CN1279243A - Cancer suppressing action of antisense nucleic acid to leukemia gene of bursa-dependent lymphocyte - Google Patents
Cancer suppressing action of antisense nucleic acid to leukemia gene of bursa-dependent lymphocyte Download PDFInfo
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- CN1279243A CN1279243A CN 99109364 CN99109364A CN1279243A CN 1279243 A CN1279243 A CN 1279243A CN 99109364 CN99109364 CN 99109364 CN 99109364 A CN99109364 A CN 99109364A CN 1279243 A CN1279243 A CN 1279243A
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Abstract
The present invention relates to chemically synthesize a series of antisense nucleic acids with specific sequence. Its base sequence is complemented with a sequence segment of human B cell's leukemia-2 gene (Bcl-2). These oligonucleotides can be specifically hybridized with the mRNA of human Bcl-2 gene in cell, so inhibiting the expression of Bcl-2 protein coded by Bcl-2 gene to suppress the growth of tumor cells. These antisense olignucleotides can be used to prepare the medicine to suppress growth of human-tumors.
Description
The present invention relates to suppress the oligonucleotide of human body tumour cell growth, relate to the Lymphocytic leukemia-2 (B cell leukemia-2 that suppresses the human tumor growth specifically at the capsule dependence, Bcl-2) oligomeric deoxynucleotide and contain the composition of described oligonucleotide, and the purposes that described oligonucleotide is used to prepare antitumor drug.
Oligonucleotide set forth in the present invention, the one section sequence complementation with human body Bcl-2 gene that puts in order of its base, these oligonucleotide are hybridized with the messenger RNA(mRNA) (mRNA) of human body Bcl-2 gene in cell specifically, thereby Bcl-2 protein expression that stops Bcl-2 genes encoding, reach effect (the Shinichi kitada that suppresses growth of tumour cell, Antisense Research and Development.4:71-79,1994).
Bcl-2 gene mainly is present in the organoids such as the mitochondrial inner membrane, nuclear membrane, endoplasmic reticulum of cell, and it is very wide to distribute, and is stored in lymphocyte, immature hemopoietic stem cell, epithelial cell, neurocyte etc.Bcl-2 gene is found from the lymphomatosis human B lymphocyte in Tsujimoto in 1984.Bcl-2 gene pairs apoptosis has the obvious suppression effect, and closely related with the resistance of tumour.Have now found that many and gene Bcl-2 dna homolog, Bcl-2, Bcl-XL, BAX, BAK, AI, MCL1, LMWS-HL, BHRF1, CED-9 etc. are arranged.The common ground of this gene family is that two conserved sequence: BH1, BH2 (Bcl-2homologuel 1 and 2) are arranged, substitute the tryptophane of 155 of 145 glycine of BH1 and BH2 with the point mutation technology, the Bcl after the change-2 loses the ability that radioprotective and Antiglucocorticoid cause apoptosis.
Bcl-2 gene has 3 exons: I, II, III, and wherein I is not translated, and the open reading frame of Bcl-2 is positioned at the land of II and III, and the intron of 220bp is arranged between I and II, and the intron of 370bp is arranged between II and III.5 ' of splice site end all has promotor in exon II and the exon, can produce the different mRNA that are made up of I, II or I, II, III with different promotors.
Bcl-2 gene can quicken cell fission, cell proliferation, safeguards cells survival, prolong cell survival, causes cell number to increase.Experiment shows: Bcl-2 big leap ahead of cells transfected survival time, Bcl-2 anti-apoptotic mechanism shows its adjustable ganglion cell's redox state, be combined into heterodimer with BAX, suppress the pro-apoptotic effect of BAX, influence the redistribution of metabolic defect in cellular calcium ion.Calcium ion activated endogenic restriction endonuclease and transglutaminase suppress cytochrome C and are discharged into endochylema from plastosome.Cytochrome C promotes to transfer dies, and in natural death of cerebral cells, mitochondrial membrane has unpolarizing, permeability changes, thus cytochrome C flows out outside the mitochondrial membrane, enters endochylema, thus promote necrocytosis.
Bcl-2 molecule abnormality is expressed the effect that promotes that tumour forms, and is common in the lymphadenomatous generating process of T, B.Among 90% the B cell follicular lymphoma patient t (14 is arranged; 18) transposition, on Ig heavy chain Jh fragment gene on the 14q32 and the 18q21 Bcl-2 gene of transcriptional activity is arranged like this and put, make Bcl-2 gene high expression level under the regulation and control of Ig heavy chain Jh section, and Bcl-2 overexpression in transgenic animal, can disturb the generation of superoxide and the peroxidation of adipose membrane, thus the necrocytosis of inhibited oxidation thing stress-induced.
Bcl-2 is expressed in the human tumor of many types and promotes it to transcribe, and 26K dalton Bcl-2 albumen is closed and can causes programmed cell death (Michael.L.cleary, Cell (47) 10.1986.19-28).Bcl-2 expresses on non Hodgkin lymphoma usually and can guide the opposing programmed cell death and promote tumor growth (Webb.D.Cunningham.TheLancet.349.April.19.1997,1137-1141).If attack the mRNA of Bcl-2 with antisense oligonucleotide, Bcl-2 is expressed descend, thereby programmed cell death is increased, can effectively suppress growth of tumor (Fionna.J.Keith, Leukemia 1995.9.131-138) like this.
Bcl-2 gene is relevant with rheumatoid arthritis, systemic lupus erythematous, kidney injury, cardiovascular system diseases, tumour etc., as: mesentery proliferous type glomerulonephritis in the glomerulonephritis, find that cell count and the cell proliferation of the suppressor gene Bcl-2 of energy express cell apoptosis has tangible dependency.Evidence, Bcl-2 overexpression can make that the mesangial cell number increases in the glomerulonephritis, and generation time prolongs.
The research that above-mentioned relevant Bcl-2 gene works in tumour takes place makes people with the target spot of Bcl-2 gene as the oncotherapy medication.A large amount of screening operations show, can filter out many molecules, and wherein great majority act on the reactive site or the regulatory site of organoid.Wish that the molecule filter out can be used as cancer therapy drug, can bring into play the drug action mechanism different, and these molecules can produce the cell growth-inhibiting but do not produce cytotoxicity with existing anticarcinogen.Though yet very big drawback is these compounds is specific to Bcl-2 gene sometimes, and other genes can not shown very strong selective power.Bcl-2 gene is also further carrying out in the further investigation aspect tumour generation and the adjusting and controlling growth, whether can suppress growth of tumor by regulation and control and also will constantly study, the irrelevant side effect that occurs do not wished at Bcl-2 gene regulating a lot of take place otherwise can cause with tumour at Bcl-2 gene.
Our selected antisense nucleic acid is by hybridizing with the mRNA of Bcl-2 is complementary, suppressing mRNA by number of mechanisms and express Bcl-2 albumen.Because antisense nucleic acid can specific combining target mRNA, can design a kind of antisense nucleic acid, require itself and tumour that the mRNA complementation of relevant Bcl-2 takes place, the specificity selection of height also can make it not produce the toxic side effect that present other drug produces.
The present invention filters out the antisense nucleic acid special to Bcl-2 in cell cultures, be incorporated into mRNA 3 ' the end non-translational region of Bcl-2 gene, demonstrates the inhibition to the mRNA marking protein of Bcl-2.Studies show that at present Bcl-2 genetic expression descends, can promote programmed cell death, and then suppress tumor growth, reach therapeutic purpose.
One of purpose of the present invention is that a kind of antisense dna oligo (DNA) or Yeast Nucleic Acid (RNA) sequence can be provided, but it is characterized in that the different zones of specificity in conjunction with the mRNA of Bcl-2 gene.
Another object of the present invention provides the pharmaceutical composition that contains antisense oligonucleotide of the present invention.
Another object of the present invention has provided the purposes that antisense oligonucleotide of the present invention is used to prepare antitumor drug.
The present invention has also designed the antisense nucleic acid molecule of different zones of the mRNA of a series of Bcl-2 that can be incorporated into Bcl-2 gene, the antisense nucleic acid that screening suppresses Bcl-2 genetic expression specificity in cell cultures.Cell cultures adopts the A549 cell, sets up a negative control PAC23b, two positive control PAC14, PAC66, and all antisense nucleic acid molecule length of screening are 20 Nucleotide.The selection result shows that wherein nine antisense nucleic acides all have the characteristic of inhibition human body tumour cell growth in various degree, they are named respectively be PAC60, PAC61, PAC62, PAC63, PAC64, PAC67, PAC68, PAC69, PAC70, wherein PAC63 can suppress the activity of human body tumour cell growth the biglyyest.
It is as follows to have active PAC series antisense oligonucleotide based composition of the human body tumour cell growth of inhibition and order:
PAC60： 5’—AGT?CCG?GTA?TTC?GCA?GAA?GT—3’ 20mer
PAC61： 5’—CAA?TGT?ATT?AAG?CTG?CCT?GG—3’ 20mer
PAC62： 5’—GTC?TAC?TTC?CTC?TGT?GAT?GC—3’ 20mer
PAC63： 5’—CGC?GGA?ACA?CTT?GAT?TCT?GG—3’ 20mer
PAC64： 5’—AGG?AGT?TAT?AAT?CCA?GCT?AT—3’ 20mer
PAC67： 5’—CAG?TAA?ATA?GCT?GAT?TCG?AC—3’ 20mer
PAC68： 5’—TCC?GTC?TGC?TCT?TCA?GAT?GG—3’ 20mer
PAC69： 5’—CGA?TCT?CGG?ACC?TGT?GGC?CT—3’ 20mer
PAC70：?5’—TTC?GCC?GGC?TCC?ACA?GCC?TC—3’?20mer
Set up two of positive controls, PAC14C and PAC66, its sequence is as follows:
PAC14：?5’—TCC?CGC?CTG?TGA?CAT?GCA?TT—3’?20mer
PAC66：?5’—TCT?CCC?AGC?GTG?CGC?CAT—3’ 18mer
Set up PAC23 of negative control, its sequence is as follows:
PAC23：?5’—CAC?GGT?GCG?TCG?ACG?CAC?TA—3’?20mer
More than designed antisense oligonucleotide except that PAC66 is 18 Nucleotide, all the other all are that length is 20 Nucleotide and through the oligonucleotides of thio-modification, target of attack is the messenger RNA(mRNA) (mRNA) of human body Bcl-2 gene.This section antisense nucleotide can combine with messenger RNA(mRNA) (mRNA) specificity of Bcl-2 gene in tenuigenin, thereby the proteic expression of Bcl-2 of blocking-up Bcl-2 genes encoding, promote programmed cell death, can finally reach the effect that suppresses tumor growth like this.The designed antisense base sequences of the present invention is by bringing into play its distinctive biological characteristics with the specific hybrid of the mRNA of Bcl-2 gene.At present, the hybridization avidity of RNA and mRNA has very high status than the hybridization avidity height of DNA and mRNA in the nucleic acid hybridization on the medical science pharmaceutical use.But because the artificial-synthetic DNA is again far away than low many of the cost of synthetic RNA, according to the needs of medical market and the cost requirement of clinical application, the antisense drug of using in clinical trial is DNA at present.The cost of synthetic RNA is high, and along with the continuous progress of science and technology and the improvement of technology, the cost of synthetic RNA will decrease in future, be expected to use clinically.Synthetic oligomerization Yeast Nucleic Acid (RNA) will be developed as new drug with the chimeric antisense nucleic acid that forms that links to each other of RNA (ribonucleic acid) monomer and thymus nucleic acid (DNA) monomer.
The antisense nucleic acid medicament of the present invention's design, it is active that its sequence has specific biological.For a certain gene locus complementary antisense nucleic acid and complementary length much relations are arranged, longer as complementary, then biologic activity is just higher, and the effect of curing the disease will be better, but the cost of the antisense nucleic acid of synthetic will increase, and does not so just meet antisense nucleic acid as medicinal purpose.The anti sense nucleotide sequence through 20 base length of screening is all adopted in this invention, increase or reduce an antisense nucleic acid that is complementary to same gene locus to several bases, equally also can have biologic activity in various degree, also can reach the effect of curing the disease in various degree.Being complementary near the homologous genes site antisense nucleic acid combining site simultaneously has in various degree overlapping, and these can think to have in various degree sequence homology, have identical biologic activity in various degree too.
Various chemical modification methods are a lot of in the antisense nucleic acid research now, why adopt the antisense nucleic acid of thio-modification, the preclinical studies such as pharmacology, pharmacokinetics, toxicology that mainly are the antisense nucleic acid of sulfo-are that research is the most comprehensive in the antisense nucleic acid of various chemically modifieds.The sulfo-antisense nucleic acid can prevent effectively that in vivo a large amount of exonucleases are cut Degradation to the enzyme of antisense nucleic acid in the human body, makes antisense nucleic acid keep its due biologic activity.Therefore the antisense nucleic acid of sulfo-also can excite the activity of RNA enzyme in addition, and degraded is the RNA chain of hybridization with it, selects for use the antisense nucleic acid of the particular design of thio-modification all to have in various degree biologic activity.
In sum, the antisense nucleic acid that is complementary to Bcl-2 gene mRNA different zones has the characteristic that suppresses human body tumour cell growth, treats tumour clinically and has using value very widely.Filter out the different lengths and the anti sense nucleotide sequence of homology in various degree through chemically modified, can develop into antitumor drug with clinical value.Antisense nucleic acid of the present invention can be combined with pharmaceutical carrier, be used for animal (comprising Mammals and people).Used pharmaceutical carrier can be phosphate buffered saline buffer (PH7.4) and other carrier and damping fluids commonly used.Marginal data:
Among the figure: antisense nucleic acid PAC series is to the inhibition result (WesternBlotting) of Bcl-2 genetic expression.
The A549 cell strain is divided into control group (non-processor) and antisense nucleic acid treatment group, in containing HAM ' S S/F-12 nutrient solution of 10% foetal calf serum, cultivate, being cultured to total cellular score is 100,000 o'clock, change cell culture fluid into serum-free medium, the not homotactic sulfo-antisense nucleic acid that the liposome of all the other group adding 20ug/ml and final concentration are 800 nM except that control group was handled 5 hours, change common nutrient solution again into, recovered 20 hours.Extract albumen with the NP40 lysis buffer,, be transferred on the Hybond nylon membrane at 200V voltage, 12% the last electrophoresis of SDS-PAGE.Western analyzes, and one resists the antibody with mouse-anti Bcl-2, and two is anti-with sheep anti-mouse antibodies (top in conjunction with AP), the colour developing BCIP/NBT of Promega company.
No. 0 sample is the non-processor blank among the figure, No. 23 negative contrasts of sample, and other are the sample of different PAC series antisense nucleic acid.As seen from the figure, PAC series antisense nucleic acid has different inhibition abilities to the expression of Bcl-2, and wherein the inhibition degree with PAC60 is the highest.
Fig. 2: antisense nucleic acid PAC series is to human body tumour cell strain A549 growth-inhibiting
The A549 cell is divided into blank group, positive controls, negative control group and PAC series antisense nucleic acid treatment group.Using respectively and being cultured to total cellular score in HAM ' S S/F-12 nutrient solution of 10% foetal calf serum is 100,000 o'clock, change serum-free medium into, except that blank, the liposome that all the other groups add 10ug/ml respectively and final concentration are that 800 different sulfo-antisense nucleic acides were handled 5 hours, change the nutrient solution that contains 10% foetal calf serum normally again into, continue down to cultivate at 37 ℃.Carry out cell counting after 66 hours, measure the cell extent of growth with mtt assay.With blank group numerical value is 100%, make cell growth-inhibiting figure, as seen from the figure, the representative of black cylinder is at the positive antisense nucleic acid result of Bcl-2 gene, the negative contrast antisense nucleic acid of grey cylinder result, white cylinder is the PAC series antisense nucleic acid result at Bcl-2 gene.Wherein PAC66 is the positive control antisense nucleic acid result at Bcl-2 gene, and the antisense nucleic acid cell growth of the PAC series among the figure all has inhibition in various degree, and it is the highest wherein to suppress degree with PAC63.
Embodiment 1: antisense nucleic acid is attacked the synthetic of determining of target position target and antisense nucleic acid
391-04 type DNA-RNA synthesizer that uses U.S. applying biological Account Dept of PE company (Applied Biosystems) to make.Antisense nucleic acid with phosphorous acid acid amides solid phase synthesis sulfo-.
Synthesis material is available from U.S. Glen company, and main raw material has:
Four kinds of di-isopropyl-cyanoethyl phosphorous acid amide monomers: Desoxyadenosine (dA), pancreatic desoxyribonuclease (dG), Deoxyribose cytidine (dC), deoxythymidine (dT).
Four kinds of control aperture glass powder (CPG).Solid phase synthesis post (A, C, G, T), scale is 10 micromoles.
Block reagent:
Block reagent A: diacetyl oxide, lutidine, tetrahydrofuran (THF)
Block reagent B: monomethyl imidazoles, tetrahydrofuran (THF)
Thio reaction reagent: Beaucage reagent, acetonitrile
Detritylation reagent: trichoroacetic acid(TCA), methylene dichloride
Liquid reagent: acetonitrile, trichloromethane
Synthetic scale is 10 micromoles, and the base monomer is dissolved in the anhydrous acetonitrile, and concentration is 100 mmoles, and other reagent concentration and consumption all carry out according to the instrument handbook.
Itself can use time variable control the building-up process instrument.
Synthetic product cuts down with strong aqua (28%) and is collected in the withstand voltage vial of sealing, handles various hydroxy-protective groups and the amido protecting group of sloughing on the antisense nucleic acid molecule in 15 hours in 55 ℃.After deprotection finishes; most of ammonia is removed in volatilization; freeze-drying gets the white powder crude product; again be dissolved in the damping fluid; carry out purifying with Pharmacia Source 30Q chromatography column and AKTA chromatographic system; purification of samples G-15 molecular sieve sephadex chromatography post desalination, freeze-drying gets the pure product of white cotton-shaped antisense nucleic acid, is stored in one 30 ℃.The sample that takes a morsel carries out following analysis:
1, surveys its purity and molecular weight with capillary electrophoresis;
2, survey its purity with high performance liquid chromatography (HPLC);
3, the dna sequencing method is measured its sequence accuracy;
4, P
31NMR (nucleus magnetic resonance) method is measured its degree of thioation;
Capillary electrophoresis and high-efficient liquid phase technique purity check result show that the pure product purity of Pac series antisense nucleic acid is all greater than 90%.
Sequencing proof Pac series anti sense nucleotide sequence is identical with implementation sequence.
P
31NMR analyzes and shows that Pac series antisense nucleic acid sulfo-rate is greater than 98.5%.
Embodiment 2: at the Pac series antisense nucleic acid of Bcl-2
The inhibition of in culturing cell, Bcl-2 being expressed
Select for use human body lung cancer cell line A549 as the screening experiment cell strain, document (Miwa.W.et with reference to Miwa.W etc., al.1994 Biologicai Chemistry Lipopeseyler, 375 (10): 705-709), test is grouped into: blank non-processor group, negative control group, positive controls, antisense nucleic acid treatment group.The A549 cell strain is cultivated in containing HAM ' S S/F-12 nutrient solution of 10% foetal calf serum, being cultured to total cellular score is 100,000 o'clock, change cell culture fluid into serum-free medium, the not homotactic sulfo-antisense nucleic acid that the liposome of all the other group adding 20ug/ml and final concentration are 800 nM except that control group was handled 5 hours, change common nutrient solution again into, recovered 20 hours.Extract albumen with the NP40 lysis buffer,, be transferred on the Hybond nylon membrane at 200V voltage, 12% the last electrophoresis of SDS-PAGE.The Molecular Cloning:A Loboratory Manual.2 that the Western hybridization analysis is write with reference to Sambrook.J etc.
Nd.ed ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y..One is anti-with mouse-anti Bcl-2 antibody in the hybridization, and two is anti-with sheep anti-mouse antibody (top in conjunction with AP), develops the color with the BCIP/NBT of Promega company.
The result of hybridization is quantitatively drawn by Pharmcia Image Master scanner.Bcl-2 hybrid belt content with the non-processor cellular control unit is 100%, and the result as shown in Figure 1.No. 0 sample is the non-processor blank among the figure, No. 23 negative contrasts of sample, and other is the sample of different PAC series antisense nucleic acid.As seen from the figure, PAC series antisense nucleic acid has different inhibition abilities to the expression of Bcl-2, and wherein the inhibition degree with PAC60 is the highest.
The structure of chemical composition of embodiment 3:Pac63
With reference to Nucleic Acids Research.1995, Vol:24, the method for No:2.P4219-4223 is carried out sequencing to Pac63, and the result is as follows:
PAC63：5’—CGC?GGA?ACA?CTT?GAT?TCT?GG—3’
Consistent with implementation sequence, with the molecular weight of capillary electrophoresis mensuration Pac63, the result is 6416, with theoretical molecular 6418 approximately equals.
Pac63 is the antisense nucleic acid that 20 deoxynucleosides link to each other with the phosphoric acid thioester bond, and the target target of its attack is the mRNA of Bcl-2 gene.
Embodiment 4:MTT method is measured antisense nucleic acid Pac series
Inhibition to human body tumour cell strain A549 growth
The A549 cell is divided into blank group, positive controls and Pac series antisense nucleic acid treatment group, and being cultured to total cellular score respectively in containing HAM ' s S/F-12 nutrient solution of 10% foetal calf serum is 100,000.Change serum-free medium into, except that the blank group, the liposome that all the other groups add 10ug/ml respectively and final concentration are that the different separately antisense nucleic acid of 800nM was handled 5 hours, change the nutrient solution that normally contains 10% foetal calf serum into, continue to cultivate at 37 ℃.Carry out cell counting after 66 hours, measure the cell extent of growth with mtt assay.With blank group numerical value is 100%, makes cell growth-inhibiting figure.Among the figure as seen, the representative of black cylinder is at the positive control antisense nucleic acid result of Bcl-2 gene, the negative contrast antisense nucleic acid of grey cylinder result, remaining white cylinder is the Pac series antisense nucleic acid result at Bcl-2 gene, and wherein Pac66 is at Bcl-2 gene masculine contrast antisense nucleic acid result.Pac series antisense nucleic acid cell growth among the figure all has inhibition in various degree, and wherein Pac63 inhibition degree is the highest.
Embodiment 5: the pharmaceutical composition that contains antisense nucleic acid
Antisense nucleic acid medicine composition as the animal vivo medicine-feeding is composed as follows: antisense nucleic acid Pac63 10mg/ml one hypophosphite monohydrate one hydrogen sodium 1.73mg/ml seven hypophosphite monohydrate disodium hydrogen 14.33mg/ml sodium-chlor 4.4mg/ml waters for injection are an amount of
Other several antisense oligonucleotides also adopt same prescription among the present invention.
Do not use superfluous words, antisense nucleic acid of the present invention to a great extent can by different chemically modifieds, adopt different length and in various degree the sequence of homology develop into antitumor drug with clinical value.It can use separately or unite use with other medicines and carry out tumor treatment, and these it seems it is conspicuous to one skilled in the relevant art.
( 1 ) 、 ( ⅲ ) ：9 ( 1 ) ．1 ( ⅰ ) ． ( A ) ．：20 ( B ) ．： ( C ) ．： ( D ) ．： ( ⅱ ) ．： ( ⅳ ) ．： ( ⅹⅰ ) ．：1 PAC60 AGT CCG GTA TTC GCA GAA GT 20 ( 2 ) ．2 ( ⅰ ) ． ( A ) ．：20 ( B ) ．： ( C ) ．： ( D ) ．： ( ⅱ ) ．： ( ⅳ ) ．： ( ⅹⅰ ) ．：2 PAC61 CAA TGT ATT AAG CTG CCT GG 20 ( 3 ) ．3 ( ⅰ ) ． ( A ) ．：20 ( B ) ．： ( C ) ．： ( D ) ．： ( ⅱ ) ．： ( ⅳ ) ．： ( ⅹⅰ ) ．：3 PAC62 GTC TAC TTC CTC TGT GAT GC 20 ( 4 ) ．4 ( ⅰ ) ． ( A ) ．：20 ( B ) ．： ( C ) ．： ( D ) ．： ( ⅱ ) ．： ( ⅳ ) ．： ( ⅹⅰ ) ．：4 PAC63 CGC GGA ACA CTT GAT TCT GG 20 ( 5 ) ．5 ( ⅰ ) ． ( A ) ．：20 ( B ) ．： ( C ) ．： ( D ) ．： ( ⅱ ) ．： ( ⅳ ) ．： ( ⅹⅰ ) ．：5 PAC64 AGG AGT TAT AAT CCA GCA GCT AT 20 ( 6 ) ．6 ( ⅰ ) ． ( A ) ．：20 ( B ) ．： ( C ) ．： ( D ) ．： ( ⅱ ) ．： ( ⅳ ) ．： ( ⅹⅰ ) ．：6 PAC67 CAG TAA ATA GCT GAT TCG AC 20 ( 7 ) ．7 ( ⅰ ) ． ( A ) ．：20 ( B ) ．： ( C ) ．： ( D ) ．： ( ⅱ ) ．： ( ⅳ ) ．： ( ⅹⅰ ) ．：7 PAC68 TCC GTC TGC TCT TCA GAT GG 20 ( 8 ) ．8 ( ⅰ ) ． ( A ) ．：20 ( B ) ．： ( C ) ．： ( D ) ．： ( ⅱ ) ．： ( ⅳ ) ．： ( ⅹⅰ ) ．：8 PAC69 CGA TCT CGG ACC TGT GGC CT 20 ( 9 ) ．9 ( ⅰ ) ． ( A ) ．：20 ( B ) ．： ( C ) ．： ( D ) ．： ( ⅱ ) ．： ( ⅳ ) ．： ( ⅹⅰ ) ．：9 PAC70 TTC GCC GGC TCC ACA GCC TC 20
Claims (6)
1, a kind of oligomerization antisense thymus nucleic acid (DNA) or Yeast Nucleic Acid (RNA), but it is characterized in that the different zones of specificity in conjunction with human body B cell leukemia-2 (Bcl-2) gene, and described oligonucleotide sequence is selected from:
1．?PAC60： 5’—AGT?CCG?GTA?TTC?GCA?GAA?GT—3’
2．?PAC61： 5’—CAA?TGT?ATT?AAG?CTG?CCT?GG—3’
3．?PAC62： 5’—GTC?TAC?TTC?CTC?TGT?GAT?GC—3’
4．?PAC63： 5’—CGC?GGA?ACA?CTT?GAT?TCT?GG—3’
5．?PAC64： 5’—AGG?AGT?TAT?AAT?CCA?GCT?AT—3’
6．?PAC67： 5’—CAG?TAA?ATA?GCT?GAT?TCG?AC—3’
7．?PAC68： 5’—TCC?GTC?TGC?TCT?TCA?GAT?GG—3’
8．?PAC69： 5’—CGA?TCT?CGG?ACC?TGT?GGC?CT—3’
9. PAC70:5 '-TTC GCC GGC TCC ACA GCC TC-3 ' and homologous sequence thereof.
2, the antisense nucleic acid in the claim 1, wherein said homologous sequence and sequence 1-9 have at least 70% homology.
3, the antisense nucleic acid in the claim 1, wherein said homologous sequence and sequence 1-9 have at least 80% homology.
4, the antisense nucleic acid in the claim 1, wherein said homologous sequence and sequence 1-9 have at least 90% homology.
5, contain anti sense nucleotide sequence in the claim 1 and the medicine of pharmaceutically acceptable carrier.
6, the antisense nucleic acid in the claim 1 is used to prepare the purposes of antitumor drug.
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