CN1317389C - Structure of antisense digonucleotide for inhibiting fibronectin expression and use - Google Patents
Structure of antisense digonucleotide for inhibiting fibronectin expression and use Download PDFInfo
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Abstract
The present invention relates to a structure and a purpose of a new medicine for resisting hepatitis B virus target marks and corresponding antisense oligonucleotide, particularly to a structure and a purpose of target fibronectin for resisting hepatitis B virus (HBV) antisense oligonucleotide. Fibronectin is the extracellular matrix constituent of sinusoid of human livers and can be combined with a previous S2 region of hepatitis B viruses by the report of literatures by a specific method. The laboratory discovers good specificity and drug properties of the fibronectin by applying gene chip technology by sieving and verification, so the laboratory synthesizes antisense oligonucleotide of the target fibronectin, adopts HepG2.2.15 cells, and sieves and evaluates the activity of five pieces of antisense oligonucleotide. The present invention discovers that oligonucleotide FN1 and FN5 complementary with a fibronectin mRNA specific function region can effectively inhibit the copying and the expression of hepatitis B in the HepG2.2.15 cells, so the present invention relates to a sequence and a structure of antisense oligonucleotide of target fibronectin mRNA resisting HBV infection and a new medicine for treating hepatitis B virus correlation diseases and decreasing the damage of the hepatitis B correlation diseases.
Description
Technical field:
The present invention relates to the biotechnology pharmaceutical field, sequence, structure and the medicine thereof of the antisense oligonucleotide (ASODN, antisense oligodexynucleotide) that specifically a kind of target fibronectin (fibronectin) treatment hepatitis B virus (HBV) infects.
Background technology:
HBV infects the serious threat human beings'health, is the major reason that causes hepatitis, liver cirrhosis, liver cancer.Still lack especially effectively medicine at present, so novel anti HBV medicine have important social and economic benefit.
Fibronectin in the foreign literature report people liver may combine (Budkowska A, Bedossa P, Groh F. with the antigenic determinant that HBV encodes by preceding S2 district in the restrictive mode of a kind of kind in vivo; J Virol 1995 Feb; 69 (2): 840-8); Discovery HBxAg such as P.A.Norton can activate fibronectin expression (P.A.Norton, H.M.G.P.V.Reis, Journal of Viral Hepatitis, 2004,11,332-341).This laboratory finds that by screening and checking but fibronectin has the excellent specificity and the property of medicine in anti HBV infecting, can develop into the latent effect target spot of anti-HBV treatment.
ASODN is the oligonucleotide fragment of a class through synthetic, and length mostly is 15～30 Nucleotide.By the principle of base complementrity, disturb transcribing and translating of genes involved, perhaps duplicating of whole genome, its advantage is its theoretic height target-specific, is that a kind of ideal has accurately optionally gene target medicine.Owing to the high degree of specificity of ASODN effect, therefore be considered to have the antitumor and new antiviral drug of potentiality.One of external more existing famous pharmacy corporations have been developed antisense drug as its new drug research emphasis direction.
The objective of the invention is, according to disclosed fibronectin gene mRNA sequence, design is at the ASODN of fibronectin, by suppressing the expression of fibronectin, stop HBV to infect, suppress hbv replication and expression, for the treatment chronic HBV infection provides new specific medicine.
Summary of the invention:
To the effect that of the present invention: by the nucleic acid sequence data storehouse among the retrieval GeneBank, the fibronectin mRNA reference sequences X02761 that selects NCBI to announce, to its computer simulation of carrying out the RNA secondary structure, selected the action target spot of 5 unsettled loop-stem structures as ASODN.By with the online blast sequence alignment of GeneBank, selected target sequence all has excellent specificity, can not disturb the expression of human other normal gene.Synthetic sulfo-Antisensedigonucleotsequence sequence (S-ASODN) on automatic dna synthesizer.Adopt transfection that HBV DNA is arranged, but stably express HBV albumen and complete DaneShi particulate HepG2.2.15 cell model carry out screening active ingredients and evaluation to above-mentioned ASODNs.The result shows among 5 ASODNs, FN1, FN5 when 0.8 μ mol/L to HBV DNA, HBsAg, HBeAg, fibronectin albumen has the obvious suppression effect, and to HBV DNA, HBsAg, the inhibition activity of HBeAg is greater than the inhibition activity of lamivudine.In 0.2-0.8 μ mol/L concentration range, FN1, FN5 be to HBV DNA, HBsAg, and HBeAg and fibronectin albumen have specific dose-dependent inhibition activity.
Sulfo-Antisensedigonucleotsequence sequence and character
| Numbering | Title | Target position (nt) | Length (nt) | Character | Antisense sequences (5 '-3 ') |
| 1 2 3 4 5 6 7 | FN1 FN2 FN3 FN4 FN5 FN1s FN5s | 2188-2207 6478-6497 20-40 4704-4723 6607-6626 2188-2207 6607-6626 | 20 20 20 20 20 20 20 | A A A A A S S | GCT CAT CTC CCT CCT CAC TC TTC GTT CCC ACT CAT CTC CA CTG GGG CTG AAC CAT TTG CT GCC TTC AAT AGT CAT TTC TG GAC GGT CCC ACT TCT CTC CA GAG TGA GGA GGG AGA TGA GC TG GAG AGA AGT GGG ACC GTC |
A: antisense; S: justice
According to the present invention, but the expression specificity of inhibition fibronectin suppresses duplicating and expressing of hepatitis B virus, and fibronectin might become the newtype drug action target spot of treatment and prevention HBV relative disease.
According to the present invention, can specificity suppress duplicating and expressing of hepatitis B virus at the ASODNs of fibronectin mRNA, might become the novel biological engineering medicine of treatment and prevention HBV relative disease.
According to the present invention, the length of antisense oligonucleotide and its cell permeability are relevant with factors such as target sequence binding affinity and effect specificitys, FN1, the length of FN5 determines that according to experiment the present invention has comprised and FN1, and FN5 has any length oligonucleotide of identical sequence.
According to the present invention, for strengthening nuclease resistance, bioavailability and the tissue target tropism etc. of antisense oligonucleotide, the present invention has comprised FN1, the thio-modification of FN5.
According to the present invention, oligonucleotide of the present invention and modifier thereof can be made into the preparation of parenterai administration by means known in the art.
According to the present invention, the treatment of oligonucleotide of the present invention and modifier thereof is formed can use independent effective constituent or composition forms comprises other antisense oligonucleotides of associating and derivative form thereof.
According to the present invention, treatment of the present invention is formed, comprise pharmacokinetics, pharmacokinetics, mode of administration, route of administration, the receptor's of certain drug age, body weight, hepatic and renal function state, character, degree and the treatment time limit etc. of disease according to different situations, with the appropriate dosage administration.
Enforcement of the present invention has important social benefit and economic benefit to the hepatitis B of serious harm human health and the treatment of relative disease thereof.
Description of drawings:
Fig. 1 fibronectin mRNA is in HepG2 cell, HepG2.2.15 cell and the HepG2.2.15 cell expression changing conditions before and after drug treating
Fig. 2 fibronectin albumen is in HepG2 cell, HepG2.2.15 cell and the HepG2.2.15 cell expression changing conditions before and after drug treating
Fig. 3 sulfo-fibronectin Antisensedigonucleotsequence sequence is to the restraining effect of HBV DNA in the HepG2.2.15 cell
Fig. 4 sulfo-fibronectin Antisensedigonucleotsequence sequence is to the restraining effect of HBsAg in the HepG2.2.15 cell
Fig. 5 sulfo-fibronectin Antisensedigonucleotsequence sequence is to the restraining effect of HBeAg in the HepG2.2.15 cell
Fig. 6 sulfo-fibronectin Antisensedigonucleotsequence sequence FN1, FN5 is to the proteic restraining effect of fibronectin in the HepG2.2.15 cell
Fig. 7 sulfo-fibronectin Antisensedigonucleotsequence sequence FN1 is to the influence of HepG2.2.15 cell proliferation
Fig. 8 sulfo-fibronectin Antisensedigonucleotsequence sequence FN5 is to the influence of HepG2.2.15 cell proliferation
Fig. 9 sulfo-fibronectin Antisensedigonucleotsequence sequence FN1, FN5 and just sequence thereof are to the proteic restraining effect of fibronectin in the HepG2.2.15 cell
Figure 10 sulfo-fibronectin Antisensedigonucleotsequence sequence FN1, FN5 and just sequence thereof are to the restraining effect of HepG2.2.15 emiocytosis HBsAg
Figure 11 sulfo-fibronectin Antisensedigonucleotsequence sequence FN1, FN5 and just sequence thereof are to the restraining effect of HepG2.2.15 emiocytosis HBeAg
Figure 12 sulfo-fibronectin Antisensedigonucleotsequence sequence FN1 is dose-dependently to the proteic restraining effect of fibronectin in the HepG2.2.15 cell
Figure 13 sulfo-fibronectin Antisensedigonucleotsequence sequence FN5 is dose-dependently to the proteic restraining effect of fibronectin in the HepG2.2.15 cell
Figure 14 sulfo-fibronectin Antisensedigonucleotsequence sequence FN1 is dose-dependently to the restraining effect of HepG2.2.15 emiocytosis HBV DNA, HBsAg, HBeAg
Figure 15 sulfo-fibronectin Antisensedigonucleotsequence sequence FN5 is dose-dependently to the restraining effect of HepG2.2.15 emiocytosis HBV DNA, HBsAg, HBeAg
Figure 16 5 sulfo-fibronectin Antisensedigonucleotsequence sequence and character
Figure 17 sulfo-antisense oligonucleotide FN1, just sequence and the character of FN5
Embodiment:
Embodiment one
Materials and methods
1. medicine preparation
It is 10mmol/L that lamivudine is dissolved to final concentration with PBS; It is 100mmol/L that the lamivudine medicine is dissolved to final concentration with DMSO, is diluted to 1mmol/L with nutrient solution during dosing, is not higher than 0.1% to guarantee the concentration of DMSO in nutrient solution; It is 250mg/mL that IBE5 is dissolved to final concentration with DMSO.
2. cell cultures
Used cell is the HepG2.2.15 cell strain that hepatoma cell line HepG2 cell strain and transfection have HBV DNA.The HepG2.2.15 cell derives from the HepG2 cell, contains the HBV DNA of integration, sustainable stably secretion DaneShi particle and HBsAg in nutrient solution in cell cultivation process, HBV DNA etc.(the HepG2.2.15 cell is with containing 10% foetal calf serum for FBS, DMEM cell culture fluid cultivation Gibco), and the MEM cell culture fluid of 380 μ g/mlG418 (Promega) is cultivated with containing 10% foetal calf serum for the HepG2 cell.
3. cell dosing
Treat that the HepG2.2.15 cell covers with the back and goes down to posterity at 1: 3, use the MEM nutrient solution that contains 2%FBS after 48 hours instead, add lamivudine simultaneously, lamivudine or IBE5.The lamivudine final concentration is 25 μ mol/L, and the lamivudine final concentration is 1 μ mol/L, and the IBE5 final concentration is 250 μ g/ml, sets up corresponding cellular control unit simultaneously.Changed the cell culture fluid that contains the isoconcentration medicine, the 8th day collecting cell after the dosing in 4 days.
4.RT-PCR detect fibronectin mRNA at HepG2, HepG2.2.15 cell and the expression in the HepG2.2.15 of drug treating cell
Treat that HepG2 cell, HepG2.2.15 cell cover with or the dosing of HepG2.2.15 cell after the 8th day, nutrient solution is removed in suction, after twice of PBS cleaning, extract cell total rna according to Trizol test kit (Invitrogen) specification sheets, ultraviolet spectrophotometer is quantitative, OD260/280 is between 1.8-2.0, and RNA denaturing formaldehyde electrophoresis showed does not have degraded.Carry out reverse transcription then, concrete grammar is as follows: respectively get total RNA 1 μ g, OligodT (15) 0.5 μ g, RNA enzyme inhibitors (40U) 0.1 μ l, totally 10.3 μ l, mixing, 70 ℃ of incubation 10min, cooled on ice immediately.Add the first chain reaction damping fluid, 5 μ l, DTT 2.5 μ l, RNA enzyme inhibitors 0.7 μ l, dNTP (A, G, C, T10mM) 1.0 μ l, mixing, 42 ℃ of reaction 2min, add reversed transcriptive enzyme superscriptII0.5 μ l, 42 ℃ of incubation 1h, last 70 ℃ of sex change 15min.Reverse transcription product is got the template of 0.5 μ l as pcr amplification, carries out double PCR with house-keeping gene GAPDH as interior mark.Target gene fibronectin upstream primer is 5 ' TAGCCCTGTCCAGGAGTTCA3 ', and downstream primer is 5 ' CTGCAAGCCTTCAATAGTCA3 ', and the amplification segment is 307bp.The upstream primer of GAPDH is 5 ' ACCACAGTCCATGCCATCAC3 ', and downstream primer is 5 ' TCCACCACCCTGTTGCTGTA3 ', amplification segment 449bp.PCR reaction system cumulative volume 20 μ l, upstream and downstream primer final concentration are 1 μ m, Mg
2+Concentration 1.5mM adds reverse transcription product 0.5 μ l, Taq 1U.The PCR loop parameter is 94 ℃ of pre-sex change 2min, 94 ℃ of 20s, and 61 ℃ of 30s, 72 ℃ of 20s circulate 22 times, and last 72 ℃ are extended 2min.2% sepharose (Sigma) electrophoresis detection.
5.Western the trace method detects fibronectin at HepG2 cell, HepG2.2.15 cell and the expression in the HepG2.2.15 of drug treating cell
Treat that HepG2 cell, HepG2.2.15 cell cover with or the dosing of HepG2.2.15 cell after the 8th day, inhale and remove nutrient solution, clean twice with PBS after, RIPA-PICT (Pharmacia) method is extracted total protein of cell, behind the protein quantification each extract is transferred to same concentrations.Every hole 30-50 μ g total protein behind 10% polyacrylamide gel electrophoresis, adopts half-dried transfer printing that albumen is gone to nitrocellulose filter (PROTRAN BA-S 83 Reinforced NC, Schleicher ﹠amp; Schuell) on.4 ℃ of sealings are spent the night, and confining liquid is formed: 10% skim-milk, 1 * TBST.Combine 1h with mouse-anti people fibronectin antibody (Santa cruz) or mouse-anti people β-actin antibody (Sigma) then, TBST washes film 3 times, each 10min, combine 1h with the anti-mouse two anti-(middle mountain) of horseradish peroxidase mark again, TBST washes film 3 times, each 10min, the colour developing of ECL (Pharmacia) Color Appearance System, the exposure of X-ray sheet.
The result
1.Fibronectin mRNA is at HepG2 cell, HepG2.2.15 cell and the expression in the HepG2.2.15 of drug treating cell
Behind the HepG2 cell of equivalent, the HepG2.2.15 cell total rna reverse transcription PCR, 2% agarose gel electrophoresis detects expression, and house-keeping gene GAPDH is as interior mark.The result almost detects the expression less than fibronectin mRNA as shown in fig. 1 in the HepG2 cell, and its expression level and GAPDH expression level are approaching in the HepG2.2.15 cell.After 25 μ mol/L lamivudines and 250 μ g/mlIBE5 processing, fibronectin mRNA expresses obviously downward modulation in the HepG2.2.15 cell.But faint downward modulation only takes place in fibronectin mRNA expression in the HepG2.2.15 cell after 1 μ M lamivudine is handled.Control represents the relative expression situation of fibronectin mRNA in cellular control unit among Fig. 1; Lamivudine, adefovir and IBE5 represent the fibronectin mRNA relative expression's situation in three kinds of drug treating group cells respectively; HepG2, HepG2.2.15 represent the relative expression situation of fibronectin mRNA in HepG2, HepG2.2.15 cell.
2.Fibronectin albumen is at HepG2 cell, HepG2.2.15 cell and the expression in the HepG2.2.15 of drug treating cell
Show among Fig. 2 that fibronectin albumen relative expression quantity in the HepG2.2.15 cell is higher, and almost detects the proteic expression less than Fibronectin in the HepG2 cell.Fibronectin albumen was obviously reduced after 25 μ mol/L lamivudines and 250 μ g/mlIBE5 handled the HepG2.2.15 cell, and 1 μ mol/L lamivudine is handled the faint downward modulation of Fibronectin protein expression in the HepG2.2.15 cell of back.
Conclusion
Fibronectin infects the back up-regulated at HBV, and expresses obviously downward modulation after drug intervention, therefore may become the newtype drug action target spot of treatment and prevention HBV relative disease.
Embodiment two
Materials and methods
1.S-ASODN design and synthetic
Nucleic acid sequence data storehouse among the retrieval GeneBank, the fibronectin mRNA reference sequences X02761 that selects NCBI to announce, by it being carried out computer aided design (CAD), selected the action target spot of 5 unsettled loop-stem structures as ASODN based on many predictions RNA secondary structures.By with the online blast sequence alignment of GeneBank, selected target sequence all has excellent specificity, can not disturb the expression (seeing Figure 16) of human other normal gene.All oligonucleotide all adopt ABI8909 type automatic dna synthesizer to synthesize and carry out thio-modification when synthetic, process is as follows: with sulfo-reagent (Beaucage regent, Transgenomic) be dissolved in that to make its final concentration in the anhydrous acetonitrile be 1g/100ml, place the AUX position of dna synthesizer, adopt the DNA sulfuration program that provides on the synthesizer to carry out synthetic automatically G 3139.Synthetic 55 ℃ of cuttings of strong aqua and the deprotection of finishing be after 15 hours, through the anti-phase purification column of Micro PureII (Oligo Prep OP120, SAVANT) purifying, the quantitative final vacuum drying of ultraviolet ,-20 ℃ of preservations are standby.
2.ASODN transfection
The Hep2.2.15 cell is in the MEM nutrient solution that contains 10% foetal calf serum (Gibco) and 380 μ g/ml, in 37 ℃, 5%CO
2Cultivate in the incubator.The observation of cell growth conditions is good, is cultured to the logarithmic growth after date, with Hep2.2.15 cell inoculation 6 orifice plates, 1.5 * 10
5Cells/well, 37 ℃, 5%CO2 was hatched 48-72 hour, after waiting to grow to the 40-60% cell and converging, under the serum-free state, adopted liposome Lipofectin (Invitrogen, 1mg/ml) reagent and with reference to specification sheets operation carrying out transfection.The concentration of antisense oligonucleotide is respectively 0.2 μ M, 0.4 μ M, 0.8 μ M and establishes cell contrast, liposome contrast.After the transfection 22 hours, change normal cell nutrient solution (the MEM cell culture fluid that contains 10%FBS), 37 ℃, 5%CO2 was hatched 72 hours.The collecting cell nutrient solution ,-20 ℃ of preservations are standby.(Trizol RNA extracts test kit, and Invitrogen) and the Hep2.2.15 total protein of cell ,-20 ℃ of preservations are standby to extract the Hep2.2.15 cell total rna.
3.ASODN the restraining effect to Hep2.2.15 emiocytosis HBV DNA detects
Get cell culture fluid, 100 ℃ are boiled 15min, the centrifugal 10min of 12000r/min, get the template of supernatant as quantitative fluorescent PCR, experimentation is operated (He Yunyan, Wang Shengqi etc., Chinese hepatopathy magazine by the method for the combined probe PCR detection by quantitative HBV that this laboratory is set up, 2001, V9N6:376-377).The primer sequence of detection by quantitative HBV DNA is: P1:5 '-GGAGTA TGG ATT CGC ACT CCT C-3 '; P2:5 '-TTG TTG TTG TAG GGG ACC TGC CT-3 '; Fluorescent probe sequence F:5 '-ACT TCC GGA AAC TAC TGT TAG ACG A-3 '; Cancellation probe sequence Q:5 '-GTA GTT TCCGGA AGT-3 '.20 μ l reaction systems contain the 200nmol/L primer, 670nmol/L fluorescent probe F, 180nmol/L cancellation probe, 200 μ mol/LdNTP, the Mg of 4.0mmol/L
2+, 2 μ l templates are put into the automatic PCR instrument of iCycle with each reaction tubes with the typical curve reaction tubes behind the mixing and are increased, amplification condition is: 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 30s, totally 40 circulations, reaction finish the back and calculate quantitative result automatically by computer.
4.ASODN to Hep2.2.15 emiocytosis HBsAg, the restraining effect of HBeAg detects
Get the good cell culture fluid of collection, according to HBsAg, HBeAgELISA detection kit (magnificent company) specification sheets operation steps detects.Get in the 96 antigen coated orifice plates of 50 μ l cell culture fluids adding hepatitis B surface antigen or e, every hole adds the enzyme mark binding substances of 50 μ l surface antigens or e antigen correspondence, 37 ℃ hatch 1h after, wash plate 5 times with washing lotion, add colour developing liquid A50 μ l, add colour developing liquid B50 μ l again, hatch 15min for 37 ℃, add stop buffer 50 μ l, at multiple labeling inspection enzyme-linked immunosorbent assay instrument (VICTOR
TMWallac 1420 Multilabel Counter Wallac) go up the mensuration 450nm 1s of place light absorption value A.According to IR=(A450
Contrast-A450
Administration)/A450
ContrastCalculate inhibiting rate.
5.ASODN the proteic restraining effect of fibronectin in the Hep2.2.15 cell is detected
RIPA-PICT (Pharmacia) extracts total protein of cell, behind the protein quantification each extract is transferred to same concentrations.Carry out the western Blot experiment then.Experimental technique reference example one.
The result
1.ASODN restraining effect to Hep2.2.15 emiocytosis HBV DNA
Five antisense sequences FN1-FN5 of target ASGPR mRNA, handle the Hep2.2.15 cell with 0.8 μ M administration respectively, set up the cell contrast simultaneously, liposome contrast and positive drug lamivudine control group, collecting cell nutrient solution after 72 hours, fluorescence quantitative PCR detection is respectively organized excretory HBV DNA copy number in the cell, according to formula IR=(C
The dosing group-C
The cell control group)/C
The cell control groupCalculate inhibiting rate, IR represents inhibiting rate in the formula, and C represents HBV DNA copy number in the detected cell culture fluid.The triplicate experiment, the mean value of calculating inhibiting rate.C represents the cell control group among Fig. 3, and LIP represents the liposome control group, and it near 0, does not have the obvious suppression effect to the inhibiting rate of HBVDNA.LAM10, LAM25 represent 10 μ M, the 25 μ M lamivudines restraining effect to HepG2.2.15 emiocytosis HBV DNA respectively.FN1-FN5 represents five antisense sequences restraining effect to HBVDNA when 0.8 μ M, can show from figure, and FN1, FN5 are to the restraining effect of the HBV DNA restraining effect more than or equal to HBV DNA in the 25 μ M lamivudine pair cell nutrient solutions.
2.ASODN restraining effect to Hep2.2.15 emiocytosis HBsAg
Five antisense sequences FN1-FN5 of target ASGPR mRNA, handle the HepG2.2.15 cell with 0.8 μ M dosing respectively, set up cell contrast, liposome contrast and positive drug lamivudine control group simultaneously, collecting cell nutrient solution after 72 hours, the HBsAgELISA detection kit detects the expression of HBsAg in the Hep2.2.15 cell culture fluid, according to formula IR=(A
The dosing group-A
The cell control group)/A
The cell control groupCalculate inhibiting rate, IR represents inhibiting rate in the formula, and the A representative respectively detects the absorbancy of hole at 450nm.The triplicate experiment, the mean value of calculating inhibiting rate is seen Fig. 4.LIP represents the restraining effect of liposome control group pair cell secretion HBsAg among the figure, and showing does not have obvious restraining effect.LAM represents the restraining effect of 25 μ M lamivudine pair cells secretion HBsAg, and inhibiting rate is greater than 50%.FN1-FN5 represents the restraining effect of five antisense sequences pair cells secretion HBsAg, and FN1 wherein, the average inhibiting rate of FN5 be greater than 50%, and greater than the restraining effect of 25 μ M lamivudine pair cells secretion HBsAg.
3.ASODN restraining effect to Hep2.2.15 emiocytosis HBeAg
Five antisense sequences FN1-FN5 of target ASGPR mRNA, handle the Hep2.2.15 cell with 0.8 μ M administration respectively, collecting cell nutrient solution after 72 hours, the HBeAgELISA detection kit detects the expression of HBeAg in the Hep2.2.15 cell culture fluid, according to formula IR=(A
The dosing group-A
The cell control group)/A cell control group calculates inhibiting rate, and IR represents inhibiting rate in the formula, and the A representative respectively detects the absorbancy of hole at 450nm.The triplicate experiment, the mean value of calculating inhibiting rate is seen Fig. 5.Among the figure, LIP represents the restraining effect of liposome control group pair cell secretion HBeAg, and showing does not have obvious restraining effect.LAM represents the restraining effect of 25 μ M lamivudine pair cells secretion HBsAg, and inhibiting rate is greater than 50%.FN1-FN5 represents the restraining effect of five antisense sequences pair cells secretion HBsAg, and FN1 wherein, the average inhibiting rate of FN5 be greater than 50%, and approaching with the inhibiting rate of 25 μ M lamivudine pair cells secretion HBsAg.
4.ASODN the proteic restraining effect of pair cell fibronectin
Five antisense sequences FN1-FN5 of target fibronectin mRNA, handle the Hep2.2.15 cell with 0.8 μ M administration respectively, extract total protein behind the 72h, behind 10% polyacrylamide gel electrophoresis, adopt half-dried transfer printing that albumen is gone on the nitrocellulose filter, with β actin (43kD) is contrast, detects the influence of sulfo-antisense oligonucleotide to target protein ASGPR expression amount by Western Blot method.As seen from Figure 6, FN1, FN5 can significantly suppress the proteic expression of fibronectin, FN2, FN4 does not have obvious restraining effect to fibronectin albumen, and FN3 is to fibronectin albumen unrestraint effect.
Conclusion
1. the expression that suppresses fibronectin can suppress duplicating and expressing of HBV in the HepG2.2.15 cell
2. in five sulfo-fibronectin Antisensedigonucleotsequence sequences, FN1, FN5 have the effect of hbv replication and expression in the obvious suppression HepG2.2.15 cell.
Embodiment three
Materials and methods
1.ASODN design with synthetic
According to embodiment two results, select effect Antisensedigonucleotsequence sequence FN1, FN5 preferably, synthetic its positive MODN (seeing Figure 17).Synthesizing of all G 3139s with embodiment two.
2. sulfo-Antisensedigonucleotsequence sequence FN1, the cytotoxicity of FN5 detects
The Hep2.2.15 cell is in the MEM substratum that contains 10% foetal calf serum (Gibco) and 380 μ g/ml, in 37 ℃, 5%CO
2Cultivate in the incubator.The observation of cell growth conditions is good, is cultured to the logarithmic growth after date, inoculates 96 orifice plates, 0.75 * 10
5Cells/well, 37 ℃, 5%CO2 was hatched 48-72 hour, after waiting to grow to the 40-60% cell and converging, under the serum-free state, adopted liposome Lipofectin (Invitrogen, 1mg/ml) reagent and with reference to specification sheets operation carrying out transfection.Antisense oligonucleotide FN1, the concentration of FN5 is respectively 0.2 μ M, 0.4 μ M, 0.8 μ M, 1.6 μ M, 10 μ M and establishes the cell contrast, and every concentration repeats three holes.After the transfection 22 hours, change normal cell nutrient solution (the MEM cell culture fluid that contains 10%FBS), 37 ℃, after 5%CO2 is hatched 72 hours, with reference to MTS (Promega) working instructions, every hole adds MTS20 μ l/100 μ l nutrient solution, and 37 ℃ of lucifuges were hatched 1.5 hours, and 490nm detects absorbancy at multiple labeling inspection enzyme-linked immunosorbent assay instrument.Simultaneously, behind the transfection ASODN every day observation of cell form under inverted microscope.
3. sulfo-Antisensedigonucleotsequence sequence FN1, the specificity of FN5 is investigated
With 0.8 μ M transfection HepG2.2.15 cell, transfection method is with embodiment two respectively for sulfo-Antisensedigonucleotsequence sequence FN1, FN5 and just sequence FN1s thereof, FN5s.Collect nutrient solution, extract total RNA and total protein, with reference to the method for embodiment one, example two, detect FN1, HBsAg in FN5 and the just sequence pair cell nutrient solution thereof, the restraining effect of HBeAg, FN1, FN5 and just sequence thereof are to the proteic restraining effect of fibronectin.
4. sulfo-Antisensedigonucleotsequence sequence FN1, the dose-dependently of FN5 detects
Sulfo-Antisensedigonucleotsequence sequence FN1, FN5 is with 0.2 μ M, 0.4 μ M, 0.8 μ M transfection HepG2.2.15 cell, and transfection method is with embodiment one.Collect nutrient solution, extract total protein,, detect FN1, HBV DNA in the FN5 different concns pair cell nutrient solution, HBsAg, the restraining effect of HBeAg and the proteic restraining effect of fibronectin with reference to the method for embodiment one, example two.
The result
1. sulfo-antisense oligonucleotide FN1, FN5 is to the value-added influence of HepG2.2.15 cell
Sulfo-antisense oligonucleotide FN1, FN5 handles in the cell processes with 0.2 μ M, 0.4 μ M, 0.8 μ M, 1.6 μ M, 10 μ M, and every day is the observation of cell form under inverted microscope, and dosing group cellular form and cellular control unit form do not have noticeable change.The cell proliferation experiment detected result is seen Fig. 7, Fig. 8.Show among the figure that in 0.2 μ M-10 μ M scope, each dosage group OD value of FN1, FN5 and normal cell contrast are basic identical.
2. sulfo-Antisensedigonucleotsequence sequence FN1, the specificity of FN5
Sulfo-Antisensedigonucleotsequence sequence FN1, FN5 and just sequence FN1s thereof, FN5s is respectively with 0.8 μ M transfection HepG2.2.15 cell, collect nutrient solution, the ELISA detection kit detects the expression of HBsAg, HBeAg in the nutrient solution, if the content of HBsAg, HBeAg is 100% in the control cells nutrient solution, the content of HBsAg, HBeAg then is in the experimental group: A
Experimental group/ A
The cell control group* 100%, wherein A is illustrated in the absorbancy of 450nm.The results are shown in Figure 10,11.Show FN1 among the figure, the just G 3139 sequence of FN5 (FN1s, FN5s) HBsAg, HBeAg and normal cell contrast no significant difference (P 〉=0.05) in the treatment group cell culture fluid.And sulfo-Antisensedigonucleotsequence sequence FN1, FN5 has good restraining effect (inhibiting rate 〉=50%).Equally, at sulfo-Antisensedigonucleotsequence sequence FN1, FN5 and just sequence FN1s thereof, FN5s is respectively with behind the 0.8 μ M transfection HepG2.2.15 cell 72 hours, extract total protein, the western detected result shows FN1s, and FN5s handles groups of cells fibronectin protein expression and control group basically identical, and FN1, FN5 demonstrates the effect (see figure 9) of good restraining fibronectin protein expression.
3. sulfo-Antisensedigonucleotsequence sequence FN1, FN5 is to the hbv replication and the dose-dependently of expressing influence.
Sulfo-Antisensedigonucleotsequence sequence FN1, after FN5 handles the HepG2.2.15 cell with 0 μ M, 0.2 μ M, 0.4 μ M, 0.8 μ M administration, collect nutrient solution, the copy number of HBV DNA in the fluorescence quantitative PCR detection nutrient solution, the expression that the ELISA detection kit detects HBsAg, HBeAg in the nutrient solution calculates inhibiting rate respectively according to the formula among the embodiment two.The triplicate experiment, the mean value of calculating inhibiting rate.As Figure 14 and Figure 15, FN1, HBV DNA in the FN5 pair cell nutrient solution, HBsAg, the restraining effect of HBeAg is along with sulfo-Antisensedigonucleotsequence sequence FN1, and the increase of FN5 concentration and successively decreasing demonstrates dose-dependence clearly.Equally, at 0 μ M, 0.2 μ M, 0.4 μ M, 0.8 μ M FN1, FN5 handled behind the cell 72 hours, extracted total protein, and the western engram technology detects the proteic expression of fibronectin, the result is shown in Figure 12,13, along with FN1, the increase of FN5 concentration, the fibronectin expressing quantity reduces gradually, 0.8 during μ M, FN5 handles groups of cells and almost detects the proteic expression less than fibronectin.
Conclusion
1. sulfo-Antisensedigonucleotsequence sequence FN1, FN5 is to the not significantly influence of propagation of HepG2.2.15 cell
2. sulfo-Antisensedigonucleotsequence sequence FN1, FN5 have the effect of duplicating and expressing of HBV in the sequence-specific inhibition HepG2.2.15 cell
3. in 0.2-0.8 μ mol/L concentration range, FN1, FN5 be to HBV DNA, HBsAg, and HBeAg and fibronectin albumen have specific dose-dependent inhibition activity.
Sequence table
＜110〉INST OF EMISSION ﹠ RADIATION M
＜120〉structure and the purposes of the antisense oligonucleotide of inhibition fibronectin expression
<130>
<160>5
<170>Patent version 3.1
<210>1
<211>20
<212>DNA
<213>
<400>1
gct cat ctc cct cct cac tc 20
<210>2
<211>20
<212>DNA
<213>
<400>2
ttc gtt ccc act cat ctc ca 20
<210>3
<211>20
<212>DNA
<213>
<400>3
ctg ggg ctg aac cat ttg ct 20
<210>4
<211>20
<212>DNA
<213>
<400>4
gcc ttc aat agt cat ttc tg 20
<210>5
<211>20
<212>DNA
<213>
<400>5
gac ggt ccc act tct ctc ca 20
Claims (5)
1. with fibronectin mRNA5 ' non-coding region and coding region complementary antisense oligonucleotide, the sequence of described antisense oligonucleotide be selected from one of following:
1)FN1：5’-GCT CAT CTC CCT CCT CAC TC-3’；
2)FN2：5’-TTC GTT CCC ACT CAT CTC CA-3’；
3)FN3：5’-CTG GGG CTG AAC CAT TTG CT-3’；
4)FN4：5’-GCC TTC AAT AGT CAT TTC TG-3’；
5)FN5：5’-GAC GGT CCC ACT TCT CTC CA-3’。
2. antisense oligonucleotide according to claim 1, it is one of following to it is characterized in that described Antisensedigonucleotsequence sequence structure is selected from:
1)FN1：5’-GCT CAT CTC CCT CCT CAC TC-3’；
5)FN5：5’-GAC GGT CCC ACT TCT CTC CA-3’。
3. antisense oligonucleotide according to claim 1 is characterized in that this antisense oligonucleotide is through the different chemical modification.
4. antisense oligonucleotide according to claim 3, its chemically modified are thio-modification.
5. the purposes of the arbitrary antisense oligonucleotide described in the claim 1,2,3,4 in preparation treatment hepatitis B and diseases related medicine thereof.
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| BR112012019881A2 (en) * | 2010-02-18 | 2017-06-27 | Bristol Myers Squibb Co | fibronectin-based structural domain proteins that bind to il-23 |
| EP2773659A2 (en) | 2011-10-31 | 2014-09-10 | Bristol-Myers Squibb Company | Fibronectin binding domains with reduced immunogenicity |
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Non-Patent Citations (4)
| Title |
|---|
| 不同基因区反义抑制肝炎病毒基因表达的比较 钟森，王升启，中华传染病杂志，第16卷第1期 1998 * |
| 不同基因区反义抑制肝炎病毒基因表达的比较 钟森，王升启，中华传染病杂志，第16卷第1期 1998;反义寡核苷酸抗乙型肝炎病毒的研究进展 丘梦标，张吉翔，国外医学*流行病学传染病学分册，第30卷第5期 2003;乙型肝炎病毒对纤维连接蛋白基因表达的调节 杨瑗 成军 赵英仁，胃肠病学和肝病学杂志，第13卷第1期 2004 * |
| 乙型肝炎病毒对纤维连接蛋白基因表达的调节 杨瑗 成军 赵英仁，胃肠病学和肝病学杂志，第13卷第1期 2004 * |
| 反义寡核苷酸抗乙型肝炎病毒的研究进展 丘梦标，张吉翔，国外医学*流行病学传染病学分册，第30卷第5期 2003 * |
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