CN109182381A - A kind of the novel oncolytic virus and its construction method of selectively killing colon cancer cell - Google Patents
A kind of the novel oncolytic virus and its construction method of selectively killing colon cancer cell Download PDFInfo
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Abstract
The invention discloses a kind of novel oncolytic of selectively killing colon cancer cell virus and its construction methods, by the way that the ICP4 gene promoter in I herpes simplex virus type 17+ pnca gene group is replaced as colon cancer specificity promoter, again by ICP34.5 gene knockout in I herpes simplex virus type 17+ pnca gene group and it is inserted into IL-12 expressed sequence, obtains HSVCEA+IL‑12.The present invention provides a kind of novel oncolytic of selectively killing colon cancer cell virus and its construction method, novel oncolytic virus HSVCEA+IL‑12It is bred in colon cancer cell with high selectivity using CEA promoter and the building method realization for rejecting ICP34.5, it is ensured that the virus does not infect normal tissue cell；Meanwhile insertion IL-12 expressed sequence enhances novel oncolytic virus HSVCEA+IL‑12Immunoregulation effect, it is ensured that impaired colon cancer cell is thoroughly removed completely by the immunocyte of body itself.
Description
Technical field
The present invention relates to biotechnologys and field of gene, and it is thin to particularly relate to a kind of selectively killing colon cancer
The novel oncolytic virus and its construction method of born of the same parents.
Background technique
Oncolytic viral therapy be it is a kind of using virus-specific then killing tumor cell is replicated in tumour cell, and
Body is stimulated to generate the novel tumor treatment method of specificity antineoplastic immunity reaction.Compared to other tumor therapeuticing methods, oncolytic
Virus therapy has the characteristics that duplication is efficient, fragmentation effect is good and toxic side effect is small, has become oncotherapy research field
New hot spot.
I type herpe simplex recombinant virus T-VEC (talimogenelaherparepvec) of Amgen company of the U.S. is being controlled
It treats and shows good oncotherapy effect in III clinical trial phase of advanced melanoma patient, and become the first acquisition U.S.
The oncolytic virus class therapeutic agent of FDA approval listing.The success that oncolytic virus obtains in melanoma treatment causes science
Family widely pays close attention to oncolytic viral therapy, and the research of oncolytic virus has obtained further promotion.So far it is controlled for oncolytic
The virus for the treatment of is up to tens of kinds, including herpessimplexvirustypeⅰ (herpes simplexvirustype 1, HSV-1), adenopathy
Poison, reovirus, newcastle disease virus, poliovirus, Coxsackie virus, measles virus, human immunodeficiency virus,
Mumps virus, vaccinia virus, vesicular stomatitis virus (vesicularstomatitis virus, VSV) and influenza virus
Deng.Oncolytic virus is broadly divided into 4 seed types by development course: (1) wild-type strain or natural attenuated strain, such as Newcastle Disease
Poison, Coxsackie virus and reovirus etc.；(2) genetic engineering selectivity attenuated strain mainly deletes the certain crucial bases of virus
Thus realize the tumor-selective of virus replication, such as ONYX-015, G207；(3) gene loaded type Strain, mainly preceding
It states and loads exogenous therapeutic gene on the basis of two kinds of oncolytic virus, such as load granulocyte macrophage colony stimulating factor
The JX-594 and T-VEC of (granulocytemacrophage colony stimulating factor, GM-CSF) etc.；(4)
Targeted viral strain is transcribed, i.e., is inserted into tissue before viral indispensable gene or tumor-specific promoters exists to control oncolytic virus
Duplication in tumour cell, such as G92A.
Colon cancer is a kind of common cancer.Carcinomebryonic antigen (Carcinoembryonic antigen, CEA) is a kind of
Mainly in the secreted protein of colon cancer cell expression, the present invention utilizes building selection the characteristics of colon cancer specific expressed CEA
Property colon cancer cell infection breeding novel oncolytic virus HSVCEA+IL-12.The virus do not infect normal colon mucosa cell and
Other normal tissues, can intravenously administrable, have many advantages, such as efficiently, high specificity.To the circulation colon cancer cell in blood and turn
Shifting property colon cancer stove has killing effect.
Summary of the invention
To solve the problems mentioned above in the background art, the purpose of the present invention is to provide a kind of selectively killing colons
The novel oncolytic virus and its construction method of cancer cell.
To achieve the above object, the technical scheme adopted by the invention is as follows:
The invention discloses a kind of novel oncolytic virus construction methods of selectively killing colon cancer cell, including following step
It is rapid:
Step 1: building pdICP4-promoter plasmid: PCR expands 17+ plants of ICP4 bases of I herpes simplex virus type respectively
After the flanking sequence of promoter upstream and downstream, ICP4 gene is obtained after being digested respectively with restriction enzyme EcoRI/SpeI
It the upstream flanking sequence of promoter, downstream flanking sequence and removes 17+ plants of I herpes simplex virus type of ICP4 promoter, is used in combination
Complementary ligase Linker 1 and Linker 2 is by the upstream flanking sequence of the ICP4 gene promoter and downstream flank sequence
Column connect, and EcoRI the and SalI restriction enzyme site of the product cloning after connection to pBluescript is created plasmid
pdICP4-promoter；
Step 2: building pdICP4-CEA-promoter plasmid: with the double digested mode of EcoRI/XhoI by
The people CEA promoter sequence of CMV promoter control is released from plasmid pcDNA3.1-CEA-promoter, more with T4DNA
Poly- enzymatic treatment rear clone enters at the restriction enzyme EcoHV restriction enzyme site of plasmid pdICP4-promoter obtained by step 1, from
And construct plasmid pdICP4-CEA-promoter；
Step 3: 17+ plants of homologous recombination I herpes simplex virus type in bhk cell: ICP4 promoter will be removed in step 1
17+ plants of I herpes simplex virus type transfect bhk cell jointly with plasmid pdICP4-CEA-promoter in step 2, make this two
In bhk cell homologous recombination occurs for person, obtains the recombinant viral vector 17-d4-CEA-promoter of expression CEA promoter；
Step 4: building pdICP34.5 plasmid: PCR expands ICP34.5 gene in 17+ plants of I herpes simplex virus type respectively
Upstream and downstream flanking sequence, obtained two segments of ICP34.5 upstream and downstream flanking sequence are connected with over-lap PCR
It connects, then the product insertion after connection is used in advance in the postdigestive plasmid pSP72 of restriction enzyme BamHI/XhoI
The cohesive end of T4DNA polymerase filling-in plasmid, obtained plasmid are named as pdICP34.5；
Step 5: building plasmid pdICP34.5-hIL-12: using hIL-12 gene substitution plasmid pcDNA3.1-CEA-
CEA-promoter in promoter generates plasmid pcDNA3.1-hIL-12；In plasmid pcDNA3.1-hIL-12
HIL-12 expression cassette be cloned at the site Afel of plasmid pdICP34.5, create plasmid pdICP34.5-hIL-12；
Step 6: the plasmid pdICP34.5-hIL-12 in step 5 is used to delete recombinant viral vector 17- in step 3
ICP34.5 gene in d4-CEA-promoter, to construct oncolytic virus HSVCEA+IL-12。
In above-mentioned technical proposal, in the ICP4 promoter in step 1,
The primer pair of upstream flanking sequence includes: that upstream sequence ICP4-promoterUSf is AAAAGAATTCGATACAC
ATCGTTCAGACGGAGC；
Downstream sequence ICP4-promoterUSr is AAAAACTAGTGATCGATCTCGCACATGGCCT；
The primer pair of downstream flanking sequence includes: that upstream sequence ICP4-promoterDSf is
AAAAAAGCTTTCACGCGCATGCTCTTCTC；
Downstream sequence ICP4-promoterDSr is AAAACAGCTGCACCGTGCCCGTGATGAA.
In above-mentioned technical proposal, in the ICP34.5 gene in step 4,
The primer pair of upstream flanking sequence includes: that upstream sequence ICP34.5USf is
CTCTGACCTGAGATTGGCGGCACTG；
Downstream sequence ICP34.5USr is GCGGCCGCAGCGCTGCGGCCGCCGCGGGCGCGTCCTGACCGCGGG；
The primer pair of downstream flanking sequence includes: that upstream sequence ICP34.5DSf is GCGGCCGCAGCGCTGCGGCCGCC
AGCGCGGCGGGGCCCGGCCAACCA；
Downstream sequence ICP34.5DSr is TTCTTCCCTCTTCTCCCGCCCTCCA.
In above-mentioned technical proposal, in step 1, the primer sequence of Linker 1 is CTAGTGAATTCTAGTGGATCCCCC
GGGCTGCAGGAATTCGATATCA；
The primer sequence of Linker 2 is AGCTTGATATCGAATTCCTGCAGCCCGGGGGATCCACTAGAATTCA.
In above-mentioned technical proposal, recombinant viral vector 17-d4-CEA-promoter passes through the thermophilic poison of four-wheel picking in step 3
The method of spot is purified.
The invention also discloses a kind of novel oncolytic of selectively killing colon cancer cell viruses, using following building sides
Method: the ICP4 gene promoter in I herpes simplex virus type 17+ pnca gene group is replaced as colon cancer specificity promoter CEA
Promoter, then by ICP34.5 gene knockout in I herpes simplex virus type 17+ pnca gene group and it is inserted into the building of IL-12 expressed sequence
Obtain novel oncolytic virus HSVCEA+IL-12。
In above-mentioned technical proposal, the colon cancer specificity promoter is CEA promoter.
In above-mentioned technical proposal, the construction method is a kind of novel oncolytic virus structure of selectively killing colon cancer cell
Construction method described in construction method.
Compared with prior art, the beneficial effects of the present invention are:
The present invention is by setting the ICP4 gene promoter on 17+ plants of I herpes simplex virus type (the female virus of HSV) genomes
Change colon cancer specificity promoter (Carcinoembryonic Antigen CEA promoter) into, again will be in I herpes simplex virus type 17+ pnca gene group
ICP34.5 gene knockout is simultaneously inserted into IL-12 expressed sequence, obtains HSVCEA+IL-12。
Nearly all colon cancer cell can express CEA, therefore CEA promoter can guarantee HSVCEA+IL-12Selectively tying
Breeding in colon-cancer cell.ICP34.5 can fight the effect of interferon anti-reflecting virus in normal cell, and about 90% or more tumour is thin
The interferon anti-reflecting virus effect of born of the same parents has different degrees of defect, rejects ICP34.5 gene and makes viral HSVCEA+IL-12It cannot be
The growth and breeding in tumour cell is only capable of in normal cell, it is ensured that the virus does not infect normal tissue cell.Meanwhile it being inserted into
IL-12 expressed sequence can enhance the immunoregulation effect of the virus, it is ensured that impaired colon cancer cell is by the immune of body itself
Cell thoroughly removes completely.
Detailed description of the invention
Fig. 1 is recombinant virus HSV in the present inventionCEA+IL-12Schematic diagram；
Fig. 2 is HSVCEA+IL-12The bar chart of the survival ability of selective depression colon cancer cell COLO；
Fig. 3 is HSVCEA+IL-12The bar chart of the clonality of selective depression colon cancer cell COLO；
Fig. 4 is HSVCEA+IL-12The bar chart of the transfer ability of selective depression colon cancer cell COLO；
Fig. 5 is HSVCEA+IL-12The bar chart of the invasive ability of selective depression colon cancer cell COLO.
Specific embodiment
To be easy to understand the technical means, the creative features, the aims and the efficiencies achieved by the present invention, below with reference to
The drawings and specific embodiments, how the present invention is further explained implements.
In the present invention, restriction enzyme EcoRI/SpeI: it is purchased from Thermo Scientific；PBluescript: purchase
In Stratagene；PcDNA3.1-CEA-promoter: win profit biology YRGENE, China are purchased from；Restriction enzyme BamHI/
XhoI: it is purchased from Thermo Scientific；PSP72: it is purchased from Promega company；HIL-12 gene: Invitrogen public affairs are purchased from
Department.
The invention discloses a kind of novel oncolytic virus construction methods of selectively killing colon cancer cell, including following step
It is rapid:
Step 1: building pdICP4-promoter plasmid: PCR expands 17+ plants of ICP4 bases of I herpes simplex virus type respectively
Because promoter upstream (up-stream, US) and downstream (down-stream, DS) flanking sequence (flanking regions,
FLRs after), the upstream flanking sequence of the ICP4 gene promoter obtained after being digested respectively with restriction enzyme EcoRI/SpeI
US FLRs, downstream flanking sequence DS FLRs and remove 17+ the plant of I herpes simplex virus type of ICP4 promoter, and with complementation
Ligase Linker 1 and Linker 2 is by the upstream flanking sequence US FLRs and downstream flanking sequence DS of ICP4 gene promoter
FLRs is connected, EcoRI the and SalI restriction enzyme site of the product cloning after connection to pBluescript is then created plasmid
pdICP4-promoter；
Step 2: building pdICP4-CEA-promoter plasmid: with the double digested mode of EcoRI/XhoI by
The people CEA promoter sequence of CMV promoter control is released from plasmid pcDNA3.1-CEA-promoter, more with T4DNA
Poly- enzymatic treatment rear clone enters at the restriction enzyme EcoHV restriction enzyme site of plasmid pdICP4-promoter obtained by step 1, from
And construct plasmid pdICP4-CEA-promoter；
Step 3: 17+ plants of homologous recombination I herpes simplex virus type in bhk cell: ICP4 promoter will be removed in step 1
17+ plants of I herpes simplex virus type transfect bhk cell jointly with plasmid pdICP4-CEA-promoter in step 2, make this two
In bhk cell homologous recombination occurs for person, obtains the recombinant viral vector 17-d4-CEA-promoter of expression CEA promoter；
Step 4: building pdICP34.5 plasmid: PCR expands ICP34.5 gene in 17+ plants of I herpes simplex virus type respectively
Upstream flanking sequence US FLRs and downstream flanking sequence DS FLRs, obtained ICP34.5US FLRs and ICP34.5DS
Two segments of FLRs are attached with over-lap PCR, and restriction enzyme BamHI/ is then used in the product insertion after connection in advance
In the postdigestive plasmid pSP72 of XhoI, with the cohesive end of T4DNA polymerase filling-in plasmid, obtained plasmid is named as
pdICP34.5；
Step 5: building plasmid pdICP34.5-hIL-12: using hIL-12 gene substitution plasmid pcDNA3.1-CEA-
CEA-promoter in promoter generates plasmid pcDNA3.1-hIL-12；In plasmid pcDNA3.1-hIL-12
HIL-12 expression cassette be cloned at the site Afel of plasmid pdICP34.5, create plasmid pdICP34.5-hIL-12；Step
Six, the plasmid pdICP34.5-hIL-12 in step 5 is used to delete recombinant viral vector 17-d4-CEA- in step 3
The ICP34.5 gene of promoter, to construct oncolytic virus HSVCEA+IL-12。
As shown in table 1, in the ICP4 promoter in step 1, the primer pair of upstream flanking sequence includes: upstream sequence
ICP4-promoterUSf is AAAAGAATTCGATACACATCGTTCAGACGGAGC (SEQ ID NO:1)；
Downstream sequence ICP4-promoterUSr be AAAAACTAGTGATCGATCTCGCACATGGCCT (SEQ ID NO:
2)；
The primer pair of downstream flanking sequence includes: that upstream sequence ICP4-promoterDSf is
AAAAAAGCTTTCACGCGCATGCTCTTCTC (SEQ ID NO:3)；
Downstream sequence ICP4-promoterDSr is AAAACAGCTGCACCGTGCCCGTGATGAA (SEQ ID NO:4).
In ICP34.5 gene in step 4, the primer pair of upstream flanking sequence includes: that upstream sequence ICP34.5USf is
CTCTGACCTGAGATTGGCGGCACTG (SEQ ID NO:5)；
Downstream sequence ICP34.5USr is GCGGCCGCAGCGCTGCGGCCGCCGCGGGCGCGTCCTGACCGCGGG (SEQ
ID NO:6)；
The primer pair of downstream flanking sequence includes:
Upstream sequence ICP34.5DSf is GCGGCCGCAGCGCTGCGGCCGCCAGCGCGGCGGGGCCCGGCCAACCA
(SEQ ID NO:7)；
Downstream sequence ICP34.5DSr is TTCTTCCCTCTTCTCCCGCCCTCCA (SEQ ID NO:8).
In step 1, the primer sequence of Linker 1 is CTAGTGAATTCTAGTGGATCCCCCGGGCTGCAGGAATTC
GATATCA (SEQ ID NO:9)；
The primer sequence of Linker 2 is AGCTTGATATCGAATTCCTGCAGCCCGGGGGATCCACTAGAATTCA
(SEQ ID NO:10).
Table 1. constructs primer used in plasmid pdICP34.5 and pdICP4-promoter
Note: genome sequence is marked with underscore, shuttle plasmid restriction enzyme site bold Italic used when constructing
It indicates.ICP34.5USr with carried out in ICP34.5DSf repeat PCR connect ICP34.5US and DS FLRs complementary series use
Runic marks.
In the present invention, recombinant viral vector 17-d4-CEA-promoter passes through the side of the thermophilic malicious spot of four-wheel picking in step 3
Method is purified.
The invention also discloses a kind of novel oncolytic of selectively killing colon cancer cell viruses, using following building sides
Method: the ICP4 gene promoter in I herpes simplex virus type 17+ pnca gene group is replaced as colon cancer specificity promoter (cancer
Embryonal antigen CEA promoter), then by ICP34.5 gene knockout in I herpes simplex virus type 17+ pnca gene group and it is inserted into IL-12 table
Novel oncolytic virus HSV is obtained up to sequence constructCEA+IL-12；Wherein, the colon cancer specificity promoter is CEA promoter；Such as figure
Shown in 1.
In the present invention, the construction method is a kind of novel oncolytic virus construction method of selectively killing colon cancer cell
Described in construction method.
HSVCEA+IL-12Oncolytic effect confirmatory experiment:
In the present invention, novel oncolytic virus HSVCEA+IL-12To the selective inhibiting effect of colon cancer cell, do not infect normal
Colon epithelial cell, infection rate is also very low in other tumour cells.
1, MTT experiment
MTT is the good method for reflecting cell viability measurement.The present invention had detected under different virus titre using MTT (0,
0.001,0.01,0.1,1) HSVCEA+IL-12To the shadow of the survival ability of colon cancer cell COLO, normal colon epithelial cells FHC
It rings, finds this novel oncolytic virus HSVCEA+IL-12The survival ability that colon cancer cell COLO can be significantly inhibited, to normal knot
The survival ability of enterocyte FHC does not influence (see Fig. 2).
2, plate clone forms experiment
Clonality is a kind of monopolizing characteristic of malignant cell.Plate clone forms experiment and can be well reflected
The clonality of tumour cell.The present invention forms experiment detection HSV using plate cloneCEA+IL-12To colon cancer cell
The influence of COLO, normal colon epithelial cells FHC clonality find this novel oncolytic virus HSVCEA+IL-12It can be with
The clonality for significantly inhibiting colon cancer cell COLO does not have the clonality of normal colon epithelial cells FHC
It influences (see Fig. 3).
3, the cell Transwell migration experiment
Transfer ability is a kind of characteristic of malignant tumour, reflects the ability of metastases.The migration of the cell Transwell is real
It tests and is able to detect this transfer ability.The present invention uses the cell Transwell migration experiment detection HSVCEA+IL-12It is thin to colon cancer
The influence of born of the same parents COLO, normal colon epithelial cells FHC transfer ability find this novel oncolytic virus HSVCEA+IL-12It can show
The transfer ability for inhibiting colon cancer cell COLO is write, the transfer ability of normal colon epithelial cells FHC is not influenced (see figure
4)。
4, the cell Transwell Matrigel
Invasion are the exclusive characteristics of malignant tumour, and the cell Transwell Matrigel is able to reflect this invasive ability.This
Invention detects HSV using the cell Transwell MatrigelCEA+IL-1To colon cancer cell COLO, normal colon epithelial cells FHC
The influence of invasive ability finds this novel oncolytic virus HSVCEA+IL-1The invasion energy of colon cancer cell COLO can be significantly inhibited
Power does not influence (see Fig. 5) invasive ability of normal colon epithelial cells FHC.
The present invention is detected by MTT, plate clone forms experiment, the migration experiment of the cell Transwell and Transwell are small
Room Matrigel shows new virus HSV provided by the inventionCEA+IL-1With selectively inhibition colon cancer cell COLO's
Survival, Clone formation, migration and invasion ability, illustrate HSVCEA+IL-1The property of can choose kills colon cancer cell.
In the present invention, CEA promoter sequence (SEQ ID NO:11):
The promoter (SEQ ID NO:12) of ICP4 gene:
The herpes simplex virus type upstream pnca gene group ICP34.5 17+ I (up-stream, US) and downstream (down-
Stream, DS) flanking sequence:
ICP34.5 upstream flanking sequence (SEQ ID NO:13):
ICP34.5 downstream flanking sequence (SEQ ID NO:14):
125334 cgcgggg
125341 gtcgcggggg tcgcgggggt cgcgggggtc gcgggggtcg cggggg
The herpes simplex virus type pnca gene upstream group ICP4 promoter 17+ I (up-stream, US) and downstream (down-
Stream, DS) flanking sequence:
The flanking sequence (SEQ ID NO:15) of the upstream ICP4 promoter:
The flanking sequence (SEQ ID NO:16) in the downstream ICP4 promoter:
Finally, it is stated that the above examples are only used to illustrate the technical scheme of the present invention and are not limiting, although referring to compared with
Good embodiment describes the invention in detail, those skilled in the art should understand that, it can be to skill of the invention
Art scheme is modified or replaced equivalently, and without departing from the objective and range of technical solution of the present invention, should all be covered at this
In the scope of the claims of invention.
SEQUENCE LISTING
<110>Hubei University of Science and Technology
<120>the novel oncolytic virus and its construction method of a kind of selectively killing colon cancer cell
<130> 2018
<160> 16
<170> PatentIn version 3.5
<210> 1
<211> 33
<212> DNA
<213>artificial synthesized
<220>
<221> primer
<222> (1)..(33)
<400> 1
aaaagaattc gatacacatc gttcagacgg agc 33
<210> 2
<211> 31
<212> DNA
<213>artificial synthesized
<220>
<221> primer
<222> (1)..(31)
<400> 2
aaaaactagt gatcgatctc gcacatggcc t 31
<210> 3
<211> 29
<212> DNA
<213>artificial synthesized
<220>
<221> primer
<222> (1)..(29)
<400> 3
aaaaaagctt tcacgcgcat gctcttctc 29
<210> 4
<211> 28
<212> DNA
<213>artificial synthesized
<220>
<221> primer
<222> (1)..(28)
<400> 4
aaaacagctg caccgtgccc gtgatgaa 28
<210> 5
<211> 25
<212> DNA
<213>artificial synthesized
<220>
<221> primer
<222> (1)..(25)
<400> 5
ctctgacctg agattggcgg cactg 25
<210> 6
<211> 45
<212> DNA
<213>artificial synthesized
<220>
<221> primer
<222> (1)..(45)
<400> 6
gcggccgcag cgctgcggcc gccgcgggcg cgtcctgacc gcggg 45
<210> 7
<211> 47
<212> DNA
<213>artificial synthesized
<220>
<221> primer
<222> (1)..(47)
<400> 7
gcggccgcag cgctgcggcc gccagcgcgg cggggcccgg ccaacca 47
<210> 8
<211> 25
<212> DNA
<213>artificial synthesized
<220>
<221> primer
<222> (1)..(25)
<400> 8
ttcttccctc ttctcccgcc ctcca 25
<210> 9
<211> 46
<212> DNA
<213>artificial synthesized
<220>
<221> primer
<222> (1)..(46)
<400> 9
ctagtgaatt ctagtggatc ccccgggctg caggaattcg atatca 46
<210> 10
<211> 46
<212> DNA
<213>artificial synthesized
<220>
<221> primer
<222> (1)..(46)
<400> 10
agcttgatat cgaattcctg cagcccgggg gatccactag aattca 46
<210> 11
<211> 960
<212> DNA
<213>artificial synthesized
<220>
<221> promoter
<222> (1)..(960)
<400> 11
ggggctcacc gagctgaaac ctggtagcac tttggcataa catgtgcatg acccgtgttc 60
aatgtctaga gatcagtgtt gagtaaaaca gcctggtctg gggccgctgc tgtccccact 120
tccctcctgt ccaccagagg gcggcagagt tcctcccacc ctggagcctc cccaggggct 180
gctgacctcc ctcagccggg cccacagccc agcagggtcc accctcaccc gggtcacctc 240
ggcctacgtc ctcctcgccc tccgagctcc tcacacggac tctgtcagct cctccctgca 300
gcctatcggc cgcccacctg aggcttgtcg gccgcccact tgaggcctgt cggctgccct 360
ctgcaggcag ctcctgtccc ctacaccccc tccttccccg ggctcagctg aaagggcgtc 420
tcccagggca gctccctgtg atctccagga cagctcagtc tctcacaggc tccgacgccc 480
cctatgctgt cacctcacag ccctgtcatt accattaact cctcagtccc atgaagttca 540
ctgagcgcct gtctcccggt tacaggaaaa ctctgtgaca gggaccacgt ctgtcctgct 600
ctctgtggaa tcccagggcc cagcccagtg ctgacacgga acagatgctc cataaatact 660
ggttaaatgt gtgggagatc tctaaaaaga aacatatcac ctccgtgtgg cccccagcag 720
tcagagtctg ttccatgtgg acacaggggc actggcacca gcatgggagg aggccagcaa 780
gtgcccgcgg ctgccccagg aatgaggcct caacccccag agcttcagaa gggaggacag 840
aggcctgcag ggaatagatc tccggcctga ccctgcagcc taatccagag ttcagggtca 900
gctcacacca cgtcgaccct ggtcagcatc cctagggcag ttccagacaa ggccggaggt 960
<210> 12
<211> 515
<212> DNA
<213>artificial synthesized
<220>
<221> promoter
<222> (1)..(515)
<400> 12
cccgggcccc gcccccggcc cgttcctcgt tagcatgcgg aacggaagcg gaaaccaccg 60
gatcgggcgg taatgagatg ccatgcgggg cggggcgcgg gcccacccgc cctcgcgccc 120
cgcccatggc agatggcgcg gatgggcggg gccgggggtt cgaccaacgg gccgcggcca 180
cgggcccccg gcgtgccggc gtcggggcgg ggtcgtgcat aatggaattc cgttcggggc 240
gggcccgcct ggggggcggg gggccggcgg cctccgctgc tcctccttcc cgccggcccc 300
tgggactata tgagcccgag gacgccccga tcgtccacac ggagcgcggc tgccgacacg 360
gatccacgac ccgacgcggg accgccagag acagaccgtc agacgctcgc cgcgccggga 420
cgccgatacg cggacgaagc gcgggagggg gatcggccgt ccctgtcctt tttcccaccc 480
aagcatcgac cggtccgcgc tagttccgcg tcgac 515
<210> 13
<211> 159
<212> DNA
<213>artificial synthesized
<220>
<221> promoter
<222> (1)..(159)
<400> 13
ctgtatatat aaagtcaggg ggtcacatgg cgacccccaa cagggcgacc ccggtccctg 60
tatatatagg gtcagggggt tccgcacccc ctaacatggc gcccccggtc cctgtatata 120
tagtgtcacg gggttccacg ccccctaaca tggcgcccc 159
<210> 14
<211> 53
<212> DNA
<213>artificial synthesized
<220>
<221> promoter
<222> (1)..(53)
<400> 14
cgcgggggtc gcgggggtcg cgggggtcgc gggggtcgcg ggggtcgcgg ggg 53
<210> 15
<211> 333
<212> DNA
<213>artificial synthesized
<220>
<221> promoter
<222> (1)..(333)
<400> 15
ccgcccctcg ccccctcccg cccctcgccc cctcccgccc ctcgccccct cccgcccctc 60
gccccctccc gcccctcgcc ccctcccgcc cctcgccccc tcccgcccct cgccccctcc 120
cgcccctcgc cccctcccgc ccctcgcccc ctcccgcccc tcgccccctc ccgcccctcg 180
ccccctcccg cccctcgccc cctcccgccc ctcgccccct cccgcccctc gccccctccc 240
gcccctcgcc ccctcccgcc cctcgccccc tcccgcccct cgccccctcc cgcccctcgc 300
cccctcccgc ccctcgcccc ctcccgcccc tcg 333
<210> 16
<211> 126
<212> DNA
<213>artificial synthesized
<220>
<221> promoter
<222> (1)..(126)
<400> 16
gggcggagga gggggggacg cgggggcgga ggagggggga cgcgggggcg gaggaggggg 60
gacgcggggg cggaggaggg gggacgcggg ggcggaggag gggggacgcg ggggcggagg 120
aggggg 126
Claims (8)
1. a kind of novel oncolytic virus construction method of selectively killing colon cancer cell, which comprises the following steps:
Step 1: building pdICP4-promoter plasmid: PCR expands 17+ plants of ICP4 genes of I herpes simplex virus type respectively and opens
After the flanking sequence of mover upstream and downstream, ICP4 gene promoter is obtained after being digested respectively with restriction enzyme EcoRI/SpeI
The upstream flanking sequence of son, downstream flanking sequence and remove 17+ the plant of I herpes simplex virus type of ICP4 promoter, and with complementary
Ligase Linker 1 and Linker 2 upstream flanking sequence of the ICP4 gene promoter and downstream flanking sequence are connected
It picks up and, and EcoRI the and SalI restriction enzyme site of the product cloning after connection to pBluescript is created plasmid pdICP4-
promoter；
Step 2: building pdICP4-CEA-promoter plasmid: being opened with the double digested mode handle of EcoRI/XhoI by CMV
The people CEA promoter sequence of mover control is released from plasmid pcDNA3.1-CEA-promoter, with T4DNA polymerase
Processing rear clone enters at the restriction enzyme EcoHV restriction enzyme site of plasmid pdICP4-promoter obtained by step 1, thus structure
Build plasmid pdICP4-CEA-promoter；
Step 3: 17+ plants of homologous recombination I herpes simplex virus type in bhk cell: the I of ICP4 promoter will be removed in step 1
Both 17+ plants of herpes simplex virus type transfect bhk cell with plasmid pdICP4-CEA-promoter in step 2 jointly, make
Homologous recombination occurs in bhk cell, obtains the recombinant viral vector 17-d4-CEA-promoter of expression CEA promoter；
Step 4: building pdICP34.5 plasmid: PCR expands the upper of ICP34.5 gene in 17+ plants of I herpes simplex virus type respectively
Trip and downstream flanking sequence, obtained two segments of ICP34.5 upstream and downstream flanking sequence are attached with over-lap PCR, with
The product insertion after connection is used in the postdigestive plasmid pSP72 of restriction enzyme BamHI/XhoI in advance afterwards, it is more with T4DNA
The cohesive end of poly- enzyme filling-in plasmid, obtained plasmid are named as pdICP34.5；
Step 5: building plasmid pdICP34.5-hIL-12: using hIL-12 gene substitution plasmid pcDNA3.1-CEA-promoter
In CEA-promoter, generate plasmid pcDNA3.1-hIL-12；The hIL-12 in plasmid pcDNA3.1-hIL-12
Expression cassette is cloned at the site Afel of plasmid pdICP34.5, creates plasmid pdICP34.5-hIL-12；
Step 6: the plasmid pdICP34.5-hIL-12 in step 5 is used to delete recombinant viral vector 17-d4- in step 3
ICP34.5 gene in CEA-promoter, to construct oncolytic virus HSVCEA+IL-12。
2. a kind of novel oncolytic virus construction method of selectively killing colon cancer cell according to claim 1, special
Sign is, in the ICP4 promoter in step 1,
The primer pair of upstream flanking sequence includes: that upstream sequence ICP4-promoterUSf is AAAAGAATTCGATACACATCG
TTCAGACGGAGC；
Downstream sequence ICP4-promoterUSr is AAAAACTAGTGATCGATCTCGCACATGGCCT；
The primer pair of downstream flanking sequence includes: that upstream sequence ICP4-promoterDSf is
AAAAAAGCTTTCACGCGCATGCTCTTCTC；
Downstream sequence ICP4-promoterDSr is AAAACAGCTGCACCGTGCCCGTGATGAA.
3. a kind of novel oncolytic virus construction method of selectively killing colon cancer cell according to claim 1, special
Sign is, in the ICP34.5 gene in step 4,
The primer pair of upstream flanking sequence includes: that upstream sequence ICP34.5USf is CTCTGACCTGAGATTGGCGGCACTG；
Downstream sequence ICP34.5USr is GCGGCCGCAGCGCTGCGGCCGCCGCGGGCGCGTCCTGACCGCGGG；
The primer pair of downstream flanking sequence includes: that upstream sequence ICP34.5DSf is GCGGCCGCAGCGCTGCGGCCGCCAGCG
CGGCGGGGCCCGGCCAACCA；
Downstream sequence ICP34.5DSr is TTCTTCCCTCTTCTCCCGCCCTCCA.
4. a kind of novel oncolytic virus construction method of selectively killing colon cancer cell according to claim 1, special
Sign is, in step 1, the primer sequence of Linker 1 is CTAGTGAATTCTAGTGGATCCCCCGGGCTGCAGGAATTCG
ATATCA；
The primer sequence of Linker 2 is AGCTTGATATCGAATTCCTGCAGCCCGGGGGATCCACTAGAATTCA.
5. a kind of novel oncolytic virus construction method of selectively killing colon cancer cell according to claim 1, special
Sign is that recombinant viral vector 17-d4-CEA-promoter is carried out pure by the method for the thermophilic malicious spot of four-wheel picking in step 3
Change.
6. a kind of novel oncolytic virus of selectively killing colon cancer cell, which is characterized in that use following construction methods: by I
ICP4 gene promoter in herpes simplex virus type 17+ pnca gene group is replaced as colon cancer specificity promoter, then by I type list
In pure herpesviral 17+ pnca gene group ICP34.5 gene knockout and be inserted into IL-12 expressed sequence construct to obtain novel oncolytic virus
HSVCEA+IL-12。
7. a kind of novel oncolytic virus of selectively killing colon cancer cell according to claim 6, which is characterized in that institute
Stating colon cancer specificity promoter is CEA promoter.
8. a kind of novel oncolytic virus of selectively killing colon cancer cell according to claim 6 or 7, feature exist
In the construction method is construction method of any of claims 1-5.
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