CN101054579A - A siRNA capable of preventing and curing hair loss and accelerating hair growth, and preparation method and application thereof - Google Patents

Abstract

The present invention belongs to biological preparation technology field, more specificly a siRNA for preventing hair loss and promoting hair growth and its preparation method and uses. It comprises: constructing long double-chain RNA(dsRNA) expression vector pET-SRAR related to preventing hair loss and promoting hair growth and expressing highly in E.Coli, transforming E.Coli and fermenting to produce long double-chain RNA, obtaining dsRNA, homogenizing for destroying cell wall and extracting E.Coli total RNA from fermented E.coli, purifying extract and obtaining dsRNA, hydrolyzing and purifying to obtain small interference RNA (siRNA),embedding siRNA using liposome, obtaining siRNA for preventing hair loss and promoting hair growth. The present invention has obvious effect on promoting hair growth.

Description

A kind of siRNA that prevents and treats alopecia and promotion hair tonic and its production and application

Technical field

The invention belongs to the biologic product technology field, be specifically related to a kind of siRNA that prevents and treats alopecia and promotion hair tonic and preparation method thereof, and the application of this siRNA aspect control alopecia and promotion hair tonic.

Technical background

(1) about alopecia

Change, the pressure of routine work and the generation of transgenation of pollution, people life style and the standard of living of producing along with modern industrialization, alopecia has become more and more general at whole human society, more and more serious, too high cost also is difficult to realize the control to alopecia, cause various forms of baldnesses, had a strong impact on people's physical and mental health and quality of life.Modal baldness is a male baldness, and in every year, about 1,500,000,000 U.S. dollars of the U.S. 6,000 ten thousand people (2/3 male sex) cost are conquered baldness.

The reason that causes alopecia is a lot: mainly can ascribe two aspects to: (1) cause of disease alopecia; cause by body metabolism disease or the change of local skin disease; as circular alopecia, seborrheic alopecia disease, atrophic alopecia, scar alopecia etc.; some diseases such as syphilis, lupus erythematosus, dysthyreosis, hypoferric anemia, malnutrition etc. also can cause alopecia; also have, the use unusual or some drugs (as anticarcinogen, oral contraceptive etc.) of the change of environment and diet, emotional stress also can cause alopecia in various degree.(2) gene baldness, result of study show that testosterone is changed into dihydro testosterone (DHT) under the effect of steroid reductase enzyme, and the latter is toxigenicity in cell, makes cell can not get blood supply and death.5-testosterone reductase enzyme (steroid 5 α-reductase in the hair follicle of hair loss patient, EC1.3.99.5) concentration or hyperactivity, especially at patient's forehead, the hair follicle at position, the crown, the activity of enzyme is high especially, when male hormones (testosterone) are secreted, this kind of enzyme can be the testosterone metabolism dihydro testosterone DHT, the DHT of sufficiently long time and sufficient dosage stays in the hair follicle, the toxicity of DHT can allow the vasoconstriction of hair follicle periphery, blood flow reduces, make hair follicle under-nutrition and atrophy gradually, downright bad, become fine or hair stops growing fully, so that present the baldness of various degree.The effect of DHT also comprises: increase health and facial hair, acne and acne, alopecia areata (hair minimizing), prostatomegaly.

Growth has active effect to male sex hormone to bald and chaeta, lacks male sex hormone and can cause the growth that scalps of hair composing type, and chaeta is grown and also depended on male sex hormone.(Androgenetic alopecia AGA) often is used to describe the forfeiture of masculinity and femininity heredity responsive type medelling hair of scalp to the alopecia of male sex hormone origin, be also referred to as male sex's sample baldness (MPHL), or the male sex is bald, women's sample baldness (FPHL).It is androgenic unusual responsive to round-robin that MPHL and FPHL betide the scalp hair follicle.

The dihydro testosterone is to greater than testosterone 5 times of the avidity of androgen receptor.2 classes, 5 α R are arranged, and the first kind comes across in the elaeodochon, and the second class distribution and urinary tract and hair follicle are all by the liver synthesis secretion.Thereby also there is certain positive relationship in the activity of androgen receptor with alopecia

FDA only ratifies 2 kinds of treatment baldness medicines, and a kind of is minoxidil (Upjhon production), and the user is few; Another kind is to use Finasteride (Propacia that Merck Co. produces), and effect is obvious, but can cause the female reproduction defective, only for the male sex uses, can suppress the generation of DHT (dihydrotestosterone) (dihydro testosterone).Two kinds of medicines all are the inhibitor of reductase enzyme, realize preventing and treating alopecia by the generation that suppresses DHT, and then reach the purpose that promotes hair tonic, but the both has certain side effect.

The product of control alopecia and promotion hair tonic is multifarious, and most products have certain side effect, use trouble, and it is poor to take effect.Seek specificity, safety, nothing or low toxic side effect, control alopecia easy to use and promote the method for hair tonic and product is the target always pursued of medical science, health care, beauty industry in recent years.The siRNAs of the control alopecia of the present invention's preparation and promotion hair tonic has clear and definite, special, the water-soluble convenient advantage of using of target site.

(2) about the RNA perturbation technique

The mRNA molecule that RNA disturbs (RNAi) to refer to that double-stranded RNA (dsRNA) molecule can bring out specifically with its homologous sequence is degraded, and causes the phenomenon of corresponding gene expression inhibiting, is gene silencing (PTGS) phenomenon after a kind of special transcribing.The RNAi technology is based on the RNAi phenomenon and Protocols in Molecular Biology that the inhibition specific gene developed is expressed.The report of relevant RNAi phenomenon appears at nineteen ninety the earliest, report the common inhibition phenomenon in the transgenic plant simultaneously by two different research groups, in nearly all eukaryotes such as nematode, fruit bat, zebra fish and mouse, observed the RNAi phenomenon again later on.1999, Hamilton and Baulcombe have detected length in the plant that the RNAi phenomenon takes place be the RNA fragment of 21-25 Nucleotide, and it is necessary that these RNA fragments are proved to be RNAi, is called small molecules interference RNA (siRNA).Achievement in research in the fruit bat has been illustrated the mechanism of RNAi substantially, when the dsRNA of long-chain enters cell, it is discerned and is sheared into the siRNA of 21-25nt by a kind of Dicer hydrolase nucleic acid, double-stranded siRNA is unwind by the RNA desmolase, form with strand combines formation RNA combined enzyme agent with another hydrolase nucleic acid, single stranded RNA in the complex body guides combined enzyme agent recognition sequence complementary mRNA and with its hydrolysis with it as guide, thus the accurate translation of suppressor gene specifically.In nematode and plant, the siRNA of strand also can be used as the primer of polyreaction except playing " guide " effect.Under the effect of the RNA polymerase (RdRP) that RNA relies on, be template with mRNA, the siRNA of strand is the synthetic complementary strand of primer, makes the mRNA of strand become double-stranded RNA, new synthetic dsRNA is then cut into siRNA by nuclease again.The effect of RdRP makes the signal of RNAi obtain amplifying, and suppresses as long as the dsRNA of denier just can cause intensive genetic expression.The characteristic of the efficient and single-minded ground of RNAi inhibition of gene expression at first is widely used in the nematode functional genome research.In mammalian cell, the dsRNA that surpasses 30bp can cause the inhibition and the non-specific RNA degraded of translation initiation by activating PKR system and RNase L, finally causes nonspecific genetic expression to suppress.This mechanism has hindered the application of RNAi technology in mammalian cell.The complementary double-stranded siRNA of the 21nt of human synthetic such as Elbashir has brought out RNAi mechanism in multiple mammalian cell, and PKR system and RNase L have been avoided, suppressed to specificity the expression of goal gene, shown that the RNAi technology can successfully be applied to Mammals.

It is preparation siRNA that the RNAi technology is applied to mammiferous key.SiRNA preparation at present has several different methods: use intestinal bacteria III type RNase or Dicer hydrolysis method etc. behind chemical synthesis, cell inner expression method and the in-vitro transcription long segment dsRNA.But above method costs an arm and a leg, not easy to operate and popularization difficulty.

Summary of the invention

The object of the present invention is to provide a kind of control alopecia efficient, easy, with low cost and promote siRNAs molecule of hair tonic and its production and application.

The artificial gene that the present invention is formed by connecting testosterone reductase gene fragment (SR) and androgen receptor gene fragment (AR) is building up to express to be had on the expression vector pET-SAAK of neck ring structure SR-AR double-stranded RNA (dsRNA); Ferment behind the transformed into escherichia coli, express and produce dsRNA; Centrifugal collection coli somatic with the broken bacterium of clarifixator crush method, obtains the RNA extract, and this extract adsorbs with the CF-11 resin, flush away single stranded RNA and dna molecular, and wash-out is collected the dsRNA part, and elutriant reclaims by alcohol precipitation and obtains dsRNA; With RNase III the dsRNA of long-chain is cut into the siRNAs (small molecules interference RNA) of 18-25bp again, removes the siRNAs that RNase III obtains the control alopecia and promotes hair tonic.Pass through (Yelkin TTS etc.) lipid again and siRNAs is carried out liposome embedded, be prepared into the siRNA liposome.This siRNA liposome is smeared at the scalp of alopecia place, can prevent and treat alopecia and promote hair tonic.

Among the present invention, cause the enzyme of hair follicle necrosis with the catalysis male hormone metabolism--the 5-testosterone reductase enzyme (steroid5 α-reductase in the hair follicle, EC1.3.99.5) (SR) and androgen receptor (Androgen receptor) (AR) gene be target position, screen suitable gene order synthetic oligonucleotide fragment; And be connected to the purpose artificial gene by molecular biology method, and the specific limited restriction enzyme site is introduced in design, carry out molecular cloning with the specific limited restriction enzyme site, obtain a recombination SRAR (SEQ.ID.NO1), and the coli expression carrier pET-SRAR of construction expression SR-AR double-stranded RNA.

The SR-AR dsrna expression vector that makes up is a kind of neck ring structure, can express the dsRNA of target gene fragment in intestinal bacteria, and by fermentation expression, expression level reaches 400-500 milligram/100 gram wet thallus.Thalline after the fermentation is by the clarifixator broken wall extracting RNA that homogenizes, and dsRNA obtains dsRNA solution through CF-11 resin absorption purifying, further concentrates the purpose dsRNA that obtains purifying with alcohol precipitation or with propyl carbinol.

The present invention is in the method for the dsRNA of expression in escherichia coli control alopecia and promotion hair tonic, comprise and make up a kind of expression vector pET-SRAR (seeing Fig. 1,2) that can in intestinal bacteria, efficiently express dsRNA, carrier is the plasmid that sets out with pET-22b (available from U.S. Novagen company), between Bam HI and Eco RI restriction enzyme site, insert the external source fragment of one section non-bacterial origin as loop, obtain the pET-loop plasmid, structure as shown in Figure 1.The basic comprising element comprises forward sequence A fragment, inverted repeats B fragment, wherein A, B fragment form (SRAR) by testosterone reductase gene fragment (SR) and androgen receptor gene fragment (AR) through reorganization, and two segmental gene orders are seen SEQ.ID.NO1.Concrete clone's flow process is seen shown in Figure 2, inserts rightabout SRAR gene fragment in pET-loop plasmid both sides with proper restriction site, and the expression vector of the double-stranded RNA that builds is called as pET-SRAR.Calcium Chloride Method by routine is transformed into expression vector in the intestinal bacteria [BL21-CodonPlus  (DE3)-ril].Single colony inoculation of choosing conversion is in the LB substratum that contains 100 μ g/mL penbritins, in 37 ℃ of shaking tables with the rotating speed overnight incubation of 250rpm, getting 20 μ L seed liquor is inoculated in the fresh LB substratum that contains 100 μ g/mL penbritins of 2mL, similarity condition continues shaking culture down, as the 600nm of bacterium liquid light absorption value (OD ₆₀₀) when reaching near 1, add IPTG and induce to final concentration 0.1mM, the same terms continues to cultivate collected thalline in 3 hours later on.Method by alkaline lysis extracting plasmid DNA is extracted nucleic acid from thalline, extract is handled with commercially available RNaseA, with 1% sepharose to handling sample electrophoretic analysis, EB staining analysis expression of results.Because loop reaches two ends for repeating complementary sequence, expression product can form the complementary long dsrna naturally under physiological condition.Large scale fermentation production result shows that the expression vector of the expression dsRNA that present method makes up ferments in intestinal bacteria, the coli somatic of 100g weight in wet base can obtain the dsRNA sample of 400-500mg.

Among the present invention, the sequence of segmental two gene fragments of recombination respectively by the synthetic oligonucleotide fragment through the artificial gene technology obtain and order-checking correct, with specific limited endonuclease restriction enzyme site clone serve as series connection reorganization A segment (450bp) and with the A fragment complementation the opposite B fragment (450bp) of orientation.

Among the present invention, the intestinal bacteria ril of fermentative production long dsrna (1.5g/300L) induces fermentation by lactose (6kg/300L) or isopropylthio-(ITPG) after transforming cultivation, express output with special raising.

The siRNAs of the present invention's preparation, specificity ground suppresses to cause metabolism key enzyme (5-α-testosterone reductase enzyme) gene of trichomadesis and the expression of acceptor gene by RNA interferential mechanism is effective, reaches the effect of control alopecia and promotion hair tonic.

Among the present invention, step by liposome embedded siRNAs is as follows: according to the injection stage soybean lecithin: cholesterol (AR): vitamin-E (AR)=100: the prescription of 8-12: 0.8-1.2, take by weighing above lipid, be dissolved in an amount of chloroform, in the pyriform round-bottomed flask under 35-40 ℃ of temperature through rotation decompression thin film concentration method, evaporation forms adipose membrane, removes remaining chloroform with pure nitrogen gas.Under the 35-40 ℃ of condition of bleeding, the granulated glass sphere number that adds the 5-8mm diameter, the phosphate buffered saline buffer (containing 0.5% tween 80,10mg vitamins C and the siRNAs that treats embedding) that adds 23-27 milliliter pH7.0-7.4 with 1 gram adipose membrane is rotated aquation 15-20 minute to oyster white to adipose membrane.Under the ice-water bath condition hydrating fluid is carried out supersound process 2-3 time, each 3-5 minute.Use (Sephadex G-100) sieve chromatography purifying to obtain the liposome of embedding siRNAs again.

The liposome siRNAs of the present invention's preparation smears through alopecia place scalp and penetrates into skin, reaches the purpose of control alopecia and promotion hair tonic.

Advantage of the present invention:

(1) testosterone reductase gene and the necessary androgen receptor gene of DHT performance biological action that directly produces with catalysis dihydro testosterone (DHT) serves as to disturb target position, by Protocols in Molecular Biology the gene fragment of two genes is recombinated, utilize the cheap production system fermentative production of microorganism to prepare the necessary dsRNA of siRNAs, dsRNA is applied to the siRNAs of biologic activity effect is arranged through RNase III hydrolysis, and utilize the effect of microRNA interferential special and suppress the expression of testosterone reductase gene and androgen receptor gene efficiently, reaching the formation that suppresses DHT and the DHT purpose of necessary androgen receptor that plays a role, thereby realize the control alopecia and promote hair tonic.

(2) made up the expression vector that can in intestinal bacteria, efficiently express dsRNA.Utilize a series of Protocols in Molecular Biology, design and clonal expression element have made up the expression vector pET-SRAR that is suitable for specifically expressing dsRNA in intestinal bacteria.

(3) provide the method for effective siRNA of preparation.The enzymatic property that utilizes RNase III is to through extracting and the dsRNA of purifying is hydrolyzed, and what obtain 18-25bp can be used for microRNA interferential siRNAs.

(4) provide the method for liposome embedded siRNAs, improved stability and the transdermal characteristic of siRNAs, made product preferably at external preparation for skin.

Description of drawings

Fig. 1: dsrna expression vector pET-loop structural representation.

Fig. 2: pET-SRAR clones structural representation.

Fig. 3: SRAR dsRNA is at the electrophoretic analysis figure of expression in escherichia coli.

Caption: M1:1Kb dna molecular amount mark

1: handle through RNase A without the nucleic acid in the IPTG inductive expression strain;

2:IPTG induces the nucleic acid in the expression strain of back to handle through RNase A.

The dsRNA of arrow indication for expressing.

Fig. 4: the DEAE post separates the elution curve and the electrophoretic analysis figure of RNase III and hydrolysate thereof.

Caption: A, elution curve; B, top are 15% non-sex change PAGE, and the lower section is 10% SDS-PAGE electrophoretic analysis figure, and protein electrophorese dyes with Coomassie brilliant blue, and nucleic acid electrophoresis dyes with EB.Peak1 is the elution peak (having only the protein colour developing) of RNaseIII, and peak2 is blended oligonucleotide fragment (having only nucleic acid colour developing peak).

Fig. 5: Superdex 75 sieve chromatography separation and purification esiRNA.

Caption: A is the elution peak when contacting separation with two Superdex75 posts; B is the pairing purification of samples electrophorogram of A peak figure.The sample that 15% non-sex change PAGE electrophoretic analysis is collected.S is a sample solution, and 1-6 is effusive each component of molecular sieve.EB dyeing.The 2-4 elution peak that purifying is collected among the figure is the purpose siRNAs of 21-25bp.

Embodiment:

1. the clone of each Expression element: the human testicle hormone reductase enzyme of announcing according to Genebank and the sequence of androgen receptor gene, select the associated clip sequence respectively, design and synthetic oligonucleotide fragment, connect the artificial gene fragment SRAR (for SEQ.ID.NO1) that each oligonucleotide fragment obtains two gene fragments, and introducing specific limited endonuclease digestion site is used to clone required, press the strategy clone dsrna expression vector pET-SRAR of Fig. 1 and Fig. 2, the expression plasmid behind the clone is cut with PCR reaction and enzyme and is reacted evaluation.

2. fermentative production: pET-SRAR is transformed in the intestinal bacteria [BL21-CodonPlus  (DE3)-RIL] by conventional calcium chloride transformation, picking list colony inoculation is in the LB substratum that contains 100 μ g/mL penbritins, in 37 ℃ of shaking tables with the rotating speed overnight incubation of 250rpm, respectively getting 20 μ L seed liquor is inoculated in the fresh LB substratum that contains 100 μ g/mL penbritins of 2mL, similarity condition continues shaking culture down, as the 600nm of bacterium liquid light absorption value (OD ₆₀₀) when reaching near 1.0, adding IPTG to final concentration 0.1mM, the same terms continues to cultivate after 3 hours collects thalline.Method with alkaline process extracting plasmid DNA is extracted nucleic acid from thalline, extract is handled with RNase A, carry out electrophoretic analysis with 1% sepharose to handling sample, the EB coloration result of Fig. 3 shows, from through inducing and without inductive cultivate extracting the bacterial strain to nucleic acid after RNase A handles, have only to have transformed in the cellular lysate liquid of expressing the hairpin structure plasmid to have tangible RNA band through the IPTG inductive, and without the RNA in the inductive cellular lysate liquid after RNase A effect almost without any band.RNase A is the specific RNA lytic enzyme of a kind of strand, and the result shows, is not the dsRNA of process abduction delivering by the small molecular weight RNA of RNase A hydrolysis.The pET-SRAR carrier that makes up in the invention can be expressed the purpose double-stranded RNA.Large scale fermentation production result shows that the expression vector that the present invention makes up can be expressed the purpose double-stranded RNA preferably in intestinal bacteria, and it is 400-500 milligram purpose double-stranded RNA that expression amount reaches per 100 gram wet thallus.

3.dsRNA extract: the Escherichia coli fermentation thalline 30-50 gram of getting cold storage, fully be suspended in phosphoric acid salt (PBS) damping fluid of 500-600mL, clarifixator homogeneous broken wall, 68-72 ℃ of water-bath heated 30 minutes, the 7000rpm low-temperature centrifugation was removed the precipitating proteins of sex change in 30 minutes, and it is 20% that supernatant is partly added ethanol to final concentration.

4.dsRNA purifying: CF-11 resin (every 500mL broken wall supernatant with 30 gram resins) is with after the 20% ethanol balance, is added in the broken wall supernatant treatment solution to stir, and mixture fully adsorbs on ice bath and spends the night.The adsorption liquid that spends the night is removed supernatant liquor by suction filtration, and with 18% spirituous solution filtering and washing of ice bath precooling 3-4 time, fully flush away DNA and single stranded RNA.Then collect double-stranded RNA with the STE damping fluid suction filtration wash-out that is preheated to 60-70 ℃.Add the dehydrated alcohol of 2 times of volumes and the 3M NaAc of 0.1 times of volume in the dsRNA suction filtration liquid of collecting,-20 ℃ precipitate 30 minutes, 10000rpm got precipitation in centrifugal 20 minutes, precipitate cold ethanol suspension flush away salt with 70%, centrifugal stream is got precipitation, precipitation is dissolved the dsRNA that obtains purifying ,-40 ℃ of refrigerator preservations with the TE of 10mM pH8.0.Fig. 4 is the agarose gel electrophoretogram of the RNA sample before and after the purifying, and as can be seen from the figure the sample that obtains of purifying is single long-chain dsRNA.The OD260/OD280=2.0 of the dsRNA that CF-11 chromatography column purifying obtains shows the purity height of sample.The 100g coli somatic obtains dsRNA sample 400-500mg altogether.

5.siRNA acquisition: through dsRNA that CF-11 chromatography column purifying obtains after quantitatively, by every microgram ds RNA with the RNase III in 0.02-0.03 microgram intestinal bacteria source 37 ℃ of hydrolysis 3 hours.Contain 1mM DTT in the reaction soln, 50mMTris-HCl, 10mM MnCl ₂, 50mM NaCl, pH7.5.Add the EDTA termination reaction in the reaction solution, electrophoresis detection result shows that hydrolysis promptly reached good hydrolysis effect in 1 hour, saw shown in Figure 4.

6.siRNA purifying: double-stranded RNA by intestinal bacteria RNase III hydrolysis after, add in the solution NaCl to final concentration be 250mM, go up sample then to using sample-loading buffer (10mM Tris-HCl, 250mM NaCl, 1mM EDTA, pH7.5) on the DEAE post that balance is crossed (1 * 10cm resin), continue to be washed till baseline, then the hydrolysate that is adsorbed with the NaCl concentration gradient wash-out of 250mM to 550mM with sample-loading buffer.In the hydrolysate of DEAE column purification, include the oligonucleotide fragment of the different sizes of siRNA of each target fragment, further select for use sieve chromatography to separate this mixture, with the BIOLOGIC DUOFLOW of BioRad company with other ^TMThe 25mL Superdex75 FPLC column of CHROMATOGRAPHY SYSTEM and pharmacia, damping fluid is PBS, eluted product is collected size after 15% polyacrylamide gel electrophoresis (PAGE) detection be the product of 21-25bp, frozen standby in-20 ℃ of storages after the packing.

7.siRNAs liposome embedded, according to Yelkin TTS: cholesterol: the prescription of vitamin-E=100: 8-12: 0.8-1.2, weigh respectively, be dissolved in an amount of chloroform, 35-40 ℃ of decompression rotary evaporation forms adipose membrane in the pyriform round-bottomed flask, removes residual solvent with the pure nitrogen gas circulation.The granulated glass sphere that adds ten several 5-8mm diameters, 35-40 ℃ of temperature and under the inflated with nitrogen condition, the phosphate buffered saline buffer (contain 0.5% tween 80 and treat the siRNAs of embedding) that adds 25 milliliters of pH7.2 with 1 gram adipose membrane is rotated aquation 15-20 minute to oyster white to adipose membrane.Under the ice-water bath condition hydrating fluid is carried out supersound process 2-3 time, each 3-5 minute.Sephadex G-100 sieve chromatography purifying obtains liposome siRNAs.

8. dip in finger and get the siRNAs liposome solutions a little carries out the administration of smearing of scalp in alopecia scalp district, each once used week back alopecia quantity obviously to reduce, and was the alopecia extinction tests gradually sooner or later every day.Use after one month, unusual alopecia phenomenon no longer occurs, the tiny hair of local visible new growth continues to use to 2 months, and newborn hair can obviously appear in alopecia scalp place.80% user's use result shows that the siRNA of the present invention's preparation has tangible control alopecia and promotes hair regrowth.

Sequence table

SEQ.ID.NO1：

cgcgaagtga?tccagaaccc?gggccccagg?cacccagagg?ccgcgagcgc?agcacctccc ggcgccagtt?tgctgctgct?gcagcagcag?cagcagcagc?agcagcagca?gcagcagcag cagcagcagc?agcagcagca?gcagcagcag?cagcagcaag?agactagccc?caggcagcag cagcagcagc?agggtgagga?tggttctccc?caagcccatc?gacggcgacg?gcgacggcgg tggtggagga?gcgcctgctg?gctgcgttcg?cctaccttca?gtgcgccgtg?ggctgcgcgg tcttcgctcg?gaatcgtcag?acgaactcag?tgtacagccg?ccacgcgcca?cccagccgca ggctccgagt?gccggcgcgg?gccacccggg?tggtgcagaa?gctgccctca?ctggccctgc cgctctacca?gtacaccagt?gagtccaccc

Claims (5)

1, a kind of siRNA that prevents and treats alopecia and promote hair tonic is characterized in that it is obtained by following method: the artificial gene that gene segment SR and AR are formed by connecting is building up to expression to be had on the expression vector pET-SAAK of neck ring structure SR-AR double-stranded RNA; Ferment behind the transformed into escherichia coli, express and produce dsRNA; Centrifugal collection coli somatic with the broken bacterium of clarifixator crush method, obtains the RNA extract, and this extract adsorbs with the CF-11 resin, flush away single stranded RNA and dna molecular, and wash-out is collected the dsRNA part, and elutriant reclaims by alcohol precipitation and obtains dsRNA; With RNaseIII the dsRNA of long-chain is cut into the siRNAs of 18-25bp again, removes RNase III, obtain the control alopecia and promote the siRNAs of hair tonic; Wherein, SR is a testosterone reductase gene fragment, and AR is the androgen receptor gene fragment.

2, a kind of expression vector pET-SRAR at expression in escherichia coli dsRNA, it is characterized in that carrier is the plasmid that sets out with pET-226, between Bam HI and Eco RI restriction enzyme site, insert the external source fragment of one section non-bacterial origin as loop, obtain the pET-loop plasmid, the basic comprising element comprises forward sequence A fragment, inverted repeats B fragment, wherein A, B fragment are formed through reorganization by testosterone reductase gene fragment and androgen receptor gene fragment, and two segmental gene orders are SEQ.ID.NO1.

3, a kind of siRNA liposome is characterized in that by lipid the described siRNAs of claim 1 being carried out liposome embedded back obtains.

4, the preparation method of a kind of siRNAs claimed in claim 1 is characterized in that concrete steps are as follows:

Make up the described expression vector pET-SRAR of claim 2, the artificial gene that gene fragment SR and gene fragment AR are formed by connecting is building up to express to be had on the expression vector pET-SAAK of neck ring structure SR-AR double-stranded RNA; Ferment behind the transformed into escherichia coli, express and produce dsRNA; Centrifugal collection coli somatic with the broken bacterium of clarifixator crush method, obtains the RNA extract, and this extract adsorbs with the CF-11 resin, flush away single stranded RNA and dna molecular, and wash-out is collected the dsRNA part, and elutriant reclaims by alcohol precipitation and obtains dsRNA; With RNase III the dsRNA of long-chain is cut into the siRNAs (small molecules interference RNA) of 18-25bp again, removes RNase III, obtain the control alopecia and promote the siRNAs of hair tonic.

5, a kind of preparation method of siRNAs liposome as claimed in claim 3, it is characterized in that concrete steps are as follows: according to the injection stage soybean lecithin: cholesterol: the prescription of vitamin-E=100: 8-12: 0.8-1.2, take by weighing above lipid, be dissolved in the chloroform, under 35-40 ℃ of temperature through rotation decompression thin film concentration method, evaporation forms adipose membrane, remove remaining chloroform with nitrogen, under the 35-40 ℃ of condition of bleeding, add the granulated glass sphere number of 5-8mm diameter, adding 23-27 milliliter pH value with 1 gram adipose membrane is 0.5% tween 80 that contains of 7.0-7.4,10mg vitamins C and treat that the phosphate buffered saline buffer of the siRNAs of embedding is rotated aquation 15-20 minute to oyster white to adipose membrane; Under the ice-water bath condition hydrating fluid is carried out supersound process 2-3 time, each 3-5 minute; Obtain the liposome of embedding siRNAs again with the sieve chromatography purifying.

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