CN108004216A - The applications and recombinant herpes simplex virus of TSPO in glioma is treated and its preparation method and application - Google Patents
The applications and recombinant herpes simplex virus of TSPO in glioma is treated and its preparation method and application Download PDFInfo
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Abstract
The present invention relates to genetic engineering field, discloses a kind of recombinant herpes simplex virus, the preparation method and application of the recombinant herpes simplex virus.Specifically, recombinant herpes simplex virus includes carrier and exogenous nucleotide sequence, for carrier to lack the herpes simplex virus of the gene and at least one of optional gene for lacking coding ICP6, TK and UNG that encode ICP34.5 and ICP47, the insertion point of exogenous nucleotide sequence is at least one in the position for the gene that coding ICP34.5, ICP47, ICP6, TK and UNG are lacked on carrier；Exogenous nucleotide sequence is included with the nucleotide sequence that can reduce TSPO gene expression doses.And reduce application of the expression of TSPO genes in brain glioblastoma cell in the medicine for preparing treatment glioma.The recombinant herpes simplex virus of the present invention has good anti-glioma effect.
Description
Technical field
The present invention relates to genetic engineering field, and in particular to the expression for reducing TSPO genes in brain glioblastoma cell exists
Prepare the application for being used to treat in the medicine of glioma, and recombinant herpes simplex virus and its preparation method and application.
Background technology
Glioma is the most common tumour of central nervous system, the World Health Organization (World Health
Organization, WHO) glioma is divided into I~IV 4 ranks, I, II is Low grade glioma, and III, IV is high-level
The invasion of glioma, wherein High Grade Gliomas is strong, and patient's prognosis is worse, its median survival interval is only respectively 18 months (III)
And 12 months (IV).The treatment generally use surgery excision of glioma, aids in complex treatments such as radiation and chemotherapies；But brain glue
The operation of matter knurl is difficult to cut off completely, and recurrence rate is higher, easily tolerance is produced to chemicotherapy, so Patients with gliomas life cycle
Short, poor prognosis, great threat is constituted to the health of the mankind.Although a variety of emerging therapeutic means of glioma are continuous in recent years
Make progress, but its prognosis is still not fully up to expectations, and patient survival does not still have clear improvement.
Oncolytic virus treatment is one of novel method for treating glioma.Oncolytic virus is by deleting viral genome
On specific gene selectively replicate amplification ability in tumour cell so as to assign oncolytic virus, obtain tumor-killing effect,
Changing the oncolytic virus after structure at the same time can not replicate in normal cell, thus possess higher security.Oncolytic virus is adapted to control
Treatment glioma is primarily due to glioma and almost occurs to be conducive to oncolytic virus without shifting inside an organ and exist
Amplification in partial copy and knurl.In addition, being nearly all the cell after mitosis around glioma, oncolytic virus is more prone to
Replicated in vigorous tumour cell is divided.HSV-1 oncolytic viruses are the virus applied earliest on glioma.1991
Martuza et al. deletes the TK genes of HSV-1 viruses, and experiment proves that HSV-1 viruses being capable of selective killing brain glioblastoma cell
System, suppresses plantation in the growth of the glioma of mouse and improves mouse survival rate.Hereafter 20 years, 15 kinds of oncolytic viruses is there are about and existed
Tested on glioma, wherein 7 kinds enter clinical experimental stage, including HSV-1 oncolytic viruses 1716, G207, G47 Δ and
M032.HSV-1 oncolytic viruses become progress it is most fast, into the most oncolytic virus of clinical test.Clinical test results show
HSV-1 oncolytic viruses have good security in glioma treatment.
But HSV-1 oncolytic viruses validity cannot still meet Treatment need.Cause HSV-1 oncolytic virus curative effects relatively low
The reason for the fast-growth including glioma cause the low of oncolytic virus relative populations and replication capacity.Therefore, one is sought
Kind the glioma speed of growth can be reduced to provide the sufficient time for HSV-1 oncolytic viruses to kill brain glioblastoma cell
Method it is imperative.
The content of the invention
The purpose of the invention is to overcome drawbacks described above existing in the prior art, there is provided reduce in brain glioblastoma cell
The expression of TSPO genes is preparing the application in being used to treat the medicine of glioma, and a kind of recombinant herpes simplex disease
Poison and its preparation method and application.Recombinant herpes simplex virus provided by the invention can effectively reduce the growth of glioma
Speed, and it is small using rear side reaction, have a good application prospect.
To achieve these goals, in a first aspect, the present invention provides a kind of recombinant herpes simplex virus, the restructuring are single
Pure herpesviral includes carrier and exogenous nucleotide sequence, the carrier for missing coding ICP34.5 and ICP47 gene, with
And the herpes simplex virus of at least one of gene of optional missing coding ICP6, TK and UNG, the extraneous nucleotide sequence
The insertion point of row in the position of the gene of missing coding ICP34.5, ICP47, ICP6, TK and UNG on the carrier extremely
At few one；
Wherein, the exogenous nucleotide sequence is included with the nucleotide sequence that can reduce TSPO gene expression doses
Fragment.
Second aspect, present invention also offers a kind of method for preparing herpes simplex virus as described above, this method bag
Include：By in herpes simplex virus encode ICP34.5 and ICP47 gene knockout and alternatively knock out coding ICP6, TK and
At least one of gene of UNG, and exogenous nucleotide sequence is inserted into, wherein, the insertion point of the exogenous nucleotide sequence
To be knocked out on the carrier at least one in the position of the gene of coding ICP34.5, ICP47, ICP6, TK and UNG；
Wherein, the exogenous nucleotide sequence is included with the nucleotide sequence that can reduce TSPO gene expression doses.
The third aspect, present invention also offers the expression for reducing TSPO genes in brain glioblastoma cell to be used in preparation
Treat the application in the medicine of glioma.
Fourth aspect, present invention also offers recombinant herpes simplex virus as described above and/or by side as described above
The recombinant herpes simplex virus that method is prepared is preparing the application in being used to treat the medicine of glioma.
The present invention by with lack encode infected cell polypeptides 34.5 (infected cell polypetide 34.5,
ICP34.5 gene and the gene of coding ICP47 and gene, the encoding thymidine kinase of selectable missing coding ICP6)
The gene of (thymidine kinase, TK) and coding uracil N-glycosylase (uracil-N-glycosylase, UNG)
The herpes simplex virus of at least one of gene is carrier, and has and can reduce in the position insertion of missing said gene
The fragment of the nucleotide sequence of TSPO gene expression doses, is further preferably inserted into CD genes, and it is simple to obtain the restructuring stablized and expressed
Herpesviral.Also, the recombinant herpes simplex virus can effectively reduce the glioma speed of growth, so as to be HSV-1
Oncolytic virus provides the sufficient time to kill brain glioblastoma cell, improves the killing-efficiency of glioma, and after use
Side reaction is small, has a good application prospect.
Brief description of the drawings
Fig. 1 shows that siTSPO#1 and siTSPO#2 strike low-level to TSPO genes.
Fig. 2 shows infection with being uninfected by the expression of CD genes in the recombinant herpes simplex virus of insertion CD genes.
Fig. 3, which is shown, to be combined with the embodiment 2 and comparative example 1-2 for not combining 5-FC to brain glioblastoma cell U87MG forms
Influence.
Fig. 4 shows joint with not combining the work of the embodiment 2 and comparative example 1-2 of 5-FC to brain glioblastoma cell U87MG
The influence of property.
Fig. 5 shows joint with not combining the embodiment 1-2 and comparative example 1-2 of 5-FC to brain glioblastoma cell U87MG shapes
The influence of state.
Fig. 6 shows joint with not combining the embodiment 1-2 and comparative example 1-2 of 5-FC to brain glioblastoma cell U87MG's
The influence of activity.
Fig. 7 shows joint with not combining the embodiment 1-2 and comparative example 1-2 of 5-FC to brain glioblastoma cell U251 forms
Influence.
Fig. 8 shows joint with not combining the work of the embodiment 1-2 and comparative example 1-2 of 5-FC to brain glioblastoma cell U251
The influence of property.
Embodiment
The endpoint of disclosed scope and any value are not limited to the accurate scope or value herein, these scopes or
Value should be understood to comprising the value close to these scopes or value.For number range, between the endpoint value of each scope, respectively
It can be combined with each other between the endpoint value of a scope and single point value, and individually between point value and obtain one or more
New number range, these number ranges should be considered as specific open herein.
There is the expression that document report strikes the TSPO genes in low rat C6 spongiocytes to increase its invasive ability (The
Peripheral-Type Benzodiazepine Receptor and Tumorigenicity:Isoquinoline
Binding Protein (IBP) Antisense Knockdown in the C6Glioma Cell Line, Evgeny
Levin, et al., Biochemistry 2005,44,9924-9935), separately there is document report to strike low U118MG glioma cells
The expression of middle TSPO genes can improve the growth of cell and the generation of blood vessel and increase the transfer ability (The of oncocyte
18kDa translocator protein influences angiogenesis,as well as aggressiveness,
adhesion,migration,and proliferation of glioblastoma cells,Bode et al.,
Pharmacogenetics and Genomics 2012,Vol 22No 7,538-550).And the present inventor is studying
Middle discovery, the expression by suppressing TSPO genes in brain glioblastoma cell can reduce the metaboilic level of brain glioblastoma cell, from
And the glioma speed of growth is effectively reduced, can be with when it is combined with HSV-1 oncolytic viruses for treatment with glioma
The sufficient time is provided for HSV-1 oncolytic viruses to kill brain glioblastoma cell.
Based on discovery as above, the first aspect of the present invention provides the expression for reducing TSPO genes in brain glioblastoma cell
It is horizontal to prepare the application in being used to treat the medicine of glioma.
Second aspect, the present invention provides a kind of recombinant herpes simplex virus, the recombinant herpes simplex virus includes carrying
Body and exogenous nucleotide sequence, gene and optional missing coding of the carrier for missing coding ICP34.5 and ICP47
The herpes simplex virus of at least one of the gene of ICP6, TK and UNG, the insertion point of the exogenous nucleotide sequence is institute
State at least one in the position for the gene that coding ICP34.5, ICP47, ICP6, TK and UNG are lacked on carrier；
Wherein, the exogenous nucleotide sequence is included with the nucleotide sequence that can reduce TSPO gene expression doses
Fragment.
Applicant wishes explanation herein, and the insertion point of the exogenous nucleotide sequence is in the position of missing gene
At least one at.If for example, encoding the gene delection of ICP34.5, ICP47, ICP6 and TK, the gene for encoding UNG does not lack,
Then insertion point is at any one place at missing, rather than the gene of coding UNG.When in the carrier also inserted with other cores
During nucleotide sequence, for example, CD genes are mentioned below, (TSPO gene expression doses can be reduced to two kinds of nucleotide sequences by having
The fragment and CD genes of nucleotide sequence) can connect after be inserted at same site, different loci can also be inserted into respectively.It is preferred that
Ground, two kinds of nucleotide sequences are inserted into same site after connection.
In the present invention, the gene of the coding ICP34.5, the gene for encoding ICP47, the gene of coding ICP6, coding
The gene of TK and the gene of coding UNG are known to those skilled in the art, and also can be by logging in relevant data
Storehouse is found, for example, can be by logging in GenBank data base queryings to relevant nucleotide sequence, these are this area skills
The conventional technical means that art personnel have, details are not described herein by the present invention.
A kind of preferred embodiment according to the present invention, the carrier are the gene of missing coding ICP34.5 and ICP47
Herpes simplex virus, the insertion point of the exogenous nucleotide sequence for missing coding ICP34.5 on the carrier and/or
The position of the gene of ICP47.
According to the present invention, in order to effectively carry out independently translating and expressing to the exogenous nucleotide sequence, this
The exogenous nucleotide sequence of invention further preferably includes promoter and terminator sequence, exogenous nucleotide sequence preferably include promoter,
Optional initiation codon and terminator sequence (can be terminator codon) and optional catenation sequence and/or PolyA sequences,
In the case of so preferably, when the recombinant herpes simplex virus infects host cell and carries out the expression of autogene,
Independent and complete purpose mRNA fragments can be transcribed out.
In another preferred embodiment of the present invention, (e.g., carried when in the same site of carrier or different loci
On body missing coding ICP34.5 gene position, and/or on carrier missing coding ICP47 gene position) insertion described in
Exogenous nucleotide sequence, and when exogenous nucleotide sequence is at least 2 kinds, for example, with TSPO gene expression doses can be reduced
Nucleotide sequence fragment and CD genes mentioned below, in order to make 2 kinds of exogenous nucleotide sequences each single expression, each
Exogenous nucleotide sequence each has independent expression cassette, and each independent expression cassette has promoter respectively.According to the present invention,
To the species of the promoter, there is no particular limitation, as long as the transcription of exogenous nucleotide sequence can be controlled, preferred
In the case of, as the shRNA of antisense DNA or RNA or TSPO genes that exogenous nucleotide sequence is TSPO genes, the startup
Son is U6 promoters or H1 promoters.When exogenous nucleotide sequence is other genes, the promoter is CMV promoter, also
Further preferably include initiation codon and terminator codon.In the present invention, described " reduction " can refer to compared to not intervening
Brain glioblastoma cell in TSPO gene expressions expression, scheme using the present invention intervene after brain glioblastoma cell in
TSPO gene expression doses decline, and after can also referring to scheme intervention using the present invention, are no longer expressed in brain glioblastoma cell
TSPO genes.
Wherein, described " having the nucleotide sequence that can reduce TSPO gene expression doses " can be that can be used in reducing
Any nucleotide sequence of TSPO gene expression doses.It is for instance possible to use the method for RNA interference, specifically, can use
The antisense DNA or RNA of TSPO genes can also use the siRNA or shRNA of TSPO genes to reduce TSPO gene expression doses
To reduce TSPO gene expression doses.
Present invention preferably employs shRNA to reduce TSPO gene expression doses, the preferred embodiment of the shRNA includes SEQ
ID No：1 or SEQ ID No：Nucleotide sequence shown in 2.The nucleotide sequence of the homologous siRNA such as SEQ ID with shRNA
No：3 or SEQ ID No：Shown in 4.
In the present invention, the TSPO genes have SEQ ID No：Nucleotide sequence (Gene ID shown in 5:706).
5 FU 5 fluorouracil (5-FU) is the relatively broad antineoplastic chemotherapy medicine of clinical practice, its mechanism of action is in cell
5 FU 5 fluorouracil deoxynucleotide (5F-dUMP) is inside converted into, suppresses the effect of deoxythymidine acid enzyme, prevents BrdU
Sour (dUMP), which methylates, generates deoxythymidylic acid (dTMP), so as to influence DNA synthesis, achievees the purpose that to kill cell.5-FU exists
After conversion in the body is 5F-dUMP, it can also mix in RNA, other each phase cells are also had inhibitory action by interferencing protein synthesis.
5-FU is effective to kinds of tumors, particularly preferable to alimentary tract cancer and breast cancer curative effect；To oophoroma, uterine neck
Cancer, chorioepithelioma, carcinoma of urinary bladder etc. are also effective.Its adverse reaction is mainly gastrointestinal reaction, and severe one courage and uprightness are dropped and dead；Bone
Marrow suppression, alopecia, incoordination etc.；Because irritation can cause phlebitis or thromboendarteritis；Accidental Liver and kidney function infringement.
5-FU has normal cell and tumour cell a lethal effect, but its 5 Flucytosine of prodrug (5-FC) to cell without
Effect.
5-FU is transformed by 5-FC deaminizatings, this process can be by the cytosine deaminase of certain micro-organisms
(cytosine deaminase, CD) is catalyzed, and lacks such deaminase in human body.Cytosine deaminase be (CD genes
Expression product), originally nontoxic pro-drug 5-FC can be changed into the cytotoxic chemotherapeutics 5-FU of tool.
It was found by the inventors of the present invention that by the way that 5-FC and cytosine deaminase are applied in the treatment of glioma, take
Obtained good effect.
Therefore, a kind of preferred embodiment according to the present invention, the exogenous nucleotide sequence further includes CD genes, described
CD genes preferably have SEQ ID No：Nucleotide sequence shown in 6.
In the present invention, the exogenous nucleotide sequence can also include marker gene (for example, encoding ss-galactosidase glycosides
Enzyme, luciferase, the gene of green fluorescent protein or other fluorescins).Also, the exogenous nucleotide sequence can also wrap
Include usually with the relevant associated retroviral regulating and controlling sequence of transcription sequence, for example, site of polyadenylation, Kozak sequences, WPRE and under
Swim enhancer element.These are known to those skilled in the art, and details are not described herein by the present invention.
In the present invention, to the species of the herpes simplex virus, there is no particular limitation, can be the routine of this area
Selection, but in order to which the purpose of the stable expression cell factor is better achieved, the herpes simplex virus is preferably the simple blister of I types
Exanthema virus.Also there is no particular limitation to the source of the herpes simplex virus by the present invention, can by conventional commercially available,
Acquisition can also be voluntarily separated by laboratory.
Second aspect, present invention also offers a kind of preparation method of recombinant herpes simplex virus as above, this method bag
Include：By in herpes simplex virus encode ICP34.5 and ICP47 gene knockout and alternatively knock out coding ICP6, TK and
At least one of gene of UNG, and exogenous nucleotide sequence is inserted into, wherein, the insertion point of the exogenous nucleotide sequence
To be knocked out on the carrier at least one in the position of the gene of coding ICP34.5, ICP47, ICP6, TK and UNG；
Wherein, the exogenous nucleotide sequence is included with the nucleotide sequence that can reduce TSPO gene expression doses
Fragment.
In the present invention, the knockout of the gene of the above coding ICP34.5, ICP47, ICP, TK and UNG can use ability
The various methods of domain routine, the present invention are not particularly limited this, for example, by way of homologous recombination, pass through fixed point
The mode of knockout；Alternatively, knockout can also be pinpointed by way of CRISPY.Understanding the goal of the invention of the present invention and sheet
On the premise of inventing the viral vector used, the conventional technical means that those skilled in the art grasp according to it can be realized to such as
The knockout of upper gene.
In the present invention, the insertion of exogenous nucleotide sequence can also use the various methods of this area routine, described to insert
The mode entered can be directly to be inserted into the aim sequence in selected insertion point, for example, can be by way of CRISPY
Insertion, can also replace number of base sequence so as to be inserted into the aim sequence, the present invention is preferably by way of homologous recombination
The latter.
In the present invention, recombinant herpes simplex virus is as normal herpes simplex virus, it is also desirable in host cell
Complete its history of life, therefore the passage propagation of the recombinant virus needs to carry out in host cell.The host cell can be with
It is the various host cells that can cultivate viral vector of the present invention and/or recombinant virus, for example, African green monkey kidney cell (Vero
Cell), hamster kidney cell (bhk cell), Primary rabbit kidney cell, chick-embryo cell, amnion cell, (Hela is thin for human cervical carcinoma cell
Born of the same parents) and human embryonic lung diploid fibroblast (WI -38 cell).
The third aspect, is prepared present invention also offers above-mentioned recombinant herpes simplex virus and/or by the above method
Recombinant herpes simplex virus is preparing the application in being used to treat the medicine of glioma.
The present invention will be described in detail by way of examples below.
In the following Examples and Comparative Examples：
shTSPO#1：5‘-TGAGAAGGCTGTGGTTCCCCTTCAAGAGAGGGGAACCACAGCCTTC TCTTTTTTC-
3’(SEQ ID No：1)；shTSPO#2：5‘-TCACTCAACTACTGCGTATGTTCAAGAGACATACGCAGTAGTTGAGT
GTTTTTTC-3’(SEQ ID No：2)；siTSPO#1：5’-GAGAAGGCUGUGGUUCCCC-3’(SEQ ID No：3)；
siTSPO#2：5’-CACUCAACUACUGCGUAUG-3’(SEQ ID No：4) had with CD genes by Suzhou gold only intelligence biotechnology
Limit company synthesizes；
Vero cells are purchased from ATCC, article No. CCL-81；
Glioma cell line U87MG is bought from ATCC, article No. HTB-14, wherein high efficient expression TSPO genes；
U251 cells are bought from ATCC, article No. HTX-1725, wherein high efficient expression TSPO genes.
Test case 1
This test case is used to illustrate that siRNA interference effects are identified
SiRNA (siTSPO#1 and siTSPO#2) is transfected into U87MG cells respectively and (is used
3000 transfection reagents, purchased from Thermo companies, method is with reference to specification), and in 37 DEG C, 5%CO2After cultivating 72h, collect thin
Born of the same parents, Western blot detect the expression of TSPO genes, so as to obtain the efficiency of siRNA interference TSPO gene expressions.Wherein, with
The normal U87MG cells of untransfected siRNA are positive control, with actin (Actin) for reference protein.
The results are shown in Figure 1, wherein, siTSPO#1 and siTSPO#2 can strike the expression of low TSPO genes, siTSPO#2
Effect is good compared with siTSPO#1.
Embodiment 1
The present embodiment is used for the structure for illustrating recombinant herpes simplex virus provided by the invention.
According to application number 2004100064921, the method described in the patent application of Authorization Notice No. CN1283803C will
Wild type HSV-1 virus (No. GenBank of its gene order：ICP34.5 genes and ICP47 genes NC_001806, similarly hereinafter)
Knock out, and exogenous nucleotide sequence is inserted into the position of the knockout ICP34.5 genes in HSV-1 viruses, except that this implementation
Example uses Vero cells as host cell.The exogenous nucleotide sequence includes successively from 5 ' ends to 3 ' ends：U6 promoters,
ShTSPO#2, terminator sequence (TTTTTT).List is properly inserted into Suzhou Jin Weizhi companies sequencing identification exogenous nucleotide sequence
In pure herpesvirus vector, to obtain recombinant viral vector.Build successful recombinant viral vector on Vero host cells in
37 DEG C, 5%CO2Under the conditions of breed, infection multiplicity 0.1, removes cell fragment, Ran Hou after harvest with 0.65 μm of filter
High speed centrifugation purifies under the conditions of 13000rpm, obtains titre as 1 × 108The viral suspension of pfu/mL, for cell experiment.
Embodiment 2
The present embodiment is used for the structure for illustrating recombinant herpes simplex virus provided by the invention.
According to application number 2004100064921, the method described in the patent application of Authorization Notice No. CN1283803C will
The ICP34.5 genes and ICP47 gene knockouts of wild type HSV-1 viruses, also, in the knockout ICP34.5 genes of HSV-1 viruses
Position insertion artificial chemistry synthesis exogenous nucleotide sequence 1, HSV-1 viruses knockout ICP47 genes position be inserted into
The exogenous nucleotide sequence 2 of artificial chemistry synthesis, except that the present embodiment uses Vero cells as host cell.Wherein,
Exogenous nucleotide sequence 1 includes successively from 5 ' ends to 3 ' ends：U6 promoters, shTSPO#2, terminator sequence (TTTTTT).External source core
Nucleotide sequence 2 includes successively from 5 ' ends to 3 ' ends：CMV promoter, gene, the BGH PolyA for encoding CD.In Suzhou, gold only intelligence is public
Department's sequencing identification exogenous nucleotide sequence is properly inserted into herpes simplex virus vector, to obtain recombinant viral vector.Structure
Successful recombinant viral vector is on Vero host cells in 37 DEG C, 5%CO2Under the conditions of breed, infection multiplicity 0.1, harvest
Remove cell fragment with 0.65 μm of filter afterwards, then high speed centrifugation purifies under the conditions of 13000rpm, obtain titre for 1 ×
108The viral suspension of pfu/mL, for cell experiment.
Embodiment 3
The present embodiment is used for the structure for illustrating recombinant herpes simplex virus provided by the invention.
The structure of recombinant herpes simplex virus is carried out according to the method in embodiment 1, unlike, by wild type HSV-1
ICP34.5 genes, ICP47 genes and the ICP6 gene knockouts of virus, to obtain recombinant viral vector.
Embodiment 4
The present embodiment is used for the structure for illustrating recombinant herpes simplex virus provided by the invention.
The structure of recombinant herpes simplex virus is carried out according to the method in embodiment 1, unlike, exogenous nucleotide sequence
Insertion point be HSV-1 viruses knockout ICP47 genes position.
Embodiment 5
The present embodiment is used for the structure for illustrating recombinant herpes simplex virus provided by the invention.
The structure of recombinant herpes simplex virus is carried out according to the method in embodiment 2, unlike, exogenous nucleotide sequence
1 and exogenous nucleotide sequence 2 insertion point be HSV-1 viruses knockout ICP34.5 position.
Comparative example 1
This comparative example is used for the structure for illustrating the recombinant herpes simplex virus of reference.
The structure of recombinant herpes simplex virus is carried out according to the method in embodiment 1, unlike, the exogenous nucleotide of insertion
Acid sequence is only the CD genes in embodiment 2.
Comparative example 2
This comparative example is used for the structure for illustrating the recombinant herpes simplex virus of reference.
The structure of recombinant herpes simplex virus is carried out according to the method in embodiment 1, unlike, only by wild type HSV-
The ICP34.5 genes and ICP47 gene knockouts of 1 virus, are not inserted into exogenous nucleotide sequence.
Test case 1
This test case is used for the expression for illustrating CD genes in embodiment 2 and comparative example 1-2.
By U87MG cell inoculations into 24 orifice plates, 37 DEG C, 5%CO2Lower Multiplying culture, corresponding oncolytic is added after adherent
The recombinant herpes simplex virus (infection multiplicity MOI=0.01) of viral embodiment 2 and comparative example 1 structure, it is thin to infect U87MG respectively
Born of the same parents, 37 DEG C, 5%CO2Cell is collected after culture 24h, Western blot detect the expression of CD genes in cell.Wherein, with not
The U87MG cells of infection are used as reference protein as negative control, actin (Actin).
The results are shown in Figure 2, and CD bases are expressed in the U87MG cells of the recombinant herpes simplex virus of infection insertion CD genes
Because, and it is uninfected by can't detect the expression of CD genes in the viral cell of insertion CD genes.
Test case 2
This test case is used to illustrate that recombinant herpes simplex virus is to brain glioblastoma cell in embodiment 2 and comparative example 1-2
Killing situation.
By U87MG cell inoculations into 24 orifice plates, 37 DEG C, 5%CO2Under the conditions of Multiplying culture, be separately added into reality after adherent
The recombinant herpes simplex virus and 5-FC (50 μ g/ml) of example 2 and comparative example 1-2 are applied, while the group for being added without 5-FC is set respectively
It Zuo Wei not compare, be then placed in incubator and continue to cultivate.48 it is small when after, micro- Microscopic observation cellular morphology (100 ×), find
Do not add in the group of 5-FC, add comparative example 1-2 recombinant virus group oncolytic effect indifferences, add 2 recombinant virus group of embodiment
The lethal effect to glioma it is obvious, the result is shown in Fig. 3 and Fig. 5.And when with 5-FC use in conjunction, add comparative example 2 and recombinate
Virus group oncolytic effect does not improve, and adds comparative example 1 and 2 recombinant virus group of embodiment to brain glioblastoma cell U87MG's
Lethal effect is obviously improved, and the result is shown in Fig. 3 and Fig. 5.
U251 cells are tested after the same method, the results are shown in Figure 7 (does not combine the comparative example 1 of 5-FC
As a result it is not shown).
Electronic cell counter counts cell concentration, so as to measure cytoactive.U87MG cell concentrations are adjusted to 3000
It is a/100 microlitres.Above-mentioned 100 microlitres of cell suspensions are added in 96 orifice plates per hole, are set to 5 groups, 4 multiple holes of every group of setting, 37
DEG C, 5%CO2Cultivate it is adherent after, exhaust orifice plate in cell culture fluid, then into every hole add 100 microlitres of basal medium (DMEM
Add 10%FBS) and 10 microlitres of CCK8 solution (attention avoids generation bubble, and influences the reading of OD values).Orifice plate is placed again into
When incubation 1 is small in incubator.It is separately added into comparative example 1-2 and embodiment 2 and comparative example 1 and embodiment 2 combines 5-FC, uses enzyme
Cytoactive when absorbance detection incubation 0 is small at instrument measure 450nm is marked, is then placed in 37 degree of incubators and is incubated 48h, detect
Cytoactive.
As a result as shown in Figure 4 and Figure 6, comparative example 1-2 and the recombinant virus of embodiment 2 suppress cytoactive, when comparative example 1
After the recombinant virus joint 5-FC of embodiment 2, the inhibition of cytoactive becomes apparent from.
U251 cells are tested after the same method, the results are shown in Figure 8 (does not combine the comparative example 1 of 5-FC
As a result it is not shown).
Test case 3
Illustrate killing situation of the recombinant herpes simplex virus to brain glioblastoma cell in embodiment 1,3-5.
According to the method detection embodiment 1 of test case 2, in 3-5 recombinant herpes simplex virus respectively to U87MG cells and
The killing situation of U251 cells, unlike, it is added without 5-FC.
Wherein, U87MG cells, the result of embodiment 1 as shown in Figure 5 and Figure 6, the effect of the effect of embodiment 5 than embodiment 1
Fruit is good, and the effect of embodiment 4 is suitable with embodiment 1, and 3 effect of embodiment is slightly worse.
Wherein, U251 cells, the result of embodiment 1 as shown in Figure 7 and Figure 8, the effect of the effect of embodiment 5 than embodiment 1
Fruit is good, and the effect of embodiment 4 is suitable with embodiment 1, and 3 effect of embodiment is slightly worse.
By the way that above example 1-5 is compared with the result of comparative example 1-2, the simple blister of restructuring provided by the invention
Exanthema virus shows good anti-glioma effect, has a good application prospect.
The preferred embodiment of the present invention described in detail above, still, the present invention is not limited thereto.In the skill of the present invention
In art concept, technical scheme can be carried out a variety of simple variants, including each technical characteristic with it is any its
Its suitable method is combined, these simple variants and combination should equally be considered as content disclosed in this invention, belong to
Protection scope of the present invention.
SEQUENCE LISTING
<110>Beijing Inst. of Neurosurgery
Beijing Shen Yuan morals bio tech ltd
<120>The applications and recombinant herpes simplex virus of TSPO in glioma is treated and preparation method thereof
And application
<130> I48120SOD
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 55
<212> DNA
<213> Artificial
<400> 1
tgagaaggct gtggttcccc ttcaagagag gggaaccaca gccttctctt ttttc 55
<210> 2
<211> 55
<212> DNA
<213> Artificial
<400> 2
tcactcaact actgcgtatg ttcaagagac atacgcagta gttgagtgtt ttttc 55
<210> 3
<211> 19
<212> RNA
<213> Artificial
<400> 3
gagaaggcug ugguucccc 19
<210> 4
<211> 19
<212> RNA
<213> Artificial
<400> 4
cacucaacua cugcguaug 19
<210> 5
<211> 510
<212> DNA
<213> Artificial
<400> 5
atggccccgc cctgggtgcc cgccatgggc ttcacgctgg cgcccagcct ggggtgcttc 60
gtgggctccc gctttgtcca cggcgagggt ctccgctggt acgccggcct gcagaagccc 120
tcgtggcacc cgccccactg ggtgctgggc cctgtctggg gcacgctcta ctcagccatg 180
gggtacggct cctacctggt ctggaaagag ctgggaggct tcacagagaa ggctgtggtt 240
cccctgggcc tctacactgg gcagctggcc ctgaactggg catggccccc catcttcttt 300
ggtgcccgac aaatgggctg ggccttggtg gatctcctgc tggtcagtgg ggcggcggca 360
gccactaccg tggcctggta ccaggtgagc ccgctggccg cccgcctgct ctacccctac 420
ctggcctggc tggccttcac gaccacactc aactactgcg tatggcggga caaccatggc 480
tggcgtgggg gacggcggct gccagagtga 510
<210> 6
<211> 1284
<212> DNA
<213> Artificial
<400> 6
atgtcgaata acgctttaca aacaattatt aacgcccggt taccaggcga agaggggctg 60
tggcagattc atctgcagga cggaaaaatc agcgccattg atgcgcaatc cggcgtgatg 120
cccataactg aaaacagcct ggatgccgaa caaggtttag ttataccgcc gtttgtggag 180
ccacatattc acctggacac cacgcaaacc gccggacaac cgaactggaa tcagtccggc 240
acgctgtttg aaggcattga acgctgggcc gagcgcaaag cgttattaac ccatgacgat 300
gtgaaacaac gcgcatggca aacgctgaaa tggcagattg ccaacggcat tcagcatgtg 360
cgtacccatg tcgatgtttc ggatgcaacg ctaactgcgc tgaaagcaat gctggaagtg 420
aagcaggaag tcgcgccgtg gattgatctg caaatcgtcg ccttccctca ggaagggatt 480
ttgtcgtatc ccaacggtga agcgttgctg gaagaggcgt tacgcttagg ggcagatgta 540
gtgggggcga ttccgcattt tgaatttacc cgtgaatacg gcgtggagtc gctgcataaa 600
accttcgccc tggcgcaaaa atacgaccgt ctcatcgacg ttcactgtga tgagatcgat 660
gacgagcagt cgcgctttgt cgaaaccgtt gctgccctgg cgcaccatga aggcatgggc 720
gcgcgagtca ccgccagcca caccacggca atgcactcct ataacggggc gtatacctca 780
cgcctgttcc gcttgctgaa aatgtccggt attaactttg tcgccaaccc gctggtcaat 840
attcatctgc aaggacgttt cgatacgtat ccaaaacgtc gcggcatcac gcgcgttaaa 900
gagatgctgg agtccggcat taacgtctgc tttggtcacg atgatgtctt cgatccgtgg 960
tatccgctgg gaacggcgaa tatgctgcaa gtgctgcata tggggctgca tgtttgccag 1020
ttgatgggct acgggcagat taacgatggc ctgaatttaa tcacccacca cagcgcaagg 1080
acgttgaatt tgcaggatta cggcattgcc gccggaaaca gcgccaacct gattatcctg 1140
ccggctgaaa atgggtttga tgcgctgcgc cgtcaggttc cggtacgtta ttcggtacgt 1200
ggcggcaagg tgattgccag cacacaaccg gcacaaacca ccgtatatct ggagcagcca 1260
gaagccatcg attacaaacg ttga 1284
Claims (10)
1. a kind of recombinant herpes simplex virus, it is characterised in that the recombinant herpes simplex virus includes carrier and exogenous nucleotide
Acid sequence, gene and optional missing coding ICP6, TK and UNG of the carrier for missing coding ICP34.5 and ICP47
At least one of gene herpes simplex virus, the insertion point of the exogenous nucleotide sequence is lacks on the carrier
Encode at least one in the position of the gene of ICP34.5, ICP47, ICP6, TK and UNG；
Wherein, the exogenous nucleotide sequence includes the piece with the nucleotide sequence that can reduce TSPO gene expression doses
Section.
2. recombinant herpes simplex virus according to claim 1, wherein, the carrier for missing coding ICP34.5 and
The herpes simplex virus of the gene of ICP47, the insertion point of the exogenous nucleotide sequence encode to be lacked on the carrier
The position of the gene of ICP34.5 and/or ICP47.
3. recombinant herpes simplex virus according to claim 1 or 2, wherein, the exogenous nucleotide sequence, which further includes, to be opened
Mover and terminator sequence.
4. recombinant herpes simplex virus according to claim 3, wherein, the promoter is selected from U6 promoters, H1 starts
At least one of son and CMV promoter, are preferably U6 promoters and/or H1 promoters.
5. according to the recombinant herpes simplex virus described in any one in claim 1-4, wherein, it is described to have and reduce
The fragment of the nucleotide sequence of TSPO gene expression doses includes：
The antisense DNA or RNA of-TSPO genes；And
The shRNA of-TSPO genes；
Preferably, the shRNA of TSPO genes has SEQ ID No：1 or SEQ ID No：Nucleotide sequence shown in 2.
6. according to the recombinant herpes simplex virus described in any one in claim 1-4, wherein, the TSPO genes have
SEQ ID No：Nucleotide sequence shown in 5.
7. recombinant herpes simplex virus according to claim 1, wherein, the exogenous nucleotide sequence further includes CD bases
Cause, the CD genes preferably have SEQ ID No：Nucleotide sequence shown in 6.
A kind of 8. method for preparing the herpes simplex virus in claim 1-7 described in any one, it is characterised in that this method
Including：By in herpes simplex virus encode ICP34.5 and ICP47 gene knockout and alternatively knock out coding ICP6, TK and
At least one of gene of UNG, and exogenous nucleotide sequence is inserted into, wherein, the insertion point of the exogenous nucleotide sequence
To be knocked out on the carrier at least one in the position of the gene of coding ICP34.5, ICP47, ICP6, TK and UNG；
Wherein, the exogenous nucleotide sequence includes the piece with the nucleotide sequence that can reduce TSPO gene expression doses
Section.
9. the expression for reducing TSPO genes in brain glioblastoma cell is being prepared for treating answering in the medicine of glioma
With.
10. recombinant herpes simplex virus in claim 1-7 described in any one and/or as the method described in claim 8
The recombinant herpes simplex virus being prepared is preparing the application in being used to treat the medicine of glioma.
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| CN108841796A (en) * | 2017-06-15 | 2018-11-20 | 杭州睿可特生物科技有限公司 | Recombinant herpes simplex virus and its preparation method and application |
| CN111850126A (en) * | 2020-08-06 | 2020-10-30 | 北京市神经外科研究所 | Application of oncolytic virus in treatment of uveal melanoma, marker of treatment effect and detection reagent thereof |
| CN113943752A (en) * | 2020-07-15 | 2022-01-18 | 东莞市东阳光生物药研发有限公司 | Constructs, oncolytic viruses with improved sensitivity and uses thereof |
| CN116334010A (en) * | 2023-05-30 | 2023-06-27 | 中义(北京)健康研究院 | Recombinant herpes simplex virus and preparation method and application thereof |
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| CN107354136A (en) * | 2017-06-15 | 2017-11-17 | 杭州睿可特生物科技有限公司 | Recombinant herpes simplex virus and its preparation method and application |
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| CN107354136A (en) * | 2017-06-15 | 2017-11-17 | 杭州睿可特生物科技有限公司 | Recombinant herpes simplex virus and its preparation method and application |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108841796A (en) * | 2017-06-15 | 2018-11-20 | 杭州睿可特生物科技有限公司 | Recombinant herpes simplex virus and its preparation method and application |
| WO2018228538A1 (en) * | 2017-06-15 | 2018-12-20 | 杭州睿可特生物科技有限公司 | Recombinant herpes simplex virus, preparation method therefor, and application thereof |
| CN113943752A (en) * | 2020-07-15 | 2022-01-18 | 东莞市东阳光生物药研发有限公司 | Constructs, oncolytic viruses with improved sensitivity and uses thereof |
| CN111850126A (en) * | 2020-08-06 | 2020-10-30 | 北京市神经外科研究所 | Application of oncolytic virus in treatment of uveal melanoma, marker of treatment effect and detection reagent thereof |
| CN116334010A (en) * | 2023-05-30 | 2023-06-27 | 中义(北京)健康研究院 | Recombinant herpes simplex virus and preparation method and application thereof |
| CN116334010B (en) * | 2023-05-30 | 2023-08-29 | 中义(北京)健康研究院 | Recombinant herpes simplex virus and preparation method and application thereof |
| WO2024244306A1 (en) * | 2023-05-30 | 2024-12-05 | 中义（苏州）生物制药有限公司 | Recombinant herpes simplex virus, and preparation method therefor and use thereof |
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