CN103540616B - Express slow virus carrier system and the preparation and application thereof of Diphtheria toxin A fragment - Google Patents
Express slow virus carrier system and the preparation and application thereof of Diphtheria toxin A fragment Download PDFInfo
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Abstract
The invention discloses a kind of slow virus carrier system and the preparation and application thereof of expressing Diphtheria toxin A fragment, slow virus carrier system energy coordinate expression bicistronic mRNA GFP/RFP and DTA that the present invention builds, drive Diphtheria toxin A fragment high expression in tumour cell, the expression of fluorescin can be used for the validity of the DTA expressed by checking, meanwhile, the expression of fluorescin also can be used for the packaging effect of monitoring slow virus.The synthesis of protein in the slow virus carrier system energy inhibition tumor cell growth that the present invention builds and cell, especially has specific killing effect to colorectal cancer cell system SW620 and breast cancer cell line ZR7530, can be used for therapy of tumor.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of slow virus carrier system and the preparation and application thereof of expressing Diphtheria toxin A fragment.
Background technology
Diphtheria toxin (diphtheriatoxin, DT) only produces in the lysogeny thalline of diphtheria corynebacterium, because only have the structure gene (Tox gene) of lysogen just containing this albumen of coding.Diphtheria toxin mechanism of toxication mainly can suppress the intracellular protein of eukaryotic cells synthesize and show toxicity, NAD in its energy catalysis eucaryon kytoplasm
+on ADP ribosyl to translation elongation factor-2 (EF-2) transfer, thus consume NAD needed for biological normal metabolic processes
+and block eukaryotic cell protein synthesis and to cell-lethal.Meanwhile, the toxicity of diphtheria toxin has very strong specificity, is in particular in two aspects: first, its catalyzing N AD
+upper ADP ribosyl shifts to EF-2, to NAD
+analogue is as α-NAD
+, NADP
+then this shift reaction is there is not with NADH; Secondly, only Eukaryotic EF-2 is as the acceptor of this reaction.
The toxicity of diphtheria toxin all concentrates on Diphtheria toxin A fragment (DTA), and Segment A can express ADP ribosyl transfer activity independently after entering cell, and the mrna length of encode fragment A approximately only has 800 bases, is conducive to the operation of gene therapy aspect.
Ku80 albumen (XRCC5 genes encoding) high abundance in tumour cell is expressed, and gene expression abundance is very low in normal tissue cell, simultaneously this albumen can also strengthen propagation, the migration and invasion ability of esophageal cancer cell, these all show the unconventionality expression of this gene and relation between tumor tight.In esophageal cancer cell, RNA interference is carried out to Ku80 albumen, find interference propagation capable of inhibiting cell and strengthen the susceptibility of cell to ray and ametycin.
For realizing safe gene therapy, slow virus integration ability should be eliminated as far as possible.Nonconformable lentiviral vectors due to virus assembly, reverse transcription, proviral enter the integration process of core and virus all separate, therefore can not only avoid by integrating the insertion mutation risk caused, and still there is the advantage of mediated gene transfer.
Summary of the invention
The object of the present invention is to provide a kind of slow virus carrier system and the preparation and application thereof of expressing Diphtheria toxin A fragment, this slow virus carrier system a kind ofly has remarkable treatment ability to tumour cell, can in tumour cell efficient coordinate expression CMV promoter or the fluorescin of XRCC5 promoters driven and the bicistronic mRNA non-integration slow virus vector system of Diphtheria toxin A fragment.
The invention discloses a kind of slow virus carrier system of expressing Diphtheria toxin A fragment, PMDL(D64A intergrase D64 being sported A), REV, VSVG and expression vector corotation enter the 293T cell of overexpression sudden change EF-2, namely in host cell, packaging obtains slow virus carrier system;
Described expression vector comprises pPRIME-CMV-GFP-2A-DTA-FF3, pPRIME-CMV-RFP-2A-DTA-FF3, pPRIME-XRCC5p-GFP-2A-DTA-FF3, pPRIME-XRCC5p-RFP-2A-DTA-FF3;
Described slow virus carrier system is the non-integration slow virus vector system of the Diphtheria toxin A fragment that a kind of composing type and tumour cell specific promoter drive.
Described slow virus carrier system utilizes CMV promoter or XRCC5 promoters driven Diphtheria toxin A fragment and fluorescin coordinate expression, and its carrier that sets out is pPRIME-CMV-GFP-FF3.
Described expression vector energy coordinate expression bicistronic mRNA GFP/RFP and DTA, the Lentiviral wherein driven by CMV is as positive control, and the expression vector of XRCC5 promoters driven is the lentiviral vectors of target tumor gene therapy.
As a preparation method for the slow virus carrier system of above-mentioned expression Diphtheria toxin A fragment, comprise the following steps:
The clone of step 1) fluorescence protein gene: with pPRIME-CMV-GFP-FF3 or pPRIME-CMV-dsRed-FF3 plasmid for template, GFP forward primer: 5 '-GC
aCCGGTgCCACCATGGTGAGCAAGGGCGAGG-3 ' (SEQ ID NO:1), end is AgeI restriction enzyme site (base that underscore represents), GFP reverse primer: 5 '-ATAGTTTA
gCGGCCGCgGGCCCTCTAGACTTGTACAGCTCGTCCATGC-3 ' (SEQ ID NO:2), end is NotI restriction enzyme site (base that underscore represents); RFP forward primer: 5 '-GC
aCCGGTgCCACCATGGCCTCCTCCGAGGAC-3 ' (SEQ ID NO:3), end is AgeI restriction enzyme site (base that underscore represents), RFP reverse primer: 5 '-ATAGTTTA
gCGGCCGCgGGCCCTCTAGACAGGAACAGGTGGTGGCG-3 ' (SEQ ID NO:4), end is NotI restriction enzyme site (base that underscore represents), high-fidelity PCR amplification GFP and RFP gene, PCR reaction conditions is: 95 ° of C sex change 2min, 95 ° of C sex change 20sec-60 ° of C annealing 20sec-72 ° of C 30sec extends repetition 35 circulation, 72 ° of C extend 5min, and are cloned into T/A cloning vector;
Step 2) DTA clone: with the plasmid of pBSDTA-II for template, forward primer: 5 '-GC
gGGCCCaTGGACCCTGATGATGTTG-3 ' (SEQ ID NO:5), end is ApaI restriction enzyme site (base that underscore represents), reverse primer: 5 '-ATAGTTTA
gCGGCCGCtTAGAGCTTTAAATCTCTGTA-3 ' (SEQ ID NO:6), end is NotI restriction enzyme site (base that underscore represents), high-fidelity clone DTA fragment, PCR reaction conditions is: 95 ° of C sex change 2min, 95 ° of C sex change 20sec-60 ° of C annealing 20sec-72 ° of C 1min extends repetition 35 circulation, 72 ° of C extend 5min, and PCR primer is cloned in the T/A cloning vector of step 1) with ApaI and NotI enzyme by double digestion method of attachment, obtain the T/A cloning vector of GFP/RFP-DTA;
The design of step 3) foot and mouth disease virus 2A sequence and synthesis: after the Nucleotide translation of synthesis, corresponding aminoacid sequence is: SRAPVKQTLNFDLLKLAGDVESNPGP(SEQ ID NO:15), according to this 2A small peptide aminoacid sequence, design oligonucleotides fragment, by annealing, connect inserting step 2) between GFP/RFP and DTA of T/A cloning vector that obtain, obtain the T/A cloning vector of GFP/RFP-2A-DTA;
Step 4) promotor is cloned: with the 293T cell genomic dna extracted for template, forward primer: 5 '-GC
gGGCCCcTGCAGAGTAGAATCTCCCTTC-3 ' (SEQ ID NO:7), end is ApaI restriction enzyme site (base that underscore represents), reverse primer: 5 '-CTA
gCTAGCgTTGCCGGTCCTCAGGCGCT-3 ' (SEQ ID NO:8), end is NheI restriction enzyme site (base that underscore represents), high-fidelity clone gene promotor, PCR reaction conditions is: 95 ° of C sex change 2min, 95 ° of C sex change 20sec-55 ° of C annealing 30sec-72 ° of C 45sec extends repetition 35 circulation, 72 ° of C extend 5min, PCR primer is cloned into the linear pPRIME-CMV-GFP-FF3 lentiviral vectors formed with endonuclease digestion of the same race by double digestion method of attachment with ApaI and NheI enzyme, be connected to form pPRIME-XRCC5p-GFP-FF3 expression vector;
Step 5) utilizes enzyme to cut method of attachment and the GFP/RFP-2A-DTA fragment in GFP/RFP-2A-DTA T/A cloning vector is transferred to pPRIME-CMV-GFP-FF3 or pPRIME-XRCC5p-GFP-FF3 Lentiviral respectively, obtain pPRIME-CMV-GFP-2A-DTA-FF3, pPRIME-CMV-RFP-2A-DTA-FF3, pPRIME-XRCC5p-GFP-2A-DTA-FF3 or pPRIME-XRCC5p-RFP-2A-DTA-FF3;
The clone of step 6) EF-2 gene: the cDNA formed with the RNA reverse transcription of 293T cell for template, forward primer: 5 '-CG
gAATTCgCCACCATGGTGAACTTCACGGTAGAC-3 ' (SEQ ID NO:9), end is EcoRI restriction enzyme site (base that underscore represents), reverse primer: 5 '-CG
gGATCCcTACAATTTGTCCAGGAAGTTG-3 ' (SEQ ID NO:10), end is BamHI restriction enzyme site (base that underscore represents), high-fidelity amplification EF-2 gene, PCR reaction conditions is: 95 ° of C sex change 2min, 95 ° of C sex change 20sec-57 ° of C annealing 20sec-72 ° of C 1.5min extends repetition 35 circulation, and 72 ° of C extend 5min;
The sudden change of step 7) EF-2 717 amino acids and the structure of lentiviral vectors: the method utilizing over-lap PCR, be arginine CGA by 717 of EF-2 glycine GGA rite-directed mutagenesises, mutant primer is: forward primer: 5 '-GACGCCATCCACCGC
cgAGGGGGCCAGATCA-3 ' (SEQ ID NO:11), reverse primer: 5 '-TGATCTGGCCCCCTC
ggCGGTGGATGGCGTC-3 ' (SEQ ID NO:12), with the amplified production in step 6) for template carries out high-fidelity amplification, PCR reaction conditions is: 95 ° of C sex change 2min, 95 ° of C sex change 20sec-62 ° of C annealing 20sec-72 ° of C 2.5min extends repetition 30 circulation, 72 ° of C extend 5min, by double digestion method of attachment, PCR variants is inserted into pCDH-CMV-MCS-EF1-Puro lentiviral vectors with EcoRI and BamHI enzyme again, forms pCDH-CMV-mEF-2-EF1-Puro;
The foundation of the clone of step 8) stably express sudden change EF-2: by packaged pCDH-CMV-mEF-2-EF1-Puro, with PHR, VSVG carrier in proportion corotation enter 293T clone, collect vial supernatant and carry out cell infection, recycle 1.5 μ g/ml tetracyclines to screen cells infected, obtain the 293T clone of sudden change EF-2 overexpression;
The packaging of step 9) slow virus: before transfection, non-serum starved 2 hours are carried out to 293T cell, to intergrase D64 will be positioned at sport the PMDL(D64A of A), REV, after Lentiviral mixing in VSVG and step 5), mix with homemade PEI transfection reagent again, jointly add in the 293T packing cell of overexpression sudden change EF-2 in step 8), after 6 hours, substratum is replaced with the substratum of serum containing 2%, continuous collection 3 days supernatants, utilize ultracentrifuge concentrating virus, obtain the pPRIME-CMV-GFP-2A-DTA-FF3 of circles, pPRIME-CMV-RFP-2A-DTA-FF3, pPRIME-XRCC5p-GFP-2A-DTA-FF3 or pPRIME-XRCC5p-RFP-2A-DTA-FF3 slow virus.
Described slow virus carrier system is a kind of bicistronic mRNA non-integration slow virus vector system being applied to therapy of tumor.
Described therapy of tumor is for colorectal cancer cell system SW620 and breast cancer cell line ZR7530, and its reconstruction method is as follows:
The clone of step 1) luciferase gene and the structure of retroviral vector: with pGL3-Basic plasmid for template, forward primer: 5 '-CG
gAATTCgCCACCATGGAAGACGCCAAAAACA-3 ' (SEQ ID NO:13), end is EcoRI restriction enzyme site (base that underscore represents), reverse primer: 5 '-GA
aGATCTtTACACGGCGATCTTTCCG-3 ' (SEQ ID NO:14), end is BglII restriction enzyme site (base that underscore represents), high-fidelity amplification luciferase gene, PCR reaction conditions is: 95 ° of C sex change 2min, 95 ° of C sex change 20sec-60 ° of C annealing 20sec-72 ° of C 1min extends repetition 35 circulation, 72 ° of C extend 5min, luciferase gene is inserted into PLXSN3 retroviral vector with EcoRI and BglII enzyme by double digestion method of attachment by PCR primer, obtains pLXSN3-Luciferase retroviral vector;
Step 2) establishment method of SW620 and ZR7530 clone of stably express sudden change EF-2 is: packaged pCDH-CMV-mEF-2-EF1-Puro is infected SW620 and ZR7530 cell respectively, the tetracycline being 1 μ g/ml and 1.5 μ g/ml with concentration respectively again screens cells infected, obtains the SW620(mEF-2 of sudden change EF-2 overexpression) and ZR7530(mEF-2) clone;
The foundation of SW620 and the ZR7530 clone of step 3) stably express luciferase gene: by pLXSN3-Luciferase retroviral vector respectively transfection to SW620, ZR7530, SW620(mEF-2), ZR7530(mEF-2) in clone, respectively SW620 or ZR7530 clone is screened with the G418 of 2 μ g/ μ l and 1.2 μ g/ μ l, obtains corresponding clone: W620(Luc), ZR7530(Luc), SW620(Luc+mEF-2), ZR7530(Luc+mEF-2).
beneficial effect of the present invention is:
(1) from tumour cell, be effectively separated the XRCC5 promotor of high expression;
(2) by the mode of vector construction, construct the lentiviral vectors by the separately-driven fluorescin GFR/RFP of CMV and XRCC5 promotor and DTA coordinate expression, between GFR/RFP and DTA, carry out lotus root connection by the 2A fragment of foot and mouth disease virus;
(3) construct the Lentiviral of protein translation elongation factor (EF-2) Specific amino acid sudden change, and carried out the foundation of viral packaging and 293T stable cell lines, thus form the slow virus package cell line of specific DTA;
(4) utilize and be positioned at intergrase D64 and sport the PMDL (D64A) of A, REV, VSVG and Lentiviral, the slow virus of DTA and GFP/RFP bicistronic mRNA coordinate expression is packed, obtains nonconformable slow virus carrier system;
(5) by the comparison of the survival ability to protein synthesis capacity (ability to express of fluorescin and luciferase) and tumour cell, find that the lentiviral vectors of coordinate expression fluorescin and the diphtheria toxin built effectively can not only monitor the wrapping process of virus, meanwhile, the slow virus that XRCC5 drives has targeting to tumour cell;
(6) the Lentiviral system that the present invention builds has specific killing ability to tumour cell, the generation using fluorescence albumen of this system virus carries out Real-Time Monitoring, confirm through experiment in vitro, the constructed synthesis of slow virus carrier system energy arrestin matter and the growth of cell.
Accompanying drawing explanation
Fig. 1: CMV or the lentiviral vectors design of graphics of XRCC5 promoters driven fluorescin and Diphtheria toxin A fragment coordinate expression;
Fig. 2: the packaging of slow virus and tumour remove the schema of model;
Fig. 3: the fluorescin monitoring packaging of slow virus and the foundation of Viral packaging cell system;
The lentiviral vectors of Fig. 4: overexpression DTA infects the uciferase activity analysis of overexpression luciferase gene.
Embodiment
Expressing the slow virus carrier system of Diphtheria toxin A fragment is intergrase D64 is sported the 293T cell that PMDL-D64A, REV, VSVG of A and expression vector corotation enter overexpression sudden change EF-2, and namely in host cell, packaging obtains slow virus carrier system; Its expression vector comprises pPRIME-CMV-GFP-2A-DTA-FF3, pPRIME-CMV-RFP-2A-DTA-FF3, pPRIME-XRCC5p-GFP-2A-DTA-FF3, pPRIME-XRCC5p-RFP-2A-DTA-FF3.
embodiment 1
The preparation method expressing the slow virus carrier system of Diphtheria toxin A fragment comprises the following steps:
The clone of step 1) green fluorescence protein gene: with pPRIME-CMV-GFP-FF3 plasmid for template, forward primer: 5 '-GC
aCCGGTgCCACCATGGTGAGCAAGGGCGAGG-3 ' (SEQ ID NO:1), end is AgeI restriction enzyme site (base that underscore represents), reverse primer: 5 '-ATAGTTTA
gCGGCCGcGGGCCCTCTAGACTTGTACAGCTCGTCCATGC-3 ' (SEQ ID NO:2), end is NotI restriction enzyme site (base that underscore represents), high-fidelity PCR amplification GFP gene, and is cloned into T/A cloning vector;
Step 2) DTA clone: with the plasmid of pBSDTA-II for template, forward primer: 5 '-GC
gGGCCCaTGGACCCTGATGATGTTG-3 ' (SEQ ID NO:5), end is ApaI restriction enzyme site (base that underscore represents), reverse primer: 5 '-ATAGTTTA
gCGGCCGCtTAGAGCTTTAAATCTCTGTA-3 ' (SEQ ID NO:6), end is NotI restriction enzyme site (base that underscore represents), high-fidelity clone DTA fragment, PCR primer is cloned in the T/A cloning vector of step 1) with ApaI and NotI enzyme by double digestion method of attachment, obtains the T/A cloning vector of GFP-DTA;
The design of step 3) foot and mouth disease virus 2A sequence and synthesis: after the Nucleotide translation of synthesis, corresponding aminoacid sequence is: SRAPVKQTLNFDLLKLAGDVESNPGP(SEQ ID NO:15), according to this 2A small peptide aminoacid sequence, design oligonucleotides fragment, by annealing, connect inserting step 2) between GFP and DTA of T/A cloning vector that obtain, obtain the T/A cloning vector of GFP-2A-DTA;
Step 4) utilizes enzyme to cut method of attachment and the GFP-2A-DTA fragment in GFP-2A-DTA T/A cloning vector is transferred to pPRIME-CMV-GFP-FF3 Lentiviral, obtains pPRIME-CMV-GFP-2A-DTA-FF3;
The clone of step 5) EF-2 gene: the cDNA formed with the RNA reverse transcription of 293T cell for template, forward primer: 5 '-CG
gAATTCgCCACCATGGTGAACTTCACGGTAGAC-3 ' (SEQ ID NO:9), end is EcoRI restriction enzyme site (base that underscore represents), reverse primer: 5 '-CG
gGATCCcTACAATTTGTCCAGGAAGTTG-3 ' (SEQ ID NO:10), end is BamHI restriction enzyme site (base that underscore represents), high-fidelity amplification EF-2 gene;
The sudden change of step 6) EF-2 717 amino acids and the structure of lentiviral vectors: the method utilizing over-lap PCR, be arginine CGA by 717 of EF-2 glycine GGA rite-directed mutagenesises, mutant primer is: forward primer: 5 '-GACGCCATCCACCGC
cgAGGGGGCCAGATCA-3 ' (SEQ ID NO:11), reverse primer: 5 '-TGATCTGGCCCCCT C
ggCGGTGGATGGCGTC-3 ' (SEQ ID NO:12), with the amplified production in step 1) for template carries out high-fidelity amplification, by double digestion method of attachment, PCR variants is inserted into pCDH-CMV-MCS-EF1-Puro lentiviral vectors with EcoRI and BamHI enzyme again, forms pCDH-CMV-mEF-2-EF1-Puro;
The foundation of the clone of step 7) stably express sudden change EF-2: by packaged pCDH-CMV-mEF-2-EF1-Puro, with PHR, VSVG carrier in proportion corotation enter 293T clone, collect vial supernatant and carry out cell infection, recycle 1.5 μ g/ml tetracyclines to screen cells infected, obtain the 293T clone of sudden change EF-2 overexpression;
The packaging of step 8) slow virus: before transfection, non-serum starved 2 hours are carried out to 293T cell, by be positioned at intergrase D64 sport A PMDL-D64A, REV, VSVG and pPRIME-CMV-GFP-2A-DTA-FF3 expression vector mixing after, mix with homemade PEI transfection reagent again, jointly add in the 293T packing cell of overexpression sudden change EF-2, after 6 hours, substratum is replaced with the substratum of serum containing 2%, continuous collection 3 days supernatants, utilize ultracentrifuge concentrating virus, obtain the pPRIME-CMV-GFP-2A-DTA-FF3 slow virus of circles.
The reconstruction method of colorectal cancer cell system SW620 is as follows:
The clone of step 1) luciferase gene and the structure of retroviral vector: with pGL3-Basic plasmid for template, forward primer: 5 '-CG
gAATTCgCCACCATGGAAGACGCCAAAAACA-3 ' (SEQ ID NO:13), end is EcoRI restriction enzyme site (base that underscore represents), reverse primer: 5 '-GA
aGATCTtTACACGGCGATCTTTCCG-3 ' (SEQ ID NO:14), end is BglII restriction enzyme site (base that underscore represents), high-fidelity amplification luciferase gene, luciferase gene is inserted into PLXSN3 retroviral vector with EcoRI and BglII enzyme by double digestion method of attachment by PCR primer, obtains pLXSN3-Luciferase retroviral vector;
Step 2) establishment method of SW620 clone of stably express sudden change EF-2 is: packaged pCDH-CMV-mEF-2-EF1-Puro is infected SW620 cell, the tetracycline being 1 μ g/ml with concentration again screens cells infected, obtains the SW620(mEF-2 of sudden change EF-2 overexpression) clone;
The foundation of the SW620 clone of step 3) stably express luciferase gene: by the transfection of pLXSN3-Luciferase retroviral vector to SW620, SW620(mEF-2) in two clones, with the G418 of 2 μ g/ μ l, SW620 clone is screened, obtains corresponding 2 clone: SW620(Luc), SW620(Luc+mEF-2).
embodiment 2
The preparation method expressing the slow virus carrier system of Diphtheria toxin A fragment comprises the following steps:
The clone of step 1) green fluorescence protein gene: with embodiment 1;
Step 2) DTA clone: with embodiment 1;
The design of step 3) foot and mouth disease virus 2A sequence and synthesis: with embodiment 1;
Step 4) XRCC5 promotor is cloned: with the 293T cell genomic dna extracted for template, forward primer: 5 '-GC
gGGCCCCtGCAGAGTAGAATCTCCCTTC-3 ' (SEQ ID NO:7), end is ApaI restriction enzyme site (base that underscore represents), reverse primer: 5 '-CTA
gCTAGCgTTGCCGGTCCTCAGGCGCT-3 ' (SEQ ID NO:8), end is NheI restriction enzyme site (base that underscore represents), the promotor of high-fidelity clone XRCC5 gene, PCR primer is cloned into the linear pPRIME-CMV-GFP-FF3 lentiviral vectors formed with endonuclease digestion of the same race by double digestion method of attachment with ApaI and NheI enzyme, be connected to form pPRIME-XRCC5p-GFP-FF3 expression vector;
Step 5) utilizes enzyme to cut method of attachment and the GFP-2A-DTA fragment in GFP-2A-DTA T/A cloning vector is transferred to pPRIME-XRCC5p-GFP-FF3 Lentiviral, obtains pPRIME-XRCC5p-GFP-2A-DTA-FF3;
The clone of step 6) EF-2 gene: with embodiment 1;
The sudden change of step 7) EF-2 717 amino acids and the structure of lentiviral vectors: with embodiment 1;
The foundation of the clone of step 8) stably express sudden change EF-2: with embodiment 1;
The packaging of step 9) slow virus: before transfection, non-serum starved 2 hours are carried out to 293T cell, to intergrase D64 will be positioned at sport the PMDL-D64A of A, REV, after the mixing of VSVG and pPRIME-XRCC5p-GFP-2A-DTA-FF3 expression vector, mix with homemade PEI transfection reagent again, jointly add in the 293T packing cell of overexpression sudden change EF-2 in step 8), after 6 hours, substratum is replaced with the substratum of serum containing 2%, continuous collection 3 days supernatants, utilize ultracentrifuge concentrating virus, obtain the pPRIME-XRCC5p-GFP-2A-DTA-FF3 slow virus of circles.
The reconstruction method of colorectal cancer cell system SW620 is with embodiment 1.
embodiment 3
The preparation method expressing the slow virus carrier system of Diphtheria toxin A fragment comprises the following steps:
The clone of step 1) red fluorescent protein gene: with pPRIME-CMV-dsRed-FF3 plasmid for template, forward primer: 5 '-GC
aCCGGTgCCACCATGGCCTCCTCCGAGGAC-3 ' (SEQ ID NO:3), end is AgeI restriction enzyme site (base that underscore represents), reverse primer: 5 '-ATAGTTTA
gCGGCCGCgGGCCCTCTAGACAGGAACAGGTGGTGGCG-3 ' (SEQ ID NO:4), end is NotI restriction enzyme site (base that underscore represents), high-fidelity PCR amplification RFP gene, and is cloned into T/A cloning vector;
Step 2) DTA clone: with the plasmid of pBSDTA-II for template, forward primer: 5 '-GC
gGGCCCaTGGACCCTGATGATGTTG-3 ' (SEQ ID NO:5), end is ApaI restriction enzyme site (base that underscore represents), reverse primer: 5 '-ATAGTTTA
gCGGCCGCtTAGAGCTTTAAATCTCTGTA-3 ' (SEQ ID NO:6), end is NotI restriction enzyme site (base that underscore represents), high-fidelity clone DTA fragment, PCR primer is cloned in the T/A cloning vector of step 1) with ApaI and NotI enzyme by double digestion method of attachment, obtains the T/A cloning vector of RFP-DTA;
The design of step 3) foot and mouth disease virus 2A sequence and synthesis: after the Nucleotide translation of synthesis, corresponding aminoacid sequence is: SRAPVKQTLNFDLLKLAGDVESNPGP(SEQ ID NO:15), according to this 2A small peptide aminoacid sequence, design oligonucleotides fragment, by annealing, connect inserting step 2) between RFP and DTA of T/A cloning vector that obtain, obtain the T/A cloning vector of RFP-2A-DTA;
Step 4) utilizes enzyme to cut method of attachment and the RFP-2A-DTA fragment in RFP-2A-DTA T/A cloning vector is transferred to pPRIME-CMV-GFP-FF3 Lentiviral, obtains pPRIME-CMV-RFP-2A-DTA-FF3;
The clone of step 5) EF-2 gene: with embodiment 1;
The sudden change of step 6) EF-2 717 amino acids and the structure of lentiviral vectors: with embodiment 1;
The foundation of the clone of step 7) stably express sudden change EF-2: with embodiment 1;
The packaging of step 8) slow virus: before transfection, non-serum starved 2 hours are carried out to 293T cell, to intergrase D64 will be positioned at sport the PMDL-D64A of A, REV, after the mixing of VSVG and pPRIME-CMV-RFP-2A-DTA-FF3 expression vector, mix with homemade PEI transfection reagent again, jointly add in the 293T packing cell of overexpression sudden change EF-2 in step 8), after 6 hours, substratum is replaced with the substratum of serum containing 2%, continuous collection 3 days supernatants, utilize ultracentrifuge concentrating virus, obtain the pPRIME-CMV-RFP-2A-DTA-FF3 slow virus of circles.
The reconstruction method of breast cancer cell line ZR7530 is:
The clone of step 1) luciferase gene and the structure of retroviral vector: with pGL3-Basic plasmid for template, forward primer: 5 '-CG
gAATTCgCCACCATGGAAGACGCCAAAAACA-3 ' (SEQ ID NO:13), end is EcoRI restriction enzyme site (base that underscore represents), reverse primer: 5 '-GA
aGATCTtTACACGGCGATCTTTCCG-3 ' (SEQ ID NO:14), end is BglII restriction enzyme site (base that underscore represents), high-fidelity amplification luciferase gene, luciferase gene is inserted into PLXSN3 retroviral vector with EcoRI and BglII enzyme by double digestion method of attachment by PCR primer, obtains pLXSN3-Luciferase retroviral vector;
Step 2) establishment method of ZR7530 clone of stably express sudden change EF-2 is: packaged pCDH-CMV-mEF-2-EF1-Puro is infected ZR7530 cell, the tetracycline being 1.5 μ g/ml with concentration again screens cells infected, obtains the ZR7530(mEF-2 of sudden change EF-2 overexpression) clone;
The foundation of the ZR7530 clone of step 3) stably express luciferase gene: by the transfection of pLXSN3-Luciferase retroviral vector to ZR7530, ZR7530(mEF-2) in two clones, with the G418 of 1.2 μ g/ μ l, ZR7530 clone is screened, obtains corresponding 2 clone: ZR7530(Luc), ZR7530(Luc+mEF-2).
embodiment 4
The preparation method expressing the slow virus carrier system of Diphtheria toxin A fragment comprises the following steps:
The clone of step 1) red fluorescent protein gene: with embodiment 3;
Step 2) DTA clone: with embodiment 3;
The design of step 3) foot and mouth disease virus 2A sequence and synthesis: with embodiment 3;
Step 4) XRCC5 promotor is cloned: with embodiment 2;
Step 5) utilizes enzyme to cut method of attachment and the RFP-2A-DTA fragment in RFP-2A-DTA T/A cloning vector is transferred to pPRIME-XRCC5p-GFP-FF3 Lentiviral, obtains pPRIME-XRCC5p-RFP-2A-DTA-FF3.
The clone of step 6) EF-2 gene: with embodiment 1;
The sudden change of step 7) EF-2 717 amino acids and the structure of lentiviral vectors: with embodiment 1;
The foundation of the clone of step 8) stably express sudden change EF-2: with embodiment 1; ;
The packaging of step 9) slow virus: before transfection, non-serum starved 2 hours are carried out to 293T cell, to intergrase D64 will be positioned at sport the PMDL-D64A of A, REV, after the mixing of VSVG and pPRIME-XRCC5p-RFP-2A-DTA-FF3 expression vector, mix with homemade PEI transfection reagent again, jointly add in the 293T packing cell of overexpression sudden change EF-2 in step 8), after 6 hours, substratum is replaced with the substratum of serum containing 2%, continuous collection 3 days supernatants, utilize ultracentrifuge concentrating virus, obtain the pPRIME-XRCC5p-RFP-2A-DTA-FF3 slow virus of circles.
The reconstruction method of breast cancer cell line ZR7530 is with embodiment 3.
Lentiviral is to the mensuration of protein synthesis rejection ability: (1) as shown in Figure 3, in the 293T cell of expressing wild-type translation elongation factor EF-2, does not see the expression of fluorescin; And in the 293T stable cell lines of overexpression sudden change EF-2, the remarkable expression of fluorescence can be seen, show that the synthesis of this lentiviral vectors to protein has significant restraining effect.(2) the concentrated slow virus of expressing DTA is infected the SW620 with expressing luciferase gene ability, ZR7530, SW620 (mEF-2), ZR7530 (mEF-2) stable cell lines respectively.Analyzed from Fig. 4 uciferase activity, at stably express luciferase gene SW620, in ZR7530 clone, extremely low luciferase gene expression detected; And the SW620 (mEF-2) of the EF-2 that suddenlys change at overexpression, in ZR7530 (mEF-2) tumor cell line, the expression of luciferase gene significantly can be detected, and the DTA expression lentiviral vectors that XRCC5 drives is stronger to the rejection ability of luciferase gene expression in tumour cell.
The foregoing is only preferred embodiment of the present invention, all equalizations done according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.
SEQUENCE LISTING
<110> Fuzhou General Hospital, Nanjing Military Area, PLA
<120> expresses slow virus carrier system and the preparation and application thereof of Diphtheria toxin A fragment
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<170> PatentIn version 3.3
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gcaccggtgc caccatggtg agcaagggcg agg 33
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atagtttagc ggccgcgggc cctctagact tgtacagctc gtccatgc 48
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gcaccggtgc caccatggcc tcctccgagg ac 32
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atagtttagc ggccgcgggc cctctagaca ggaacaggtg gtggcg 46
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gcgggcccat ggaccctgat gatgttg 27
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atagtttagc ggccgcttag agctttaaat ctctgta 37
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gcgggcccct gcagagtaga atctcccttc 30
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ctagctagcg ttgccggtcc tcaggcgct 29
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cggaattcgc caccatggtg aacttcacgg tagac 35
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cgggatccct acaatttgtc caggaagttg 30
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gacgccatcc accgccgagg gggccagatc a 31
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tgatctggcc ccctcggcgg tggatggcgt c 31
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Ser Arg Ala Pro Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu
1 5 10 15
Ala Gly Asp Val Glu Ser Asn Pro Gly Pro
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Claims (2)
1. express the slow virus carrier system of Diphtheria toxin A fragment, it is characterized in that: intergrase D64 is sported the 293T cell that PMDL-D64A, REV, VSVG of A and expression vector corotation enter overexpression sudden change EF-2, namely in host cell, packaging obtains slow virus carrier system;
Described expression vector is selected from pPRIME-CMV-GFP-2A-DTA-FF3, pPRIME-CMV-RFP-2A-DTA-FF3, pPRIME-XRCC5p-GFP-2A-DTA-FF3, or pPRIME-XRCC5p-RFP-2A-DTA-FF3, its energy coordinate expression bicistronic mRNA GFP/RFP and DTA, the Lentiviral wherein driven by CMV is as positive control, and the expression vector of XRCC5 promoters driven is the lentiviral vectors of target tumor gene therapy;
Described slow virus carrier system utilizes CMV promoter or XRCC5 promoters driven Diphtheria toxin A fragment and fluorescin coordinate expression, its carrier that sets out is pPRIME-CMV-GFP-FF3, be the non-integration slow virus vector system of the Diphtheria toxin A fragment that a kind of composing type and tumour cell specific promoter drive, be applied to therapy of tumor;
The preparation method of described slow virus carrier system specifically comprises the following steps:
The clone of step 1) fluorescence protein gene: with pPRIME-CMV-GFP-FF3 or pPRIME-CMV-dsRed-FF3 plasmid for template, high-fidelity PCR amplification GFP and RFP gene, and be cloned into T/A cloning vector;
Step 2) DTA clone: with the plasmid of pBSDTA-II for template, High fidelity PCR clone DTA fragment, PCR primer is cloned in the T/A cloning vector of step 1) with ApaI and NotI enzyme by double digestion method of attachment, obtains the T/A cloning vector of GFP/RFP-DTA;
The design of step 3) foot and mouth disease virus 2A sequence and synthesis: according to 2A small peptide aminoacid sequence corresponding after the Nucleotide translation of synthesis, design oligonucleotides fragment, by annealing, connect inserting step 2) between GFP/RFP and DTA of T/A cloning vector that obtain, obtain the T/A cloning vector of GFP/RFP-2A-DTA;
Step 4) promotor is cloned: with the 293T cell genomic dna extracted for template, High fidelity PCR clone gene promotor, PCR primer is cloned into the linear pPRIME-CMV-GFP-FF3 lentiviral vectors formed with endonuclease digestion of the same race by double digestion method of attachment with ApaI and NheI enzyme, be connected to form pPRIME-XRCC5p-GFP-FF3 expression vector;
Step 5) utilizes enzyme to cut method of attachment and the GFP/RFP-2A-DTA fragment in GFP/RFP-2A-DTA T/A cloning vector is transferred to pPRIME-CMV-GFP-FF3 or pPRIME-XRCC5p-GFP-FF3 Lentiviral respectively, obtain pPRIME-CMV-GFP-2A-DTA-FF3, pPRIME-CMV-RFP-2A-DTA-FF3, pPRIME-XRCC5p-GFP-2A-DTA-FF3 or pPRIME-XRCC5p-RFP-2A-DTA-FF3;
The clone of step 6) EF-2 gene: the cDNA formed with the RNA reverse transcription of 293T cell for template, high-fidelity PCR amplification EF-2 gene;
The sudden change of step 7) EF-2 717 amino acids and the structure of lentiviral vectors: the method utilizing over-lap PCR, be arginine CGA by 717 of EF-2 glycine GGA rite-directed mutagenesises, with the amplified production in step 6) for template carries out high-fidelity PCR amplification, by double digestion method of attachment, PCR variants is inserted into pCDH-CMV-MCS-EF1-Puro lentiviral vectors with EcoRI and BamHI enzyme again, forms pCDH-CMV-mEF-2-EF1-Puro;
The foundation of the clone of step 8) stably express sudden change EF-2: by packaged pCDH-CMV-mEF-2-EF1-Puro, with PHR, VSVG carrier in proportion corotation enter 293T clone, collect vial supernatant and carry out cell infection, recycle 1.5 μ g/ml tetracyclines to screen cells infected, obtain the 293T clone of sudden change EF-2 overexpression;
The packaging of step 9) slow virus: before transfection, non-serum starved 2 hours are carried out to 293T cell, to intergrase D64 will be positioned at sport the PMDL-D64A of A, REV, after Lentiviral mixing in VSVG and step 5), mix with homemade PEI transfection reagent again, jointly add in the 293T packing cell of overexpression sudden change EF-2 in step 8), being replaced with by substratum containing volume fraction after 6 hours is the substratum of 2% serum, continuous collection 3 days supernatants, utilize ultracentrifuge concentrating virus, obtain the pPRIME-CMV-GFP-2A-DTA-FF3 of circles, pPRIME-CMV-RFP-2A-DTA-FF3, pPRIME-XRCC5p-GFP-2A-DTA-FF3 or pPRIME-XRCC5p-RFP-2A-DTA-FF3 slow virus.
2. the slow virus carrier system of expression Diphtheria toxin A fragment according to claim 1, is characterized in that: described therapy of tumor is for colorectal cancer cell system SW620 and breast cancer cell line ZR7530.
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| CN104099370A (en) * | 2014-07-16 | 2014-10-15 | 中国人民解放军***福州总医院 | Nonconformable slow virus carrier system targeting therapy of tumors and preparation method and application |
| CN105671082B (en) * | 2016-01-18 | 2020-08-07 | 武汉淼灵生物科技有限公司 | Lentiviral vector for expressing exosome marker and construction method and application thereof |
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