CN101921744B - Interfering target site sequence of P gene, small interfering RNA and composition and application thereof - Google Patents

Abstract

The invention provides an interfering target site sequence of a P gene, small interfering RNA which is used for inhibiting the expression of the P gene, a cosmetic composition containing the small interfering RNA and application of the small interfering RNA to preparing the cosmetic composition for inhibiting melanin from generating and depositing or removing the melanin. The small interfering RNA has high activity of inhibiting the expression of the P gene and can effectively inhibit the melanin from generating and depositing.

Description

Interference target site sequence of P gene, small interfering nucleic acid, composition and application

technical field

The present invention relates to the interference target site sequence of the P gene, the small interfering nucleic acid and its composition and application, more specifically, the present invention relates to the interference target site sequence of the P gene, the small nucleic acid for inhibiting the expression of the P gene, and The cosmetic composition containing the small interfering nucleic acid and the application of the small interfering nucleic acid in the preparation of a cosmetic composition for inhibiting melanin production and deposition or removing melanin.

Background technique

Melanin (melanin) is a biopolymer synthesized by melanocytes, which is produced by melanocytes in the skin, hair follicles, iris and retina, and is divided into black-brown eumelanin and red-yellow pheomelanin. The skin and hair color of animals and humans are directly related to the number, size and distribution of melanosomes. Excessive melanin can darken the skin, while unevenly distributed melanin can lead to melasma and freckles.

With the improvement of living standards and quality of life, people pay more and more attention to beauty, and whitening and speckle removal are one of the most important contents. Therefore, it is an urgent problem to develop a product that can inhibit melanin production and deposition or remove melanin.

Contents of the invention

The first object of the present invention is to provide the interference target site sequence of P gene.

The second object of the present invention is to provide a small interfering nucleic acid for inhibiting the expression of P gene.

The third object of the present invention is to provide a cosmetic composition containing the siRNA.

The fourth object of the present invention is to provide the application of the small interfering nucleic acid in the preparation of a cosmetic composition for inhibiting melanin production and depositing or removing melanin.

The emergence of RNA interference technology provides a new way to inhibit melanin production and deposition or remove melanin. RNA interference (RNA interference, RNAi) is a process in which double-stranded RNA (double-stranded RNA, dsRNA) molecules shut down the expression of the corresponding gene at the mRNA level or silence the gene. RNA interference technology, also known as gene knockdown (knock-down) or gene silencing (gene silencing), is a typical post-transcriptional gene regulation method, also known as post-transcriptional gene silencing (PTGS). ). The earliest report on RNA interference appeared in 1990. Two different research groups simultaneously reported the phenomenon of RNA interference in transgenic plants, and later observed it in almost all eukaryotic organisms such as nematodes, fruit flies, zebrafish, and mice. to the phenomenon of RNA interference. In 1999, Hamilton and Baulcombe detected RNA fragments with a length of 21-25 nucleotides in plants where RNA interference occurred. These RNA fragments were proved to be necessary for RNA interference, called small interfering RNA (small interfering nucleic acid ). Double-stranded small interfering nucleic acid forms an RNA-induced silencing complex (RISC) with cell-derived related enzymes and proteins. In the process of RNA interference, the sense strand in the double-stranded small interfering nucleic acid is excluded from the complex, and the antisense strand guides RISC to bind to the corresponding site of the target mRNA, and then the ribonuclease III in the complex degrades the target mRNA, thereby Turn off the expression of the target gene.

The inventors have conducted detailed research on the synthesis and deposition of melanin, and found that melanin is synthesized by a series of complex physiological and biochemical processes coordinated by multiple genes. These genes related to pigmentation are involved in the synthesis, transportation and distribution of melanin . Among a series of genes related to melanin production, P gene is one of the most important genes.

The P gene, also known as the OCA2 gene, is responsible for the synthesis of P protein. P protein is a 110kDa transmembrane protein, consisting of 838 amino acid residues, containing 12 transmembrane regions, 6 glycosylation sites, located on the melanosome membrane, and related to the integrity of the melanosome membrane, is Protein necessary for the production of melanosomes. Another study showed that the protein encoded by the P gene has significant homology with some membrane transport proteins involved in anion transport. Regulate to make the local environment neutral, which is conducive to maintaining the stability of the polymer tyrosine-TYRP1-TYRP2 complex, enhancing the activity of tyrosinase, and increasing the production and deposition of melanin.

In addition, the mutation of the P gene will lead to a decrease in the number of melanocytes and a decrease in the synthesis of melanin in the melanocytes. The encoded product of the P gene is necessary for the synthesis of eumelanin. Therefore, the P gene can be specifically inhibited by RNA interference The expression of P gene, thereby silencing the activity of P gene, is an effective way to inhibit melanin synthesis and improve skin color.

The present invention provides an interference target site sequence of the P gene, wherein the target sequence has a nucleotide sequence as shown in one of SEQ ID Nos: 2-16.

The present invention also provides a small interfering nucleic acid, wherein the small interfering nucleic acid includes a nucleotide sequence capable of hybridizing with the interference target site sequence under stringent conditions.

The present invention also provides a cosmetic composition for inhibiting melanin production and depositing or removing melanin, wherein the cosmetic composition contains the small interfering nucleic acid as an active ingredient.

The present invention also provides the application of the small interfering nucleic acid in the preparation of a cosmetic composition for inhibiting melanin production and deposition or removing melanin.

The small interfering nucleic acid provided by the invention has high inhibitory activity on P gene expression, and can effectively inhibit melanin production and deposition. For example, the small interfering nucleic acids PGENE-1 to PGENE-30 provided by the present invention can efficiently inhibit the expression of the P gene, which shows that the small interfering nucleic acids provided by the present invention are used to inhibit the expression of the P gene, and the inhibitory activity on the expression of the P gene higher.

Detailed ways

The present invention provides an interference target site sequence of the P gene, wherein the target sequence has a nucleotide sequence as shown in one of SEQ ID Nos: 2-16. Preferably, the target sequence has a nucleotide sequence as shown in SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 7, SEQ ID No: 10 or SEQ ID No: 12.

The present invention also provides a small interfering nucleic acid, wherein the small interfering nucleic acid can inhibit the expression of the P gene by acting on the target site sequence described in claim 1, and the GC content of the small interfering nucleic acid is 35-60% .

The P gene includes 24 exons and 23 introns. Exon 1 encodes the 5' untranslated sequence of P gene mRNA, and the translation initiation site is located in exon 2. The P gene encodes a 110kDa transmembrane protein, which is necessary for the synthesis of eumelanin. It consists of 838 amino acid residues, contains 12 transmembrane regions and 6 glycosylation sites, and is located on the melanosome membrane , and the integrity of the melanosome membrane.

The small interfering nucleic acid of the present invention is a double-stranded molecule comprising a sense strand and an antisense strand, and the lengths of the sense strand and the antisense strand are respectively 15-27 nucleotides; preferably, the sense strand and the antisense The sense strands were 19-21 nucleotides in length, respectively.

In a preferred embodiment of this aspect of the present invention, the small interfering nucleic acid has PGENE-1, PGENE-2, PGENE-3, PGENE-4, PGENE-5, PGENE-6, PGENE-7, PGENE-8, PGENE -9, PGENE-10, PGENE-11, PGENE-12, PGENE-13, PGENE-14 or the nucleotide sequence shown in PGENE-15, or have a pair of PGENE-1, PGENE-2, PGENE-3, PGENE Nuclei indicated by -4, PGENE-5, PGENE-6, PGENE-7, PGENE-8, PGENE-9, PGENE-10, PGENE-11, PGENE-12, PGENE-13, PGENE-14 or PGENE-15 A nucleotide sequence obtained by chemically modifying the nucleotide sequence, wherein,

PGENE-1 sense strand: 5'-GCCAGGAGUUUGCUUCAUUdTdT-3'

  Antisense strand: 5'-AAUGAAGCAAACUCCUGGCdTdT-3';

PGENE-2 sense strand: 5'-GGAGGUCUCACUCUUCUUUdTdT-3'

Antisense strand: 5'-AAAGAAGAGUGAGACCUCCdTdT-3';

PGENE-3 sense strand: 5'-CCUUAUCACCGCUGGAGAAdTdT-3'

Antisense strand: 5'-UUCUCCAGCGGUGAUAAGGdTdT-3';

PGENE-4 sense strand: 5'-GCCUGUGACCAUAAGGUUGdTdT-3'

  Antisense strand: 5'-CAACCUUAUGGUCACAGGCdTdT-3';

PGENE-5 sense strand: 5'-GCAGAAGUGAUCUUCACAAdTdT-3'

Antisense strand: 5'-UUGUGAAGAUCACUUCUGCdTdT-3';

PGENE-6 sense strand: 5'-CCGGAUUCACUGCACACAUdTdT-3'

Antisense strand: 5'-AUGUGUGCAGUGAAUCCGGdTdT-3';

PGENE-7 sense strand: 5'-GGAGACCAAUAUCCAAGAAdTdT-3'

Antisense strand: 5'-UUCUUGGAUAUUGGUCUCCdTdT-3';

PGENE-8 sense strand: 5'-CCCUGGCAUUCAUCUUGAUdTdT-3'

Antisense strand: 5'-AUCAAGAUGAAUGCCAGGGdTdT-3';

PGENE-9 sense strand: 5'-GGUGCCAUCUGGUUGCUAAdTdT-3'

  Antisense strand: 5'-UUAGCAACCAGAUGGCACCdTdT-3';

PGENE-10 sense strand: 5'-GGGCUUCCCAAUGAUGGUUdTdT-3'

  Antisense strand: 5'-AACCAUUCAUUGGGAAGCCCdTdT-3';

PGENE-11 sense strand: 5'-GGUUGUGUCCUGCACUGUUdTdT-3'

  Antisense strand: 5'-AACAGUGCAGGACACAACCdTdT-3';

PGENE-12 sense strand: 5'-GCACUGUUGGGAUGUGUUAdTdT-3'

  Antisense strand: 5'-UAACACAUCCCAACAGUGCdTdT-3';

PGENE-13 sense strand: 5'-GCUUACCCUGGAGAUGCUAdTdT-3'

  Antisense strand: 5'-UAGCAUCUCCAGGGUAAGCdTdT-3';

PGENE-14 sense strand: 5'-GGAAUAGACCUUGACACUAdTdT-3'

Antisense strand: 5'-UAGUGUCAAGGUCUAUUCCdTdT-3';

PGENE-15 sense strand: 5'-UCAAUUCUCCUGAGGCUAAdTdT-3'

Antisense strand: 5'-UUAGCCUCAGGAGAAUUGAdTdT-3'.

Preferably, the small interfering nucleic acid has a nucleotide sequence represented by PGENE-2, PGENE-3, PGENE-6, PGENE-9 or PGENE-11.

According to the present invention, the chemical modification is at least one of the following modifications:

(1) modification of the phosphodiester bond connecting nucleotides in the nucleotide sequence of the small interfering nucleic acid;

(2) modification of the 2'-OH of ribose in the nucleotide sequence of the small interfering nucleic acid;

(3) Modification of bases in the nucleotide sequence of the small interfering nucleic acid.

The chemical modification is well known to those skilled in the art. The modification of the phosphodiester bond refers to the modification of the oxygen in the phosphodiester bond, including the modification of phosphorothioate, as shown in formula 1; and borylation of phosphoric acid Salt modification, as shown in formula 2. Both modifications can stabilize the structure of small interfering nucleic acids and maintain high specificity and high affinity of base pairing.

The ribose modification refers to the modification of the 2'-OH in the nucleotide pentose, that is, the introduction of certain substituents at the hydroxyl position of the ribose, for example, 2'-fluoro modification, as shown in formula 3; 2' -Oxymethyl modification, as shown in formula 4; 2'-oxyethylene methoxy modification, as shown in formula 5; 2,4'-dinitrophenol modification, as shown in formula 6; locked nucleic acid ( LNA), as shown in formula 7; 2'-amino modification, as shown in formula 8; 2'-deoxy modification, as shown in formula 9.

The base modification refers to the modification of the base of the nucleotide, for example, 5'-bromouracil modification, as shown in formula 10; 5'-iodouracil modification, as shown in formula 11; N-methyl Modified with uracil, as shown in formula 12; modified with 2,6-diaminopurine, as shown in formula 13.

Preferably, the modification enhances the ability of the modified small interfering nucleic acid to resist nuclease hydrolysis in cells.

In addition, in order to promote the entry of small interfering nucleic acids into cells, on the basis of the above modifications, a lipophilic group such as cholesterol can be introduced at the end of the sense strand of small interfering nucleic acids to facilitate passage through the cell membrane composed of lipid bilayers and the intracellular mRNA takes effect.

The preparation method of the small interfering nucleic acid provided by the present invention includes the design of the small interfering nucleic acid sequence and the preparation of the small interfering nucleic acid.

The design of the siRNA sequence refers to selecting a 19 bp nucleotide sequence within the range of 1-3136 bp of the P gene cDNA sequence (Genbank accession number: NM-000275). The selection of 19bp nucleotide sequence mainly refers to the following principles: (1) GC content is between 35-55%, (2) avoid repeat sequences or low-complexity sequence regions, (3) avoid more than 4 (4) Avoid containing single nucleotide polymorphism sites, (5) Avoid being within the 50-100bp region of the start codon and stop codon of the reading frame, in addition, Analyze the compositional and thermodynamic properties of nucleotide sequences. Through BLAST analysis, the candidate small interfering nucleic acid target site is homologously compared with the human gene sequence, and the sequence with a large sequence homology (more than 16 bases) with other genes is excluded to ensure the candidate small interference The nucleic acid target site will not inhibit other unrelated genes, but only has a specific inhibitory effect on the P gene.

Finally, two deoxythymine nucleotides are added to the 3' end of the 19bp nucleotide sequence obtained in this way as the sense strand of the small interfering nucleic acid sequence, and two deoxythymine nucleotides are added to the 3' end of the complementary sequence of the 19bp nucleotide sequence. Deoxythymidine nucleotides serve as the antisense strand of the siRNA sequence.

According to the present invention, the preparation method of the siRNA is well known to those skilled in the art, for example, the siRNA can be obtained by chemical synthesis, or expressed by plasmid and/or virus vector.

The synthesis of the siRNA sequence can be done by chemical synthesis, or entrusted to a biotechnology company specialized in nucleic acid synthesis, such as Shanghai GenePharma Company.

In general, the method of chemical synthesis includes the following four processes:

(1) synthesis of oligoribonucleotides; (2) deprotection; (3) purification and separation; (4) desalting.

For example, the specific steps of the chemical synthesis of the small interfering nucleic acid with the nucleotide sequence shown in PGENE-1 are as follows:

(1) Synthesis of oligoribonucleotides: the synthesis of oligoribonucleotides is carried out on an automatic DNA/RNA synthesizer (for example, Applied Biosystems EXPEDITE8909), according to the nucleotide sequence shown in PGENE-1 ( Sense strand: 5'-GCCAGGAGUUUGCUUCAUUdTdT-3', antisense strand: 5'-AAUGAAGCAAAUCUCCUGGCdTdT-3') and connect corresponding nucleotides one by one. Since the small interfering nucleic acid is composed of 19 oligoribonucleotides and 2 deoxythymidylic acid. Therefore, the starting material is 5'-O-p-dimethoxy-thymidine linked to the solid phase, and each specific cycle synthesis can be divided into four steps to complete. The first step is to combine the thymidine with the fixed link The protecting group at the 5' position is eluted under the action of trichloroacetic acid; in the second step, under the action of the active catalyst S-ethyltetrazole, the 5'-O-p-dimethoxytrityl-thymidine The phosphoramidite is coupled to the last thymidine that has been deprotected to form a dimhymidine phosphite triester. The coupling time and the number of couplings are all completed according to the procedures provided by the instrument manufacturer; the third step is to combine the coupled dimhymidine Under the action of 0.05M iodine water, the phosphite triester is oxidized into dimhymidine phosphate triester; the fourth step is acetylation, and a small amount of unreacted active groups (for example, hydroxyl and amine groups) on the solid phase are reacted in ethyl Under the action of acid anhydride, ester or amide is formed, so as to achieve the blocking effect, so as to reduce the generation of overall by-products, and repeat this cycle until the synthesis of all nucleic acid sequences is completed.

(2) Deprotection

Put the synthesized solid-phase siRNA into a sealable vial, add 1 ml of ethanol/amine (volume ratio 1:3), seal it, place it in a 55-70°C incubator, and incubate After 2-30 hours, the solution was taken out, and the solid phase was rinsed with double distilled water again, and the eluate was collected and dried to remove the solvent. Then, 1 ml of tetrabutylammonium fluoride tetrahydrofuran solution (1M) was added, left at room temperature for 4-12 hours, and then subjected to ethanol precipitation to obtain a crude product of small interfering nucleic acid.

(3) Purification and separation

The crude product of small interfering nucleic acid was dissolved in 2 ml of ammonium acetate aqueous solution, and then separated by reaction C18 high pressure liquid chromatography, and the method of gradient elution was used to collect the main product of small interfering nucleic acid (eluent A: 0.1M acetic acid Ammonium; Eluent B: 20% of 0.1M ammonium acetate and 80% of acetonitrile), remove the solvent in the main product, and add 5 ml of 80% aqueous acetic acid solution, let stand at room temperature for 15 minutes, then remove the solution Anion-exchange separation (DEAE-5PW, anion-exchange column) can obtain small interfering nucleic acids with a purity of more than 90% (gradient elution, eluent A: 0.025M Tris-HCl, 0.025M NaCl, pH= 8, 5% acetonitrile; eluent B: 0.025M Tris-HCl, 2.0M NaCl, pH=8, 5% acetonitrile).

(4) Desalination

The purified sinucleic acid is dialyzed to remove salt, and then the sinucleic acid solution is filtered and sterilized and dried for crystallization. Then the oligoribonucleic acid of the sense strand and the antisense strand is annealed to form a stable double-stranded small interfering nucleic acid. The method is to mix and dissolve the oligoribonucleotides of the sense strand and the antisense strand in 1-2 ml buffer (10mM Tris, pH=7.5-8.0, 50mM NaCl), heat the solution to 95°C, then slowly cool the solution to room temperature, and finally store the solution in a refrigerator at 4°C for ready use at any time use.

In addition to chemical synthesis, small interfering nucleic acids can also be obtained through the expression of plasmids and/or viral vectors to obtain shRNA with a hairpin structure, and its length is 50-90 nucleotides. The structure of shRNA is:

5'-GATCCG-sense strand GAAGCTTG-antisense strand TTTTTTGGAATT-3'

Both ends are restriction sites (such as BamHI and EcoRI), and the middle is a loop sequence (such as GAAGCTTG), which is inserted into a vector digested with the corresponding restriction endonuclease by cloning technology, and then integrated into the chromosome, namely Stable expression of small interfering nucleic acids.

According to the cosmetic composition for inhibiting melanin generation and depositing or removing melanin provided by the present invention, the cosmetic composition contains the small interfering nucleic acid provided by the present invention as an active ingredient.

According to the invention, the cosmetic composition also contains a carrier.

In the present invention, the content of the small interfering nucleic acid and the carrier can vary in a wide range, preferably, relative to 100 parts by weight of the small interfering nucleic acid, the content of the carrier is 1000-10000000 parts by weight; more preferably , relative to 100 parts by weight of small interfering nucleic acid, the content of the carrier is 2000-100000 parts by weight.

In the present invention, the carrier is not particularly limited, it can be any carrier used in cosmetic compositions, for example, it can be one or more of animal and vegetable oils, hydrocarbon oils, esters, higher fatty acids and higher fatty alcohols; for example , the animal and vegetable oil can be one or more of mink oil, turtle oil, soybean oil, sesame oil, corn oil, rapeseed oil, cottonseed oil, olive oil and castor oil; the hydrocarbon oil can be paraffin oil, One or more of isohexadecane and petrolatum; the esters can be isopropyl palmitate, butyl stearate, hexyl laurate, isononyl isononanoate, 2-ethylhexyl palmitate One or more of esters, 2-hexyldecyl laurate and 2-octyl dodecyl myristate; the higher fatty acid can be palmitic acid, stearic acid, linoleic acid and linoleic acid One or more of the acids; higher fatty alcohols can be cetyl alcohol and/or stearyl alcohol.

According to the invention, the cosmetic composition also contains other ingredients for cosmetic compositions.

In the present invention, the content of the small interfering nucleic acid and other ingredients used in the cosmetic composition can vary in a wide range. Preferably, relative to 100 parts by weight of the small interfering nucleic acid, the other ingredients used in the cosmetic composition The content of the component is 1000-10000000 parts by weight; more preferably, relative to 100 parts by weight of the small interfering nucleic acid, the content of the other components used in the cosmetic composition is 2000-100000 parts by weight.

In the present invention, there is no particular limitation on the other ingredients used in the cosmetic composition, for example, it can be one or more of moisturizing agents, coloring agents, preservatives, thickeners, antioxidants, surfactants and adjuvants Several kinds.

In the present invention, the moisturizing agent is not particularly limited, and can be various moisturizing agents used in cosmetic compositions, for example, the moisturizing agent can be propylene glycol, dipropylene glycol, polypropylene glycol, polyethylene glycol, sorbitol , hexanediol, 1,3-butanediol and glycerin in one or more.

In the present invention, the coloring agent is not particularly limited, and can be various coloring agents used in cosmetic compositions, for example, the coloring agent can be carmine, talc, mica, magnesium carbonate, calcium carbonate, silicic acid One or more of magnesium, silicon dioxide, zinc oxide and ultramarine blue.

In the present invention, the thickener is not particularly limited, it can be various thickeners used in cosmetic compositions, for example, the thickener can be beeswax, candelilla wax, spermaceti, microcrystalline One or more of wax, starch, xanthan gum, gum arabic, guar gum, pectin and bentonite.

In the present invention, the preservative is not particularly limited, and may be various preservatives used in cosmetic compositions, for example, the preservative may be benzoic acid, salicylic acid, methyl paraben, p-hydroxy One or more of propyl benzoate and resorcinol.

In the present invention, the antioxidant is not particularly limited, and may be various antioxidants used in cosmetic compositions, for example, the antioxidant may be ascorbic acid, vitamin A, β-carotene, vitamin B, cysteine One or more of amino acid, glutathione, catalase, citric acid and catechol.

In the present invention, the surfactant is not particularly limited, and may be various surfactants used in cosmetic compositions, for example, the surfactant may be fatty acid glycerides, polyoxyethylene hydrogenated castor oil, alkanes One or more of sodium phenyl sulfonate, dialkyl sulfosuccinate, N-acyl glutamic acid and sodium polyoxyethylene alkyl sulfate.

The type of the adjuvant is not particularly limited, and can be various adjuvants that can be used in cosmetic compositions, for example, the adjuvant can be vitamin B3, seaweed extract, azone, trehalose, bis-imidazolidinyl One or more of urea and hyaluronic acid.

The cosmetic composition can be in the form of various conventional cosmetic compositions, for example, it can be emulsion, ointment, liquid, aerosol or powder. The cosmetic composition is an external preparation that can be applied to the skin of various parts of the body, such as the face, neck, arms, trunk (chest, abdomen and back), legs, hands and feet.

The present invention also provides the application of the small interfering nucleic acid in the preparation of a cosmetic composition for inhibiting melanin production and deposition or removing melanin.

The P gene interference target site, small interfering nucleic acid, cosmetic composition and application provided by the present invention can be used to prevent, improve, and eliminate skin pigmentation, so as to prevent, improve, and eliminate unsatisfactory or unhealthy skin and The purpose of whitening the skin. Moreover, the small interfering nucleic acid of the present invention can specifically, efficiently, and stably specifically inhibit the expression of P protein, and the small interfering nucleic acid provided by the present invention can be widely used in the preparation of various products used to prevent, improve, and remove skin pigmentation. medicines or cosmetics. And the small interfering nucleic acid, cosmetic composition, etc. provided by the present invention can be directly applied or transferred to the site of action.

The present invention will be further described below in conjunction with the examples. Unless otherwise specified, the reagents and culture media used in the present invention are commercially available.

Example 1

Synthesis of small interfering nucleic acids

A nucleotide sequence with a length of 19 bp was selected within the range of 1-3136 bp of the cDNA sequence of the P gene (Genbank accession number: NM-000275). The selection of 19bp nucleotide sequence mainly refers to the following principles: (1) GC content is between 35-55%, (2) avoid repeat sequences or low-complexity sequence regions, (3) avoid more than 4 (4) Avoid containing single nucleotide polymorphism sites, (5) Avoid being within the 50-100bp region of the start codon and stop codon of the reading frame, in addition, Analyze the compositional and thermodynamic properties of nucleotide sequences. Through BLAST analysis, the candidate small interfering nucleic acid target site is homologously compared with the human gene sequence, and the sequence with a large sequence homology (more than 16 bases) with other genes is excluded to ensure the candidate small interference The nucleic acid target site will not inhibit other unrelated genes, but only has a specific inhibitory effect on the P gene.

Finally, two deoxythymine nucleotides are added to the 3' end of the 19bp nucleotide sequence obtained in this way as the sense strand of the small interfering nucleic acid sequence, and two deoxythymine nucleotides are added to the 3' end of the complementary sequence of the 19bp nucleotide sequence. Deoxythymidine nucleotides serve as the antisense strand of the siRNA sequence.

The designed small interfering nucleic acids were chemically synthesized by Shanghai GenePharma Company to obtain small interfering nucleic acids PGENE-1 to PGENE-15. The nucleotide sequences of the small interfering nucleic acids PGENE-1 to PGENE-15 are shown in Table 1 shown.

Table 1

The attack range refers to the corresponding position of the small interfering nucleic acid in sequence No. 1 of the sequence listing.

Example 2

Inhibition activity detection of P gene expression

(1) Culture of melanocytes

Use MEM complete medium containing 10% fetal bovine serum, 2mM L-glutamine, 100U/ml penicillin, 100μg/ml streptomycin, in a six-well cell culture plate at a density of 5× ¹⁰⁶ cells/well Inoculate SK-MEL-3 melanoma cells (Beijing Baixing Technology Development Co., Ltd.), culture in an incubator with a temperature of 37°C and a _CO2 content of 5%, passage every 48 hours, and replace fresh medium.

2) Transfection of small interfering nucleic acids

The small interfering nucleic acids PGENE-1 to PGENE-15 obtained in Example 1 were respectively transfected with Lipofectamine ^™ 2000 liposomes from Invitrogen Company, with irrelevant small interfering nucleic acids (sense strand: 5-UUCUCCGAACGUGUCACGUTT-3; antisense strand: 5-ACGUGACACGUUCGGAGAATT-3) served as a negative control. The specific operation steps are as follows: the small interfering nucleic acid is dissolved in RNase-free sterile water to prepare a solution with a concentration of 20 μmol/L. SK-MEL-3 melanoma cells were inoculated into 24-well plates, diluted with OptiMEM I low serum medium (Invitrogen, 31985-062) to a concentration of 8×10 ⁵ cells/ml, 500 μl per well. Dilute 1.5 μl small interfering nucleic acid (20 μmol/L) in 50 μl Opti-MEM I low serum medium (Invitrogen, 31985-062), and dilute 1 μl Lipofectamine ^TM 2000 liposomes in 50 μl Opti-MEM I low serum culture medium (Invitrogen Company, 31985-062), then incubate the above two solutions at room temperature for 5 minutes and then mix them. in a 24-well plate. The final concentration of siRNA was 50 nM. Cells were cultured at 37°C for 4 hours, then 1ml of MEM complete medium containing 10% fetal bovine serum, 2mM L-glutamine, 100U/ml penicillin, 100μg/ml streptomycin was added, and then cultured at 37°C for another 24 hours .

(3) Detection of inhibitory activity

RT-PCR was used to detect the expression of P gene mRNA in SK-MEL-3 melanoma cells transfected with small interfering nucleic acids PGENE-1 to PGENE-15, and the specific steps were:

The SK-MEL-3 melanoma cells transfected with small interfering nucleic acid were lysed with 1ml TRIzol (GIBCOL company), and the total RNA was extracted. ₂ Culture in an incubator with 5% content for 24 hours, collect the cells by centrifugation, remove the supernatant, and wash once with pre-cooled PBS; the composition of the PBS is: NaCl 137mmol/L, KCl2.7mmol/L, Na ₂ HPO44 .3mmol/L, KH ₂ PO4 1.4mmol/L; add 1ml Trizol to each well to lyse the cells for 3 minutes; pipette the cells, transfer the lysate to a 1.5ml EP tube, and let stand at room temperature for 10 minutes; add 200μl chloroform and shake on the shaker Shake vigorously for 15 seconds, and place at room temperature for 10 minutes; centrifuge at 12,000 rpm at 4°C for 20 minutes; take about 300-500 μl of the supernatant and put it in a new EP tube, add an equal volume of isopropanol, and precipitate overnight at -30°C; 12,000 rpm, Centrifuge at 4°C for 10 minutes. Remove the supernatant, wash once with 1ml 75% ethanol; centrifuge at 12,000rpm, 4°C for 10 minutes; aspirate the supernatant, dry the RNA pellet for 10 minutes; add 20μl DEPC water to dissolve the RNA.

2 units of DNase I (RNase-free) (TakaRa Company) were added to the total RNA, and left at 37°C for 30 minutes to remove residual DNA in the total RNA. After being treated with DNase I, the total RNA was purified, and the specific steps of purification were: (using the PureLink Micro-to-Midi Total RNA Purification Kit of Invitrogen Company), adding an equal volume of 70% ethanol to the total RNA, shaking and mixing evenly, and Transfer the mixture to a purification column, centrifuge at 12,000 g for 15 seconds at room temperature, discard the filtrate, add 700 μl of washing buffer I, centrifuge at 12,000 g for 15 seconds at room temperature, discard the filtrate, add 500 μl of washing buffer II, centrifuge at 12,000 g for 15 seconds at room temperature, and discard the filtrate , then add 500 μl of washing buffer II, centrifuge at 12,000 g at room temperature for 15 seconds, discard the filtrate, and centrifuge at 12,000 g at room temperature for 1 minute, transfer the purification column to an RNA collection tube, add 30 μl of DEPC water, place at room temperature for 1 minute, and centrifuge at 13,000 g for 2 minutes at room temperature. Discard the purification column and store the RNA sample at -80°C.

The purified total RNA was subjected to a reverse transcription reaction. In the reverse transcription reaction, the reverse transcriptase used was Promega's M-MLV reverse transcriptase. The specific steps of the reverse transcription reaction were: mix 1 ug of the purified total RNA with 0.5ug of Oligo dT was mixed in the test tube, and the total volume was made up to 16.25 μl with DEPC water, and the test tube was heated. The heating conditions included a heating temperature of 70°C and a heating time of 5 minutes; then the test tube was rapidly cooled to 0 ℃, and add buffer (5×MLV buffer 5μl, 10mM Dntp 1.25μl, RNasin 0.5μl, M-MLV 1μl), incubate at 42℃ for 1 hour to obtain cDNA.

Real-time PCR reaction was carried out using the obtained cDNA as a template for PCR reaction. Real-time PCR reaction system is: ddH ₂ O 17.5 μl, 10mM Dntp 0.5 μl, 10×Taq buffer 2.5 μl, Taq 0.5 μl, F primer 0.5 μl, R primer 0.5 μl, Syber Green I 1 μl, cDNA 2 μl; The conditions are: 94 degrees for 2 minutes, 94 degrees for 15 seconds, 60 degrees for 30 seconds, a total of 40 cycles. The inhibitory activity of the small interfering nucleic acid was calculated according to the following formula, and the results are shown in Table 2.

Small interfering nucleic acid inhibitory activity=[1-(copy number of P gene after small interfering nucleic acid transfection/GAPDH copy number after small interfering nucleic acid transfection)/(control hole P gene copy number/control hole GAPDH copy number) ] × 100%.

GAPDH: Glyceraldehyde-3-phosphate dehydrogenase gene. Glyceraldehyde 3-phosphate dehydrogenase gene is a housekeeping gene in cells, which is stably expressed in cells and is not affected by other external factors, so it is used as an internal reference for fluorescent quantitative PCR reactions.

Table 2

As can be seen from Table 2, the small interfering nucleic acids PGENE-1 to PGENE-30 provided by the present invention can efficiently express the P gene, especially the small interfering nucleic acids PGENE-2, PGENE-3, PGENE-6, PGENE-9 and The inhibitory activity of PGENE-11 to the P gene reaches 85%, 82%, 82%, 81% and 82% respectively, indicating that when the small interfering nucleic acid provided by the present invention is used to inhibit the expression of the P gene, the inhibitory activity to the expression of the P gene high.

Example 3

Inhibition of melanin content in melanocytes

(1) Culture of melanocytes

Use MEM complete medium containing 15% fetal bovine serum, 2mM L-glutamine, 100U/ml penicillin, 100μg/ml streptomycin in an incubator with a temperature of 37°C and _a CO content of 5% for SK - MEL-3 melanoma cells were cultured, passaged every 48 hours, and fresh medium was replaced.

(2) Detection of melanin content

The inhibitory effects of small interfering nucleic acids PGENE-1 to PGENE-15 on melanin content were detected respectively, and the specific steps were as follows:

2×10 ⁵ cells/1 mL per well were inoculated in a 6-well culture plate, siRNA and Lipofectamine ^™ 2000 liposomes (Invitrogen) were added to each well for transfection, and the final concentration of siRNA was 50 nM. An irrelevant small interfering nucleic acid: (sense strand: 5-UUCUCCGAACGUGUCACGUTT-3; antisense strand: 5-ACGUGACACGUUCGGAGAATT-3) was used as a negative control. Discard the medium 72 hours after transfection, wash twice with PBS of pH 7.4, the composition of the PBS is: NaCl 137mmol/L, KCl 2.7mmol/L, Na ₂ HPO 44.3mmol/L, KH ₂ PO 41.4mmol /L; then add 500 μL of TritonX-100 solution with a concentration of 1% by weight to each hole, the composition of the TritonX-100 solution is 99ml water, 1ml TritonX-100; quickly placed in -80 ° C, frozen for 30 minutes; then Thaw at room temperature to completely rupture the cells, centrifuge, wash the pellet once with 10% by weight trichloroacetic acid and absolute ethanol, centrifuge at 12000g for 5 minutes, discard the supernatant, and finally add 1M NaOH (containing 10% volume DMSO) The absorbance value was measured at 405nm and the inhibition rate of melanin content was calculated after being incubated in a water bath at 80° C. for 2 hours. The results are shown in Table 3.

Inhibition rate of melanin content=[1-[(absorbance value of well after siRNA transfection-absorbance value of blank well)/(absorbance value of control well-absorbance value of blank well)]]×100%.

table 3

As can be seen from Table 3, the small interfering nucleic acids PGENE-1 to PGENE-30 provided by the present invention can significantly improve the inhibition rate of melanin content, especially through the small interfering nucleic acids PGENE-2, PGENE-3, PGENE-6, PGENE -9 and PGENE-11 inhibition, the inhibition rate of melanin content in melanocytes reaches more than 45%, indicating that the small interfering nucleic acid provided by the present invention can significantly improve the inhibition rate of melanin content.

Example 4

Test of whitening effect on human skin

In order to enhance the stability of small interfering nucleic acids, Shanghai Gemma Company was commissioned to synthesize chemically modified small interfering nucleic acids PGENE-1 to PGENE-15, wherein chemical modification refers to the U The 2'-OH of the pentose sugars of , C and G nucleotides were modified with 2'-fluorine, and the 2'-OH of the pentose sugars of U and C nucleotides of the antisense strand were modified with 2'-oxymethyl. Then mix the components according to the composition shown in Table 4, and then stir evenly to obtain compositions 1-30, so as to evaluate the clinical whitening effect of the compositions containing the small interfering nucleic acid provided by the present invention as an active ingredient.

Select 20 healthy female volunteers aged between 18 and 55, and use the GS2006 solar simulator (Beijing Aohua Equipment Co., Ltd., China) to induce skin darkening on the volunteers' buttocks. The time for inducing darkening is After 1 hour, a blackened spot with a diameter of 1.0cm is formed, which is obviously darker than the skin color of the non-blackened skin; after that, use the composition 1-15 to apply to the darkened skin, once in the morning and once in the evening, each time the dosage is 0.5ml , and observe the change of melanized skin color on the last day of each month after application, the results are shown in Table 5.

Table 4

table 5

Whitening effect evaluation criteria:

no effect: nothing changes;

Slight effect: the skin tone of melanized skin is slightly whiter, but still slightly darker than that of non-melanized skin;

Significant effect: the skin color of melanized skin is obviously whitened, which is basically the same as that of non-melanized skin.

As can be seen from Table 5, the compositions containing the small interfering nucleic acids PGENE-1 to PGENE-15 provided by the present invention have a significant whitening effect on darkened skin after being used for 5 months, especially by containing PGENE-2, The composition of PGENE-3, PGENE-6, PGENE-9 and PGENE-11 has a slight whitening effect on melanized skin after 1 month of use, and has a significant whitening effect on melanized skin after 3 months of use .

SEQUENCE LISTING

<110>Suzhou Ruibo Biotechnology Co., Ltd.

<120> Interference target site sequence and small interfering nucleic acid of P gene and composition and application

<130>I10627SRB

<160>31

<170>PatentIn version 3.4

<210>1

<211>3136

<212>DNA

<213> Human (Homo sapiens)

<400>1

tctgagttct tacttcgaag gctgtgctcc gctcaccatc cagagcggag gtgcggacct 60

taaactcact cctggagaaa gatctgcaag tgcgcagaga gaagactggc agtggagcat 120

gcatctggag ggcagagacg gcaggcggta ccccggcgcg ccggcggtgg agctcctgca 180

gacgtccgtg cccagcggac tcgctgaact tgtggccggc aagcgcaggc ttcctcgggg 240

agccggtgga gctgacccct cgcactcctg ccccaggggg gctgccgggc agagctcttg 300

ggctcctgca ggccaggagt ttgcttcatt cctcacaaaa gggaggtctc actcttcttt 360

gccccagatg tccagctcca ggtctaaaga ttcctgcttt acagaaaaca ctcctttgct 420

gaggaattcc ttacaggaga aagggtcacg gtgcatacct gtttaccatc cagagttcat 480

cactgctgaa gagtcttggg aagacagctc tgctgactgg gagcgaagat acctgctaag 540

cagggaggtg tctggtctgt ctgcatctgc ctcctccgag aagggagacc ttctggacag 600

cccgcacatc cgactccgtc tttccaagct gaggcgctgt gtgcagtggc tgaaagtcat 660

gggcctgttt gcctttgtgg tgctgtgttc tattttgttc agcctatatc cggatcaagg 720

aaagctctgg cagctgttgg ccttatcacc gctggagaac tactccgtga accttagcag 780

ccacgtggac tccacgctgc tgcaggtgga cctggcaggg gccctagtgg ccagtgggcc 840

gagtcgtcct gggagggaag agcacatcgt ggtggagctg accccaggatg acgctttggg 900

ctccaggtgg cggcggccac agcaggtcac tcacaactgg acggtgtatt taaatccgag 960

gagaagcgag cactcagtga tgagcaggac ctttgaggta ctgaccagag agacggtgtc 1020

catcagcatc cgggcctccc tgcagcagac ccaggctgtc cctcttttga tggctcatca 1080

gtacctccgc ggaagtgtag aaacccaggt gaccatcgcg acggccatcc tcgcgggcgt 1140

ctacgcgctg atcatatttg agatcgtgca cagaactctg gcggccatgc tgggttccct 1200

tgcagcactg gcagcactgg ctgtgattgg cgatagaccc agcctgaccc atgtggtgga 1260

gtggattgat tttgagacgc tggccctgct gtttggcatg atgatcttag tagccatatt 1320

ttcagaaacg ggatttttcg attattgtgc tgtaaaggca taccggctct cccggggacg 1380

ggtgtgggcc atgatcatca tgctctgtct catcgcggcc gtcctctctg ccttcttgga 1440

caacgtcacc accatgctcc tcttcacgcc tgtgaccata aggttgtgtg aggtgctcaa 1500

ccttgatcca aagacaagtcc tgattgcaga agtgatcttc acaaacattg gaggagctgc 1560

cactgccatc ggggaccctc caaatgtcat tattgtttcc aaccaagagc tgaggaagat 1620

gggcctggac tttgccggat tcactgcaca catgttcatt gggatttgcc ttgttctcct 1680

ggtctgcttt ccgctcctca gactccttta ctggaacaga aagctttata acaaggaacc 1740

cagtgagatt gttgaactga agcacgagat tcacgtctgg cgcctgactg ctcagcgcat 1800

cagcccggcc agccgcgagg agacagctgt gcgccgcctg ctgctgggga aggtgctggc 1860

actggagcac ctgctcgccc ggaggctgca caccttccac aagacagatct cacaggagga 1920

caaaaattgg gagaccaata tccaagaact ccaaaaaaag cataggatat ctgacgggat 1980

tctgctcgcc aaatgcctga cagtgttggg atttgttatc ttcatgtttt tcctcaattc 2040

gtttgtccct ggcattcatc ttgatcttgg atggattgct attctgggtg ccatctggtt 2100

gctaatttta gctgatattc atgattttga gataattcta cacagagtgg aatgggcaac 2160

ccttctgttt tttgcagcgc tctttgttct gatggaggca ttggcacatc tccacttaat 2220

agaatatgtt ggagaacaaa ctgctttgct aataaagatg gtccccagagg agcagcgcct 2280

catagccgcc attgtcctgg tggtgtgggt ctcagccctg gcgtcgtccc tgattgacaa 2340

catcccgttc actgctacca tgattcccgt gctcctgaac ctgagccacg accctgaggt 2400

tggcctgccc gcaccgccgc tcatgtatgc cctggccttc ggtgcttgcc tgggaggcaa 2460

cgggacactg attggcgcgt cggcaaacgt cgtgtgtgca gggattgcag aacagcatgg 2520

atatgggttc tccttcatgg aatttttcag gctgggcttc ccaatgatgg ttgtgtcctg 2580

cactgttggg atgtgttatc tccttgtggc tcatgtggtg gtgggatgga attaatagac 2640

atccatctat tgctcgaaga ctaaaggaaa cttcatccat cacaacccat tagtcataaa 2700

actaccctga ccccactgtt tgaagaagaa aaggtgctta ccctggagat gctacagaga 2760

cacagtggaa tagaccttga cactaacact ctaattcaag cgaatgttgg aacaccatga 2820

cctcctctgt gtgtcctttc tccccaagga caaaatgtag aaagatgtga gataacttac 2880

tcaagattcc cctccagaaa aatacgtatg tttaaaaacc cttcctgcta tacataggaa 2940

aagacacaca tccacctaaa attgactgta ctgtttaact gtcaattctc ctgaggctaa 3000

acacagtttg tttttcttgt aatcactttt catgttaaaa taatcagcat tcaaattgta 3060

tgctttctga atatagactt tctgggaaaa ggtttatactgc tcgtaaggaa aattttatg 3120

tattaaaata aactgt 3136

<210>2

<211>19

<212>DNA

<213> Human (Homo sapiens)

<400>2

gccaggagt ttgcttcatt 19

<210>3

<211>19

<212>DNA

<213> Human (Homo sapiens)

<400>3

ggaggtctcactcttcttt 19

<210>4

<211>19

<212>DNA

<213> Human (Homo sapiens)

<400>4

ccttatcaccgctggagaa 19

<210>5

<211>19

<212>DNA

<213> Human (Homo sapiens)

<400>5

gcctgtgaccataaggttg 19

<210>6

<211>19

<212>DNA

<213> Human (Homo sapiens)

<400>6

gcagaagtgatcttcacaa 19

<210>7

<211>19

<212>DNA

<213> Human (Homo sapiens)

<400>7

ccggattcactgcacacat 19

<210>8

<211>19

<212>DNA

<213> Human (Homo sapiens)

<400>8

ggagaccaatatccaagaa 19

<210>9

<211>19

<212>DNA

<213> Human (Homo sapiens)

<400>9

ccctggcattcatcttgat 19

<210>10

<211>19

<212>DNA

<213> Human (Homo sapiens)

<400>10

ggtgccatctggttgctaa 19

<210>11

<211>19

<212>DNA

<213> Human (Homo sapiens)

<400>11

gggcttcccaatgatggtt 19

<210>12

<211>19

<212>DNA

<213> Human (Homo sapiens)

<400>12

ggttgtgtcctgcactgtt 19

<210>13

<211>19

<212>DNA

<213> Human (Homo sapiens)

<400>13

gcactgttgggatgtgtta 19

<210>14

<211>19

<212>DNA

<213> Human (Homo sapiens)

<400>14

gcttaccctggagatgcta 19

<210>15

<211>19

<212>DNA

<213> Human (Homo sapiens)

<400>15

ggaatagaccttgacacta 19

<210>16

<211>19

<212>DNA

<213> Human (Homo sapiens)

<400>16

tcaattctcctgaggctaa 19

<210>17

<211>21

<212> RNA

<213> Artificial sequence

<400>17

aaugaagcaa acuccuggct t 21

<210>18

<211>21

<212> RNA

<213> Artificial sequence

<400>18

aaagaagagu gagaccucct t 21

<210>19

<211>21

<212> RNA

<213> Artificial sequence

<400>19

uucuccagcg gugauaaggt t 21

<210>20

<211>21

<212> RNA

<213> Artificial sequence

<400>20

caaccuuaug gucacaggct t 21

<210>21

<211>21

<212> RNA

<213> Artificial sequence

<400>21

uugugaagau cacuucugct t 21

<210>22

<211>21

<212> RNA

<213> Artificial sequence

<400>22

augugugcag ugaauccggt t 21

<210>23

<211>21

<212> RNA

<213> Artificial sequence

<400>23

uucuuggaua uuggucucct t 21

<210>24

<211>21

<212> RNA

<213> Artificial sequence

<400>24

aucaagauga augccagggt t 21

<210>25

<211>21

<212> RNA

<213> Artificial sequence

<400>25

uuagcaacca gauggcacct t 21

<210>26

<211>21

<212> RNA

<213> Artificial sequence

<400>26

aaccaucauu gggaagccct t 21

<210>27

<211>21

<212> RNA

<213> Artificial sequence

<400>27

aacagugcag gacacaacct t 21

<210>28

<211>21

<212> RNA

<213> Artificial sequence

<400>28

uaacacaucc caacagugct t 21

<210>29

<211>21

<212> RNA

<213> Artificial sequence

<400>29

uagcaucucc aggguaagct t 21

<210>30

<211>21

<212> RNA

<213> Artificial sequence

<400>30

uagugucaag gucuauucct t 21

<210>31

<211>21

<212> RNA

<213> Artificial sequence

<400>31

uuagccucag gagaauugat t 21

Claims (9)

1. A small interfering nucleic acid, wherein the sequence of the small interfering nucleic acid is the nucleotide sequence shown in PGENE-3, or the sequence of the small interfering nucleic acid is chemically modified to the nucleotide sequence shown in PGENE-3 The resulting nucleotide sequence, wherein, PGENE-3 sense strand: 5'-CCUUAUCACCGCUGGAGAAdTdT-3' Antisense strand: 5'-UUCUCCAGCGGUGAUAAGGdTdT-3'. 2. The small interfering nucleic acid according to claim 1, wherein the chemical modification is at least one of the following modifications: (1) Modification of the phosphodiester bond connecting the nucleotides in the nucleotide sequence of the small interfering nucleic acid; (2) Modification of the ribose sugar in the nucleotide sequence of the small interfering nucleic acid; (3) Modification of bases in the nucleotide sequence of small interfering nucleic acids. 3. A cosmetic composition for inhibiting melanin generation and deposition or removing melanin, characterized in that the cosmetic composition contains the small interfering nucleic acid as claimed in claim 1 or 2 as an active ingredient. 4. The cosmetic composition according to claim 3, wherein the cosmetic composition also contains a carrier, and the carrier is selected from one or more of auto vegetable oil, hydrocarbon oil, ester, higher fatty acid and higher fatty alcohol. 5. The cosmetic composition according to claim 4, wherein, relative to 100 parts by weight of the small interfering nucleic acid, the content of the carrier is 1000-10000000 parts by weight. 6. The cosmetic composition according to claim 4 or 5, wherein the cosmetic composition also contains other ingredients for the cosmetic composition, and the other ingredients for the cosmetic composition are selected from moisturizing agents, coloring agents , preservatives, thickeners, antioxidants and surfactants in one or more. 7. The cosmetic composition according to claim 6, wherein, relative to 100 parts by weight of the small interfering nucleic acid, the content of the other ingredients for the cosmetic composition is 1000-10000000 parts by weight. 8. The cosmetic composition according to claim 3, wherein the cosmetic composition is emulsion, ointment, liquid, aerosol or powder. 9. The application of the small interfering nucleic acid according to claim 1 or 2 in the preparation of a cosmetic composition for inhibiting melanin production and deposition or removing melanin.