CN101225383B - RNA interference sequence and recombinant interference plasmid of fucosyl transferase I and IV for suppressing synthesis of LeY carbohydrate antigen - Google Patents
RNA interference sequence and recombinant interference plasmid of fucosyl transferase I and IV for suppressing synthesis of LeY carbohydrate antigen Download PDFInfo
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Abstract
The present invention relates to a RNA interference sequence and a restructuring interference plasmid of the fucosyltransferase I and IV used for inhibiting the synthesis of LeY saccharide antigen, which mainly comprises: the DNA sequence of seven FUT1/FUT4 SiRNA templates of fucosyltransferase I and seven FUT1/FUT4 SiRNA templates of fucosyltransferase IV are screened, seven FUT1 and seven FUT4 restructuring interference plasmid are respectively constructed on the base of polyclonal specific enzyme cutting sites of the RNA interference carried (pSilencer-4.1-CMV neo), and the interference plasmid is used as antitumor drugs. The RNA interference sequence and a restructuring interference plasmid of the fucosyltransferase I and IV used for inhibiting the synthesis of LeY saccharide antigen has the advantages of obvious function of inhibiting tumor growing, high efficiency, high safety and economic tumor gene therapeutic mode.
Description
Technical field the present invention relates to a kind of special dna sequence dna and according to the reorganization interfere plasmid of this sequence construct.
The background technology malignant tumour is a kind of disease of comparatively refractory, does not still have the ideal methods of treatment.At present, mainly be to take comprehensive means such as surgical operation, radiotherapy and chemotherapy to the tumor treatment strategy, but each method still have limitation and can produce to a certain degree toxic side effect.
Summary of the invention the object of the present invention is to provide a kind of according to the tomour specific carbohydrate antigen synthetic and tumor growth, infiltration, transfer equimolecular mechanism constructed, the RNA that is used to suppress LeY tumour oligosaccharides antigen synthetic fucosyl transferase I and IV interferes sequence and reorganization interfere plasmid.The present invention mainly is: application RNAi (RNAi, RNA interference) technology is a target site with the special carbohydrate antigen of tumour, and it is antigenic synthetic to suppress cell surface Lewis Y (LeY) tumour oligosaccharides.The LeY molecular structure is Fuc α 1 → 2Gal β → 4 (Fuc α 1 → 3) GlcNAc), promptly by designing and synthesizing antigenic key enzyme fucosyl transferase I (the Fucosyltransferase I of LeY tumour oligosaccharides, be called for short FUT1) and fucosyltransferase IV (Fucosyltransferase IV, abbreviation FUT4) the interference sequence of gene, and adopt the molecular cloning recombinant technology to make up reorganization FUT1 interfere plasmid (pSilencer-4.1-FUT1SiRNA) and reorganization FUT4 interfere plasmid (pSilencer-4.1-FUT4SiRNA), be used for antitumor drug and use.
Technical scheme of the present invention is as follows:
1. design and synthesize the interference sequence of FUT1 and FUT4 gene
The homologous sequence of whole people's FUT1/FUT4 gene among the GenBank of having used the DNASTAR software analysis, the total length of the encoding sequence of FUT1/FUT4 gene is respectively 1098bp and 1218bp, the dna profiling sequence of the interfere plasmid of the reorganization FUT1/FUT4 that is used for neoplasm growth that obtains from the SiRNA design software is respectively chosen 7 as forming the template DNA sequences of interfering sequences, and length is 19bp.Wherein, the interference sequence of designed FUT1 gene is: FUT1-1:TGGCCGGTTTGGTAATCAG; FUT1-2:AGACTTTGCCCTGCTCACA; FUT1-3:GAGGAGTACGCGGACTTGA; FUT1-4:CAGATCCGCAGAGAGTTCA; FUT1-5:CGGCATGGAGTGGTGTAAA; FUT1-6:TGGCCTTCCTGCTAGTCTG; FUT1-7:TTCGCCTTTCCTCCCCTGC.The position of these sequences in the FUT1 gene as shown in Figure 1.The interference sequence of designed FUT4 gene is: FUT4-1:GCCTGGCAAGTAACCTCTT; FUT4-2:GTAACCTCTTCAACTGGAC; FUT4-3:CGGACGTCTTTGTGCCTTA; FUT4-4:CATGTGACCGTGGACGTGT; FUT4-5:GTTCTACCTGGCTTTCGAG; FUT4-6:CTCGTACCTGCTTTTCCTC; FUT4-7:GCGGAGGCAGCGCTGCCTG.The position of these sequences in the FUT4 gene as shown in Figure 2.
2. make up reorganization FUT1 interfere plasmid and make up reorganization FUT4 interfere plasmid
1. obtain to be used for the dna profiling sequence of interfere plasmid of the reorganization FUT1/FUT4 of neoplasm growth according to the SiRNA design software, it is the structure that carries out interfere plasmid with the sequence of the control region of above-mentioned FUT1/FUT4 gene and coding region.
2. according to pSilencer-4.1-CMV-neo (Ambion, Inc) the dna profiling sequence of the FUT1/FUT4 interfere plasmid of carrier is inserted in the design of carrier structure information, promptly based on the polyclone restriction enzyme site (HindIII, BamHI) of interference vector, design the dna sequence dna that the FUT1/FUT4SiRNA template of identical sticky end is arranged with interference vector, make up 7 FUT1 interfere plasmids respectively: pSilencer-4.1-FUT1-1/FUT1-2/FUT1-3/FUT1-4/FUT1-5/FUT1-6/ FUT1-7; The interfere plasmid of 7 FUT4: pSilencer-4.1-FUT4-1/FUT4-2/FUT4-3/FUT4-4/FUT4-5/FUT4-6/ FUT4-7.The 5` end of every chain contains the restriction enzyme site (underscore marks) of HindIII or BamH I.Concrete sequence is as follows:
FUT1-1(5’-3’)
GATCCTGGCCGGTTTGGTAATCAGTTCAAGAGACTGATTACCAAACCGGCCAGGA
AGCTTCCTGGCCGGTTTGGTAATCAGTCTCTTGAACTGATTACCAAACCGGCCAG
FUT1-2(5’-3’)
GATCCAGACTTTGCCCTGCTCACATTCAAGAGATGTGAGCAGGGCAAAGTCTGGA
AGCTTCCAGACTTTGCCCTGCTCACATCTCTTGAATGTGAGCAGGGCAAAGTCTG
FUT1-3(5’-3’)
GATCCGAGGAGTACGCGGACTTGATTCAAGAGATCAAGTCCGCGTACTCCTCGGA
AGCTTCCGAGGAGTACGCGGACTTGATCTCTTGAATCAAGTCCGCGTACTCCTCG
FUT1-4(5’-3’)
GATCCCAGATCCGCAGAGAGTTCATTCAAGAGATGAACTCTCTGCGGATCTGGGA
AGCTTCCCAGATCCGCAGAGAGTTCATCTCTTGAATGAACTCTCTGCGGATCTGG
FUT1-5(5’-3’)
GATCCCGGCATGGAGTGGTGTAAATTCAAGAGATTTACACCACTCCATGCCGGGA
AGCTTCCCGGCATGGAGTGGTGTAAATCTCTTGAATTTACACCACTCCATGCCGG
FUT1-6(5’-3’)
GATCCTGGCCTTCCTGCTAGTCTGTTCAAGAGACAGACTAGCAGGAAGGCCAGGA
AGCTTCCTGGCCTTCCTGCTAGTCTGTCTCTTGAACAGACTAGCAGGAAGGCCAG
FUT1-7(5’-3’)
GATCCTTCGCCTTTCCTCCCCTGCTTCAAGAGAGCAGGGGAGGAAAGGCGAAGGA
AGCTTCCTTCGCCTTTCCTCCCCTGCTCTCTTGAAGCAGGGGAGGAAAGGCGAAG
FUT4-1(5’-3’)
GATCCGCCTGGCAAGTAACCTCTTCTCAAGAGAAAGAGGTTACTTGCCAGGCGGA
AGCTTCCAGCCTGGCAAGTAACCTCTTTCTCTTGAGAAGAGGTTACTTGCCAGGCG
FUT4-2(5`-3`)
GATCCGTAACCTCTTCAACTGGACTTCAAGAGAGTCCAGTTGAAGAGGTTACGGA
AGCTTCCGTAACCTCTTCAACTGGACTCTCTTGAAGTCCAGTTGAAGAGGTTACG
FUT4-3(5’-3’)
GATCCCGGACGTCTTTGTGCCTTATTCAAGAGATAAGGCACAAAGACGTCCGGGA
AGCTTCCCGGACGTCTTTGTGCCTTATCTCTTGAATAAGGCACAAAGACGTCCGG
FUT4-4(5’-3’)
GATCCCATGTGACCGTGGACGTGTTTCAAGAGAACACGTCCACGGTCACATGGGA
AGCTTCCCATGTGACCGTGGACGTGTTCTCTTGAAACACGTCCACGGTCACATGG
FUT4-5(5’-3’)
GATCCGTTCTACCTGGCTTTCGAGTTCAAGAGACTCGAAAGCCAGGTAGAACGGA
AGCTTCCGTTCTACCTGGCTTTCGAGTCTCTTGAACTCGAAAGCCAGGTAGAACG
FUT4-6(5’-3’)
GATCCCTCGTACCTGCTTTTCCTCTTCAAGAGAGAGGAAAAGCAGGTACGAGGGA
AGCTTCCCTCGTACCTGCTTTTCCTCTCTCTTGAAGAGGAAAAGCAGGTACGAGG
FUT4-7(5’-3’)
GATCCGCGGAGGCAGCGCTGCCTGTTCAAGAGACAGGCAGCGCTGCCTCCGCGGA
AGCTTCCGCGGAGGCAGCGCTGCCTGTCTCTTGAACAGGCAGCGCTGCCTCCGCG
3. after the special sequence synthetic (Takara company), form the double chain DNA sequence of the SiRNA template of 55bp (containing sticky end HindIII, BamH I) through sex change, annealing.Concrete grammar is: two chains of template DNA sequence that synthetic are used to disturb the SiRNA of FUT1/FUT4 gene, use the dissolving of 10-100 μ l deionized water or TE (Tris-HCl EDTA) damping fluid respectively, and the light absorption value of mensuration 260nm, calculate its DNA concentration.With deionized water or TE dilution (10-500ng/ μ l).The strand sample of drawing dilution adds the DNA annealing buffer, and (contain 10mM Tris (pH7.4), 50mM NaCl 1mMEDTA) in 90-98 ℃ of sex change 3-10 minute, is cooled to 37 ℃ and kept 1 hour, preserves this annealing product in-20 ℃ of temperature, and is standby.
4. pSilencer-4.1-FUT1/FUT4SiRNA construction of recombinant plasmid
Get pSilencer-4.1-CMV-neo (10-500ng) and carried out HindIII and BamHI double digestion 1-3 hour in 37 ℃, enzyme is cut the back purified product and is connected with the product of annealing.Ligation is as follows: get 1-20 μ l annealing product, 1-20 μ l carrier enzyme and cut back purified product, 1-20 μ l ligase enzyme, 1-20 μ l and connect damping fluid, add at last deionized water to 10-200 μ l in 16 ℃ of connections of spending the night.Connect product and transform DH5 α intestinal bacteria, utilize amicillin resistance label screening positive colony, enzyme was cut evaluation after a small amount of was extracted plasmid, as Fig. 3 and Fig. 4.To identify that correct clone's carries out large quantity extracting plasmid.
Studies show that: the unusual synthetic major cause that is malignant tumour propagation, soaks into, shifts of tumour carbohydrate antigen, and be one of important symbol of its vicious transformation.Special tumour carbohydrate antigen synthetic is by the specificity decision of glycosyltransferase, and the synthetic level of carbohydrate antigen is relevant with the expression regulation of glycosyltransferase gene.FUT1/FUT4 is the key enzyme of synthetic LeY tumour oligosaccharides.FUT1 is α 1, and the fucosylated glycosyltransferase of 2-(α 1,2-FUT), and synthetic relevant with terminal α 1,2 fucosido of LeY tumour oligosaccharides antigen, catalyzed synthesizing alpha 1,2-Fucose glycosidic bond; And FUT4 is α 1, the fucosylated glycosyltransferase (α of 3-
1,3-FUT), catalyzed synthesizing alpha 1,3-Fucose glycosidic bond.LeY tumour oligosaccharides antigen is the sugar chain composition in the cell surface saccharide complex (glycoprotein, glycolipid and proteoglycan), have been found that its resulting anomaly in kinds of tumors increases, closely related with tumor proliferation, infiltration, transfer, clinical stages (classification) and prognosis.Therefore, will be effective target spot to reduce the antigenic resultant quantity of LeY tumour oligosaccharides by downward modulation FUT1 and FUT4 expression level, the research of this aspect does not appear in the newspapers at present both at home and abroad as yet.The present invention utilizes the RNA interference technique, makes up reorganization FUT1 and FUT4 interfere plasmid, and by suppressing the synthetic and expression of the special fucosyltransferase of tumour cell, it is antigenic synthetic to reduce LeY tumour oligosaccharides, and then suppresses growth of tumor, infiltration and transfer.
The present invention has following advantage compared to existing technology: adopt the present invention to make up the FUT1 of reorganization and FUT interfere plasmid and provide new thinking and means for the target gene therapy of clinical tumor, not only effect is obvious but also toxic side effect is little for it, safety, patient treatment cycle weak point is a kind of efficient, safe, economic antitumor pattern simultaneously.
Description of drawings
Fig. 1 is the site plan of dna profiling sequence on gene of the interfere plasmid of FUT1 gene of the present invention.
Fig. 2 is the site plan of dna profiling sequence on gene of the interfere plasmid of FUT4 gene of the present invention.
Fig. 3 is that the interfere plasmid enzyme of FUT1 gene of the present invention is cut identification and analysis figure.
Fig. 4 is that the interfere plasmid enzyme of FUT4 gene of the present invention is cut identification and analysis figure.
Fig. 5 is a FUT1 Gene RT-PCR analysis chart behind the interfere plasmid transfection A431 tumour cell of the present invention.
Fig. 6 is a FUT4 Gene RT-PCR analysis chart behind the interfere plasmid transfection A431 tumour cell of the present invention.
Fig. 7 is that LeY resultant quantity fluidic cell detects figure behind FUT1 interfere plasmid of the present invention (FUT1-1 and FUT1-2) the transfection A431 tumour cell.
Fig. 8 is that LeY resultant quantity fluidic cell detects figure behind FUT4 interfere plasmid of the present invention (FUT4-1 and FUT4-2) the transfection A431 tumour cell.
Fig. 9 is the inhibiting figure of FUT1 interfere plasmid of the present invention (FUT1-1 and FUT1-2) transfection A431 growth of tumour cell.
Figure 10 is the inhibiting figure of FUT4 interfere plasmid of the present invention (FUT4-1 and FUT4-2) transfection A431 growth of tumour cell.
Figure 11 is the figures of the different interfere plasmids of FUT1 of the present invention to the effect of proliferating cell nuclear antigen (PCNA) expression inhibiting.
Figure 12 is the inhibiting figure that the different interfere plasmids of FUT4 of the present invention are expressed proliferating cell nuclear antigen (PCNA).
Figure 13 is that the present invention influences the analysis chart that the mouse tumor volume is respectively organized in experimentation on animals.
Figure 14 is that the present invention influences the analysis chart that mouse tumor weight is respectively organized in experimentation on animals.
Embodiment
Example 1 makes up reorganization FUT1 interfere plasmid
1. obtain to be used for the dna profiling sequence of the reorganization FUT1 interfere plasmid of neoplasm growth according to the SiRNA design software, it is the structure that carries out interfere plasmid with the sequence of the control region of above-mentioned FUT1 gene and coding region.The 5` end of every chain contains the restriction enzyme site (underscore marks) of HindIII or BamH I.Specific as follows:
FUT1-1(5’-3’)
GATCCTGGCCGGTTTGGTAATCAGTTCAAGAGACTGATTACCAAACCGGCCAGGA
AGCTTCCTGGCCGGTTTGGTAATCAGTCTCTTGAACTGATTACCAAACCGGCCAG
FUT1-2(5’-3’)
GATCCAGACTTTGCCCTGCTCACATTCAAGAGATGTGAGCAGGGCAAAGTCTGGA
AGCTTCCAGACTTTGCCCTGCTCACATCTCTTGAATGTGAGCAGGGCAAAGTCTG
2. insert the dna profiling sequence of the FUT1 interfere plasmid of carrier according to the design of pSilencer-4.1-CMV-neo carrier structure information, promptly based on the polyclone restriction enzyme site (HindIII, BamH I) of interference vector, design the dna sequence dna that the FUT1SiRNA template of identical sticky end is arranged with interference vector, make up the interfere plasmid of pSilencer-4.1-FUT1-1 and pSilencer-4.1-FUT1-2 respectively.
3. after the special sequence synthetic (Takara company), form the double chain DNA sequence of the SiRNA template of 55bp (containing sticky end HindIII, BamH I) through sex change, annealing.Two chains of template DNA sequence that synthetic are used to disturb the SiRNA of FUT1 gene, use 100 μ l deionized water dissolvings respectively, get 1 μ l deionized water by the dilution of 1: 10 volume ratio, measure the light absorption value of 260nm, calculate its DNA concentration (about 33 μ g/ml), be diluted to 80ng/ μ l with deionized water, drawing every chain 10 μ l adds 100 μ l1 * DNA annealing buffer and (contains 10mM Tris (pH7.4), 50mM NaCl, 1mM EDTA) in 98 ℃ of sex change 3 minutes, be cooled to 37 ℃ and kept 1 hour, preserve this annealing product in-20 ℃ of temperature, standby.
4. pSilencer-4.1-FUT1 reorganization interfere plasmid makes up
PSilencer-4.1-CMV-neo (90ng) was carried out HindIII and BamH I double digestion 2 hours in 37 ℃, enzyme is cut back recovery product and is connected with the annealing product, ligation is as follows: get 2 μ l annealing product, 2 μ l carrier enzymes and cut the back purifying and reclaim product, 2 μ l ligase enzymes, 2 μ l and connect damping fluid, add 2 μ l deionized water to 10 μ l at last in 16 ℃ of connections of spending the night.Connect product and transform DH5 α intestinal bacteria, utilize penbritin (Amp) resistance screening positive colony, enzyme was cut evaluation after a small amount of was extracted plasmid.The correct sub-large quantity extracting plasmid of clone will be identified.
Example 2 makes up reorganization FUT4 interfere plasmid
1. obtain to be used for the dna profiling sequence of the reorganization FUT4 interfere plasmid of neoplasm growth according to the SiRNA design software, it is the structure that carries out interfere plasmid with the sequence of the control region of above-mentioned FUT4 gene and coding region.The 5` end of every chain contains the restriction enzyme site (underscore marks) of HindIII or BamHI.Specific as follows:
FUT4-1(5’-3’)
GATCCGCCTGGCAAGTAACCTCTTCTCAAGAGAAAGAGGTTACTTGCCAGGCGGA
AGCTTCCAGCCTGGCAAGTAACCTCTTTCTCTTGAGAAGAGGTTACTTGCCAGGCG
FUT4-2(5`-3`)
GATCCGTAACCTCTTCAACTGGACTTCAAGAGAGTCCAGTTGAAGAGGTTACGGA
AGCTTCCGTAACCTCTTCAACTGGACTCTCTTGAAGTCCAGTTGAAGAGGTTACG
2. insert the dna profiling sequence of the FUT4 interfere plasmid of carrier according to the design of pSilencer-4.1-CMV-neo carrier structure information, promptly based on the polyclone restriction enzyme site (HindIII, BamH I) of interference vector, design the dna sequence dna that the FUT4SiRNA template of identical sticky end is arranged with interference vector, make up the interfere plasmid of pSilencer-4.1-FUT4-1 and pSilencer-4.1-FUT4-2 respectively.
3. after the special sequence synthetic (Takara company), form the double chain DNA sequence of the SiRNA template of 55bp (containing sticky end HindIII, BamH I) through sex change, annealing.Two chains of template DNA sequence that synthetic are used to disturb the SiRNA of FUT4 gene, use 50 μ l deionized water dissolvings respectively, get 0.5 μ l deionized water by the dilution of 1: 10 volume ratio, measure the light absorption value of 260nm, calculate its DNA concentration, be diluted to 100ng/ μ l with deionized water, drawing every chain 10 μ l adds 46 μ l1 * DNA annealing buffer and (contains 10mM Tris (pH7.4), 50mM NaCl, 1mM EDTA) in 90 ℃ of sex change 10 minutes, be cooled to 37 ℃ and kept 1 hour, preserve this annealing product in-20 ℃ of temperature, standby.
4. pSilencer-4.1-FUT4SiRNA reorganization interfere plasmid makes up
PSilencer-4.1-CMV-neo (300ng) was carried out HindIII and BamH I double digestion 3 hours in 37 ℃, enzyme is cut back recovery product and is connected with the annealing product, ligation is as follows: get 5 μ l annealing product, 5 μ l carrier enzymes and cut the back and reclaim product, 5 μ l ligase enzymes, 5 μ l and connect damping fluid, add deionization to 50 μ l at last in 16 ℃ of connections of spending the night.Connect product and transform DH5 α intestinal bacteria, utilize penbritin (Amp) resistance marker screening positive clone, enzyme was cut evaluation after a small amount of was extracted plasmid.The correct sub-large quantity extracting plasmid of clone will be identified.
Below be detected result of the present invention:
1.pSilencer-4.1-FUT1/FUT4SiRNA recombinant plasmid transfection A431 tumour cell, the cell FUT1/FUT4 that adopts RT-PCR to detect discovery transfection recombinant plasmid expresses obviously and descends, as shown in Figure 5 and Figure 6.
2.pSilencer-4.1-FUT1/FUT4SiRNA behind the recombinant plasmid transfection A431 tumour cell, adopt Flow Cytometry to detect and find that the synthetic of LeY significantly reduces, as shown in Figure 7 and Figure 8.
3.pSilencer-4.1-FUT1/FUT4SiRNA recombinant plasmid transfection A431 tumour cell detects the cell growth that shows the transfection recombinant plasmid through tetrazolium bromide (MTT) colorimetry and significantly reduces, as Fig. 9 and shown in Figure 10.
4.pSilencer-4.1-FUT1/FUT4SiRNA recombinant plasmid transfection A431 tumour cell adopts the expression amount of Western blot scientific discovery PCNA obviously to reduce, as Figure 11 and shown in Figure 12.
5.pSilencer-4.1-FUT1-1/FUT4-1 recombinant plasmid suppresses tumor growth effect in the animal model
(1) with trysinization A431 tumour cell, centrifugal collecting cell prepares cell suspension with physiological saline, and counts with trypan blue method pair cell, makes that to contain the A431 number of cells in every 0.1ml physiological saline be 2 * 10
6Get the 4-6 nude mice (n=6～8) in age in week, every right armpit skin of mouse is injected 0.1ml tumour cell suspension down.Control group equivalent injecting normal saline.
(2) experiment is divided into four groups: blank group, empty carrier group, FUT1-1 interfere plasmid group and FUT4-1 interfere plasmid group.Injection A431 tumour cell suspension next day, every 1 day injection interfere plasmid (g/ time/mouse of 150 μ), totally 10 times.(3) every day observation experiment group and control group mouse growth conditions, plantation tumour size variation, and weigh.Therapy lasted was put to death nude mice after 22 days, and measured knurl volume (table 1) and measure tumor weight (table 2).
The analysis of knurl volume is respectively organized in table 1 experiment
Annotate: * * represents P＜0.01, and difference is extremely remarkable.
Figure 13 is seen in the comparative analysis of the knurl volume of experimental group and control group.
The analysis of tumor weight is respectively organized in table 2 experiment
Annotate: * * represents (P＜0.01), and difference is extremely remarkable.
Figure 14 is seen in the comparative analysis of the tumor weight of experimental group and control group.
It is 52.8% that tumour inhibiting rate=(the average knurl of the average knurl weight-experimental group of control group is heavy)/average knurl of control group heavy * 100% obtains FUT1-1 plasmid group tumour inhibiting rate thus, with the poor heteropole of control group remarkable (P＜0.01); FUT4-1 plasmid group tumour inhibiting rate is 45.0%, with the poor heteropole of control group remarkable (P＜0.01).
Claims (4)
1. a fucosyl transferase I V who is used to suppress the constructed reorganization of LeY tumour oligosaccharides antigen synthetic fucosyl transferase I V interferes sequence FUT4-2, and it is characterized in that: its sequence is as follows: GTAACCTCTTCAACTGGAC.
2. fucosyl transferase I V interfere plasmid that is used to suppress the constructed reorganization of LeY tumour oligosaccharides antigen synthetic fucosyl transferase I V, it is characterized in that: it is the special restriction enzyme site of polyclone based on RNA interference vector pSilencer-4.1-CMV-neo, design the dna sequence dna that the FUT4SiRNA template of identical sticky end is arranged with interference vector, make up the interfere plasmid that contains FUT4-2 interference sequence of 1 FUT4: pSilencer-4.1-FUT4-2.
3. preparation method who is used to suppress the fucosyl transferase I V interfere plasmid of the constructed reorganization of LeY tumour oligosaccharides antigen synthetic fucosyl transferase I V, it is characterized in that: 1. synthetic is used to interfere the FUT4-2 of FUT4 gene to interfere the template DNA of sequence, with deionized water or the dissolving of TE damping fluid, measure the light absorption value of 260nm, and calculating DNA concentration, 2. the sample of getting after the dissolving adds in the DNA annealing buffer, in 90-98 ℃ of sex change 3-10 minute, cooling back cryopreservation is standby, 3. RNA interference vector pSilencer-4.1-CMV-neo is carried out special digestion with restriction enzyme in 37 ℃, produce and the sticky end of interfering sequences match, 4. enzyme is connected with the product of 2. middle preparation preservation after cutting product purification, 5. with above-mentioned connection product transformed competence colibacillus bacterium and utilize resistance marker screening positive clone of carrier, 6. extract and carry out enzyme behind the plasmid and cut evaluation, 7. cut and identify correct clone's preparation plasmid with enzyme.
4. the fucosyl transferase I V interfere plasmid that is used for suppressing the constructed reorganization of LeY tumour oligosaccharides antigen synthetic fucosyl transferase I V of claim 2 is in the application of preparation antitumor drug.
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| CN2010101085831A Division CN102094019B (en) | 2007-01-15 | 2007-01-15 | RNA (Ribose Nucleic Acid) interference sequence and recombined interference plasmid for inhibiting fucosyltransferase IV synthesized by LeY carbohydrate antigens |
| CN2010101089368A Division CN102094020B (en) | 2007-01-15 | 2007-01-15 | RNA (Ribose Nucleic Acid) interference sequence and recombined interference plasmid for inhibiting fucosyltransferase I synthesized by LeY carbohydrate antigens |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| EP0654082A1 (en) * | 1992-07-20 | 1995-05-24 | The Regents Of The University Of Michigan | CLONING AND EXPRESSION OF A HUMAN $g(a)(1,3)FUCOSYLTRANSFERASE, FUCT-VI |
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| EP0654082A1 (en) * | 1992-07-20 | 1995-05-24 | The Regents Of The University Of Michigan | CLONING AND EXPRESSION OF A HUMAN $g(a)(1,3)FUCOSYLTRANSFERASE, FUCT-VI |
Non-Patent Citations (2)
| Title |
|---|
| 林蓓.小鼠α1 2岩藻糖基转移酶基因的克隆及表达.分子细胞生物学报38 6.2005 |
| 林蓓.小鼠α1,2岩藻糖基转移酶基因的克隆及表达.分子细胞生物学报38 6.2005,38(6),全文. * |
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