CN108004216B - Application and recombinant herpes simplex virus and its preparation method and application of the TSPO in treatment glioma - Google Patents
Application and recombinant herpes simplex virus and its preparation method and application of the TSPO in treatment glioma Download PDFInfo
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Abstract
The present invention relates to genetic engineering field, the preparation method and application of a kind of recombinant herpes simplex virus, the recombinant herpes simplex virus are disclosed.Specifically, recombinant herpes simplex virus includes carrier and exogenous nucleotide sequence, the gene and optionally the herpes simplex virus of at least one of gene of missing coding ICP6, TK and UNG, the insertion point of exogenous nucleotide sequence are at least one in the position for the gene that missing encodes ICP34.5, ICP47, ICP6, TK and UNG on carrier that carrier is missing coding ICP34.5 and ICP47；Exogenous nucleotide sequence includes with the nucleotide sequence that can reduce TSPO gene expression dose.And reduce application of the expression of TSPO gene in brain glioblastoma cell in the drug of preparation treatment glioma.Recombinant herpes simplex virus of the invention has good anti-glioma effect.
Description
Technical field
The present invention relates to genetic engineering fields, and in particular to the expression for reducing TSPO gene in brain glioblastoma cell exists
Prepare application and the recombinant herpes simplex virus and its preparation method and application in the drug for treating glioma.
Background technique
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
Glioma, wherein the invasion of High Grade Gliomas is strong, and patient's prognosis is worse, and median survival interval is only respectively 18 months (III)
And 12 months (IV).The treatment of glioma generallys use operation excision, assists with complex treatments such as radiation and chemotherapies；But brain glue
The operation of matter tumor is difficult to cut off completely, and recurrence rate is higher, easily tolerance is generated to chemicotherapy, so Patients with gliomas life cycle
Short, poor prognosis constitutes great threat to the health of the mankind.Although a variety of emerging therapeutic means of glioma are continuous in recent years
It makes progress, but its prognosis is still not fully up to expectations, patient survival does not still have clear improvement.
Oncolytic virus treatment is one of the novel method for treating glioma.Oncolytic virus is by deleting viral genome
On specific gene selectively replicate amplification ability in tumour cell to assign oncolytic virus, obtain tumor-killing effect,
Oncolytic virus after changing structure simultaneously can not replicate in normal cell, thus have higher safety.Oncolytic virus is suitble 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 tumor.In addition, being nearly all the cell after mitosis around glioma, oncolytic virus is more prone to
It is replicated in dividing vigorous tumour cell.HSV-1 oncolytic virus is the virus applied on glioma earliest.1991
Martuza et al. deletes the TK gene of HSV-1 virus, experiments have shown that HSV-1 virus being capable of selective killing brain glioblastoma cell
System inhibits plantation in the growth of the glioma of mouse and improves mouse survival rate.Hereafter 20 years, there are about 15 kinds of oncolytic virus to exist
Tested on glioma, wherein 7 kinds enter clinical experimental stage, including HSV-1 oncolytic virus 1716, G207, G47 Δ and
M032.HSV-1 oncolytic virus become progress it is most fast, into the most oncolytic virus of clinical test.Clinical test results show
HSV-1 oncolytic virus has good safety in glioma treatment.
But HSV-1 oncolytic virus validity is not able to satisfy Treatment need still.Cause HSV-1 oncolytic virus curative effect lower
The reason of include that the fast-growth of glioma leads to the low of oncolytic virus relative populations and replication capacity.Therefore, seek one
Kind can reduce the glioma speed of growth to provide the sufficient time for HSV-1 oncolytic virus to kill brain glioblastoma cell
Method it is imperative.
Summary of the invention
The purpose of the invention is to overcome drawbacks described above of the existing technology, providing is reduced in brain glioblastoma cell
The expression of TSPO gene is preparing application and a kind of recombinant herpes simplex disease in the drug for treating glioma
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, it has a good application prospect.
To achieve the goals above, in a first aspect, the present invention provides a kind of recombinant herpes simplex virus, the recombination is single
Pure herpesviral includes carrier and exogenous nucleotide sequence, gene that the carrier is missing coding ICP34.5 and ICP47, 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 column is to be lacked in the position for encoding the gene of ICP34.5, ICP47, ICP6, TK and UNG on the carrier extremely
At few one；
Wherein, the exogenous nucleotide sequence includes with the nucleotide sequence that can reduce TSPO gene expression dose
Segment.
Second aspect, the present invention also provides a kind of method for preparing herpes simplex virus as described above, this method packets
Include: by herpes simplex virus encode ICP34.5 and ICP47 gene knockout and optionally knock out coding ICP6, TK and
At least one of gene of UNG, and it is inserted into exogenous nucleotide sequence, wherein the insertion point of the exogenous nucleotide sequence
At at least one in position to knock out the gene of coding ICP34.5, ICP47, ICP6, TK and UNG on the carrier；
Wherein, the exogenous nucleotide sequence includes with the nucleotide sequence that can reduce TSPO gene expression dose.
The third aspect, the present invention also provides the expressions for reducing TSPO gene in brain glioblastoma cell to be used in preparation
Treat the application in the drug of glioma.
Fourth aspect, the present invention also provides 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 the drug for treating glioma.
The present invention by with lack coding infected cell polypeptides 34.5 (infected cell polypetide 34.5,
ICP34.5 gene, the encoding thymidine kinase of the gene of gene and coding ICP47) and 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 in the position insertion of missing said gene and can reduce
The segment of the nucleotide sequence of TSPO gene expression dose, further preferably insertion CD gene, it is simple to obtain the recombination stablized and expressed
Herpesviral.Also, the recombinant herpes simplex virus can be effectively reduced the glioma speed of growth, so as to be HSV-1
The oncolytic virus offer sufficient time improves the killing-efficiency of glioma to kill brain glioblastoma cell, and after use
Side reaction is small, has a good application prospect.
Detailed description of the invention
Fig. 1 shows that siTSPO#1 and siTSPO#2 strike low-level to TSPO gene.
Fig. 2 shows the expression of infection with CD gene in the recombinant herpes simplex virus for being uninfected by insertion CD gene.
Fig. 3 shows joint with the embodiment 2 and comparative example 1-2 for not combining 5-FC to brain glioblastoma cell U87MG form
Influence.
Fig. 4, which is shown, to be combined and does not combine 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, which is shown, to be combined and does not combine the embodiment 1-2 and comparative example 1-2 of 5-FC to brain glioblastoma cell U87MG shape
The influence of state.
Fig. 6, which is shown, to be combined and does not combine the embodiment 1-2 and comparative example 1-2 of 5-FC to brain glioblastoma cell U87MG's
Active influence.
Fig. 7, which is shown, to be combined and does not combine the embodiment 1-2 and comparative example 1-2 of 5-FC to brain glioblastoma cell U251 form
Influence.
Fig. 8, which is shown, to be combined and does not combine the work of the embodiment 1-2 and comparative example 1-2 of 5-FC to brain glioblastoma cell U251
The influence of property.
Specific embodiment
The endpoint of disclosed range and any value are not limited to the accurate range or value herein, these ranges or
Value should be understood as comprising the value close to these ranges or value.For numberical range, between the endpoint value of each range, respectively
It can be combined with each other between the endpoint value of a range and individual point value, and individually between point value and obtain one or more
New numberical range, these numberical ranges should be considered as specific open herein.
The expression for having document report to strike the TSPO gene in low rat C6 spongiocyte will 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 cell
The expression of middle TSPO gene 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, by inhibiting the expression of TSPO gene in brain glioblastoma cell that can reduce the metaboilic level of brain glioblastoma cell, from
And the glioma speed of growth is effectively reduced, it, can be with when it to be used for the treatment with glioma in conjunction with HSV-1 oncolytic virus
The sufficient time is provided for HSV-1 oncolytic virus to kill brain glioblastoma cell.
Based on discovery as above, the first aspect of the present invention provides the expression for reducing TSPO gene in brain glioblastoma cell
Level is preparing the application in the drug for treating 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, the gene and optional missing coding that the carrier is 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 are institute
It states at least one in the position for the gene that missing encodes ICP34.5, ICP47, ICP6, TK and UNG on carrier；
Wherein, the exogenous nucleotide sequence includes with the nucleotide sequence that can reduce TSPO gene expression dose
Segment.
Applicant wishes that explanation, the insertion point of the exogenous nucleotide sequence are in the position of missing gene herein
At least one at.For example, if the gene delection of coding ICP34.5, ICP47, ICP6 and TK, the gene for encoding UNG do not lack,
Then insertion point is any one place at missing, rather than is encoded at the gene of UNG.When in the carrier also inserted with other cores
When nucleotide sequence, for example, CD gene is mentioned below, (have can reduce TSPO gene expression dose to two kinds of nucleotide sequences
The segment and CD gene 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 connecting.
In the present invention, gene, the coding of the gene of the coding ICP34.5, the gene for encoding ICP47, coding ICP6
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
Library is found, for example, can be by logging in GenBank data base querying to relevant nucleotide sequence, these are this field 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 be on the carrier missing coding ICP34.5 and/or
The position of the gene of ICP47.
According to the present invention, in order to which effectively the exogenous nucleotide sequence is independently translated and expressed, this
The exogenous nucleotide sequence of invention further preferably includes promoter and termination sequence, exogenous nucleotide sequence preferably include promoter,
Optional initiation codon and termination sequence (can be terminator codon) and optional catenation sequence and/or PolyA sequence,
In situation preferred in this way, when the recombinant herpes simplex virus infects host cell and carries out the expression of autogene,
Independent and complete purpose mRNA segment can be transcribed out.
In another preferred embodiment of the invention, (e.g., carried when in the same site of carrier or different loci
On body missing coding ICP34.5 gene position and/or carrier on missing coding ICP47 gene position) insertion described in
Exogenous nucleotide sequence, and when exogenous nucleotide sequence is at least 2 kinds, it can reduce TSPO gene expression dose for example, having
Nucleotide sequence segment and CD gene mentioned below, in order to make 2 kinds of exogenous nucleotide sequences respectively single expression, each
Exogenous nucleotide sequence respectively has independent expression cassette, and each independent expression cassette is respectively provided with promoter.According to the present invention,
To the type of the promoter, there is no particular limitation, as long as can control the transcription of exogenous nucleotide sequence, preferred
In the case where, as the shRNA of antisense DNA or RNA or TSPO gene that exogenous nucleotide sequence is TSPO gene, the starting
Son is U6 promoter or H1 promoter.When exogenous nucleotide sequence is other genes, the promoter is CMV promoter, also
It 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 expression expression, in the brain glioblastoma cell after being intervened using the solution of the present invention
The decline of TSPO gene expression dose, can also refer to after being intervened using the solution of the present invention, no longer express in brain glioblastoma cell
TSPO gene.
Wherein, described " having the nucleotide sequence that can reduce TSPO gene expression dose " can be that can be used in reducing
Any nucleotide sequence of TSPO gene expression dose.For example, can be using the method for RNA interference, specifically, can use
The antisense DNA or RNA of TSPO gene can also use the siRNA or shRNA of TSPO gene to reduce TSPO gene expression dose
To reduce TSPO gene expression dose.
Present invention preferably employs shRNA to reduce TSPO gene expression dose, and the preferred embodiment of the shRNA includes SEQ
Nucleotide sequence shown in ID No:1 or SEQ ID No:2.The nucleotide sequence of the homologous siRNA such as SEQ ID with shRNA
Shown in No:3 or SEQ ID No:4.
In the present invention, the TSPO gene has nucleotide sequence (Gene ID:706) shown in SEQ ID No:5.
5 FU 5 fluorouracil (5-FU) is the relatively broad antineoplastic chemotherapy medicine of clinical application, and mechanism of action is in cell
It is inside converted into 5 FU 5 fluorouracil deoxynucleotide (5F-dUMP), inhibits the effect of deoxythymidine acid enzyme, prevent BrdU
Sour (dUMP) methylation generates deoxythymidylic acid (dTMP), to influence DNA synthesis, achievees the purpose that kill cell.5-FU exists
It after being converted into 5F-dUMP in vivo, can also mix in RNA, interferencing protein synthesis also has inhibiting effect to other each phase cells.
5-FU is effective to kinds of tumors, especially preferable to alimentary tract cancer and breast cancer curative effect；To oophoroma, uterine neck
Cancer, chorioepithelioma, bladder cancer etc. are also effective.Its adverse reaction is mainly gastrointestinal reaction, and severe one courage and uprightness are dropped and dead；Bone
Marrow inhibition, alopecia, incoordination etc.；Because irritation can cause phlebitis or endarteritis；Accidental Liver and kidney function damage.
5-FU has a lethal effect to normal cell and tumour cell, but its 5 Flucytosine of prodrug (5-FC) to cell without
Effect.
5-FU is transformed by 5-FC deamination, 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 gene
Expression product), originally nontoxic pro-drug 5-FC can be converted to the cytotoxic chemotherapeutics 5-FU of tool.
It was found by the inventors of the present invention that being taken by the way that 5-FC and cytosine deaminase to be applied in the treatment of glioma
Obtained good effect.
Therefore, a kind of preferred embodiment according to the present invention, the exogenous nucleotide sequence further includes CD gene, described
CD gene preferably has nucleotide sequence shown in SEQ ID No:6.
In the present invention, the exogenous nucleotide sequence can also include marker gene (for example, encoding ss-galactosidase glycosides
Enzyme, luciferase, green fluorescent protein or other fluorescins gene).Also, the exogenous nucleotide sequence can also wrap
Include associated retroviral regulating and controlling sequence usually relevant to transcription sequence, for example, site of polyadenylation, Kozak sequence, 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 type of the herpes simplex virus, there is no particular limitation, can be the routine of this field
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 type
Exanthema virus.Also there is no particular limitation in source of the present invention to the herpes simplex virus, can by conventional commercially available,
Acquisition can also be voluntarily separated by laboratory.
Second aspect, the present invention also provides a kind of preparation method of recombinant herpes simplex virus as above, this method packets
Include: by herpes simplex virus encode ICP34.5 and ICP47 gene knockout and optionally knock out coding ICP6, TK and
At least one of gene of UNG, and it is inserted into exogenous nucleotide sequence, wherein the insertion point of the exogenous nucleotide sequence
At at least one in position to knock out the gene of coding ICP34.5, ICP47, ICP6, TK and UNG on the carrier；
Wherein, the exogenous nucleotide sequence includes with the nucleotide sequence that can reduce TSPO gene expression dose
Segment.
In the present invention, the knockout of the gene of 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, passing through fixed point by way of homologous recombination
The mode of knockout；Alternatively, knockout can also be pinpointed by way of CRISPY.Understanding goal of the invention and sheet of the invention
Under the premise of inventing the viral vectors used, those skilled in the art be can be realized according to its conventional technical means grasped 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 field routine, described to insert
The mode entered can be to be directly 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 by way of homologous recombination to be inserted into the aim sequence, and the present invention is preferred
The latter.
In the present invention, recombinant herpes simplex virus is as normal herpes simplex virus, it is also desirable in host cell
It completes its history of life, therefore the passage proliferation 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 vectors and/or recombinant virus of the present invention, 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, the present invention also provides above-mentioned recombinant herpes simplex virus and/or prepared by the above method
Recombinant herpes simplex virus is preparing the application in the drug for treating 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) and CD gene are had by Suzhou Jin Weizhi biotechnology
The synthesis of limit company；
Vero cell is purchased from ATCC, article No. CCL-81；
Glioma cell line U87MG purchase is from ATCC, article No. HTB-14, wherein high efficient expression TSPO gene；
The purchase of U251 cell is from ATCC, article No. HTX-1725, wherein high efficient expression TSPO gene.
Test case 1
This test case is for illustrating that siRNA interference effect is identified
SiRNA (siTSPO#1 and siTSPO#2) is transfected into respectively in U87MG cell and (is used
3000 transfection reagents are purchased from Thermo company, and method is referring 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 gene, to obtain the efficiency of siRNA interference TSPO gene expression.Wherein, with
The normal U87MG cell of untransfected siRNA is positive control, with actin (Actin) for reference protein.
As a result as shown in Figure 1, wherein siTSPO#1 and siTSPO#2 can strike the expression of low TSPO gene, siTSPO#2
Effect is good compared with siTSPO#1.
Embodiment 1
The present embodiment is used to illustrate the building of recombinant herpes simplex virus provided by the invention.
According to application number 2004100064921, the method recorded in the patent application of Authorization Notice No. CN1283803C will
The ICP34.5 gene and ICP47 gene of wild type HSV-1 viral (No. GenBank: NC_001806 of its gene order, similarly hereinafter)
It knocks out, and exogenous nucleotide sequence is inserted into the position of the knockout ICP34.5 gene in HSV-1 virus, except that this implementation
Example uses Vero cell for host cell.The exogenous nucleotide sequence from 5 ' end to 3 ' end successively include: U6 promoter,
ShTSPO#2, termination sequence (TTTTTT).In Suzhou, Jin Weizhi company sequencing identification exogenous nucleotide sequence is properly inserted into list
In pure herpesvirus vector, to obtain recombinant viral vector.Construct successful recombinant viral vector on Vero host cell in
37 DEG C, 5%CO2Under the conditions of be proliferated, infection multiplicity 0.1 removes cell fragment with 0.65 μm of filter after harvest, then exists
High speed centrifugation purifies under the conditions of 13000rpm, and obtaining titre is 1 × 108The viral suspension of pfu/mL is used for cell experiment.
Embodiment 2
The present embodiment is used to illustrate the building of recombinant herpes simplex virus provided by the invention.
According to application number 2004100064921, the method recorded in the patent application of Authorization Notice No. CN1283803C will
The ICP34.5 gene and ICP47 gene knockout of wild type HSV-1 virus, also, in the knockout ICP34.5 gene of HSV-1 virus
Position insertion artificial chemistry synthesis exogenous nucleotide sequence 1, HSV-1 virus knockout ICP47 gene position be inserted into
The exogenous nucleotide sequence 2 of artificial chemistry synthesis, except that the present embodiment uses Vero cell for host cell.Wherein,
It successively includes: U6 promoter, shTSPO#2, termination sequence (TTTTTT) that exogenous nucleotide sequence 1, which is held from 5 ' ends to 3 ',.External source core
It successively includes: CMV promoter, gene, the BGH PolyA for encoding CD that nucleotide sequence 2 is held from 5 ' ends to 3 '.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.Building
Successful recombinant viral vector is on Vero host cell in 37 DEG C, 5%CO2Under the conditions of be proliferated, 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 be 1 ×
108The viral suspension of pfu/mL is used for cell experiment.
Embodiment 3
The present embodiment is used to illustrate the building of recombinant herpes simplex virus provided by the invention.
The building of recombinant herpes simplex virus is carried out according to the method in embodiment 1, unlike, by wild type HSV-1
ICP34.5 gene, ICP47 gene and the ICP6 gene knockout of virus, to obtain recombinant viral vector.
Embodiment 4
The present embodiment is used to illustrate the building of recombinant herpes simplex virus provided by the invention.
The building of recombinant herpes simplex virus is carried out according to the method in embodiment 1, unlike, exogenous nucleotide sequence
Insertion point be HSV-1 virus knockout ICP47 gene position.
Embodiment 5
The present embodiment is used to illustrate the building of recombinant herpes simplex virus provided by the invention.
The building 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 virus knockout ICP34.5 position.
Comparative example 1
This comparative example is used to illustrate the building of the recombinant herpes simplex virus of reference.
The building 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 gene in embodiment 2.
Comparative example 2
This comparative example is used to illustrate the building of the recombinant herpes simplex virus of reference.
The building of recombinant herpes simplex virus is carried out according to the method in embodiment 1, unlike, only by wild type HSV-
The ICP34.5 gene and ICP47 gene knockout of 1 virus, are not inserted into exogenous nucleotide sequence.
Test case 1
This test case is used to illustrate the expression of CD gene in embodiment 2 and comparative example 1-2.
By U87MG cell inoculation into 24 orifice plates, 37 DEG C, 5%CO2Corresponding oncolytic is added after adherent in lower Multiplying culture
It is thin to infect U87MG respectively for the recombinant herpes simplex virus (infection multiplicity MOI=0.01) that viral embodiment 2 and comparative example 1 construct
Born of the same parents, 37 DEG C, 5%CO2Cell is collected in culture afterwards for 24 hours, and Western blot detects the expression of CD gene in cell.Wherein, with not
The U87MG cell of infection is used as reference protein as negative control, actin (Actin).
As a result as shown in Fig. 2, expressing CD base in the U87MG cell of the recombinant herpes simplex virus of infection insertion CD gene
Because, and it is uninfected by the expression that can't detect CD gene in the cell of the virus of insertion CD gene.
Test case 2
This test case is for illustrating that recombinant herpes simplex virus is to brain glioblastoma cell in embodiment 2 and comparative example 1-2
Kill situation.
By U87MG cell inoculation 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 respectively set
It Zuo Wei not compare, be then placed in and continue to cultivate in incubator.After 48 hours, microscopically observation cellular morphology (100 ×), discovery
It is not added in the group of 5-FC, comparative example 1-2 recombinant virus group oncolytic effect indifference is added, 2 recombinant virus group of embodiment is added
It is obvious to the lethal effect of glioma, as a result see Fig. 3 and Fig. 5.And when with 5-FC use in conjunction, comparative example 2 is added and recombinates
Virus group oncolytic effect does not improve, and comparative example 1 and 2 recombinant virus group of embodiment is added to brain glioblastoma cell U87MG's
Lethal effect is obviously improved, and as a result sees Fig. 3 and Fig. 5.
U251 cell is tested after the same method, as a result (does not combine the comparative example 1 of 5-FC as shown in Figure 7
As a result it is not shown).
Electronic cell counter counts cell concentration, to measure cell activity.U87MG cell concentration is adjusted to 3000
It is a/100 microlitres.Above-mentioned 100 microlitres of cell suspensions are added in every hole in 96 orifice plates, are set as 5 groups, 4 multiple holes of every group of setting, and 37
DEG C, 5%CO2Cultivate it is adherent after, exhaust orifice plate in cell culture fluid, then into every hole be added 100 microlitres of basal medium (DMEM
Add 10%FBS) and 10 microlitres of CCK8 solution (attention avoids generating bubble, and influences the reading of OD value).Orifice plate is placed again into
It is incubated for 1 hour in incubator.It is separately added into comparative example 1-2 and embodiment 2 and comparative example 1 and embodiment 2 combines 5-FC, use enzyme
The cell activity that absorbance detection at instrument measurement 450nm is incubated for 0 hour is marked, is then placed in 37 degree of incubators and is incubated for 48h, detect
Cell activity.
As a result as shown in Figure 4 and Figure 6, the recombinant virus of comparative example 1-2 and embodiment 2 inhibits cell activity, when comparative example 1
After combining 5-FC with the recombinant virus of embodiment 2, the inhibitory effect of cell activity is become apparent from.
U251 cell is tested after the same method, as a result (does not combine the comparative example 1 of 5-FC as shown in Figure 8
As a result it is not shown).
Test case 3
Illustrate embodiment 1, killing situation of the recombinant herpes simplex virus to brain glioblastoma cell in 3-5.
According to the method detection embodiment 1 of test case 2, in 3-5 recombinant herpes simplex virus respectively to U87MG cell and
The killing situation of U251 cell, unlike, it is added without 5-FC.
Wherein, the result of U87MG cell, embodiment 1 is as shown in Figure 5 and Figure 6, the effect of the effect of embodiment 5 than embodiment 1
Fruit is good, and quite, 3 effect of embodiment is slightly worse for the effect and embodiment 1 of embodiment 4.
Wherein, the result of U251 cell, embodiment 1 is as shown in Figure 7 and Figure 8, the effect of the effect of embodiment 5 than embodiment 1
Fruit is good, and quite, 3 effect of embodiment is slightly worse for the effect and embodiment 1 of embodiment 4.
By above embodiments 1-5 being compared with the result of comparative example 1-2 it is found that the simple blister of recombination provided by the invention
Exanthema virus shows good anti-glioma effect, has a good application prospect.
The preferred embodiment of the present invention has been described above in detail, and still, the present invention is not limited thereto.In skill of the invention
In art conception range, can with various simple variants of the technical solution of the present invention are made, including each technical characteristic with it is any its
Its suitable method is combined, and it should also be regarded as the disclosure of the present invention for these simple variants and combination, is belonged to
Protection scope of the present invention.
SEQUENCE LISTING
<110>Beijing Inst. of Neurosurgery
Beijing Shen Yuan moral Biotechnology Co., Ltd
<120>application and recombinant herpes simplex virus and preparation method thereof of the TSPO in treatment glioma
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 (7)
1. a kind of recombinant herpes simplex virus, which is characterized in that the recombinant herpes simplex virus includes carrier and exogenous nucleotide
Acid, the carrier are the herpes simplex virus of the gene of missing coding ICP34.5 and ICP47, the insertion of the extraneous nucleotide
Site is at least one in the position for the gene that missing encodes ICP34.5 and ICP47 on the carrier；
Wherein, the extraneous nucleotide be CD gene and can reduce TSPO gene expression dose as shown in SEQ ID No:2
Nucleotide.
2. recombinant herpes simplex virus according to claim 1, wherein the extraneous nucleotide further includes promoter and end
Only codon.
3. recombinant herpes simplex virus according to claim 2, wherein the promoter is selected from U6 promoter, H1 starts
At least one of son and CMV promoter.
4. recombinant herpes simplex virus according to claim 1, wherein the nucleotide sequence such as SEQ of the TSPO gene
Shown in ID No:5.
5. recombinant herpes simplex virus according to claim 1, wherein the nucleotide sequence of the CD gene such as SEQ ID
Shown in No:6.
6. a kind of method for preparing herpes simplex virus described in any one of claim 1-5, which is characterized in that this method
It include: the gene knockout of ICP34.5 and ICP47 will to be encoded in herpes simplex virus, and be inserted into extraneous nucleotide, wherein described
The insertion point of extraneous nucleotide is at least one in the position for knock out on the carrier gene of coding ICP34.5 and ICP47
Place；
The extraneous nucleotide is CD gene and the nucleosides as shown in SEQ ID No:2 that can reduce TSPO gene expression dose
Acid.
7. recombinant herpes simplex virus described in any one of claim 1-5 is prepared by method of claim 6
Obtained recombinant herpes simplex virus is preparing the application in the drug for treating glioma.
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| CN111850126B (en) * | 2020-08-06 | 2022-11-01 | 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 |
| 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 |
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Non-Patent Citations (2)
| Title |
|---|
| TSPO as a target for glioblastoma therapeutics;Eryn L. Werry等;《Biochem.Soc.Trans.》;20141231;第43卷;第531-536页，尤其是第531页摘要，533页右栏最后1段 |
| 胞嘧啶脱氨酶自杀基因转染U251恶性脑胶质瘤细胞及其表达研究;吕胜青等;《中国微侵袭神经外科杂志》;20061231;第11卷(第8期);第354-356页，尤其是第354页摘要部分 |
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