CN1488761A - A double-stranded DNA molecule and its use in preparing medicine for inhibiting hepatitis B virus replication - Google Patents
A double-stranded DNA molecule and its use in preparing medicine for inhibiting hepatitis B virus replication Download PDFInfo
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Abstract
The present invention relates to a method for preparing double-stranded RNA molecule for RNA interference of small interference RNA molecule by utilizing colibacillus fermentation and the application of these RNA molecules in preparation of medicine for curing hepatitis B. The Ids RNA or siRNA obtained by said invention is mixed with hepatitis B virus expression vector in proper proportion, and injected into the mouse body by vane caudalis hydraulic pressure method, and its result shows that the non-homologous sequence IdsRNA and siRNA basically do not inhibit the replication of hepatitis B virus in mouse body, and these RNA molecules are mixed with hepatitis B virus gene.
Description
Technical field
The invention belongs to molecular biology and biological medicine technology field.Be specifically related to a kind of Escherichia coli fermentation that utilizes and prepare the method for RNA interference, and these RNA molecules are used to prepare the novel drugs for the treatment of hepatitis B with double-stranded RNA (dsRNA) molecule or siRNA (siRNA) molecule.
Background technology
RNA disturbs (RNA interference, RNAi) be meant that the mRNA molecule that double-stranded RNA (dsRNA) brings out specifically with its homologous sequence is degraded, the phenomenon that causes the corresponding gene expression inhibiting, be a kind of special posttranscriptional gene expression silencing (post transcriptional gene silence, PTGS) phenomenon.The RNAi technology is meant the Protocols in Molecular Biology of the inhibition specific gene expression of developing based on the RNAi phenomenon.The report of relevant RNAi phenomenon the earliest appears at nineteen ninety, has reported common inhibition (co-suppression) 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.
The research of RNAi principle is started from genetic analysis in nematode (C. elegans), found a series of genes relevant in this way with RNAi.1999, Hamilton and Baulcombe have detected length in the plant that the RNAi phenomenon takes place be the RNA fragment of 21-25 Nucleotide (nt), it is necessary that these RNA fragments are proved to be RNAi, is called siRNA (short interfering RNA, " siRNA ").Research in fruit bat, the mechanism of RNAi is illustrated substantially: when the dsRNA of long-chain enters cell, it is discerned and is sheared into the siRNA of 21-25nt by the hydrolase nucleic acid of a kind of Dicer of being called as, 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 RdRP, 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 Dicer 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 makes it obtain using widely aspect gene functional research.With antisense nucleic acid, ribozyme with utilize homologous recombination to carry out technology such as mouse gene knockout and compare, the RNAi technology has incomparable superiority.When utilizing the expression of antisense technology and ribozyme technology suppressor gene, how from goal gene, to select effectively to suppress sequence, up to the present still not having accurate theoretical direction, can only be a process that relatively wastes time and energy by constantly attempting and improving.Utilize the RNAi technology then not have this problem, studies show that the secondary structure of goal gene mRNA and the efficient that GC content can not influence RNAi.In nematode and plant, the effect of RdRP can make the signal of RNAi obtain amplifying, and suppresses as long as the dsRNA of denier just can cause intensive genetic expression.Especially in nematode, as long as feed nematode with the intestinal bacteria that can express goal gene dsRNA, this expression of gene will be suppressed.This superiority makes the RNAi technology at first be widely used in the nematode functional genome research, and has greatly advanced the functional genome research of nematode.
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.People's such as Elbashir work has solved this problem.They have brought out RNAi mechanism with the complementary double-stranded siRNA of artificial synthetic 21nt in multiple mammalian cell, and have avoided PKR system and RNase L, have suppressed to specificity the expression of goal gene.Their work has proved that first the RNAi technology can be applied to mammalian cell.
The RNAi technology not only demonstrates irreplaceable superiority in functional genome research, and becomes possibility for some disease that is difficult to treat at present provides a kind of new treatment means.Virus disease, especially the disease that causes of retroviral infection, resemble HIV and infect the health that the AIDS that causes viral hepatitis sick and that HBV, HCV infection causes is having a strong impact on people, and present treatment means can't reach radical cure, the RNAi The Application of Technology may provide better treatment and preventive means.In the cultured cell in vitro system, realized at present utilizing the RNAi technology to suppress the infection duplication of HIV, HCV, HBV and influenza virus.Can predict the RNAi technology and can provide new treatment means for modern frequently-occurring diseases such as tumour, cardiovascular disorder and diabetes equally.
The key that the RNAi technology is applied to the Mammals system is preparation siRNA.Present nearly 3 kinds of preparation methods: chemical synthesis, cell inner expression method and in-vitro transcription long segment dsRNA (ldsRNA) back prepare siRNA with intestinal bacteria III type RNase or Dicer hydrolysis.Can only prepare siRNA with chemical synthesis at first.A lot of biotech companies all provide RNA synthetic service.As long as from goal gene mRNA sequence, select suitable base sequence, just can after sex change annealing, just obtain double-stranded siRNA by the just RNA chain and the complementary strand thereof of synthetic this sequence of biotech company.This method is fairly simple directly, and prices are rather stiff owing to the RNA synthetic, limited applying of chemical synthesis.In April, 2002, several research groups have reported simultaneously that almost the double-stranded RNA that utilizes mammalian cell III type rna polymerase promoter sublist to reach short segmental (19-21bp) band loop-stem structure can produce RNAi in the mammalian cell system in the world, the inhibiting rate that specific gene is expressed reaches more than 90%, is better than the siRNA of synthetic.The generation of this technology makes the RNAi technology of Mammals system become more easy to be more ripe.It is yet this method will be applied to clinically, be similar to gene therapy, and bigger than gene therapy difficulty.Overcome except needs the difficulties such as the required target that overcomes of gene therapy, security, it is higher to the requirement of integration efficiency, because it is the functions reversed that will increase a certain gene with general gene therapy, it need suppress the expression of specific gene.Safe and effective and easy method is to dose a patient with or inject siRNA, and this is similar to antisense nucleic acid medicament in the past.LdsRNA with intestinal bacteria RNase III or Dicer hydrolysis in-vitro transcription prepares the preparation method that siRNA is present existing most convenient.The invention provides a kind of more efficient, easy and siRNA preparation method cheaply.
Hepatitis B virus can have a strong impact on liver function after infecting the human liver, especially can cause multiple serious chronic hepatic diseases after forming chronic infection, as serious diseases such as hepatic fibrosis, liver cirrhosis and liver cancer.About 1/3rd patients have the chronic viral hepatitis B medical history in the Patients with Primary, and the serum HBsAg positive rate is 66%～80%, head and shoulders above the positive rate of normal population 10%～15%.China is the high infected zone of hepatitis B virus.HBsAg recall rate in the population of China is about 10%, and the prevalence rate that comprises anti-HBs and anti-HBc is 50-60%.Chronic asymptomatic carrier about 1.2 hundred million.The hepatitis B morbidity is about 27,70/,100,000, wherein chronic viral hepatitis B morbidity 0.1-1%.Because existing treating hepatitis B means poor effect, and face the problem of medicament-resistant mutation strain, therefore still have eager demand developing new treating hepatitis B method.At present external several research groups have reported and have successfully used the RNA perturbation technique has suppressed hepatitis B virus efficiently in cell system and experimentation on animals infection duplication.Inhibiting rate to hepatitis B surface antigen content in the mice serum can reach 94%.Viral DNA in the mouse liver and rna transcription thing also can effectively be suppressed.Although it may be very promising treating hepatitis B novel method that these data show the RNA perturbation technique, this technology is applied to clinical treatment and still need and solve some problems.The method that for example is used for obtaining suppressing the siRNA that virus infection duplicates at present is limited, and equal price costlinesses, is not suitable for mass preparation.Method provided by the invention has remedied this defective, lays a good foundation for using RNA perturbation technique treatment human body hepatitis B.
Summary of the invention
The objective of the invention is to propose a kind of method of utilizing Escherichia coli fermentation mass preparation RNAi with long-chain dsRNA (ldsRNA) and siRNA molecule, and ldsRNA or siRNA molecule are used to prepare the novel drugs for the treatment of hepatitis B virus.
The usefulness Escherichia coli fermentation mass preparation RNAi that the present invention the proposes method of ldsRNA and siRNA molecule, comprise and make up the ldsRNA expression vector, and transformed into escherichia coli host bacterium, by large scale fermentation, obtain the mixture of full RNA and plasmid DNA with alkali-SDS method extractive fermentation thalline; This mixture carries out purifying with the CF-11 post and obtains ldsRNA, and utilizes intestinal bacteria RNase III ldsRNA to be cut into the siRNA of 12-30bp.
Having proposed two kinds among the present invention can be at the expression vector of expression in escherichia coli long-chain dsRNA, specific as follows:
1. the structure of the expression vector of loop-stem structure dsRNA
The construction process of the expression vector of the ldsRNA of band neck ring structure is target gene fragment to be held 3 ' of gene with opposite direction be connected to the segmental both sides of the 3rd segment DNA, be cloned into then in the carrier that has a T7 promotor and go, the plasmid synoptic diagram is seen accompanying drawing 1.After this expression plasmid being transformed in the host bacterium that can express the T7 RNA polymerase, the DNA chain of the positive and negative both direction of goal gene and the 3rd segment DNA chain between the two just can be transcribed successively, become a successive RNA chain.Because the goal gene in this RNA chain partly is complete complementary sequence, whole piece RNA chain will form loop-stem structure under physiological condition, and the double-stranded part in the structure is exactly the ldsRNA of goal gene.
2. the structure of bidirectional promoter expression vector
The structure of bidirectional promoter expression vector is to utilize the expression vector that has the intestinal bacteria replication orgin, add resistant gene and multiple clone site in place, and add relative two promotors and terminator (as shown in Figure 2) respectively in the multiple clone site both sides, just can be after in multiple clone site, inserting target DNA fragment with positive and negative both direction transcribe rna.Because to transcribe out these two RNA chains are complete complementary sequences, thus under physiological condition, can form double-stranded, the double-stranded RNA of Here it is goal gene.
The step of utilizing Escherichia coli fermentation production and separation and purification dsRNA that proposes among the present invention is as follows:
1. thalline fermentation:The Calcium Chloride Method of dsRNA expression vector by routine is transformed in the intestinal bacteria.The intestinal bacteria colony inoculation that contains expression vector is to containing in the antibiotic nutrient solution of corresponding screening, cultivated 8-12 hour, be transferred in the fermentor tank, cultivate after 3-5 hour, add lactose or IPTG, continue to cultivate after 3-5 hour and put jar, collect thalline, carry out ldsRNA extracting and purifying with continuous centrifugal machine.
2.RNA extracting:Suspension (the 50mM glucose that adds 10-20ml in every 1g thalline, 25mM TrisHCl, 10mM EDTA, pH8.0) in, the back that fully suspends adds 2 times of lysates to suspension vol and (contains 0.2NNaOH, 1%SDS), add 1.5 times of potassium acetate solutions to suspension vol (containing the 5M acetate, the 3M potassium ion) gently behind the stirring and evenly mixing, ice bath was placed after 10-20 minute behind the stirring and evenly mixing, the centrifugal 10-30 of 10000-12000g minute, supernatant liquor is transferred in another centrifuge tube.Add and the isopyknic phenol chloroform isoamyl alcohol of supernatant liquor (25: 24: 1), behind the fierce vibration mixing the centrifugal 10-30 of 10000-12000g minute, be used for Mierocrystalline cellulose CF-11 adsorption and purification after collecting supernatant liquor.
3. the adsorption chromatography with Mierocrystalline cellulose CF-11 separates ldsRNA:Adding ethanol to final concentration to extract in the centrifuged supernatant that obtains in above-mentioned 2 is 17-30%, go up sample after the precooling to using STE solution (the 0.1M NaCl that contains same ratio alcohol, 10mM TrisHCl, 1mM EDTA is pH8.0) in the CF-11 chromatography column of pre-equilibration; Wash the CF-11 post continuously to remove single stranded RNA and DNA, until elutriant OD with containing 17-20% alcoholic acid STE solution
260Do not stop when having absorption.Use STE eluant solution double-stranded RNA then, collect effluent liquid, to OD
260There is not the end of when absorption.The collected solution that contains ldsRNA or concentrate with alcohol precipitation or with propyl carbinol, standby.
Propose among the present invention to utilize intestinal bacteria III type RNA enzymic hydrolysis ldsRNA to prepare the method for siRNA as follows: through ldsRNA that the CF-11 purifying obtains after quantitatively, by every microgram ldsRNA with the RNaseIII in 0.02-0.03 microgram intestinal bacteria source at 37 ℃ of hydrolysis 2-4 hours.Contain 1mM DTT in the reaction soln, 20mM Tris-HCl, 0.5mM EDTA, 5mM MgCl
2, 140mM NaCl, 2.7mM KCl, pH7.9.
LdsRNA and siRNA inhibition hepatitis B virus with Escherichia coli fermentation production that the present invention proposes are as follows in the intravital method of duplicating of mouse:
1. can be used for suppressing ldsRNA that hepatitis B virus infection duplicates and the preparation of siRNA
Construction process by long-chain dsRNA expression vector provided by the invention is building up to any fragment of hepatitis B virus gene group in the expression vector of bidirectional promoter expression vector or loop-stem structure ldsRNA, prepare required ldsRNA by the method for Escherichia coli fermentation production and separation and purification ldsRNA of utilizing provided by the invention then, prepare corresponding siRNA with the method for utilizing intestinal bacteria III type RNA enzymic hydrolysis long-chain ldsRNA to prepare siRNA provided by the invention at last.
2. the structure of hepatitis B virus expression vector
Hepatitis B adr hypotype ring-type genomic dna is connected in the BglII/BamHI site of pUCmT plasmid vector (available from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd) after with the BamHI linearizing, and then connects the BamHI linearizing fragment of same viral ring-type genomic dna in the BamHI site of the recombinant plasmid vector that obtains.After PCR and the evaluation of enzyme blanking method, obtain the recombinant plasmid that two copies hepatitis B virus gene group DNA arranges in end to end mode, this plasmid called after pUC-HBV.Quantitative behind this plasmid usefulness QIAGEN company plasmid purification test kit purifying with ultraviolet spectrophotometry.
3. the pUC-HBV of preparation and ldsRNA or siRNA are expelled in the mouse body with tail vein hydraulic method in the proper ratio as stated above, beginning is with the hepatitis B surface antigen content in the different time point detection mice serums after 24 hours, hepatitis B virus e antigenic content, and mouse liver function.The result shows that when the injected dose of pUC-HBV plasmid was 10 μ g/ mouse, the ldsRNA injected dose from 25 μ g to 100 μ g all can significantly suppress duplicating of hepatitis B virus, and does not see the liver function damage.In all test group, with the inhibition best results of the ldsRNA of 100 μ g coding hepatitis B virus DNA replicative enzyme partial sequence.
The above results shows that the double-stranded ldsRNA and the siRNA that are prepared by the present invention can be used for preparing the novel drugs for the treatment of hepatitis B.
Can above-mentioned ldsRNA among the present invention or siRNA be major ingredient, the auxiliary material system that is equipped with is made the injection liquid formulation.Concrete prescription is as follows:
ldsRNA 12.5-50mg
NaCl 8.6g
KCl 0.3g
CaCl
2 0.13g
Add the injection water to 1000ml
Injected dose is the 1.25-5mg/kg body weight.
Advantage of the present invention:
Compare with the in-vitro transcription method with chemical synthesis, siRNA preparation method provided by the invention is more easy, and cost is cheaper, especially is suitable for the industrially scalable siRNA preparation of medicine manufacturing purpose.The ldsRNA of the present invention preparation and siRNA all can be special and suppress hepatitis B virus efficiently at the intravital infection duplication of mouse, thereby can be used for preparing the novel drugs of efficient treatment hepatitis B.
Description of drawings
Fig. 1 expresses the plasmid vector synoptic diagram of the double-stranded RNA of band neck ring structure.
Fig. 2 bidirectional promoter expression vector synoptic diagram.
Fig. 3 pET-HBXII collection of illustrative plates.
Fig. 4 pET-2P plasmid map.
Fig. 5 CF-11 column purification dsRNA.Wherein, A: the e. coli rna extract that contains pET-SEAP2, B: the e. coli rna extract that contains pET-22b, C: the sample of the e. coli rna extract that contains pET-SEAP2 after, D: the sample of the e. coli rna extract that contains pET-22b after through the CF-11 column purification through the CF-11 column purification.
The RNaseIII hydrolysis of Fig. 6 dsRNA.Wherein, 1 is 24bp molecular weight Marker; 2 are hydrolysis 0 hour; 3 are hydrolysis 2 hours; 4 are hydrolysis 4 hours.
Fig. 7 pST plasmid map.
The relative expression quantity of hepatitis B virus surface antigen in first day mice serum after Fig. 8 transfection.
The antigenic relative expression quantity of hepatitis B virus e in first day mice serum after Fig. 9 transfection.
The relative reactivity of first day mice serum neutral and alkali phosphoesterase after Figure 10 transfection.
The relative expression quantity of hepatitis B virus surface antigen in the 4th day mice serum after Figure 11 transfection.
The antigenic relative expression quantity of hepatitis B virus e in the 4th day mice serum after Figure 12 transfection.
The relative reactivity of first day mice serum neutral and alkali phosphoesterase after Figure 13 transfection.
The relative expression quantity of hepatitis B virus surface antigen in the 7th day mice serum after Figure 14 transfection.
The antigenic relative expression quantity of hepatitis B virus e in the 7th day mice serum after Figure 15 transfection.
Embodiment
1. the building process of human hepatitis B virus (HBV virus) X protein genonema loop-stem structure dsrna expression vector is as follows:
With oligonucleotide 5 '-GGAATTC ATG GCT GCT AGG CTG TG-3 ' and 5 '-GGGGTACC GGCAGA GGT GAAAAA GTT G-3 ' is primer, with hepatitis B virus gene group DNA is template, amplify HBV virus X protein gene with PCR method, and hold at 5 ' end and 3 ' and to introduce EcoRI and KpnI site respectively, with above-mentioned restriction enzyme site this gene is cloned into pUC-118 (available from TaKaRa company) and pST respectively (as Fig. 7 then, building process is as follows: artificial synthetic oligonucleotide 5 '-ggccgcaacggtaccaccaagcttagtggatccgaattccggct-3 ', this fragment is connected between the SfiI/NotI restriction enzyme site of pSectag2a (Invitrogen company) plasmid, obtains the pST plasmid.) on the carrier, obtain pUC-HBX and pST-HBX, pST-HBX is cut with the SalI enzyme obtain the 1.5Kb segment, this segment is cloned in the SalI site of pUC-HBX, cut evaluation with the BamHI enzyme and obtain recombinant plasmid, can discharge the pulsating clone of 1Kb and be correct clone, called after pUC-HBXII.Enter with NheI and PvuII enzyme cutting clone among the NheI and SmaI site of pCI (available from Clontech company) plasmid, the clone who obtains is called pCXII.The segment of cutting the 1Kb that obtains with the EcoRI enzyme is cloned among the pET-22b (available from Novagen company), has obtained pET-HBXII, as Fig. 3.
2. the building process of bidirectional promoter expression vector pET-2P is as follows:
Promotor and T7 terminator that T7 is arranged on known pET-22b (available from the Novagen company) expression vector, usefulness PCR primer (p5:5 '-ccgctcgagttgacaattaatcatcggctcgtataatgtgcggccgcaagcttgtc-3 '; P3:5 '-aagatctggcccacccgtgaaggtgagccc gatcccgcgaaattaatacg-3 ') be to have introduced tac promotor and T3 terminator in the dna segment that goes out of template amplification with pET-22b, this segment is entered pET-22b with XhoI and BglII enzyme cutting clone, the recombinant plasmid that obtains has bidirectional promoter and terminator, with this plasmid called after pET-2P, as Fig. 4.
3. the structure of hepatitis B virus surface antigen X protein and polysaccharase part coding region gene dsRNA expression plasmid carrier
Hepatitis B virus surface antigen, X protein and polysaccharase part coding region dna fragmentation are made template with pUC-HBV, with
The following primer amplification obtains:
HBVS1Ag-sense： 5’-cg
ggatcctaccacagagtctagactcg-3’，
HBVS1Ag-antisense： 5’-cat
gtcgacgcaacataccttggtagtcc-3’，
HBX-sense 5’-g
gaattcatggctgctaggetgtg-3’
HBX-antisense 5’-gg
ggtaccggcagaggtgaaaaagttg-3’
HBVP-sense 5’-g
gaattcgtcttgggtatacatttgacc-3’，
HBVP-antisense 5’-gg
ggtaccagaggacaacagagttg-3’，
The dna fragmentation of the coding hepatitis B surface antigen that amplification obtains is cloned into the BamHI/SalI restriction enzyme site in the corresponding site of pUC118 plasmid, and the dna fragmentation of coding hepatitis B X protein and polysaccharase subregion is cloned in the corresponding site of pUC118 plasmid with the EcoRI/KpnI restriction enzyme site.The plasmid that obtains is confirmed the exactness of the sequence dna fragment be cloned into through order-checking.The dna fragmentation of correct coding hepatitis B surface antigen is cloned in the corresponding site of pET-2P with the BamHI/SalI restriction enzyme site.The correct coding X protein and the dna fragmentation of polysaccharase subregion are cloned in the corresponding site of pET-2P with the EcoRI/SalI restriction enzyme site.The expression plasmid that obtains is called after pET-HBS, pET-HBX and pET-HBVP respectively.
4.dsRNA fermentation expression, the extraction and purification process is as follows:
The thalline fermentation: BL21 (DE3) inoculation that will contain pET-HBXII (wherein containing a gene SEAP2 that can transcribe out the RNA of hairpin structure) rises to 200ml and contains in the LB nutrient solution of acillin, 37 ℃ of shaking table overnight incubation, the small fermentor that is transferred to 25 liters is cultivated in the big fermentor tank that changes 300 liters of dress liquid (molten long-pending 500 liters) after 8-9 hour again over to, 37 ℃ of fermentation culture add the lactose-induced expression of 6kg dsRNA after 3 hours, continue the GL105 type whizzer collection bacterium liquid that fermentation was produced with Shanghai whizzer institute after 3 hours, standby.
RNA extracting: in the suspension (same 2 in chatted) of 100 gram thalline with 1000ml, the lysate (institute is chatted in same 2) that adds 2000ml, add 1500ml potassium acetate solution (institute is chatted in same 2) gently behind the stirring and evenly mixing, placed 10 minutes on ice after being divided in several Erlenmeyer flasks gently behind the stirring and evenly mixing, with 10000g centrifugal 10 minutes, after being recovered in supernatant liquor in several Erlenmeyer flasks, add isopyknic phenol chloroform isoamyl alcohol (25: 24: 1), behind the fierce vibration mixing centrifugal 10 minutes with 10000g, reclaim supernatant liquor again, standby.
The purifying of dsRNA: to be contained in a diameter after the 20% ethanol STE solution soaking balance be in the glass column of 8cm with containing with 1kg CF-11 powder, is placed in the Cool Room 4.To add ethanol to final concentration 20% in the centrifugal supernatant liquor that obtains after the extracting of phenol chloroform isoamyl alcohol, place after 20 minutes in freezer on ice on sample to the CF-11 post.With 5L contain 17% alcoholic acid STE solution wash remove single stranded RNA and plasmid DNA after, reclaim dsRNA at the alcoholic acid STE solution that do not contain that is preheating to 55 ℃ with 2L.Figure below is the agarose gel electrophoresis (swimming lane A and C) of the DNA sample before and after the purifying, compares as can be known with the controlled trial result (swimming lane B and D) that common plasmid carries out, and the sample that purifying obtains (swimming lane C) is long-chain dsRNA.
5. it is as follows to utilize RNase III hydrolysis long-chain dsRNA to prepare the process of siRNA:
Get the dsRNA that 4 micrograms obtain through the CF-11 purifying, with 37 ℃ of 0.1 microgram RNase III, in the different time sampling, electrophoresis detection result as shown in Figure 6.
The experimentation on animals result
25 μ g, 50 μ g or 100 μ g coding HBsAg, HBX, dsRNA (the called after dsHBS of HBV polysaccharase part fragment and alkaline phosphatase gene, dsHBX, dsHBVP and dsAP) use tail vein hydraulic method cotransfection to mouse liver with 10 μ g hepatitis B virus expression plasmid pUC-HBV2 and 10 μ g alkaline phosphatase pSEAP2-Control plasmids, a negative control group transfection 10 μ g pUCmT plasmids, a positive control group transfection 10 μ g pUC-HBV2 and 10 μ gpSEAP2-Control plasmids.The surface antigen (HBsAg) in the positive control group mice serum and the expression amount of e antigen (HBeAg) reached the climax in 24 hours after transfection, and in the 7 day time of observation kept stable.Cotransfection dsHBS, HBsAg and HBeAg in the mice serum of dsHBX and dsHBVP experimental group are obviously suppressed, and have dose-dependence, and the alkaline phosphate ester enzymic activity in these mice serums is not suppressed..Opposite, cotransfection dsAP experimental mice serum neutral and alkali phosphate esterase active is suppressed, and HBsAg almost is not suppressed.The above results shows that duplicating of hepatitis B virus suppressed specifically.Suppress efficient after transfection first day the highest, weaken gradually later on.The inhibition efficient of dsHBVP is the most remarkable in 3 kinds of double-stranded RNAs, first day inhibiting rate to HBsAgHbeAg is respectively 98.5% and 100% behind the transfection 100 μ g dsHBVP, and remains the 7th day inhibiting rate of significant inhibition effect be respectively 88.3%and 98.6% in the experimental observation process.The mouse liver function keeps good in whole experiment, and serum albumin and gpt level are all acted normally.Concrete data see Table 1 and table 2.
Hepatitis B surface antigen inhibiting rate %
Group
*First day the 4th day the 7th day
dsAP?25 3.85 2.99 22.12
dsAP?50 10.08 -3.97 35.69
dsAP?100 15.85 9.53 6.66
dsHBS?25 73.69 11.59 2.07
dsHBS?50 84.46 58.47 28.92
dsHBS?100 91.38 79.99 53.6
dsHBVP?25 96.46 42.11 67.86
dsHBVP?50 99.07 90.22 88.3
dsHBVP?100 98.46 92.02 88.3
dsHBX 25 49.07 16.73 31.96
dsHBX 50 73.38 26.78 27.01
Table one different sorts various dose double-stranded RNA is to the inhibiting rate of hepatitis B surface antigen
Hepatitis B virus e antigen inhibiting rate %
Group
*First day the 4th day the 7th day
dsAP 25 55.53 40.79 32.68
dsAP 50 62.84 38.48 55.43
dsHBS 25 94.65 44.03 40.85
dsHBS 50 96.31 71.28 61.74
dsHBVP?25 99.45 89.39 86.35
dsHBVP?50 100 98.4 93.5
dsHBVP?100 100 100.57 98.6
dsHBX 25 86.56 59.87 57.38
dsHBX 50 96.7 71.99 41.78
Table two different sorts various dose double-stranded RNA is to the inhibiting rate of hepatitis B virus e antigen
*The double-stranded RNA of dsAP coding alkaline phosphatase; The double-stranded RNA of dsHBS coding hepatitis B surface antigen;
The double-stranded RNA of dsHBVP coding hepatitis B DNA polysaccharase; The double-stranded RNA of dsHBX coding hepatitis B X protein.
The dosage of the used double-stranded RNA of numeral (μ g).
Claims (8)
1, a kind of method of utilizing the Escherichia coli fermentation preparation to use ldsRNA and siRNA molecule with RNAi, it is characterized in that comprising structure ldsRNA expression vector, and transformed into escherichia coli host bacterium, by large scale fermentation, obtain the mixture of full RNA and plasmid DNA with alkali-SDS method extractive fermentation thalline; This mixture carries out purifying with the CF-11 post and obtains ldsRNA, and utilizes intestinal bacteria RNase III ldsRNA to be cut into the siRNA of 12-30bp.
2, preparation method according to claim 1, the method of expression vector that it is characterized in that being structured in expression in escherichia coli long-chain dsRNA is as follows: target gene fragment is connected to the segmental both sides of the 3rd segment DNA with opposite direction with 3 ' end of gene, be cloned into then in the carrier that has a T7 promotor and go, after this expression plasmid being transformed in the host bacterium that to express the T7 RNA polymerase, the DNA chain of the positive and negative both direction of goal gene and the 3rd segment DNA chain between the two just can be transcribed successively, become a successive RNA chain.
3, preparation method according to claim 1, the method of expression vector that it is characterized in that being structured in expression in escherichia coli long-chain dsRNA is as follows: utilize the expression vector that has the intestinal bacteria replication orgin, add resistant gene and multiple clone site in place, and add relative two promotors and terminator respectively in the multiple clone site both sides, just can be after in multiple clone site, inserting target DNA fragment with positive and negative both direction transcribe rna.
4,, it is characterized in that selected goal gene is any fragment in the hepatitis B virus gene group according to claim 2 or 3 described preparation methods.
5, preparation method according to claim 4 is characterized in that selected target gene fragment for to make template with pUC-HBV, the fragment that obtains with following primer amplification:
HBVP-sense 5’-g
gaattcgtcttgggtatacatttgacc-3’，
HBVP-antisense 5’-gg
ggtaccagaggacaacagagttg-3’。
6, a kind of by the method for one of claim 1-5 prepared long-chain ldsRNA molecule and siRNA molecule, the application in preparation treatment hepatitis B medicament.
7, long-chain ldsRNA molecule according to claim 6 and the siRNA molecule application in preparation treatment hepatitis B medicament, its injected dose is the 1.25-5mg/kg body weight.
8, the medicine of treatment hepatitis B according to claim 7 is characterized in that with ldsRNA molecule and siRNA molecule be major ingredient, is equipped with auxiliary material, makes the injection liquid formulation, and it is as follows specifically to fill a prescription:
ldsRNA 12.5-50mg
NaCl 8.6g
KCl 0.3g
CaCl
2 0.13g
Add injection water 1000ml.
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| CN 03142232 Expired - Fee Related CN1276086C (en) | 2003-08-13 | 2003-08-13 | A double-stranded DNA molecule and its use in preparing medicine for inhibiting hepatitis B virus replication |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100344330C (en) * | 2004-09-22 | 2007-10-24 | 广州拓谱基因技术有限公司 | Targeted small interference RNA formulation for treating viral Hepatitis B and its preparation |
| CN100344331C (en) * | 2004-09-22 | 2007-10-24 | 广州拓谱基因技术有限公司 | Targeted small interference RNA formulation for preventing and treating Hepatitis C and its preparation method |
| CN100351376C (en) * | 2004-12-30 | 2007-11-28 | 中山大学 | RNA interfered target sequence of HBV and use thereof |
| CN100395334C (en) * | 2004-08-24 | 2008-06-18 | 暨南大学 | siRNA double-chain for suppressing bc1-2 gene expression |
| CN100410373C (en) * | 2004-08-24 | 2008-08-13 | 暨南大学 | siRNA double-chain for suppressing bc1-2 gene expression |
| CN102140460B (en) * | 2010-01-29 | 2012-12-12 | 苏州瑞博生物技术有限公司 | SiRNA (Small interference ribonucleic acid) as well as medicine composition and pharmaceutical application thereof |
| CN101180394B (en) * | 2005-02-03 | 2013-01-09 | 贝尼泰克有限公司 | Rnai expression constructs |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102083983B (en) * | 2008-08-01 | 2014-04-16 | 苏州瑞博生物技术有限公司 | Small RNS interference target site sequences of hepatitis B virus and small interference RNAs and the compositions and uses thereof |
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2003
- 2003-08-13 CN CN 03142232 patent/CN1276086C/en not_active Expired - Fee Related
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100395334C (en) * | 2004-08-24 | 2008-06-18 | 暨南大学 | siRNA double-chain for suppressing bc1-2 gene expression |
| CN100410373C (en) * | 2004-08-24 | 2008-08-13 | 暨南大学 | siRNA double-chain for suppressing bc1-2 gene expression |
| CN100344330C (en) * | 2004-09-22 | 2007-10-24 | 广州拓谱基因技术有限公司 | Targeted small interference RNA formulation for treating viral Hepatitis B and its preparation |
| CN100344331C (en) * | 2004-09-22 | 2007-10-24 | 广州拓谱基因技术有限公司 | Targeted small interference RNA formulation for preventing and treating Hepatitis C and its preparation method |
| CN100351376C (en) * | 2004-12-30 | 2007-11-28 | 中山大学 | RNA interfered target sequence of HBV and use thereof |
| CN101180394B (en) * | 2005-02-03 | 2013-01-09 | 贝尼泰克有限公司 | Rnai expression constructs |
| CN102140460B (en) * | 2010-01-29 | 2012-12-12 | 苏州瑞博生物技术有限公司 | SiRNA (Small interference ribonucleic acid) as well as medicine composition and pharmaceutical application thereof |
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| CN1276086C (en) | 2006-09-20 |
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