CN106591361A - Recombinant pox oncolytic virus, and construction method and application thereof - Google Patents
Recombinant pox oncolytic virus, and construction method and application thereof Download PDFInfo
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- CN106591361A CN106591361A CN201510677281.9A CN201510677281A CN106591361A CN 106591361 A CN106591361 A CN 106591361A CN 201510677281 A CN201510677281 A CN 201510677281A CN 106591361 A CN106591361 A CN 106591361A
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Abstract
The invention belongs to the fields of biotechnology and gene therapy, and concretely relates to a recombinant pox oncolytic virus, and a construction method and an application thereof. The construction method of the recombinant pox oncolytic virus comprises the following steps: 1, constructing a pCB-Beclin-1 plasmid; 2, carrying out homologous recombination on a pox oncolytic virus in 293 cells; 3, screening the recombinant pox oncolytic virus; 4, identifying the recombinant pox oncolytic virus; and 5, amplifying and preserving the recombinant pox oncolytic virus. The pox oncolytic virus is reconstructed with an autophagic gene to reach an induced autophagic cell death and virus therapy combination purpose, and substantially improves the anti-leukemia effectiveness of the pox oncolytic virus.
Description
Technical field
The invention belongs to biotechnology and field of gene, and in particular to one kind restructuring acne oncolytic virus and its construction method and should
With.
Background technology
At present, the new treatment research of malignant tumour is one of international primary study direction.Viral therapy mainly applies molecule
The some species of virus such as adenovirus of biological means transformation becomes oncolytic virus (Oncolytic Virus), thin for killing cancer
Born of the same parents, are to develop a kind of very fast tumor biotherapy new method in recent years.Wherein, external and zoopery proves oncolytic
Virus has preferable antitumor action, and its security also confirms in I-II phase clinical researches.Therefore, oncolytic virus has very
Good industrialization prospect.
Acne oncolytic virus is third generation oncolytic virus.External and animal experiment study shows that it has advantages below：1) virus sense
8 hours virus replication products just can be discharged after dye cell, so as to infect adjacent tumor cells；48-72 hours just can be destroyed
The tumour cell of infection；2) it is wide to the targeting of tumour cell, kinds of tumor cells can be infected.Its infected tumor's cell
Mode is different with oncolytic adenovirus, is the infection cell in the way of with cell membrane fusion；3) viral DNA can not be incorporated into
In human body chromosome；4) product after virus infected cell self can be wrapped up, and seldom have virus protein exposed；5) virus can lead to
Hematogenous infection distal tumor is crossed, therefore with the effect of good systemic anti-tumor；6) clinically conventional antiviral drugs can be effective
Poxvirus infection is removed, treating once generation side effect when acne oncolytic virus can use antiviral drugs control；7) viral genome energy
Larger allogenic gene insertion is accommodated, easy applied molecular biology means build the oncolytic disease for carrying exogenous Antioncogene
Poison, enhanced virus antitumor action.But, can acne oncolytic virus be infected at present and to kill leukaemia unclear.
It is to strengthen one of the GVT of oncolytic virus, the main policies for adopting in the world " gene-virus treatment ", i.e.,
By Exogenous therapies channel genes oncolytic virus, including inducing cell apoptosis gene such as p53 and TRAIL, target tumor microenvironment
Gene and immunomodulatory gene etc..This strategy not only opens new way in viral therapy research field, is also the base of cancer
Because treatment provides new carrier, tumor biotherapy research has been widely used in it.Our preliminary in vitro and zoopery are ground
Study carefully proof, the apoptosis-induced channel genes CRAd such as TRAIL and IL-24 can be significantly increased into viral leukemia resisting action.Acne is molten
Tumor virus includes that the JX-594 of Wyeth of the U.S. mainly adopts at present GM-CSF gene.Carrying trail dna can be significantly
Strengthen acne oncolytic virus and kill colon cancer.But, because the research of acne oncolytic virus is at the early-stage, there is no other therapeutic gene
Research report.On the other hand, in order to further improve the antitumor action of acne oncolytic virus and study it to resistance to apoptosis tumour
Effect, it is tactful in the urgent need to studying new " gene-virus treatment ".
Recent study shows that autophagy cell death (Autophagic cell death) plays an important role in antineoplaston,
It is the dead important way of the resistance to apoptotic tumor cell of induction.For leukaemia, induction autophagy cell death is that anti-leukocythemia grinds
The new direction studied carefully.In leukaemia tumor-bearing mice, can fully erased tumour, targeting autophagy-gene-virotherapy be have very much before
The leukemia virus treatment new method of scape.
As can be seen here, a kind of new acne oncolytic virus can be constructed, the effect of its anti-leukocythemia and hematologic malignancies is improved,
Become those skilled in the art's technical barrier urgently to be resolved hurrily.
The content of the invention
The present invention is in order to solve above-mentioned technical barrier, there is provided one kind restructuring acne oncolytic virus and its construction method and application.The present invention
Acne oncolytic virus is transformed using autophagygene, the purpose that induction autophagy cell death and viral therapy are combined is reached, can be significantly
Improve the anti-leukocythemia effect of acne oncolytic virus.Acne oncolytic virus is transformed using autophagygene, induction autophagy cell death is reached
Combine with viral therapy, significantly improve the anti-leukocythemia effect of acne oncolytic virus.
In order to reach above-mentioned technique effect, technical scheme includes：
A kind of construction method of restructuring acne oncolytic virus, comprises the following steps：
Step one：Build pCB-Beclin-1 plasmids：Beclin-1 gene orders are cloned in pCB plasmid vectors, are constituted
PCB-Beclin-1 plasmid vectors, the Beclin-1 gene disruption TK genes of insertion, by the homologous DNA in TK genes two ends
Sequence carries out homologous recombination, and the nucleotides sequence of the pCB-Beclin-1 plasmids is classified as SEQ ID NO.1；
Step 2：293 intracellular homologous recombination acne oncolytic viruses：Inoculation wild type poxvirus treats wild type in 293 cells
After poxvirus is adsorbed onto on 293 cells, using lipofection by pCB-Beclin-1 plamid vector transfections to 293 cells,
Make the complete pathology of 293 cells；
Step 3：The screening of restructuring acne oncolytic virus：After the complete pathology of 293 cells, the restructuring acne oncolytic virus has gpt
Gene, is screened by mycophenolic acid to acne oncolytic virus of recombinating；
Step 4：The identification of restructuring acne oncolytic virus：Restructuring acne oncolytic virus to filtering out enters performing PCR identification, wherein the 1st
It is wild type acne oncolytic virus specific pairs primer to primer, the 2nd pair of primer is restructuring acne oncolytic virus specific pairs primer；
Step 5：The amplification of restructuring acne oncolytic virus and preservation：Using 293 cells amplification restructuring acne oncolytic virus, cesium chloride ladder
Degree centrifugal purification restructuring acne oncolytic virus, TCID50 methods determine purified virus titre, -80 DEG C of preservations.
Further, the nucleotides sequence of the 1st pair of primer is classified as：Upstream primer：5 '-atggaagggtctaaga-3 ' such as SEQ
Shown in ID NO.2, downstream primer：5 '-tcatttgttataaaattg-3 ' are as shown in SEQ ID NO.3；The 2nd pair of primer
Nucleotides sequence be classified as：Upstream primer：5 '-tgtgaagacgataaattaatgatc-3 ' as shown in SEQ ID NO.4, draw by downstream
Thing：5 '-gtttgccatacgctcacag-3 ' are as shown in SEQ ID NO.5.
Further, the concrete steps for expanding restructuring acne oncolytic virus using 293 cells include：293 cell densities are up to 90%
When instill oncolytic vaccinia virus, negative pressure siphons away 70% culture medium, and per 100mm culture plates, drop 0.5ml oncolytic vaccinia virus, put
Enter incubator culture 60min, plus the culture medium 5-8ml of rewarming, occur pathology effect after 24 hours, by 293 cell from ware
Bottom blowing falls, and in moving into centrifuge tube, 5000rpm is centrifuged, and 10 minutes, supernatant was abandoned in suction, and precipitation is stored in -20 DEG C or -80 DEG C.
Described culture medium is containing 5% hyclone.
One kind restructuring acne oncolytic virus, the restructuring acne oncolytic virus is the insertion induction autophagygene in acne oncolytic virus
Beclin-1, with T7 as promoter, described restructuring acne oncolytic virus is using the restructuring acne oncolytic disease constructed by above-mentioned construction method
Poison.
The application of restructuring acne oncolytic virus, described restructuring acne oncolytic virus can make in the medicine for the treatment of hematological malignancies
With.
Described hematological malignancies are leukaemia.
New acne oncolytic virus amplification technique method and quality control system are set up：1. set up and improve amplification technique：293 cells
OncoPoxV-beclin-1 is viral for amplification, and caesium chloride density gradient centrifugation purification of Recombinant virus, TCID50 methods determine purified virus titre.
Mainly set up standardization experiment flow.2. quality control system：Originate including cell line, pass on history, identification and give birth to substantially
Thing flag check；Cell line carries out carcinogenic continuity cytologic experiments；Purification process：Check Virus Pollution, nucleic acid, miscellaneous egg
Bletilla other impurities.The physical and chemical index detection of viral product.The research of different preservation condition virus activities.
Beneficial effects of the present invention include：
1. the present invention effectively combines the gene therapy of malignant tumour with viral therapy, and being prepared for can high efficient expression Beclin-1
Oncolytic vaccinia virus, enhance the killing ability to tumour relative to simple gene therapy or virus therapy.
2. neoplasm targeted therapy strategy is present invention employs, tumour cell, and proliferated specifically wherein can be effectively targeted to.
So as to greatly strengthen the security of oncolytic vaccinia virus carrier.
3. the present invention is by the way of virus replication related gene disappearance, it is ensured that the tumour internal specific of virus is replicated, and is greatly enhanced
The security of oncolytic vaccinia virus carriers.
4th, the present invention has played preferable anti-hematologic malignancies by the recombination oncolytic vaccinia virus of structure Beclin-1 genes
Effect.Complete new acne oncolytic virus and treat leukemic preclinical study, realize to the targeting and whole body to distal tumor
Antitumor action, and virus amplification and quality control system are completed, it is that further industrialization lays the foundation, the present invention has good
Good industrialization prospect.The technology of the present invention steps into first place in the world in acne oncolytic virus anti-leukocythemia research field, contributes to
Improve the academic standing of this area.
Description of the drawings
Fig. 1 show present invention induction autophagygene Beclin-1 structural representations.
Fig. 2 show mtt assay detection blood tumor cell HL-60 survival rate analysis figures of the present invention.
Fig. 3 show mtt assay detection blood tumor cell K562 survival rate analysis figures of the present invention.
Fig. 4 show mtt assay detection blood tumor cell P210 survival rate analysis figures of the present invention.
Fig. 5 show impact figure of the invention restructuring acne oncolytic virus to normal hepatocyte proliferation activity.
Fig. 6 show control group to autophagy virus RFP-GFP-LC3 cell autophagy action diagrams, and it is green that Fig. 6 A show detection cell
Color luciferase expression situation map, Fig. 6 B show detection cell red fluorescence expression figure.
Fig. 7 show unloaded recombination oncolytic vaccinia virus (VV) and promotees autophagocytosis figure to autophagy virus RFP-GFP-LC3 cells,
Fig. 7 A show detection cell green fluorescence expression figure, and Fig. 7 B show detection cell red fluorescence expression figure.
Fig. 8 show 5MOI recombination oncolytic vaccinia virus (VV-Beclin-1) and autophagy virus RFP-GFP-LC3 cells is promoted certainly
Action diagram is bitten, Fig. 8 A show detection cell green fluorescence expression figure, and Fig. 8 B show the red fluorescence expression of detection cell
Situation map.
Fig. 9 show 10MOI recombination oncolytic vaccinia virus (VV-Beclin-1) and autophagy virus RFP-GFP-LC3 cells is promoted
Autophagocytosis figure, Fig. 9 A show detection cell green fluorescence expression figure, and Fig. 9 B show detection cell red fluorescence table
Up to situation map.
Figure 10 show the expression and autophagy of the Beclin-1 genes of restructuring acne oncolytic virus infection blood tumor cell U266
The expression western blot figure of GAP-associated protein GAP P62, LC3.
Figure 11 show the expression and autophagy of the Beclin-1 genes of restructuring acne oncolytic virus infection blood tumor cell K562
The expression western blot figure of GAP-associated protein GAP P62, LC3.
Specific embodiment
The specific embodiment of the invention is described in detail below in conjunction with concrete accompanying drawing.It should be noted that described in following embodiments
The combination of technical characteristic or technical characteristic is not construed as isolated, and they can be mutually combined so as to reach preferably
Technique effect.
VV herein refers to the recombination oncolytic vaccinia virus group for not carrying Beclin-1 genes, and VV-Beclin-1 refers to carrying
The recombination oncolytic vaccinia virus group of Beclin-1 genes.
Embodiment 1
A kind of construction method of restructuring acne oncolytic virus, comprises the following steps：
Step one：Build pCB-Beclin-1 plasmids：Beclin-1 sequences are cloned in pCB plasmid vectors, are constituted
PCB-Beclin-1 plasmid vectors, the Beclin-1 gene disruption TK genes of insertion, by the homologous DNA in TK genes two ends
Sequence carries out homologous recombination；
Step 2：293 intracellular homologous recombination acne oncolytic viruses：Inoculation wild type poxvirus treats wild type in 293 cells
After poxvirus is adsorbed onto on 293 cells, using lipofection by pCB-Beclin-1 plamid vector transfections to 293 cells,
Make the complete pathology of 293 cells；
Step 3：The screening of restructuring acne oncolytic virus：After the complete pathology of 293 cells, the restructuring acne oncolytic virus has gpt
Gene, is screened by mycophenolic acid to acne oncolytic virus of recombinating；
Step 4：The identification of restructuring acne oncolytic virus：Restructuring acne oncolytic virus to filtering out enters performing PCR identification, wherein the 1st
It is wild type acne oncolytic virus specific pairs primer to primer, the 2nd pair of primer is restructuring acne oncolytic virus specific pairs primer；
Step 5：The amplification of restructuring acne oncolytic virus and preservation：Using 293 cells amplification restructuring acne oncolytic virus, concrete steps
Bag expands：The concrete steps for expanding restructuring acne oncolytic virus using 293 cells include：293 cells drip when growing to the 90% of culture medium
Enter oncolytic vaccinia virus, negative pressure siphons away 70% culture medium, per 100mm culture plates drop 0.5ml oncolytic vaccinia virus, be put into and incubate
, there is pathology effect in case culture 60min, plus the culture medium 5-8ml of rewarming after 24 hours, by 293 cell from ware bottom blowing
Fall, in moving into centrifuge tube, 5000rpm is centrifuged, 10 minutes, supernatant was abandoned in suction, and precipitation is stored in -20 DEG C or -80 DEG C；Cesium chloride
Gradient centrifugation purification restructuring acne oncolytic virus, TCID50 methods determine purified virus titre, -80 DEG C of preservations.
The nucleotides sequence of the 1st pair of primer is classified as：Upstream primer：5 '-atggaagggtctaaga-3 ', downstream primer：5’
-tcatttgttataaaattg-3’；The nucleotides sequence of the 2nd pair of primer is classified as：Upstream primer：5’-
Tgtgaagacgataaattaatgatc-3 ' downstream primers：5’-gtttgccatacgctcacag-3’.A kind of restructuring acne oncolytic virus
Using described restructuring acne oncolytic virus can be used in the medicine for the treatment of hematological malignancies, and described haematological malignant swells
Knurl disease is leukaemia.
Recombination oncolytic vaccinia virus is obtained according to above-mentioned preparation method, the restructuring acne oncolytic virus is to insert in acne oncolytic virus
Induction autophagygene Beclin-1, with T7 as promoter, as shown in figure 1, induction autophagygene Beclin-1 structural representations,
Its expression cassette is pT7-Beclin-1-polyA；Wherein pT7 represents promoter T7, and Beclin-1 represents induction autophagygene, polyA
Represent poly-A tail；The viral TK Gene Partials disappearance.In Fig. 1, TKL is TK gene left-hand components, Promoter
It is the T7 promoters of genes of interest upstream, gpt is drug screening gene, and TKR is the right-hand component of TK genes.
A kind of application of above-mentioned restructuring acne oncolytic virus, described restructuring acne oncolytic virus can be in treatment hematological malignancies
Used in medicine.Preferably, described hematological malignancies are leukaemia.
Impact of the recombination oncolytic vaccinia virus of embodiment 2 to blood tumor cell HL-60, K562 and P210 proliferation activity
Blood tumor cell HL-60, K562 and P210 (being purchased from Chinese Academy of Sciences's cell bank) are in (FBS) containing 10% hyclone
RPMI 1640 (be purchased from Gibco) in culture (37 DEG C, 5%CO2, saturated humidity), take forth generation cell by 1 × 104/ml
Concentration be inoculated with 100 μ l in 96 well culture plates.Setting control group, the recombination oncolytic vaccinia virus for not carrying Beclin-1 genes
Group (VV), recombination oncolytic vaccinia virus group (VV-Beclin-1) for carrying Beclin-1 genes, per group is done 3 multiple holes.Treat
Cell density reaches 60-70%, be separately added into VV (1MOI, 2MOI, 4MOI, 8MOI) and VV-Beclin-1 (1MOI,
2MOI, 4MOI, 8MOI), after continuing to cultivate 72 hours, add the μ l of tetramethyl azo azoles salt (MTT) 20 of 5mg/ml
Solution, after continuing to cultivate 4 hours, terminates culture, and centrifugation removes nutrient solution, and 150 μ l DMSO are added per hole, vibrates 10min
Afterwards, wavelength 490nm is selected to determine each hole light absorption value (A values) on the full-automatic ELIASAs of Thermo Varioskan Flash, it is above-mentioned
Experiment weighs 3 times.Cell inhibitory rate is calculated according to A values, computing formula is：Cell inhibitory rate (%)=(negative control group A
Value-dosing group A values)/negative control group A value × 100%.
As a result as shown in Fig. 2～4, with the increase of dosage, VV and VV-Beclin-1 is to blood tumor cell HL-60, K562
It is further notable with P210 inhibitory effects on proliferation, and VV-Beclin-1 is to blood tumor cell HL-60, K562 and P210 propagation
Depression effect is significantly stronger than VV.
Impact of the recombination oncolytic vaccinia virus of embodiment 3 to normal hepatocyte proliferation activity
Normal liver cell QSG-7701 (being purchased from Chinese Academy of Sciences's cell bank) is in the RPMI 1640 containing 10% hyclone (FBS)
Culture in (be purchased from Gibco) (37 DEG C, 5%CO2, saturated humidity), takes forth generation cell by 1 × 104The concentration inoculation of/ml
100 μ l are in 96 well culture plates.Setting control group, recombination oncolytic vaccinia virus group (VV), the restructuring for carrying Beclin-1 genes
Oncolytic vaccinia virus group (VV-Beclin-1), per group is done 3 multiple holes.Treat that cell density, up to 60-70%, is separately added into VV
(1MOI, 2MOI, 4MOI, 8MOI) and VV-Beclin-1 (1MOI, 2MOI, 4MOI, 8MOI), continues to cultivate
After 72 hours, the μ l solution of tetramethyl azo azoles salt (MTT) 20 of 5mg/ml is added, after continuing to cultivate 4 hours, terminate training
Support, centrifugation removes nutrient solution, 150 μ l DMSO are added per hole, it is complete in Thermo Varioskan Flash after vibration 10min
Automatically wavelength 490nm is selected to determine each hole light absorption value (A values) on ELIASA, above-mentioned experiment weighs 3 times.Cell is calculated according to A values
Inhibiting rate, computing formula is：Cell inhibitory rate (%)=(negative control group A value-dosing group A value)/negative control group A
Value × 100%.
As a result Fig. 5 is seen, with the increase of dosage, VV and VV-Beclin-1 is to normal liver cell QSG-7701 without substantially suppression
Effect.
Rush autophagocytosis of the recombination oncolytic vaccinia virus of embodiment 4 to blood tumor cell
5 × 10 are inoculated with six orifice plates5The U266 cells of individual/mL, and the double fluorescence indicator virus RFP-GFP-LC3 of autophagy are added,
It is separately added into the recombination oncolytic vaccinia virus (VV-Beclin-1) and zero load weight of the carrying Beclin-1 genes of 5MOI and 10MOI
Group oncolytic vaccinia virus (VV), adds the PBS, 37 DEG C, 5%CO of equivalent in control group2Culture, it is common using laser after 48h
Focusing microscope detection cell green fluorescence (GFP) and red fluorescent protein (RFP) expression.
As a result as shown in Fig. 6～9, VV-Beclin-1 energy Induces Autophagies, and high concentration VV-Beclin-1 promotees autophagocytosis more
Plus significantly.
The detection of the immunoblot experiment of embodiment 5 carries the recombination oncolytic vaccinia virus infection blood tumor cell of Beclin-1 genes
The expression of the expression and autophagy GAP-associated protein GAP P62, LC3 of K562 and U266, Beclin-1 gene.
The recombination oncolytic vaccinia virus (VV-Beclin-1) of Beclin-1 genes is carried respectively with 1MOI, 2MOI, 4MOI
Dosage infection K562 and U266 cells, 37 DEG C, 5%CO2Under the conditions of cultivate 24 hours, according to standard Western Blot grasp
Make method protein lysate pretreatment cell and collect cell and its supernatant in 1.5ml centrifuge tubes.After -20 DEG C preserve or are quantitative
Directly use.By protein sample with BCA protein quantification kits after, per hole add 10 μ g total proteins carry out SDS-PAGE
Electrophoresis, electrophoresis is transferred to electrophoresis product on NC films by semidry method after terminating.5%BSA room temperatures close 3h, plus one and resist (1 ratio
1000 dilutions), 2h is incubated at room temperature, TBST is washed behind film 10min × 3 time, plus fluorescence two anti-(1 to 15000 dilution), room temperature
Incubation 1h, TBST are washed behind film 10min × 3 time, infrared scanner (odessay infrared imaging syetem) scanning purpose
The expression of albumen.
As a result as shown in Figure 10～11, in K562 and U266 cells, as VV-BECN1 dosage increases, Beclin-1 tables
Significantly raised up to level, P62 albumen is significantly reduced, and LC3II protein expressions rise.
The present invention has played the effect of preferable anti-hematologic malignancies by the recombination oncolytic vaccinia virus of structure Beclin-1 genes
Really.Complete new acne oncolytic virus and treat leukemic preclinical study, realize and the targeting and whole body to distal tumor is resisted
Function of tumor, and virus amplification and quality control system are completed, it is that further industrialization lays the foundation, the present invention has good
Industrialization prospect.The technology of the present invention steps into first place in the world in acne oncolytic virus anti-leukocythemia research field, contributes to carrying
The academic standing of high this area.
Above-mentioned detailed description is illustrating for the possible embodiments for invention, and the embodiment simultaneously is not used to limit the special of the present invention
Sharp scope, all equivalence enforcements or change without departing from the present invention, should be contained in the scope of the claims of the present invention.
In addition, those skilled in the art can be also done in other forms and details in the claims in the present invention scope of disclosure and spirit
Various modifications, addition and replace.Certainly, the changes such as various modifications, addition and the replacement that these are made according to present invention spirit,
All should be included within scope of the present invention.
Claims (7)
1. it is a kind of restructuring acne oncolytic virus construction method, it is characterised in that comprise the following steps：
Step one：Build pCB-Beclin-1 plasmids：Beclin-1 gene orders are cloned in pCB plasmid vectors, are constituted
PCB-Beclin-1 plasmid vectors, the Beclin-1 gene disruption TK genes of insertion, by the homologous DNA in TK genes two ends
Sequence carries out homologous recombination, and the nucleotides sequence of the pCB-Beclin-1 plasmids is classified as SEQ ID NO.1；
Step 2：293 intracellular homologous recombination acne oncolytic viruses：Inoculation wild type poxvirus treats wild type in 293 cells
After poxvirus is adsorbed onto on 293 cells, using lipofection by pCB-Beclin-1 plamid vector transfections to 293 cells,
Make the complete pathology of 293 cells；
Step 3：The screening of restructuring acne oncolytic virus：After the complete pathology of 293 cells, the restructuring acne oncolytic virus has gpt
Gene, is screened by mycophenolic acid to acne oncolytic virus of recombinating；
Step 4：The identification of restructuring acne oncolytic virus：Restructuring acne oncolytic virus to filtering out enters performing PCR identification, wherein the 1st
It is wild type acne oncolytic virus specific pairs primer to primer, the 2nd pair of primer is restructuring acne oncolytic virus specific pairs primer；
Step 5：The amplification of restructuring acne oncolytic virus and preservation：Using 293 cells amplification restructuring acne oncolytic virus, cesium chloride ladder
Degree centrifugal purification restructuring acne oncolytic virus, TCID50 methods determine purified virus titre, -80 DEG C of preservations.
2. it is according to claim 1 it is a kind of restructuring acne oncolytic virus construction method, it is characterised in that described 1st pair is drawn
The nucleotides sequence of thing is classified as：Upstream primer：5 '-atggaagggtctaaga-3 ', downstream primer：5’-tcatttgttataaaattg-3’；
The nucleotides sequence of the 2nd pair of primer is classified as：Upstream primer：5 '-tgtgaagacgataaattaatgatc-3 ' downstream primers：5’
-gtttgccatacgctcacag-3’。
3. it is according to claim 1 it is a kind of restructuring acne oncolytic virus construction method, it is characterised in that using 293 cells
The concrete steps of amplification restructuring acne oncolytic virus include：Oncolytic vaccinia virus, negative pressure are instilled when 293 cell densities are up to 90%
70% culture medium is siphoned away, per 100mm culture plates drop 0.5ml oncolytic vaccinia virus, incubator culture 60min, plus rewarming is put into
Culture medium 5-8ml, occur pathology effect after 24 hours, 293 cell is fallen from ware bottom blowing, move into centrifuge tube in, from
Heart 5000rpm, 10 minutes, supernatant was abandoned in suction, and precipitation is stored in -20 DEG C or -80 DEG C.
4. it is according to claim 3 it is a kind of restructuring acne oncolytic virus construction method, it is characterised in that described culture medium
It is containing 5% hyclone.
5. a kind of restructuring acne oncolytic virus, it is characterised in that the restructuring acne oncolytic virus is the insertion induction in acne oncolytic virus
Autophagygene Beclin-1, with T7 as promoter, described restructuring acne oncolytic virus is using described in any one of Claims 1 to 4
Construction method constructed by restructuring acne oncolytic virus.
6. a kind of application of the restructuring acne oncolytic virus described in claim 5, it is characterised in that described restructuring acne oncolytic virus
Can be used in the medicine for the treatment of hematological malignancies.
7. the application of a kind of restructuring acne oncolytic virus according to claim 6, it is characterised in that described haematological malignant swells
Knurl disease is leukaemia.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108165536A (en) * | 2017-12-11 | 2018-06-15 | 浙江大学 | A kind of recombination oncolytic vaccinia virus and preparation method and application |
| CN110305850A (en) * | 2019-05-15 | 2019-10-08 | 苏州奥特铭医药科技有限公司 | A method of oncolytic virus is prepared using 293 cell productions |
| CN110564700A (en) * | 2018-06-06 | 2019-12-13 | 杭州功楚生物科技有限公司 | Oncolytic vaccinia virus carrying limulus lectin gene, construction method and application |
| CN113583979A (en) * | 2021-08-03 | 2021-11-02 | 杭州荣谷生物科技有限公司 | Recombinant oncolytic vaccinia virus, preparation method and application thereof |
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