CN110484565A - A kind of suicide gene system and its application based on gland relevant viral vector transformation - Google Patents
A kind of suicide gene system and its application based on gland relevant viral vector transformation Download PDFInfo
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Abstract
The invention belongs to genetic engineering fields, and in particular to a kind of suicide gene system and its application based on gland relevant viral vector transformation.The system includes gland relevant viral vector pAAV-SV40 enhancer-hTERT-tk, and the pAAV-SV40 enhancer-hTERT-tk carrier includes at least following characteristic: a) using SV40 as enhancer；B) hTERT is promoter；C) make the tk target gene of the thymidine kinase of GCV generation toxicity can encode；D) using AAV as carrier.The suicide gene system based on gland relevant viral vector transformation can not only high effective integration and stable expression of exogenous gene, and the low feature of AAV system immunogenicity itself is utilized, safe and efficient virus expression carrier system is provided for subsequent In vivo study and clinical trial；Demonstrating the system by cell model and animal model can assist killing cancer cell in glioma treatment, play good effect.
Description
Technical field
The invention belongs to genetic engineering fields, and in particular to a kind of suicide gene system based on gland relevant viral vector transformation
System and its application.
Background technique
Suicide gene system is a large focal spot of gene therapy.It is using technique for gene engineering that suicide gene importing tumour is thin
Script can be toxic product to the avirulent pro-drug conversion of body by the enzyme of born of the same parents, gene coding, cause cell death.More former times
Luo Wei/proganoside (Ganciclovir, GCV) is a kind of high-efficiency low-toxicity, with the wide spectrum anti-malarial virus compared with strong selectivity
Agent is treated, all herpesvirals can be fought, especially have strong inhibition effect to cytomegalovirus.Tk gene encoding thymidine kinase, can
Phosphorylation is generated to be catalyzed nucleotide analog, as GCV is converted into toxic metabolites triphosphoric acid the third oxygen bird of GCV- triphosphoric acid
Glycosides can mix in the DNA of proliferative cell, interfere the duplication of blocking dna and inhibit cell division, generate cytotoxicity and make
With.In addition, bystander effect connects phosphorylated nucleosides acid by gap between cell and cell, into not being transferred to HSV-
The cell of tk, is killed, and on this basis, by adding tumour cell specific promoter, controllable tk gene is thin in tumour
Specifically expressing in born of the same parents.
Telomere (Telomere) is a bit of DNA- protein complex of eukaryocyte linear chromosomal end, is contained
The repetitive sequence of TTAGGG shortens when homeostasis as frequency dividing cell increases, eventually leads to cell ageing, activation is withered
Die mechanism.Reverse transcriptase of telomere (Telomerase reverse transcriptase, TERT) by activation Telomerase come
The complete of telomere is maintained, so that cell can be with infinite multiplication.It does not express in TERT normal cerebral tissue, and is deposited in glioma cell
It is expressed in height.In Telomerase subunits, hTERT (human telomerase reverse transcriptase) is that the major regulatory of telomerase activation is sub- single
Position has direct correlation with telomerase activation.Therefore can using hTERT promoter as the gene therapy target of tumour,
Tk gene constructed expression carrier is being connected downstream, is imported tumour cell, is induced cell apoptosis.However in current existing report
In, transcriptional level of the hTERT promoter in most tumors cell be not high, seriously affects table of the target gene in tumour cell
It reaches.So improving transcriptional level to enhance the expression of target gene, selection starts the addition of suitable enhancer in hTERT
Before son, the carrier of the high efficient expression in tumour cell is constructed, the expression of target gene can be enhanced, reach the mesh for the treatment of
's.SV40 (vacuolating virus of monkey 40) enhancer is positioned at nucleus with promotion DNA and can be with the dual function of activated transcription, institute
Before it can be incorporated in hTERT promoter by selection, to increase foreign gene expression levels.
Cerebral glioma (Glioma) is the common primary malignancy brain tumor of central nervous system, accounts for brain tumor generation
30%, its high recurrence rate is one of tumour most fatal at present.But due to characteristics such as the pernicious infiltrative growths of glioma,
Routine operation and chemotherapy radiotherapy method, which can only seldom make the life better, quality and slightly extends life cycle of some patients, for brain glue
Five year survival rate after matter tumor is made a definite diagnosis does not have clear improvement.Characteristic based on glioma, and expectation is over the course for the treatment of
The targeting of killing to Normal healthy cells tissue, gene therapy is increasingly taken seriously.Using suicide gene and specifically open
Subsystem, so that tk genetic fragment specifically expressing in brain glioblastoma cell, then GCV is given, it can over the course for the treatment of, both
It can inhibit the growth of tumour, and can be realized the protection to healthy cell.
Adeno-associated virus (Adeno-associated virus, AAV) have host range extensively, dividing cell and overstepping one's bounds
The advantages that schistocyte can infect and foreign gene can be caused to express steadily in the long term, and it also has no pathogenicity, immunogene
Property it is low and there's almost no the characteristics such as undershooting-effect, therefore be currently viewed as a kind of higher disease with exploitation potential quality of safety
Poisonous carrier.
Although gene therapy has good targeting, the problem of carrier selects still is faced at present, i.e., how to be selected
Target gene can be transported to target cell by safe and efficient carrier, and to organism safe, low or non-immunogenicity, without cause
Characteristic of disease, this is still that those skilled in the art are badly in need of the problem captured.
Summary of the invention
The object of the present invention is to provide a kind of safe and efficient carrier and based on the suicide gene system of the vector modification with
And their construction method, they can be applied in the treatment of glioma.
Mentality of designing of the invention are as follows: carry out tissue detection, selection highly expressed promoter in samples of human glioma
HTERT constructs a kind of viral vectors, and for the carrier using SV40 as enhancer, hTERT is promoter, generates GCV can encode
The tk of the thymidine kinase of toxicity is target gene, using AAV as carrier.Also construct a kind of AAV-SV40 enhancer-hTERT-tk-
GCV system.AAV-SV40 is demonstrated by cell model (experiment in vitro) and animal model (Lump body injection, experiment in vivo) respectively
Enhancer-hTERT-tk-GCV system plays good effect in glioma treatment.
Specifically, technical scheme is as follows:
One aspect of the present invention discloses a kind of gland relevant viral vector pAAV-SV40 enhancer-hTERT-tk, described
PAAV-SV40 enhancer-hTERT-tk carrier includes at least following characteristic:
A) using SV40 as enhancer；B) hTERT is promoter；C) the thymidine kinase for making GCV generate toxicity can be encoded
Tk be target gene；D) using AAV as carrier.
Preferably, the nucleotide sequence of the pAAV-SV40 enhancer-hTERT-tk carrier is as shown in SEQ ID NO:1.
Term " nucleotide sequence " refers to nucleotide sequence, refers to putting in order for base in DNA or RNA.This means that
A in DNA, T, the arrangement of G, C A, U, G in proper order or in mRNA, the arrangement of C also include rRNA in proper order, the row of base in tRNA
Column sequence.
It should be appreciated that with the SEQ ID NO:1 shown in nucleotide sequence have the function of 90% or more homology and
The identical nucleotide sequence of property is within the scope of the present invention.
The second aspect of the present invention discloses a kind of building gland relevant viral vector pAAV-SV40 enhancer-hTERT-tk
Method, comprising the following steps:
S1: tk genetic fragment of the synthesis with restriction enzyme site and upstream and downstream homology arm；
S2: SV40 of the synthesis with restriction enzyme site and upstream and downstream homology arm enhances sub-piece；
S3: SV40 enhancing sub-piece is connected into the carrier for obtaining enhancing sub-piece containing SV40 in carrier；
S4: tk genetic fragment described in S1 is connected into the carrier containing SV40 enhancing sub-piece described in S3, mesh is obtained
Carrier pAAV-SV40 enhancer-hTERT-tk.
It should be understood that the present invention is not limited to above-mentioned steps, the step of can also including other, such as before step S1,
It also include other additional steps between step S1 and S2, between step S2 and S3, between step S3 and S4, after step S4,
Without beyond the scope of the present invention.
Term " unloaded carrier " refer to gene cloning when general we are interested by us with carriers such as plasmid, viruses
Target gene import host cell inside, then these carriers not link with target gene just referred to as zero load carrier.
Preferably, for the nucleotide sequence of the tk genetic fragment as shown in SEQ ID NO:2, the SV40 enhances sub-piece
Nucleotide sequence as shown in SEQ ID NO:3.
Preferably, in S3, the carrier is unloaded carrier pAAV-hTERT-MCS-GFP, nucleotide sequence such as SEQ
Shown in ID NO:4.
It is furthermore preferred that the nucleotide sequence of SV40 enhancing sub-piece is connected into unloaded carrier pAAV-hTERT-MCS-GFP,
Obtain nucleotide sequence carrier pAAV-SV4 enhancer-hTERT-MCS-GFP as shown in SEQ ID NO:5, the tk gene
Segment is connected into carrier pAAV-SV40 enhancer-hTERT-MCS-GFP, obtains purpose carrier pAAV-SV40 enhancer-hTERT-
Tk, nucleotide sequence such as SEQ ID NO:1.
It should be appreciated that with shown in the SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5
Nucleotide sequence with 90% or more homology and functional identical nucleotide sequence protection scope of the present invention it
It is interior.
Preferably, the S3 the following steps are included:
S31: after plasmid pAAV-hTERT-MCS-GFP digestion, then agarose gel electrophoresis, recovery purifying obtain enzyme
Cut product；
S32: the SV40 enhancing sub-piece that digestion products and S2 that S31 is obtained are obtained is seamlessly connected.
It should be understood that the present invention is not limited to above-mentioned steps, the step of can also including other, such as before step S31,
It include also other additional steps, without beyond the scope of the present invention between step S31 and S32, after step S32.
In a specific embodiment of the invention, in S31, digestion step carries out in 50 μ L endonuclease reaction systems, institute
Stating 50 μ L endonuclease reaction systems includes pAAV-hTERT-MCS-GFP 10ug, 10 × Cutsmart (NEB), 5 μ L, 2 μ of Mlu I enzyme
L complements to 50 μ L with water；Digestion 3-5h under conditions of 37 DEG C.
In a specific embodiment of the invention, in S32,50 μ L are seamless, and clone's system contains S31 digestion products respectively
102.2ng, SV40 enhancer fragment sequence 14.3ng, 1ul enzyme and 25ul buffer (buffer) complement to 50 μ L with water.
Third aspect of the present invention discloses gland relevant viral vector pAAV-SV40 enhancer-obtained by the above method
hTERT-tk。
The 4th aspect of the present invention discloses a kind of kit, and the kit includes above-mentioned gland relevant viral vector
PAAV-SV40 enhancer-hTERT-tk.
The 5th aspect of the present invention discloses a kind of suicide gene system based on gland relevant viral vector transformation, i.e. AAV-
SV40 enhancer-hTERT-tk-GCV system, the system comprises above-mentioned gland relevant viral vector pAAV-SV40 enhancers-
HTERT-tk or mentioned reagent box.
The 6th aspect of the present invention discloses above-mentioned AAV-SV40 enhancer-hTERT-tk-GCV system in tumour cell
Application in model, comprising the following steps:
(1) the pAAV-SV40 enhancer-hTERT-tk carrier in the system is constructed；
(2) the pAAV-SV40 enhancer-hTERT-tk carrier package AAV virus obtained with step (1)；
(3) the AAV virus infection tumor models after packing, and GCV is added in the medium.
It should be understood that the present invention is not limited to above-mentioned steps, the step of can also including other, such as before step (1),
It also include other additional steps between step (1) and step (2), between step (2) and step (3), after step (3), and
Without departing from protection scope of the present invention.
The 7th aspect of the present invention discloses above-mentioned AAV-SV40 enhancer-hTERT-tk-GCV system in bearing animals
Application in model, comprising the following steps:
1) the pAAV-SV40 enhancer-hTERT-tk carrier in the system is constructed；
2) the pAAV-SV40 enhancer-hTERT-tk carrier package AAV virus obtained with step 1)；
3) the AAV virus after packing is subcutaneously injected into knurl center, and GCV is given in abdominal cavity after three days, once a day, continuously
10-20 days；
4) variation of knurl is measured.
It should be understood that the present invention is not limited to above-mentioned steps, the step of can also including other, such as before step 1),
It also include it between step 1) and step 2), between step 2) and step 3), between step 3) and step 4), after step 4)
His additional step, without beyond the scope of the present invention.
Preferably, in above-mentioned application, the tumour is cerebral glioma.However, it should be appreciated by those skilled in the art that
The present invention is not limited to cerebral gliomas.
The 8th aspect of the present invention discloses above-mentioned gland relevant viral vector pAAV-SV40 enhancer-hTERT-tk and exists
Prepare the application in tumor.Preferably, the tumour is cerebral glioma.However, it is to be appreciated that the tumour
It is not limited to cerebral glioma.
On the basis of common knowledge of the art, above-mentioned each optimum condition, can any combination, and without departing from structure of the invention
Think of and protection scope.
The present invention has following remarkable advantage and effect compared with the existing technology:
(1) present invention provides a kind of AAV-SV40 enhancer-hTERT-tk-GCV system for the first time, which can not only
High effective integration and stable expression of exogenous gene, and the low feature of AAV system immunogenicity itself is also used, it is subsequent internal
Research and clinical trial provide a kind of new safe and efficient virus expression carrier system；
(2) AAV- is demonstrated by cell model (experiment in vitro) and animal model (Lump body injection, experiment in vivo) respectively
SV40 enhancer-hTERT-tk-GCV system can assist killing cancer cell in glioma treatment, play good effect.
Detailed description of the invention
Fig. 1 is the pAAV-hTERT-MCS-GFP carrier structure schematic diagram that inventive embodiments provide；
Fig. 2 is the pAAV-hTERT-intron-MCS-GFP carrier structure schematic diagram that inventive embodiments provide；
Fig. 3 is the pAAV-SV40- enhancer-hTERT-MCS-GFP carrier structure schematic diagram that inventive embodiments provide；
Fig. 4 be inventive embodiments in fluorescence schematic diagram (in figure A, B and C be respectively pAAV-hTERT-MCS-GFP carrier,
The signal of the fluorescence of pAAV-hTERT-intron-MCS-GFP carrier and pAAV-SV40- enhancer-hTERT-MCS-GFP carrier
Figure).
Fig. 5 is pAAV-SV40 enhancer-hTERT-tk carrier structure schematic diagram provided in an embodiment of the present invention.
Fig. 6 is the foundation of AAV-SV40 enhancer-hTERT-tk-GCV system provided in an embodiment of the present invention to cell membrane
The cell activity of type influences column diagram.
Fig. 7 is the foundation of AAV-SV40 enhancer-hTERT-tk-GCV system provided in an embodiment of the present invention in Animal Skin
Knurl change curve in lower tumor formation model.
Fig. 8 is expression quantity column diagram of the hTERT promoter provided in an embodiment of the present invention in samples of human glioma.
Specific embodiment
Technical solution of the present invention is described in detail with reference to the accompanying drawings and examples, but therefore will be not of the invention
It is limited among the embodiment described range.
In the following examples, the experimental methods for specific conditions are not specified, according to conventional methods and conditions, or says according to commodity
Bright book selection.The reagents and materials used in the present invention are commercially available.
Embodiment 1
Suicide gene therapy is nontoxic prodrug to be formed to toxic product in target tumor, and then kill
Tumour cell, it is common suicide system that human herpes simplex virus's thymidine kinase (HSV-TK), which combines Ganciclovir (GCV) system,
System.We are by carrying out QPCR detection to gliomatosis human tumour sample and normal cerebral tissue's sample early period, as the result is shown relatively
In normal tissue, the high expression in tumor tissues of hTERT gene, therefore we regulate and control TK gene table using hTERT promoter
It reaches, to study its effect for killing glioma.
For the expression efficiency for enhancing destination protein TK, we optimize test to existing hTERT, obtain hTERT's
The high combination of starting efficiency.
Scheme is as follows:
In order to obtain the high hTERT promoter of starting efficiency, we design the carrier of three kinds of promoters containing hTERT, carry out
Optimal inspection.These three carriers are respectively as follows: (1) hTERT promoter, i.e. building pAAV-hTERT-MCS-GFP carrier；(2) In
Introne (intron, from human beta-globin and human immunoglobulin heavy chain's gene) is inserted into behind the promoter of hTERT, i.e.,
Construct pAAV-hTERT-intron-MCS-GFP carrier；(3) enhancer of SV40, i.e. building pAAV- are added before hTERT
SV40- enhancer-hTERT-MCS-GFP carrier.We respectively by this three kinds of plasmid transfection 293T cells, by observation fluorescence come
Compare the efficiency that hTERT under these three modes starts sub-portfolio.
(1) pAAV-hTERT-MCS-GFP carrier is constructed
PAAV-hTERT-MCS-GFP carrier is constructed, structural schematic diagram is as shown in Figure 1, the specific steps are as follows:
(1) the nucleotide sequence such as SEQ ID NO:6 of hTERT segment of the synthesis with restriction enzyme site and upstream and downstream homology arm
It is shown；
(2) plasmid pAAV-CMV-MCS-GFP is subjected to digestion with XbaI and KpnI.50uL endonuclease reaction system contains pAAV-
CMV-MCS-GFP 10ug, 10 × Cutsmart (NEB) 5uL, XbaI enzyme and each 2uL of KpnI enzyme complement to 50uL with water, and 37 DEG C
1% agarose gel electrophoresis is carried out after digestion 4 hours, and large fragment is cut with blade under ultraviolet lamp and carries out recovery purifying；
(3) digestion products by the purifying that the segment of the hTERT nucleotide sequence in (1) and (2) obtain carry out seamless gram
It is grand；37 DEG C of reactions are placed on for 1 hour carries out conversion coated plate on ice；
(4) picking single colonie carries out small refer to of plasmid and identification is sequenced.Through being sequenced, expression vector pAAV-hTERT-MCS-
GFP is constructed successfully.
(2) pAAV-hTERT-intron-MCS-GFP carrier is constructed
PAAV-hTERT-intron-MCS-GFP carrier is constructed, structural schematic diagram is as shown in Figure 2, the specific steps are as follows:
(1) the nucleotide sequence such as SEQ ID NO of Intron segment of the synthesis with restriction enzyme site and upstream and downstream homology arm:
Shown in 7；
(2) plasmid pAAV-hTERT-MCS-GFP is subjected to digestion with Nhe I enzyme.50uL endonuclease reaction system contains pAAV-
HTERT-MCS-GFP 10ug, 10 × Cutsmart (NEB) 5uL, Nhe I enzyme 4uL complement to 50uL with water, and 37 DEG C of digestions 4 are small
When after carry out 1% agarose gel electrophoresis, large fragment is cut with blade under ultraviolet lamp and carries out recovery purifying；
(3) digestion products by the purifying that the Intron segment in (1) is obtained with (2) carry out seamless clone；37 DEG C of reactions 1
Hour is placed on carries out conversion coated plate on ice；
(4) picking single colonie carries out small refer to of plasmid and identification is sequenced.Through being sequenced, expression vector pAAV-hTERT-intron
MCS-GFP is constructed successfully.
(3) pAAV-SV40- enhancer-hTERT-MCS-GFP carrier is constructed
PAAV-SV40- enhancer-hTERT-MCS-GFP carrier is constructed, structural schematic diagram is as shown in figure 3, specific steps
It is as follows:
(1) the nucleotide sequence such as SEQ ID of SV40 enhancing sub-piece of the synthesis with restriction enzyme site and upstream and downstream homology arm
Shown in NO:3；
(2) plasmid pAAV-hTERT-MCS-GFP is subjected to digestion with Mlu I enzyme.50uL endonuclease reaction system contains pAAV-
HTERT-MCS-GFP 10ug, 10 × Cutsmart (NEB) 5uL, Mlu I enzyme 4uL complement to 50uL with water, and 37 DEG C of digestions 4 are small
When after carry out 1% agarose gel electrophoresis, large fragment is cut with blade under ultraviolet lamp and carries out recovery purifying；
(3) digestion products by the purifying that the SV40 enhancing sub-piece in (1) is obtained with (2) carry out seamless clone；37℃
Reaction is placed on and carries out conversion coated plate on ice for 1 hour；
(4) picking single colonie carries out small refer to of plasmid and identification is sequenced.Through being sequenced, expression vector pAAV-SV40- enhancer-
HTERT-MCS-GFP is constructed successfully.
(4) cell transfecting passes through observation fluorometric investigation starting efficiency
By the way that the plasmid transfection of above-mentioned building into cell, is judged starting efficiency by fluorescence results, specific steps are such as
Under:
(1) 293T cell is inoculated into 24 orifice plates by adherent rear 70%~80% convergence degree.
(2) second days, supernatant in 24 orifice plates is discarded, is referred to2000Reagent(Invitrogen:
11668) transfection method is transfected；It is specific as follows: to take 50uL Opti-DMEM in 1.5mL EP pipe respectively, totally 2 pipe.To
Wherein 0.8ug pAAV-hTERT-MCS-GFP, pAAV-hTERT-intron MCS-GFP or pAAV- are separately added into 1 pipe
SV40- enhancer-hTERT-MCS-GFP plasmid is soft to be uniformly mixed；1uL is added into another 1 pipe2000
Reagent is uniformly mixed, respectively static 5min.The Opti-DMEM containing liposome will be added to added with the Opti-DMEM of plasmid
In, it is soft to be uniformly mixed, stand 30 minutes.
(3) mixed liquor after standing in (2) is added into 24 holes, is placed in 37 DEG C after rolling is even, 5%CO2Incubator culture；
After (4) 6 hours, culture solution in orifice plate is discarded, 500uL DMEM complete medium is replaced in every hole；
(5) after transfecting 72 hours, fluorescence microscope is taken pictures observation.
From the fluorescence results figure fluorescence (as shown in Figure 4) for being observed that pAAV-SV40- enhancer-hTERT-MCS-GFP
Brightness is better than pAAV-hTERT-MCS-GFP and pAAV-hTERT-intron MCS-GFP, so SV40- enhancer-hTERT is opened
The combined efficiency of mover is best.
The construction method of 2 gland relevant viral vector pAAV-SV40 enhancer-hTERT-tk of embodiment
Present embodiment discloses the construction method of gland relevant viral vector pAAV-SV40 enhancer-hTERT-tk a kind of, packets
Include following steps:
(1) the nucleotide sequence such as SEQ ID NO:2 institute of tk segment of the synthesis with restriction enzyme site and upstream and downstream homology arm
Show；
(2) the nucleotide sequence such as SEQ ID of SV40 enhancing sub-piece of the synthesis with restriction enzyme site and upstream and downstream homology arm
Shown in NO:3；
(3) plasmid pAAV-hTERT-MCS-GFP (Fig. 1) is subjected to digestion with Mlu I enzyme.50 μ L endonuclease reaction systems contain
PAAV-hTERT-MCS-GFP 10ug, 10 × Cutsmart (NEB), 5 μ L, 2 μ L of Mlu I enzyme complement to 50 μ L, 37 DEG C of enzymes with water
1% agarose gel electrophoresis is carried out after cutting 4h, large fragment is cut with blade under ultraviolet lamp and carries out recovery purifying；
(4) nucleotide sequence of the digestion products for the purifying for obtaining (3) step and the SV40 of (2) step enhancing sub-piece carries out
Seamless connection.50 μ L reaction systems contain respectively: digestion products 102.2ng, SV40 the enhancer sequence 14.3ng that (2) step obtains,
1ul T4 DNA ligase, 25ul buffer (buffer) complement to 50 μ L with water, 50 DEG C of reaction 15min be placed on ice into
Row conversion coated plate；
(5) picking single colonie carries out small refer to of plasmid and identification is sequenced.Through being sequenced, expression vector pAAV-SV40 enhancer-
HTERT-MCS-GFP (Fig. 3) is constructed successfully.
(6) plasmid pAAV-SV40 enhancer-hTERT-MCS-GFP is subjected to digestion with Nhe I and Age I.50 μ L digestions
Reaction system enhancer containing pAAV-SV40-hTERT-MCS-GFP10ug, 10 × Cutsmart (NEB), 5 μ L, Nhe I and Age I
Each 1 μ L of enzyme carries out 1% agarose gel electrophoresis after complementing to 50 μ L, 37 DEG C of digestion 4h with water, will be large stretch of with blade under ultraviolet lamp
Section cuts and carries out recovery purifying；
(7) nucleotide sequence of the tk genetic fragment of the digestion products for the purifying for obtaining (6) step and (1) step carries out seamless
Clone.50 μ L reaction systems contain respectively: digestion products 102.2ng, tk genetic fragment 14.3ng, the 1ul enzyme that (6) step obtains,
25ul buffer complements to 50 μ L with water, and 50 DEG C of reaction 15min are placed on and carry out conversion coated plate on ice；
(9) picking single colonie carries out small refer to of plasmid and identification is sequenced.Through being sequenced, expression vector pAAV-SV40 enhancer-
HTERT-tk (Fig. 5) is constructed successfully.
Embodiment 3
Present embodiment discloses a kind of suicide gene system based on gland relevant viral vector transformation, i.e. AAV-SV40 enhancings
Son-hTERT-tk-GCV system, the system comprises the gland relevant viral vector pAAV- that method described in embodiment 1 obtains
SV40 enhancer-hTERT-tk.
Embodiment 4
Present embodiment discloses AAV-SV40 enhancer-hTERT-tk-GCV systems described in embodiment 3 in tumour cell
Application in model, pAAV-SV40 enhancer-hTERT-tk group specifically includes the following steps:
(1) recovery culture glioma U-87MG cell；
(2) the pAAV-SV40 enhancer-hTERT-tk carrier package AAV virus obtained with embodiment 1；
(3) the AAV virus infection U-87MG cell after packing；
(4) after virus infection 72h, GCV is added in culture medium；
(5) cell activity is detected using CTG method after 72h, assesses AAV-SV40 enhancer-hTERT-tk-GCV system to body
The lethal effect of outer culture cell；Cell killing rate=(add in 1- culture medium and be not added in cell activity/culture medium of GCV group
The cell activity of GCV group) * 100%.
By calculating, as a result as shown in fig. 6, AAV-SV40 enhancer-hTERT-tk-GCV system is to the killing rate of cell
Up to 84%.Its koilocyte group is with the pAAV-SV40 enhancer-hTERT-tk difference organized: cell is uninfected by AAV disease
Poison, and the DMSO with experimental group GCV equivalent is added in culture medium.
Embodiment 5
Present embodiment discloses AAV-SV40 enhancer-hTERT-tk-GCV systems as described in example 2 in bearing animals
Application in model, wherein experimental group specifically includes the following steps:
(1) brain glioblastoma cell U-87MG tumor formation is subcutaneously injected, makes nude mouse CDX model；
(2) inserted block is passaged to the third generation, stablizes knurl；
(3) the pAAV-SV40 enhancer-hTERT-tk carrier package AAV virus obtained with embodiment 1；
(4) by the AAV virus injection after packaging to knurl center, abdominal cavity gives GCV after three days, once a day, continuous 14
It；
(5) since after virus injection, vernier caliper measurement tumor size is used twice a week, calculates the volume of tumour,
Describe the growth curve of tumour；Tumor volume calculation formula: gross tumor volume=major diameter * minor axis * minor axis * 0.5；Knurl inhibiting rate=
(1-pAAV-SV40 enhancer-hTERT-tk group tumor volume/physiological saline group gross tumor volume) * 100%；
Knurl is taken to weigh within (6) the 15th days, assessment AAV-SV40 enhancer-hTERT-tk-GCV system is killed in body knurl
Wound effect.
Control group: unique difference with experimental group is, using physiological saline knurl center.
Knurl change curve under the Animal Skin of control group and experimental group in tumor formation model is as shown in fig. 7, by statistics meter
It calculates, AAV-SV40 enhancer-hTERT-tk-GCV system is to the knurl growth inhibition of knurl up to 81%.
Embodiment 6
The present embodiment studies expression of the hTERT promoter in samples of human glioma, and * refers to the significance of difference.Specific behaviour
Steps are as follows for work: experiment is divided into 3 groups, and one group is the tumor tissues (tumour 1) taken out from the brain tumor tissue of patient A, and one group is
From the brain tumor tissue of patient B take out tumor tissues (tumour 2), one group be taken out from the brain tissue of normal person C it is non-swell
Tumor brain tissue (control group).
Extract RNA respectively, then reverse transcription carries out qPCR, detects the hTERT promoter expression of rna level, utilizes
Student test method carries out statistical analysis, obtains P value, and * * indicates that P value < 0.01, * * * indicate P value < 0.001.As a result
As shown in figure 8, hTERT promoter expression quantity is significantly higher than non-tumour brain tissue in tumor tissues as can be drawn from Figure 8.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Sequence table
<110>and first biotechnology (Shanghai) limited liability company
<120>a kind of suicide gene system and its application based on gland relevant viral vector transformation
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 6373
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60
gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 120
actccatcac taggggttcc tgcggccgca cgcgtcttaa gggcctgaaa taacctctga 180
aagaggaact tggttaggta ccttctgagg ctgaaagaac cagctgtgga atgtgtgtca 240
gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct 300
caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca 360
aagcatgcat ctcaattagt cagcaaccat agtcccactg ttaacacgcg tgtgtctaga 420
agatctctcc gctggggccc tcgctggcgt ccctgcaccc tgggagcgcg agcggcgcgc 480
gggcggggaa gcgcggccca gacccccggg tccgcccgga gcagctgcgc tgtcggggcc 540
aggccgggct cccagtggat tcgcgggcac agacgcccag gaccgcgctt cccacgtggc 600
ggagggactg gggacccggg cacccgtcct gccccttcac cttccagctc cgcctcctcc 660
gcgcggaccc cgccccgtcc cgacccctcc cgggtccccg gcccagcccc ctccgggccc 720
tcccagcccc tccccttcct ttccgcggcc ccgccctctc ctcgcggcgc gagtttcagg 780
cagcgctgcg tcctgctgcg cacgtgggaa gccctggccc cggccacccc cgcgtgaagc 840
ttgcatgcct gcaggtcgac tctagaggat ctactagtca tatggatgag ctcgagctgc 900
accctgggag cgcgagcggc gcgcgggcgg ggaagcgcgg cccagacccc cgggtccgcc 960
cggagcagct gcgctgtcgg ggccaggccg ggctcccagt ggattcgcgg gcacagacgc 1020
ccaggaccgc gcttcccacg tggcggaggg actggggacc cgggcacccg tcctgcccct 1080
tcaccttcca gctccgcctc ctccgcgcgg accccgcccc gtcccgaccc ctcccgggtc 1140
cccggcccag ccccctccgg gccctcccag cccctcccct tcctttccgc ggccccgccc 1200
tctcctcgag ctcgagatcg gatccccggg taccgaggcg aattcggctt ctcgagccac 1260
tcttgagtgc cagcgagtag agttttctcc tccgagccgc tccgacaccg ggactgaaaa 1320
tgggtaccga aaaccccggt ccggctagcg ccaccatggc ttcgtacccc tgccatcaac 1380
acgcgtctgc gttcgaccag gctgcgcgtt ctcgcggcca tagcaaccga cgtacggcgt 1440
tgcgccctcg ccggcagcaa gaagccacgg aagtccgcct ggagcagaaa atgcccacgc 1500
tactgcgggt ttatatagac ggtcctcacg ggatggggaa aaccaccacc acgcaactgc 1560
tggtggccct gggttcgcgc gacgatatcg tctacgtacc cgagccgatg acttactggc 1620
aggtgctggg ggcttccgag acaatcgcga acatctacac cacacaacac cgcctcgacc 1680
agggtgagat atcggccggg gacgcggcgg tggtaatgac aagcgcccag ataacaatgg 1740
gcatgcctta tgccgtgacc gacgccgttc tggctcctca tatcgggggg gaggctggga 1800
gctcacatgc cccgcccccg gccctcaccc tcatcttcga ccgccatccc atcgccgccc 1860
tcctgtgcta cccggccgcg cgatacctta tgggcagcat gaccccccag gccgtgctgg 1920
cgttcgtggc cctcatcccg ccgaccttgc ccggcacaaa catcgtgttg ggggcccttc 1980
cggaggacag acacatcgac cgcctggcca aacgccagcg ccccggcgag cggcttgacc 2040
tggctatgct ggccgcgatt cgccgcgttt acgggctgct tgccaatacg gtgcggtatc 2100
tgcagggcgg cgggtcgtgg cgggaggatt ggggacagct ttcggggacg gccgtgccgc 2160
cccagggtgc cgagccccag agcaacgcgg gcccacgacc ccatatcggg gacacgttat 2220
ttaccctgtt tcgggccccc gagttgctgg cccccaacgg cgacctgtat aacgtgtttg 2280
cctgggcctt ggacgtcttg gccaaacgcc tccgtcccat gcacgtcttt atcctggatt 2340
acgaccaatc gcccgccggc tgccgggacg ccctgctgca acttacctcc gggatggtcc 2400
agacccacgt caccacccca ggctccatac cgacgatctg cgacctggcg cgcacgtttg 2460
cccgggagat gggggaggct aactaaaccg gttatcgata atcaacctct ggattacaaa 2520
atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac 2580
gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc 2640
ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt 2700
ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc 2760
tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc 2820
gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg 2880
gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc cacctggatt 2940
ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc 3000
cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt 3060
cggatctccc tttgggccgc ctccccgcat cgataccgag cgctgctcga gagatctacg 3120
ggtggcatcc ctgtgacccc tccccagtgc ctctcctggc cctggaagtt gccactccag 3180
tgcccaccag ccttgtccta ataaaattaa gttgcatcat tttgtctgac taggtgtcct 3240
tctataatat tatggggtgg aggggggtgg tatggagcaa ggggcaagtt gggaagacaa 3300
cctgtagggc ctgcggggtc tattgggaac caagctggag tgcagtggca caatcttggc 3360
tcactgcaat ctccgcctcc tgggttcaag cgattctcct gcctcagcct cccgagttgt 3420
tgggattcca ggcatgcatg accaggctca gctaattttt gtttttttgg tagagacggg 3480
gtttcaccat attggccagg ctggtctcca actcctaatc tcaggtgatc tacccacctt 3540
ggcctcccaa attgctggga ttacaggcgt gaaccactgc tcccttccct gtccttctga 3600
ttttgtaggt aaccacgtgc ggaccgagcg gccgcaggaa cccctagtga tggagttggc 3660
cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg 3720
cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg 3780
cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag 3840
caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc 3900
agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc 3960
tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg 4020
ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca 4080
cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc 4140
tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc gggctattct 4200
tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa 4260
caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc 4320
agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct 4380
gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc 4440
tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag 4500
ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg 4560
tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata 4620
cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga 4680
aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca 4740
ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat 4800
cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag 4860
agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc 4920
gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct 4980
cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca 5040
gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt 5100
ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat 5160
gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt 5220
gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta 5280
cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga 5340
ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt 5400
gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc 5460
gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct 5520
gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata 5580
ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt 5640
gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc 5700
gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg 5760
caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact 5820
ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg 5880
tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg 5940
ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac 6000
tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca 6060
cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga 6120
gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc 6180
ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct 6240
gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg 6300
agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct 6360
tttgctcaca tgt 6373
<210> 2
<211> 1131
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
atggcttcgt acccctgcca tcaacacgcg tctgcgttcg accaggctgc gcgttctcgc 60
ggccatagca accgacgtac ggcgttgcgc cctcgccggc agcaagaagc cacggaagtc 120
cgcctggagc agaaaatgcc cacgctactg cgggtttata tagacggtcc tcacgggatg 180
gggaaaacca ccaccacgca actgctggtg gccctgggtt cgcgcgacga tatcgtctac 240
gtacccgagc cgatgactta ctggcaggtg ctgggggctt ccgagacaat cgcgaacatc 300
tacaccacac aacaccgcct cgaccagggt gagatatcgg ccggggacgc ggcggtggta 360
atgacaagcg cccagataac aatgggcatg ccttatgccg tgaccgacgc cgttctggct 420
cctcatatcg ggggggaggc tgggagctca catgccccgc ccccggccct caccctcatc 480
ttcgaccgcc atcccatcgc cgccctcctg tgctacccgg ccgcgcgata ccttatgggc 540
agcatgaccc cccaggccgt gctggcgttc gtggccctca tcccgccgac cttgcccggc 600
acaaacatcg tgttgggggc ccttccggag gacagacaca tcgaccgcct ggccaaacgc 660
cagcgccccg gcgagcggct tgacctggct atgctggccg cgattcgccg cgtttacggg 720
ctgcttgcca atacggtgcg gtatctgcag ggcggcgggt cgtggcggga ggattgggga 780
cagctttcgg ggacggccgt gccgccccag ggtgccgagc cccagagcaa cgcgggccca 840
cgaccccata tcggggacac gttatttacc ctgtttcggg cccccgagtt gctggccccc 900
aacggcgacc tgtataacgt gtttgcctgg gccttggacg tcttggccaa acgcctccgt 960
cccatgcacg tctttatcct ggattacgac caatcgcccg ccggctgccg ggacgccctg 1020
ctgcaactta cctccgggat ggtccagacc cacgtcacca ccccaggctc cataccgacg 1080
atctgcgacc tggcgcgcac gtttgcccgg gagatggggg aggctaactg a 1131
<210> 3
<211> 238
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
ggcctgaaat aacctctgaa agaggaactt ggttaggtac cttctgaggc tgaaagaacc 60
agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa 120
gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc 180
cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccact 238
<210> 4
<211> 5814
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60
gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 120
actccatcac taggggttcc tgcggccgca cgcgtgtgtc tagaagatct ctccgctggg 180
gccctcgctg gcgtccctgc accctgggag cgcgagcggc gcgcgggcgg ggaagcgcgg 240
cccagacccc cgggtccgcc cggagcagct gcgctgtcgg ggccaggccg ggctcccagt 300
ggattcgcgg gcacagacgc ccaggaccgc gcttcccacg tggcggaggg actggggacc 360
cgggcacccg tcctgcccct tcaccttcca gctccgcctc ctccgcgcgg accccgcccc 420
gtcccgaccc ctcccgggtc cccggcccag ccccctccgg gccctcccag cccctcccct 480
tcctttccgc ggccccgccc tctcctcgcg gcgcgagttt caggcagcgc tgcgtcctgc 540
tgcgcacgtg ggaagccctg gccccggcca cccccgcgtg aagcttgcat gcctgcaggt 600
cgactctaga ggatctacta gtcatatgga tgagctcgag ctgcaccctg ggagcgcgag 660
cggcgcgcgg gcggggaagc gcggcccaga cccccgggtc cgcccggagc agctgcgctg 720
tcggggccag gccgggctcc cagtggattc gcgggcacag acgcccagga ccgcgcttcc 780
cacgtggcgg agggactggg gacccgggca cccgtcctgc cccttcacct tccagctccg 840
cctcctccgc gcggaccccg ccccgtcccg acccctcccg ggtccccggc ccagccccct 900
ccgggccctc ccagcccctc cccttccttt ccgcggcccc gccctctcct cgagctcgag 960
atcggatccc cgggtaccga ggcgaattcg gcttctcgag ccactcttga gtgccagcga 1020
gtagagtttt ctcctccgag ccgctccgac accgggactg aaaatgggta ccgaaaaccc 1080
cggtccggct agcgccaccg gatccggcgg atctggcatg gtgagcaagg gcgaggagct 1140
gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt 1200
cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat 1260
ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg 1320
cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc 1380
catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa 1440
gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg 1500
catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag 1560
ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat 1620
ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc 1680
catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct 1740
gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc 1800
cgggatcact ctcggcatgg acgagctgta caagggctcc ggagactaca aggatgacga 1860
tgacaaggat tacaaagacg acgatgataa ggactataag gatgatgacg acaaataaaa 1920
gctttaaacc ggttatcgat aatcaacctc tggattacaa aatttgtgaa agattgactg 1980
gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt 2040
atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc 2100
tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt 2160
ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga 2220
ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct 2280
gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat 2340
cgtcctttcc ttggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct 2400
gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc 2460
tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg 2520
cctccccgca tcgataccga gcgctgctcg agagatctac gggtggcatc cctgtgaccc 2580
ctccccagtg cctctcctgg ccctggaagt tgccactcca gtgcccacca gccttgtcct 2640
aataaaatta agttgcatca ttttgtctga ctaggtgtcc ttctataata ttatggggtg 2700
gaggggggtg gtatggagca aggggcaagt tgggaagaca acctgtaggg cctgcggggt 2760
ctattgggaa ccaagctgga gtgcagtggc acaatcttgg ctcactgcaa tctccgcctc 2820
ctgggttcaa gcgattctcc tgcctcagcc tcccgagttg ttgggattcc aggcatgcat 2880
gaccaggctc agctaatttt tgtttttttg gtagagacgg ggtttcacca tattggccag 2940
gctggtctcc aactcctaat ctcaggtgat ctacccacct tggcctccca aattgctggg 3000
attacaggcg tgaaccactg ctcccttccc tgtccttctg attttgtagg taaccacgtg 3060
cggaccgagc ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct 3120
cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg 3180
gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc 3240
cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct 3300
gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg 3360
ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg 3420
gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac 3480
ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct 3540
gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt 3600
tccaaactgg aacaacactc aaccctatct cgggctattc ttttgattta taagggattt 3660
tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt 3720
ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg 3780
ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt 3840
gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc 3900
agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat 3960
ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg 4020
gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc 4080
tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta 4140
ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg 4200
ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg 4260
gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac 4320
gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg 4380
acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt 4440
actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg 4500
ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac 4560
cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt 4620
gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag 4680
caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc 4740
aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc 4800
ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta 4860
tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg 4920
ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga 4980
ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac 5040
ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa 5100
tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat 5160
cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc 5220
taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg 5280
gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc 5340
acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg 5400
ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg 5460
ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa 5520
cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg 5580
aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga 5640
gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct 5700
gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca 5760
gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgt 5814
<210> 5
<211> 6070
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60
gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 120
actccatcac taggggttcc tgcggccgca cgcgtcttaa gggcctgaaa taacctctga 180
aagaggaact tggttaggta ccttctgagg ctgaaagaac cagctgtgga atgtgtgtca 240
gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct 300
caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca 360
aagcatgcat ctcaattagt cagcaaccat agtcccactg ttaacacgcg tgtgtctaga 420
agatctctcc gctggggccc tcgctggcgt ccctgcaccc tgggagcgcg agcggcgcgc 480
gggcggggaa gcgcggccca gacccccggg tccgcccgga gcagctgcgc tgtcggggcc 540
aggccgggct cccagtggat tcgcgggcac agacgcccag gaccgcgctt cccacgtggc 600
ggagggactg gggacccggg cacccgtcct gccccttcac cttccagctc cgcctcctcc 660
gcgcggaccc cgccccgtcc cgacccctcc cgggtccccg gcccagcccc ctccgggccc 720
tcccagcccc tccccttcct ttccgcggcc ccgccctctc ctcgcggcgc gagtttcagg 780
cagcgctgcg tcctgctgcg cacgtgggaa gccctggccc cggccacccc cgcgtgaagc 840
ttgcatgcct gcaggtcgac tctagaggat ctactagtca tatggatgag ctcgagctgc 900
accctgggag cgcgagcggc gcgcgggcgg ggaagcgcgg cccagacccc cgggtccgcc 960
cggagcagct gcgctgtcgg ggccaggccg ggctcccagt ggattcgcgg gcacagacgc 1020
ccaggaccgc gcttcccacg tggcggaggg actggggacc cgggcacccg tcctgcccct 1080
tcaccttcca gctccgcctc ctccgcgcgg accccgcccc gtcccgaccc ctcccgggtc 1140
cccggcccag ccccctccgg gccctcccag cccctcccct tcctttccgc ggccccgccc 1200
tctcctcgag ctcgagatcg gatccccggg taccgaggcg aattcggctt ctcgagccac 1260
tcttgagtgc cagcgagtag agttttctcc tccgagccgc tccgacaccg ggactgaaaa 1320
tgggtaccga aaaccccggt ccggctagcg ccaccggatc cggcggatct ggcatggtga 1380
gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg 1440
taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc 1500
tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga 1560
ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg 1620
acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg 1680
acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc 1740
gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg 1800
agtacaacta caacagccac aacgtctata tcatggccga caagcagaag aacggcatca 1860
aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact 1920
accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga 1980
gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg 2040
agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag ggctccggag 2100
actacaagga tgacgatgac aaggattaca aagacgacga tgataaggac tataaggatg 2160
atgacgacaa ataaaagctt taaaccggtt atcgataatc aacctctgga ttacaaaatt 2220
tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct 2280
gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg 2340
tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc 2400
gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt 2460
cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc 2520
gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg 2580
ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac ctggattctg 2640
cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc 2700
ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg 2760
atctcccttt gggccgcctc cccgcatcga taccgagcgc tgctcgagag atctacgggt 2820
ggcatccctg tgacccctcc ccagtgcctc tcctggccct ggaagttgcc actccagtgc 2880
ccaccagcct tgtcctaata aaattaagtt gcatcatttt gtctgactag gtgtccttct 2940
ataatattat ggggtggagg ggggtggtat ggagcaaggg gcaagttggg aagacaacct 3000
gtagggcctg cggggtctat tgggaaccaa gctggagtgc agtggcacaa tcttggctca 3060
ctgcaatctc cgcctcctgg gttcaagcga ttctcctgcc tcagcctccc gagttgttgg 3120
gattccaggc atgcatgacc aggctcagct aatttttgtt tttttggtag agacggggtt 3180
tcaccatatt ggccaggctg gtctccaact cctaatctca ggtgatctac ccaccttggc 3240
ctcccaaatt gctgggatta caggcgtgaa ccactgctcc cttccctgtc cttctgattt 3300
tgtaggtaac cacgtgcgga ccgagcggcc gcaggaaccc ctagtgatgg agttggccac 3360
tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc 3420
gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct 3480
gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa 3540
ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc 3600
gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt 3660
ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc 3720
cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt 3780
agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt 3840
aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggg ctattctttt 3900
gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa 3960
aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt 4020
acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac 4080
gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc 4140
gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc 4200
ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca 4260
ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat 4320
tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa 4380
aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt 4440
tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag 4500
ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt 4560
tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg 4620
gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag 4680
aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta 4740
agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg 4800
acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta 4860
actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac 4920
accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt 4980
actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca 5040
cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag 5100
cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta 5160
gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag 5220
ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt 5280
tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat 5340
aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta 5400
gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa 5460
acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt 5520
tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag 5580
ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta 5640
atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca 5700
agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag 5760
cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa 5820
agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga 5880
acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc 5940
gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc 6000
ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt 6060
gctcacatgt 6070
Claims (13)
1. a kind of gland relevant viral vector pAAV-SV40 enhancer-hTERT-tk, which is characterized in that the pAAV-SV40 enhancing
Son-hTERT-tk carrier includes at least following characteristic:
A) using SV40 as enhancer；B) hTERT is promoter；C) with can encode make GCV generate toxicity thymidine kinase tk
For target gene；D) using AAV as carrier.
2. gland relevant viral vector pAAV-SV40 enhancer-hTERT-tk according to claim 1, which is characterized in that institute
The nucleotide sequence of pAAV-SV40 enhancer-hTERT-tk carrier is stated as shown in SEQ ID NO:1.
3. it is a kind of construct gland relevant viral vector pAAV-SV40 enhancer-hTERT-tk method, which is characterized in that including with
Lower step:
S1: tk genetic fragment of the synthesis with restriction enzyme site and upstream and downstream homology arm；
S2: SV40 of the synthesis with restriction enzyme site and upstream and downstream homology arm enhances sub-piece；
S3: SV40 enhancing sub-piece is connected into the carrier for obtaining enhancing sub-piece containing SV40 in carrier；
S4: tk genetic fragment described in S1 is connected into the carrier containing SV40 enhancing sub-piece described in S3, purpose load is obtained
Body pAAV-SV40 enhancer-hTERT-tk.
4. according to the method described in claim 3, it is characterized in that, the nucleotide sequence of the tk genetic fragment such as SEQ ID
Shown in NO:2, the nucleotide sequence of the SV40 enhancing sub-piece is as shown in SEQ ID NO:3.
5. according to the method described in claim 3, it is characterized in that, the carrier is unloaded carrier pAAV-hTERT- in S3
MCS-GFP, nucleotide sequence is as shown in SEQ ID NO:4.
6. according to the method described in claim 5, it is characterized in that, the S3 the following steps are included:
S31: after plasmid pAAV-hTERT-MCS-GFP digestion, then agarose gel electrophoresis, recovery purifying obtain digestion production
Object；
S32: the SV40 enhancing sub-piece that digestion products and S2 that S31 is obtained are obtained is seamlessly connected.
7. according to the method described in claim 6, it is characterized in that, digestion step is in 50 μ L endonuclease reaction systems in S31
It carries out, the 50 μ L endonuclease reaction system includes pAAV-hTERT-MCS-GFP10ug, 10 × Cutsmart (NEB), 5 μ L, Mlu
2 μ L of I enzyme complements to 50 μ L with water；Digestion 3-5h under conditions of 37 DEG C.
8. the gland relevant viral vector pAAV-SV40 enhancer-obtained according to claim 3-7 any one the method
hTERT-tk。
9. a kind of kit, which is characterized in that the kit includes that claim 1-2 or gland according to any one of claims 8 are related
Viral vectors pAAV-SV40 enhancer-hTERT-tk.
10. a kind of suicide gene system based on gland relevant viral vector transformation, i.e. AAV-SV40 enhancer-hTERT-tk-GCV
System, which is characterized in that the system comprises claim 1-2, gland relevant viral vector pAAV-SV40 enhancer-described in 8
HTERT-tk or kit as claimed in claim 9.
11. AAV-SV40 enhancer-hTERT-tk-GCV system according to claim 10 is in tumor models
Using, which comprises the following steps:
(1) the pAAV-SV40 enhancer-hTERT-tk carrier in the system is constructed；
(2) the pAAV-SV40 enhancer-hTERT-tk carrier package AAV virus obtained with step (1)；
(3) the AAV virus infection tumor models after packing, and GCV is added in the medium.
12. gland relevant viral vector pAAV-SV40 enhancer-hTERT-tk described in -2 or 8 is controlled in preparation according to claim 1
Treat the application in tumour medicine.
13. application according to claim 12, which is characterized in that the tumour is cerebral glioma.
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Application publication date: 20191122 |