Target the siRNA of people RAN and its in preparing anti-viral hepatitis type C drugPurposesTechnical field
The present invention relates to pharmaceutical technology field more particularly to siRNAs, especially a kind of to be directed to RAN (Ras-Related nuclear protein) gene target siRNA and its preparing the 2 viral liver of the third type of type of therapeutic genePurposes in scorching drug.
Background technique
Hepatitis C Virus (Hepatitis C Virus, HCV) infection can cause urgency/chronic hepatitis, cirrhosis and liverCancer.There are about 1.85 hundred million HCV infection persons, infection rate is about 1-2% in the whole world at present, and the infection rate of East Asia Region is the 3.7% (worldHealth organization hepatitis practice guidelines in 2014).Annual new infections person exceedes 3,000,000, especially the most serious with developing country.Every yearThere are about 500,000 HCV infection persons death.Existing treatment is only capable of making about 50% patient to obtain continued viral response (SVR), and existsThe problems such as using taboo, individual difference and drug side-effect.There are no HCV preventative vaccines in the whole world so far, cannot blockThe propagation of HCV.Therefore, hepatitis is still the Important Infectious Diseases for threatening human health within considerable time from now on, it would be highly desirable to be openedThe new safely and effectively alternative medicine of hair.
HCV infection cell model be successfully established to small molecule compound inhibit HCV duplication research development played it is hugeBig effect.Currently, being usually used in constructing clone's system of HCV Infection in Vitro model being JFH1 plants.Common culture cell is people liverCancer cell line Huh7 and its derived cell system Huh7.5 and Huh7.5.1.The chimeric strain of the HCV 2a JFH1 and J6 that then constructPFL-JC1 confirms there is higher duplicating efficiency than JFH1 plants.These model systems allow to assess antiviral drugs to HCV RNADuplication, viral generation and infective influence, therefore also contribute to the fast development of anti-HCV medicament.
RNA interferes (RNA interference, RNAi) by causing posttranscriptional gene silencing in conjunction with specific mrnaOr target mRNA degradation.An independent technology is had become at present, and action target spot specificity is high, small to cytotoxicity, pacifies relativelyEntirely, cause the broad interest of the nearly all research field in biomedical boundary.First item clinical test is by AcuityThe siRNA that VEGF is targeted for treating age-related macular degeneration (AMD) that Pharmaceuticals company will develop in the end of the year 2004.It is Calando company for treating the CALAA01 of entity tumor that siRNA, which acts on the first clinical research of cancer, inIt obtains FDA approval in May, 2008 and enters I phase clinic.
RAN (Ras related nucleoprotein) is also referred to as GTP combination nucleoprotein, is a member of Ras superfamily.RAN is RNAIt is transported with protein by nuclear pore complex essential, also assists in control DNA synthesis and cell cycle progression.It has been found thatRAN gene mutation can destroy the synthesis of DNA.During mitosis, mitosis is spun after RAN circulation participates in chromosome separationHammer body assembling and nuclear membrane recombination.RAN or the androgen receptor co-activation factor, can be with the poly glumine of different length in heroHormone receptor difference combines.
Summary of the invention
The purpose of the present invention is to provide a kind of siRNAs and application thereof for people's RAN gene target.
Target the siRNA of people RAN:
The sequence of positive-sense strand are as follows: ACAGGAAAGUGAAGGCGAA dTdT
The sequence of antisense strand are as follows: dTdT UGUCCUUUCACUUCCGCUU
Target purposes of the siRNA of people RAN in the drug for preparing HCV-Ab IgG infection.
The siRNA of targeting people RAN is preparing the purposes in 2 type viral hepatitis type C drug of therapeutic gene.
A method of inhibit PTB to be indexed into cytoplasm by karyon, the siRNA for targeting people RAN is transfected into Huh7.5.1Cell, makes the expression silencing of RAN, and karyon-cytoplasm indexing of PTB is inhibited.
The present invention, which is experimentally confirmed, transfects Huh7.5.1 cell for the siRNA interference sequence 003 for targeting people RAN, makes itIn RAN silenced gene expression, and then the karyon of PTB-cytoplasm indexing can be inhibited, to play HCV-Ab IgG effect.It therefore can be withUsing RAN as molecular target, the siRNA interference sequence 003 of RAN is prepared into drug as active constituent, for the third type virusThe treatment of property hepatitis.
Detailed description of the invention
Fig. 1 is Q-PCR and western blot experimental result, to analyse HCV infection to RAN from mRNA and protein levelThe influence of expression.
Fig. 2 is western blot experiment, and to prove the interference effect of RAN from protein level, wherein NC is indicated unrelatedSequence, 003,004 and 005 is RAN interference sequence.Particular sequence is as follows:
The sequence of 003 positive-sense strand are as follows: ACAGGAAAGU GAAGGCGAA dTdT
The sequence of 003 antisense strand are as follows: dTdT UGUCCU UUCACUUCCGCUU
The sequence of 004 positive-sense strand are as follows: GACCUUCGUGAAACGUCAU dTdT
The sequence of 004 antisense strand are as follows: dTdT CUGGAAGCACUUUGCAGUA
The sequence of 005 positive-sense strand are as follows: GUAUGUAGCCACCUUGGGU dTdT
The sequence of 005 antisense strand are as follows: dTdT CAUACAUCGGUGGAACCCA
Behind Huh7.5.1 cell 48 hours of 003 interference JC1 infection of Fig. 3 western blot experiment display, HCV CoreProtein level significantly reduces.
Behind Huh7.5.1 cell 48 hours of 003 interference JC1 infection of Fig. 4 western blot experiment display, PTB albumenCytoplasm-karyon indexing is reduced.
Specific embodiment
In order to make it easy to understand, the present invention will be described in detail by specific drawings and examples below.It needsIt is emphasized that specific example and attached drawing are merely to explanation, it is clear that those skilled in the art can be according to hereinIllustrate, various modifications and variations are made to the present invention within the scope of the invention, these modifications and variations are also included in thisIn the range of invention.
Method therefor is conventional method unless otherwise instructed in the following example.Required material in following embodimentMaterial or reagent, are that market is bought unless otherwise specified.
RAN expression is analyzed after 1 JC1 of embodiment infection
The foundation of HCV cell infection model
First 1 day of transfection, with 1 × 107A/hole density is inoculated with 10cm culture dish, and cell fusion degree is 70%~80%.TransfectionWhen, 10 μ g HCV RNA transcriptions are taken, imported into support in the method for 260V and 25 millisecond pulse length folk prescription wave electrotransfectionsThe liver cancer cell lines Huh7.5.1 of HCV duplication.Cell conditioned medium is collected, 1500g centrifugation 5min removal cell fragment is placed on -70 DEG CIt saves.Again plantation Huh7.5.1 cell is in six porocyte culture plates, and when cell is up to 50%~60% degrees of fusion, every hole is addedThe cell conditioned medium that 2ml is collected is cleaned three times after being incubated for 24 hours with PBS, and complete medium is added and continues to cultivate.It is received after 48 hoursCollect cell and carries out subsequent experimental.
Quantitative fluorescent PCR
Utilize the intracellular HCV RNA relative quantity of fluorescence quantitative PCR detection.Cell is collected after pancreatin digestion, every pipe is addedTrizol extracts total serum IgE according to operating instruction, and ultraviolet specrophotometer measures concentration.Take 4 μ l RNA according to Reverse Transcriptase kitOperating instruction first removes genomic DNA, then carries out reverse transcription reaction, and reaction system is 20 μ l.The cDNA of synthesis is as real-timeQuantitative PCR template, positive antisense primer of the reaction system including 2 × SYBR Green Taq reaction solution, 10 μ l, HCV or GAPDH are each0.4 μ l, 0.4 μ l of Rox II, 6.8 μ l and cDNA template of sterile purified water, 22 μ l.PCR reaction condition is 95 DEG C of 30s；95 DEG C of 5s,60 DEG C of 30s, totally 40 recycle.For detecting the primer of RAN, upstream: 5 '-GTGAAGGCGAAATCCATTGT-3 ', downstream:5'-TCCTAGCAAGCCAGAGGAAG-3'；Reference gene glyceraldehyde 3-phosphate dehydro-genase (GAPDH) amplimer, upstream:5 '-GAAGGTGAAGGTCGGAGTC-3 ', downstream: 5 '-GAAGATGGTGATGGGATTTC-3 '.All samples target gene phaseExpression is calculated after reference gene GAPDH homogenization processing.
Western Blot detection
Western Blot detects HCV Core and RAN protein expression level.Extract total protein of cell and according to BCA albumenThe operating instruction of quantification kit carries out quantitative analysis.Above-mentioned 40 μ g of total protein of cell is taken to separate and be transferred to using SDS-PAGEOn nitrocellulose filter.After the closing of 5% skimmed milk power, by film and antibody HCV Core (Abcam), RAN (Abcam) and β-4 DEG C of actin (Cell signaling Technology) overnight incubations, secondary antibody use chemical illuminating reagent after being incubated for 1 hour(Pierce) it detects.
It is increased as shown in Figure 1, HCV infection can induce RAN expression.
The screening of 2 RAN interference sequence of embodiment
RNA interference
First 1 day of transfection, with 2 × 105A/hole density is inoculated with 6 porocyte culture plates.When transfection, 10 μ l siRNA (commission is takenSharp rich biosynthesis) it is diluted in 100 μ l Opti-MEM I serum free mediums, it mixes gently；Take 2 μ lLipofectamineTMRNAiMAX is diluted in 100 μ l Opti-MEM I serum free mediums, is mixed gently.It will be dilutedHCV RNA and LipofectamineTMRNAiMAX is gently mixed, and is stored at room temperature 20min.6 orifice plates are added in 200 μ l compoundsIn cell, mix gently.2mlJC1 viral supernatants infection cell is added in every hole after 24 hours, and training completely is added after being incubated for 24 hoursFeeding base continues to cultivate.Cell is collected after 48 hours carries out subsequent experimental.
Quantitative fluorescent PCR
Utilize the intracellular HCV RNA relative quantity of fluorescence quantitative PCR detection.Cell is collected after pancreatin digestion, every pipe is addedTrizol extracts total serum IgE according to operating instruction, and ultraviolet specrophotometer measures concentration.Take 4 μ l RNA according to Reverse Transcriptase kitOperating instruction first removes genomic DNA, then carries out reverse transcription reaction, and reaction system is 20 μ l.The cDNA of synthesis is as real-timeQuantitative PCR template, positive antisense primer of the reaction system including 2 × SYBR Green Taq reaction solution, 10 μ l, HCV or GAPDH are each0.4 μ l, 0.4 μ l of Rox II, 6.8 μ l and cDNA template of sterile purified water, 22 μ l.PCR reaction condition is 95 DEG C of 30s；95 DEG C of 5s,60 DEG C of 30s, totally 40 recycle.For detecting the primer of RAN, upstream: 5 '-GTGAAGGCGAAATCCATTGT-3 ', downstream:5'-TCCTAGCAAGCCAGAGGAAG-3'；Reference gene glyceraldehyde 3-phosphate dehydro-genase (GAPDH) amplimer, upstream:5 '-GAAGGTGAAGGTCGGAGTC-3 ', downstream: 5 '-GAAGATGGTGATGGGATTTC-3 '.All samples target gene phaseExpression is calculated after reference gene GAPDH homogenization processing.
Western Blot detection
Western Blot detects RAN protein expression level.Extract total protein of cell and according to BCA protein quantification kitOperating instruction carry out quantitative analysis.Above-mentioned 40 μ g of total protein of cell is taken to separate using SDS-PAGE and be transferred to nitrocelluloseOn film.After the closing of 5% skimmed milk power, by film and antibody RAN (Abcam) and β-actin (Cell signalingTechnology) 4 DEG C of overnight incubations, secondary antibody are detected after being incubated for 1 hour with chemical illuminating reagent (Pierce).
As shown in Fig. 2, 003 interference effect of RAN interference sequence is the most significant.
3 003 HCV-Ab IgG effect analysis of embodiment
Western Blot detection
Western Blot detects HCV Core Protein expression.Total protein of cell is extracted after RNA interference and according to BCAThe operating instruction of protein quantification kit carries out quantitative analysis.Above-mentioned 40 μ g of total protein of cell is taken to separate and turn using SDS-PAGEIt moves on on nitrocellulose filter.After the closing of 5% skimmed milk power, by film and antibody HCV Core (Abcam) and β-actin (CellSignaling Technology) 4 DEG C be incubated overnight, secondary antibody is detected after being incubated for 1 hour with chemical illuminating reagent (Pierce).
As shown in figure 3,003 can significantly inhibit HCV Core Protein expression.
4 003 HCV-Ab IgG mechanism analysis of embodiment
Western Blot detection
Western Blot detection PTB albumen is indexed into the level of cytoplasm by karyon.Total protein of cell is extracted after RNA interferenceAnd quantitative analysis is carried out according to the operating instruction of BCA protein quantification kit.Above-mentioned 40 μ g of total protein of cell is taken to use SDS-PAGE is separated and is transferred on nitrocellulose filter.After the closing of 5% skimmed milk power, by film and antibody PTB (Abcam), β-actin4 DEG C of (Cell signaling Technology) and PCNA (Cell signaling Technology) overnight incubations, secondary antibodyIt is detected after being incubated for 1 hour with chemical illuminating reagent (Pierce).
As shown in figure 4,003 can significantly inhibit PTB albumen and be indexed into cytoplasm by karyon
HCV-Ab IgG effect can be played by inhibiting PTB albumen to be indexed into cytoplasm by karyon by showing 003 based on the above results.