanol to precipitate RNA, and the mixture was stored in a refrigerator at-80 ℃ for about 1 hour. The RNA was collected by centrifugation at 10000g for 10min at 4 ℃. The resulting RNA precipitate was dissolved in DEPC water, centrifuged at 7500g at 4 ℃ for 10min with Ultra-2mL Centrifugal Filters, the filtrate was removed, the procedure was repeated until all solutions were centrifuged, Filters were inverted and centrifuged at 2000g for 2min, the resulting solution was collected, the concentration was determined and stored at-80 ℃.
Example 2: CCK-8 method for detecting proliferation and toxicity effects of recombinant anti-miR-129-5p on skin fibroblasts
(1) Transfection of skin fibroblasts
One day prior to transfection, cells were digested and seeded into 96-well plates at 5000/well (toxic) or 2000/well (proliferating). Transfection was started after 24 hours when the cells were fully adherent. Cell transfection was performed according to the Lipofectamine 2000 protocol. The transfection concentration of the recombinant anti-miR-129-5p is 1 mu g/mL. Grouping: negative control group (transfection negative control RNA); ② recombinant anti-miR-129-5pGroup (transfection of recombinant anti-miR-129-5p small RNA). Cells in CO 2 After incubation for 6h at 37 ℃ in an incubator, the complex was removed and the medium was replaced.
(2) Detection of proliferation and toxicity effects of recombinant anti-miR-129-5p on skin fibroblasts by CCK-8 method
Negative control and recombinant anti-miR-129-5p transfection treatment skin fibroblast, and cell activity is detected by using a multifunctional enzyme-labeling instrument at different time points.
FIG. 4 shows the effect of the CCK-8 method in the invention on the toxicity of the transfected recombinant anti-miR-129-5p on skin fibroblasts. The influence of 1 mu g/mL recombinant anti-miR-129-5p or negative control small RNA on skin fibroblast cytotoxicity is detected in multiple batches, and the result shows that the recombinant anti-miR-129-5p has no obvious toxic or side effect.
FIG. 5 shows the effect of the transfection recombinant anti-miR-129-5p on the proliferation of skin fibroblasts identified by the CCK-8 method. Through multi-batch detection of the influence of 1 mu g/mL raw material recombinant anti-miR-129-5p or negative control small RNA on the proliferation of skin fibroblasts, the result shows that the recombinant anti-miR-129-5p obviously promotes the proliferation speed of the skin fibroblasts, and the proliferation speed is increased by 29.5% after 24 hours and is increased by 85.2% after 48 hours.
Example 3: detecting effect of recombinant anti-miR-129-5p on anti-aging effect of skin fibroblasts
(1) Inducing skin fibroblast aging
One day before induction, skin fibroblasts were digested and the cells were treated at 5X 10 4 One/well was seeded in 12-well plates. After 24 hours, the cells are completely attached to the wall, etoposide is used for inducing cell senescence, the concentration is 2 mu M, and the subsequent transfection experiment is started after 48 hours of induction.
(2) The recombinant anti-miR-129-5p has the function of rescuing aged skin fibroblasts.
Skin fibroblasts were subjected to cell transfection as described in example 1 48 hours after senescence was induced with etoposide. Grouping: normal control group (cells do not induce aging and do not undergo transfection treatment); ② an aging + negative control group (cells induce aging and transfect negative control RNA); ③ aging and recombination anti-miR-129-5p group (cell induces aging and transfects recombination anti-miR-129-5p small RNA). 48 hours after transfection, cells were lysed by TRIzol, and total RNA was extracted from each group of cells by chloroform-isopropanol method, and the extracted mRNA was reverse-transcribed into cDNA using TAKARA kit. The reverse transcription conditions were: 15min at 37 ℃; 85 ℃ for 15 s.
The cDNA of each group of cells is taken as a template, GAPDH is taken as an internal reference, and the expression of senescence-associated genes P21, P53, IL-6 and the like is detected by adopting real time PCR. The primer sequences used were as follows:
GAPDH Forward Primer:5’-CCTCTGACTTCAACAGCGAC-3’
GAPDH Reverse Primer:5’-TCCTCTTGTGCTCTTGCTGG-3’
p21 Forward Primer:5’-CCTGTCACTGTCTTGTACCCT-3’
p21 Reverse Primer:5’-GCGTTTGGAGTGGTAGAAATCT-3’
p53 Forward Primer:5’-CCGCAGTCAGATCCTAGCG-3’
p53 Reverse Primer:5’-AATCATCCATTGCTTGGGACG-3’
IL-6Forward Primer:5’-ATGCAATAACCACCCCTGAC-3’
IL-6Reverse Primer:5’-AAAGCTGCGCAGAATGAGAT-3’
the Real time PCR reaction conditions were: at 95 ℃ for 30 s; 95 ℃ for 10 s; 30s at 60 ℃; 44 cycles.
Wherein, the sequence of the primer GAPDH is SEQ ID NO: 6, the sequence of the primer p21 is SEQ ID NO: 7, the sequence of the primer p53 is SEQ ID NO: 8, the sequence of the primer IL-6 is SEQ ID NO: 9.
FIG. 6 shows that the recombinant anti-miR-129-5p is detected by Real-time PCR to express the aging-related gene in the skin fibroblast. As a result, after skin fibroblasts induce aging, the expression of aging marker genes p21 (the amplification is 61.6%), p53 (the amplification is 54.2%) and IL6 (the amplification is 33.1%) is obviously increased, while the expression of aging marker genes p21 (the reduction is 77.1%), p53 (the reduction is 64.1%) and IL6 (the reduction is 45.8%) is obviously reduced relative to a negative control after recombinant anti-miR-129-5p is transfected. The anti-miR-129-5p can save the aging of skin fibroblasts. (values are expressed as "mean ± standard deviation", significance between two groups was tested by students't,. P <0.05,. P <0.01,. P < 0.001).
(3) The effect of the recombinant anti-miR-129-5p on the expression of the type I collagen gene.
Skin fibroblasts were subjected to cell transfection as described in example 1 48 hours after senescence was induced with etoposide. Grouping: normal control group (cells do not induce aging and do not undergo transfection treatment); ② an aging + negative control group (cells induce aging and transfect negative control RNA); ③ aging and recombination anti-miR-129-5p group (cell induces aging and transfects recombination anti-miR-129-5p small RNA). 48 hours after transfection, cells were lysed by TRIzol, and total RNA was extracted from each group of cells by chloroform-isopropanol method, and the extracted mRNA was reverse-transcribed into cDNA using TAKARA kit. The reverse transcription conditions were: 15min at 37 ℃; 85 ℃ for 15 s.
cDNA of each group of cells is taken as a template, GAPDH is taken as an internal reference, and real time PCR is adopted to detect the expression of the type I collagen gene. The primer sequences used were as follows:
GAPDH Forward Primer:5’-CCTCTGACTTCAACAGCGAC-3’
GAPDH Reverse Primer:5’-TCCTCTTGTGCTCTTGCTGG-3’
ColIα1Forward Primer:5’-CAGCCGCTTCACCTACAGC-3’
ColIα1Reverse Primer:5’-TTTTGTATTCAATCACTGTCTTGCC-3’
the real time PCR reaction conditions are as follows: at 95 ℃ for 30 s; 95 ℃ for 10 s; 30s at 60 ℃; 44 cycles.
Wherein, the sequence of the primer ColI alpha 1 is SEQ ID NO: 10.
FIG. 7 shows that Real-time PCR is used to detect the effect of transfection recombinant anti-miR-129-5p on the expression of type I collagen gene in skin fibroblasts. As a result, the expression of the ColI alpha 1 is obviously reduced (reduced by 42.7%) after skin fibroblasts induce aging, and the expression of the ColI alpha 1 gene is obviously increased (31.6%) relative to a negative control after the recombinant anti-miR-129-5p is transfected. The reduction of the type I collagen of the skin fibroblast cells can be saved by the recombinant anti-miR-129-5 p. (values are expressed as "mean ± standard deviation", significance between groups was tested using students't,. P < 0.001).
Example 4: patch test and results
Grouping experiments: the experimental group is carbomer gel added with recombinant anti-miR-129-5p, and the negative control is non-added carbomer gel.
Subject: 30 persons, 7 persons, 23 persons, 18-60 years old, and meeting the volunteer selection standard of the subject;
the test method comprises the following steps: selecting a qualified spot tester, adopting a closed spot test method, smearing 0.02mL of a test object in the spot tester, externally applying a special adhesive tape on the back of the test object, removing the test object after 24 hours, observing skin changes after 0.5, 24 and 48 hours of the spot test respectively, observing skin reactions according to the standard of a table 5 and recording the observation result.
TABLE 5 skin closed Patch test skin response grading Standard
And (4) judging the standard: according to cosmetic hygiene specifications 2015, 30 subjects experienced a grade 1 adverse skin reaction.
More than 5 cases, or more than 2 cases with grade 2 adverse skin reactions, or any 1 case with grade 3 or more than 3 adverse skin reactions, the tested substance is determined to have adverse reactions on human body, otherwise, the tested substance is determined to have no adverse reactions on human body.
As can be seen from Table 6, adverse skin reactions occurred in 0 of 30 human skin patch tests. Namely, the gel added with the recombinant anti-miR-129-5p does not produce skin reaction, which shows that the recombinant anti-miR-129-5p provided by the invention has safety and does not bring adverse reaction to human body.
TABLE 6 test results of human skin patches
Example 5: VISA test and results
Testing the use effect in the daily life environment:
test samples: the facial mask is prepared according to the following formula proportion.
The formula of the facial mask comprises: the technical scheme adopted by the invention is as follows: a facial mask containing small biosynthetic nucleic acids comprises the following components in percentage by mass: 2.5% of butanediol, 2% of glycerol, 1.5% of 1, 2-pentanediol, 1% of betaine, 0.25% of phenoxyethanol/ethylhexyl glycerol, 0.23% of hydroxyethyl cellulose, 0.11% of dipotassium glycyrrhizinate, 0.16% of sodium hyaluronate, 0.015% of hydrogenated phosphatidylcholine/cephalin/ribonucleic acid/propylene glycol/soybean oil amide DEA, 0.001% of tetrahydromethylpyrimidine carboxylic acid, 0.001% of carnosine, and the balance of water.
And (3) testing conditions are as follows: daily life environment (no harsh environment such as insolation, high temperature, sand blown by the wind), temperature: 20-28 ℃, humidity: 40-60% and real-time dynamic monitoring is carried out.
Test objects: 56 volunteers and females with the age of 18-45 have no pregnancy or lactation period, no serious system diseases, no immunodeficiency or autoimmune diseases, no active allergic diseases, no physique hypersensitiveness, no skin treatment on the tested part, no hormone drugs and immunosuppressants used in the last month, and no other clinical tests on the tested part in the last three months.
The testing steps are as follows: the volunteers were randomly and evenly divided into 7 groups, the facial skin was cleaned with facial cleanser, tried for 3 days continuously, each time for 15min, and the skin data (fine lines, dry lines, brightness, inflammation, brown stains, etc.) were collected with a VISA tester one time per day.
Small nucleic acid mask test results:
1. after the small nucleic acid anti-aging mask containing the recombinant anti-miR-129-5p is used, the fine grain and dry grain fading rates of testers are obviously improved, and the conditions of part of testers are specifically shown in fig. 8A and fig. 8B.
As shown in fig. 8A, the day 1 Visa data of the application shows: the fine lines and dry lines are obviously improved; the light yield is improved by 20 percent from 6 percent.
As shown in fig. 8B, the day 3 Visa data of the application shows: fine lines at the forehead are continuously improved; the light yield is improved from 20 percent to 89 percent.
2. After the small nucleic acid anti-aging mask containing the recombinant anti-miR-129-5p is used, 96% of red areas (sensitive areas) of testers with sensitive skin and damaged barriers are obviously repaired, and the efficacy conditions are shown in a figure 9A and a figure 9B.
As shown in fig. 9A, the day 1 Visa data of the application shows: the inflammation of the facial red area is obviously improved; the dilution rate is improved from 41 percent to 73 percent.
As shown in fig. 9B, the day 1 Visa data of the application shows: the inflammation of the facial red area is obviously improved; the dilution rate is improved from 22% to 64%.
3. After the small nucleic acid anti-aging mask containing the recombinant anti-miR-129-5p is used, brown spots of testers are obviously improved, and the conditions of part of testers are shown in a figure 10A and a figure 10B.
As shown in fig. 10A, the day 3 Visa data of the application shows: the brown stain index is improved; the light yield is improved from 29 percent to 38 percent.
As shown in fig. 10B, the day 3 Visa data of the application shows: the brown stain index is improved; the light yield is improved from 21 percent to 37 percent.
The results show that: the small nucleic acid anti-aging mask containing the recombinant anti-miR-129-5p can improve the dry lines of fine wrinkles, brighten the skin color and repair damaged skin, has good safety, and has a remarkable effect when being used in the mask.
The invention uses tRNA as a bracket, and a chimeric anti-miR-129-5p sequence expresses recombinant anti-miR-129-5p in escherichia coli. The produced recombinant anti-miR-129-5p can be processed and matured in skin fibroblasts and successfully expressed, simultaneously improves the activity of the skin fibroblasts, can effectively reduce the expression of senescence genes in the aging skin fibroblasts, can promote the expression of type I collagen, and further has an anti-aging effect in the skin.
And II, application embodiment. In order to prove the creativity and the technical value of the technical scheme of the invention, the part is the application example of the technical scheme of the claims on specific products or related technologies.
The recombinant anti-miR-129 can be applied to preparation of anti-aging cosmetics; the dosage forms of the anti-aging cosmetic comprise creams, emulsions, lotions and mistura.
The invention promotes the proliferation of skin fibroblasts and the expression of type I collagen, improves the expression of genes related to aging skin fibroblasts, realizes the synergistic interaction of the components, and can effectively delay skin aging, and the prepared product has the effects of resisting wrinkles and improving skin barriers.
The synthesized recombinant small RNA can be applied to biological and medical research and small nucleic acid drug development.
The above description is only for the purpose of illustrating the present invention and the appended claims are not to be construed as limiting the scope of the invention, which is intended to cover all modifications, equivalents and improvements that are within the spirit and scope of the invention as defined by the appended claims.
<110> Gibbson Biotechnology (Shaanxi) Co., Ltd
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<110> Gibbson Biotechnology (Shaanxi) Co., Ltd
<120> recombinant anti-miR-129-5p, preparation method thereof and application thereof in resisting skin aging
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Claims (10)
1. A recombinant gene, wherein the sequence of the recombinant gene is SEQ ID NO: 1.
2. the recombinant gene of claim 1, wherein the sequence of the recombinant gene is identical to SEQ ID NO: 1 with a sequence similarity of more than 90%.
3. A method for producing a recombinant gene according to claim 1, comprising:
designing an anti-miR-129-5p primer according to a genebank database and constructing a vector; designing anti-miR-129-5p, embedding the designed small interfering RNA anti-miR-129-5p into a tRNA stent, and performing recombinant expression in escherichia coli.
4. The method for preparing recombinant gene according to claim 3, wherein the sequence of the small interfering RNA anti-miR-129-5p is SEQ ID NO: 2.
5. the method for producing a recombinant gene according to claim 4, wherein the chimeric sequence is a sequence identical to SEQ ID NO: 2 sequences with more than 90% sequence similarity.
6. The method of claim 2, wherein the tRNA scaffold has a sequence that has greater than 90% similarity to a human serine tRNA sequence that is SEQ ID NO: 3.
7. the method for preparing recombinant gene according to claim 3, wherein the precursor sequence of the small interfering RNA anti-miR-129-5p is hsa-miR-34a precursor sequence of which mature sequence part is replaced by anti-miR-129-5p sequence, and the precursor sequence of the small interfering RNA anti-miR-129-5p is SEQ ID NO: 4.
8. the method for preparing the recombinant gene according to claim 3, wherein the method for preparing the recombinant anti-miR-129 specifically comprises the following steps:
designing an hsa-miR-34a precursor primer for synthesizing a chimeric anti-miR-129-5p sequence;
secondly, inserting a precursor sequence of the small interfering RNA anti-miR-129-5p into the pBSMrnaSeph plasmid by utilizing a restriction enzyme cutting site of the pBSMrnaSeph plasmid at a tRNA anticodon ring to construct an expression vector;
step three, transforming the expression vector of the chimeric target sequence into competent escherichia coli;
step four, after escherichia coli is cultured and amplified, total RNA in the bacteria is extracted, and target recombinant anti-miR-129-5p is separated and purified through FPLC.
9. An anti-aging cosmetic prepared from the recombinant gene according to claim 1.
10. The anti-aging cosmetic according to claim 9, wherein the anti-aging cosmetic is in a dosage form selected from the group consisting of creams, emulsions, lotions and mists.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015183667A1 (en) * | 2014-05-28 | 2015-12-03 | The Regents Of The University Of California | HYBRID tRNA/pre-miRNA MOLECULES AND METHODS OF USE |
| CN108753780A (en) * | 2018-06-11 | 2018-11-06 | 西安荣清畅生物科技有限公司 | It is a kind of recombination tiny RNA production method and application |
| CN109593723A (en) * | 2018-12-18 | 2019-04-09 | 中国人民解放军军事科学院军事医学研究院 | A kind of efficient mescenchymal stem cell and the preparation method and application thereof for inhibiting immune response |
| CN113134011A (en) * | 2021-05-24 | 2021-07-20 | 中国人民解放军军事科学院军事医学研究院 | Application of miR-129 in preparation of product for treating depression |
-
2022
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015183667A1 (en) * | 2014-05-28 | 2015-12-03 | The Regents Of The University Of California | HYBRID tRNA/pre-miRNA MOLECULES AND METHODS OF USE |
| CN108753780A (en) * | 2018-06-11 | 2018-11-06 | 西安荣清畅生物科技有限公司 | It is a kind of recombination tiny RNA production method and application |
| CN109593723A (en) * | 2018-12-18 | 2019-04-09 | 中国人民解放军军事科学院军事医学研究院 | A kind of efficient mescenchymal stem cell and the preparation method and application thereof for inhibiting immune response |
| CN113134011A (en) * | 2021-05-24 | 2021-07-20 | 中国人民解放军军事科学院军事医学研究院 | Application of miR-129 in preparation of product for treating depression |
Non-Patent Citations (1)
| Title |
|---|
| YUYA KAWANO等: "Royal jelly regulates the proliferation of human dermal microvascular endothelial cells through the down-regulation of a photoaging-related microRNA", DRUG DISCOV THER. * |
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