CN103205399B - Recombinant herpes simplex virus, its preparation method and application - Google Patents
Recombinant herpes simplex virus, its preparation method and application Download PDFInfo
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- CN103205399B CN103205399B CN201210337627.7A CN201210337627A CN103205399B CN 103205399 B CN103205399 B CN 103205399B CN 201210337627 A CN201210337627 A CN 201210337627A CN 103205399 B CN103205399 B CN 103205399B
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- A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
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- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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- C12N2710/16643—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention relates to a kind of recombinant herpes simplex virus and preparation method thereof, it is that the ICP4 gene promoter in the herpes simplex virus genes group containing ICP4 gene is replaced with hTERT promoter hTERTp or other tumor-specific promoters, present invention also offers the application in preparation tumor of this recombinant herpes simplex virus；Present invention also offers another kind of recombinant herpes simplex virus, it is insertion fluorescent protein expression box in aforementioned recombinant herpes simplex virus genome；Former recombinant herpes simplex virus relative to existing herpes simplex virus, can the most optionally growth and breeding in human tumor cells, and do not breed in human normal cell line, thus more effectively kill cancerous cell, protect normal cell；The another kind of recombinant herpes simplex virus of the present invention, it can be luminous in tumor cell, can realize the early diagnosis of tumor and the diagnosis of neoplasm metastasis more rapid, accurate, sensitive and wide spectrum.
Description
Technical field
The present invention relates to a kind of recombinant herpes simplex virus, particularly relate to a kind of at anti-tumor activity and tumor-targeting
On the herpes simplex virus that improves to some extent.
Background technology
Prior art having been proposed that, herpes simplex virus (herpes simplex virus, HSV) can be used for oncolytic method
Treatment cancer, and improve by rejecting ICP34.5 gene and ICP47 gene pairs herpes simplex virus so that it is alternative
Ground replicates in tumor cell, and does not injure normal cell, but this improvement can not make herpes simplex virus special effectively
Opposite sex ground only selects to replicate in tumor cell, but also has herpes simplex virus and replicate in some normal cell, thus
These normal cells are damaged.How to realize making a herpes simplex virus specifically killing tumor cell, and do not destroy
Normal cell is always the emphasis problem of those skilled in the art's research.
Telomere is a kind of special construction of eukaryote end of chromosome, and its effect is to safeguard that chromosome structure is stable, bag
Include and prevent end of chromosome from merging, protect chromosomal structural genes and avoid hereditary information to lose in duplication.Telomerase be by
MicroRNA and the reverse transcriptase of protein composition, can utilize self RNA for templated synthesis telomeric dna, make up with cell mitogenic
The telomere that division is gradually shortened.It has three key components: human telomerase RNA (human telomerase RNA,
HTR), telomerase associated proteins (telomerase-as sociated protein, TP1/TLP1), human telomerase reverse transcriptase
(human telomerase reverse transcriptase, hTERT).Telomerase RNA is all expressed in most cells,
And human telomerase reverse transcriptase is the speed limit composition of telomerase, only express in the cell of Telomerase positive, with telomerase activation
Relevant.Although some tumor cells pass through substituting lengthening of telomeres (alternative lengthening of when restructuring
Telomeres, ALT) mechanism maintains telomere length, but still has the tumor cell of more than 85% to activate by raising hTERT
Telomerase, and hTERT only expresses in only a few normal somatic cell.This just provides a good contract for oncotherapy
Machine.The infectious cell protein (infected cell proteins, ICPs) that herpes simplex virus is expressed is divided into Three Estate: just
Begin (immediate early, IE), in early days (early) and late period (late).Wherein IE albumen is the key affecting virus replication
Albumen, including: ICP0, ICP4, ICP22, ICP27 and ICP47, and wherein ICP4 is the replication-associated protein of most critical.
Have now found that kinds of tumors specificity promoter.According to its feature, can be divided into following several: (1) general tomour specific
Promoter, refers to play the promoter of its function in kinds of tumor cells, as hTERT promoter, survivin (survivin) open
Mover etc..(2) tissue-specific promoter, such as prostate specific antigen (PSA) promoter.(3) some tumor is distinctive opens
Mover, alpha-fetoglobulin as distinctive in hepatoma carcinoma cell (AFP) promoter, carcinoembryonic antigen (CEA) promoter of epithelial cell tumor
Deng.These tumor-specific promoters are widely used to therapy of tumor and diagnosing tumor field.
If ICP4 promoter to be replaced with hTERT promoter (hTERTp) or other tumor-specific promoters, make new weight
Group herpes simplex virus can only express its replication protein ICP4 in early days in the tumor cell of hTERT high expressed, thus carries out multiple
System propagation, killing tumor cell, it is expected to solve above-mentioned technical barrier.
Summary of the invention
It is an object of the invention to, it is provided that a kind of recombinant herpes simplex virus, this recombinant herpes simplex virus energy selectivity
Ground growth and breeding in human tumor cells, and do not breed in human normal cell line, thus killing tumor cell, and do not injure normal
Cell.
Another object of the present invention is to, it is provided that the preparation method of a kind of above-mentioned recombinant herpes simplex virus.
It is a further object of the present invention to provide a kind of pharmaceutical composition and answering in the medicine of preparation treatment cancer thereof
With, and the application that above-mentioned recombinant herpes simplex virus is in the medicine of preparation treatment cancer.
Another object of the present invention is to, it is provided that another kind of recombinant herpes simplex virus, its preparing the medicine of diagnosing tumour
Application in thing and a kind of tumor diagnosis kit.
The object of the invention to solve the technical problems realizes by the following technical solutions.Propose according to the present invention
Recombinant herpes simplex virus, it is to be replaced by the ICP4 gene promoter in the herpes simplex virus genes group containing ICP4 gene
It is changed to hTERT promoter hTERTp or other tumor-specific promoters.
The object of the invention to solve the technical problems also can be applied to the following technical measures to achieve further.
Aforesaid recombinant herpes simplex virus, its microbial preservation number is CGMCC No.6397.
Aforesaid recombinant herpes simplex virus, wherein this herpes simplex virus eliminates ICP34.5 gene and ICP47 gene
In one or both.
The object of the invention to solve the technical problems realizes the most by the following technical solutions.Propose according to the present invention
A kind of method preparing aforementioned recombinant herpes simplex virus, it comprises the following steps: use hTERT promoter
HTERTp replaces containing the ICP4 gene promoter in the herpes simplex virus of ICP4 gene, builds this recombinant herpes simplex virus
HSV-hTERTp_ICP4:
(1) shuttle plasmid pICP4del-hTERTp_ICP4 and pICP4del-eGFP is built:
A. this herpes simplex virus containing ICP4 gene, and its genomic DNA of purification are cultivated with bhk cell；
B. amplification ICP4 upstream region of gene flanking sequence: gained virus genom DNA is as template in step a, with following
ICP4USf forward primer and ICP4USr reverse primer:
ICP4USf forward primer: CCCTCCAGACGCACCGGAGTCGGGGG
ICP4USr reverse primer: AAGTCGACTCTAGAGGATCGATCTCTGACCTGAGATTGGCGGCACTGAGGTA
Amplify ICP4 upstream region of gene flanking sequence；
Amplification ICP4 downstream of gene flanking sequence: gained virus genom DNA is as template in step a, with following
ICP4DSf forward primer and ICP4USr reverse primer:
ICP4DSf forward primer: AAAAGTCGACCTGCAGGCATGCTAACGAGGAACGGGCAGGGGGC
ICP4DSr reverse primer: AAAAAAGCTTGCATGCCCACGTGCGCGGGGCCAGACGGGCT
Amplify ICP4 downstream of gene flanking sequence；
Upstream and downstream flanking sequence is cloned on pSP73 plasmid, builds pICP4del and pICP4del-eGFP plasmid: will
Upstream flanking sequence and the aforementioned of SalI/HindIII double digestion of the aforementioned ICP4 gene amplified of SalI enzyme action amplify
The downstream flanking sequence of ICP4 gene mix and be connected to the EcoRV/HindIII site of pSP73, obtain pICP4del；With
EcoRI/XhoI cuts, from pcDNA3.1-eGFP, the eGFP expression cassette that CMV promoter controls, through T4 archaeal dna polymerase filling-in end
After be inserted into the EcoRV site of pICP4del, obtain pICP4del-eGFP；
Three sections of sequences during PCR amplifies ICP4 gene the most in three times: first, use following primer:
ICP4-1stForward primer: TTTTTTGAATTCATGGCGTCGGAGAACAAGCAGCGCC
ICP4-1stReverse primer: TGGAGCCACCCCATGGCCTCCGCGT
ICP4-2ndForward primer: CGACGCCGCGCAGCAGTACGCCCTG
ICP4-2ndReverse primer: CGGCGGGGGCGGGCCCGGCGCACCG
ICP4-3rdForward primer: CCTCATGTTTGACCCGCGGGCCCTG
ICP4-3rdReverse primer: TTTTTTCTCGAGTTACAGCACCCCGTCCCCCTCGAAC
In step a, gained virus genom DNA is as template, amplifies three fragment gene fragments ICP4-1 respectivelyst、ICP4-
2ndAnd ICP4-3rd, following three kinds of matter are constructed in the EcoRV site that this three fragment genes fragment is inserted pSP73 plasmid the most respectively
Grain: pSP73-ICP4-1st、pSP73-ICP4-2nd、pSP73-ICP4-3rd, cut with EcoRI and BsrGI from these three kinds of plasmids
Cut out ICP4-1st, be cut out ICP4-2 with BsrGI and PvuIndAnd it is cut out ICP-3 with PvuI and XhoIrdStand-by；
D. from the plasmid containing hTERT promoter hTERTp, hTERTp sheet is cut with NruI and HindIII
Section, replaces the CMV promoter with NruI and HindIII excision from pcDNA3-NHN, obtains plasmid pcDNA3-NHN-
HTERTp, wherein, pcDNA3-NHN is to insert NheI-HapI-NheI restriction enzyme site sequence gained in the NheI site of pcDNA3；
E. by this ICP4-1 in step cst、ICP4-2ndAnd ICP4-3rdMix and be connected in step d should
EcoRI and the XhoI site of pcDNA3-NHN-hTERTp, obtains plasmid pcDNA3-NHN-hTERTp_ICP4；
F. the plasmid pICP4del containing ICP4 gene upstream and downstream flanking sequence in SalI enzyme action step b, and filling-in end are used
Stand-by, this plasmid pcDNA3-NHN-hTERTp_ICP4 obtained from step e with PmeI and HpaI is cut out hTERTp_ICP4 table
Reach box fragment, and the pICP4del plasmid that it is stand-by with after this enzyme action is connected, construct plasmid pICP4del-hTERTp_
ICP4；
G. structure BHK-ICP4 auxiliary cell: this plasmid pcDNA3-NHN-obtained from step e with EcoRI and XhoI
In hTERTp_ICP4, enzyme action goes out ICP4 gene, and is cloned into downstream EcoRI and the XhoI site of CMV promoter in pcDNA3,
To pcDNA3-CMV-ICP4 plasmid；This pcDNA3-CMV-ICP4 plasmid is transfected bhk cell, this pcDNA3-CMV-ICP4 matter
Grain DNA can recombinate in bhk cell genome, makes some BHK reconstitution cell obtain the resistance to neomycin and express ICP4, uses
Antibiotics G418 kills the bhk cell do not recombinated, and screens through several sub-clones taken turns, and filters out expression ICP4 by RT-PCR method
BHK-ICP4 assist cell；
(2) reject in genome ICP4 gene promoter and insert reverse transcriptase of telomere promoter hTERTp promoter:
A. cultivate this herpes simplex virus containing ICP4 gene with bhk cell, and extract virus genom DNA；
B. this virus genom DNA in step A is entered step with this plasmid pICP4del-eGFP corotation in step (1) b
(1) in g, this BHK-ICP4 auxiliary is intracellular, through homologous recombination, and this plasmid pICP4del-eGFP Green fluorescin GFP table
Reach box and replaced the ICP4 gene of this herpes simplex virus HSV containing ICP4 gene so that the malicious speckle green-emitting of recombinant virus
Fluorescence, through several plaque purifications taken turns, selects green fluorescence poison speckle, just can be purified into recombinant virus HSV-d4GFP；
This HSV-d4GFP virus in C, incubation step B, and extract genomic DNA；
D, by the genomic DNA of step C this recombinant virus HSV-d4GFP and step (1) f this plasmid pICP4del-
The DNA corotation of hTERTp_ICP4 enters this BHK-ICP4 and assists cell, and through homologous recombination, hTERTp_ICP4 expression cassette has replaced this
The egfp expression box GFP of recombinant virus HSV-d4GFP, makes the malicious speckle not fluoresced green of new recombinant virus, passes through
Several plaque purifications taken turns, select unstressed configuration poison speckle, just can be purified into this recombinant herpes simplex virus HSV-hTERTp_ICP4.
The object of the invention to solve the technical problems is also applied to the following technical measures to achieve further.
The preparation method of aforesaid recombinant herpes simplex virus, the most first rejects this herpes simplex containing ICP4 gene sick
Poison ICP34.5 gene and ICP47 gene in one or both, then with hTERT promoter hTERTp replace
ICP4 gene promoter；Or obtain this restructuring replacing ICP4 gene promoter with hTERT promoter hTERTp
After herpes simplex virus HSV-hTERTp_ICP4, then reject this recombinant herpes simplex virus HSV-hTERTp_ICP4's
One or both in ICP34.5 gene and ICP47 gene.
The preparation method of aforesaid recombinant herpes simplex virus, wherein substitutes this people's end with other tumor-specific promoters
Human telomerase reverse transcriptase promoter hTERTp.
The preparation method of aforesaid recombinant herpes simplex virus, wherein the method also includes carrying out the plasmid related to
Check order to confirm the step occurred without sudden change.
The preparation method of aforesaid recombinant herpes simplex virus, wherein this egfp expression box can be by hanced cyan fluorescent
Protein expression box, red fluorescent protein expression cassette, yellow fluorescence protein expression cassette or other indicator protein expression cassette are replaced.
The object of the invention to solve the technical problems realizes the most by the following technical solutions.Propose according to the present invention
Pharmaceutical composition, wherein this pharmaceutical composition comprises aforesaid recombinant herpes simplex virus, and pharmaceutically acceptable carrier or
Excipient.
The object of the invention to solve the technical problems is also applied to the following technical measures to achieve further.
Aforesaid pharmaceutical composition, wherein this pharmaceutical composition is injection, and described injection includes pharmaceutically acceptable
Carrier and aforesaid recombinant herpes simplex virus, containing 10 in every milliliter of described injection2～1010The individual simple bleb of this restructuring
Exanthema virus.
Aforesaid pharmaceutical composition, wherein this pharmaceutically acceptable carrier be pH value be the phosphoric acid buffer of 4.0～9.0
Liquid.
Aforesaid pharmaceutical composition, wherein this injection is possibly together with protective agent and/or osmotic pressure regulator；With this injection
On the basis of, described protectant content is 0.01～30% (weight), and described protective agent is in inositol, sorbitol and sucrose
One or more；Every kilogram of this injection contains 200～700 milligrams of described osmotic pressure regulators, and described osmotic pressure regulates
Agent is sodium chloride and/or potassium chloride.
The object of the invention to solve the technical problems realizes the most by the following technical solutions.Propose according to the present invention
Aforesaid recombinant herpes simplex virus, or the application that aforesaid pharmaceutical composition is in the medicine of preparation treatment tumor.
The object of the invention to solve the technical problems realizes the most by the following technical solutions.Propose according to the present invention
Another kind of recombinant herpes simplex virus, it is insertion fluorescent protein expression in aforesaid recombinant herpes simplex virus genome
Box.
The object of the invention to solve the technical problems is also applied to the following technical measures to achieve further.
Aforesaid another kind of recombinant herpes simplex virus, wherein this fluorescent protein expression box is egfp expression
Box, cyan fluorescent protein expression cassette, red fluorescent protein expression cassette, yellow fluorescence protein expression cassette and other indicator protein are expressed
In box any one.
The object of the invention to solve the technical problems realizes the most by the following technical solutions.Propose according to the present invention
Aforesaid another kind of recombinant herpes simplex virus application in the medicine preparing diagnosing tumour.
The object of the invention to solve the technical problems realizes the most by the following technical solutions.Propose according to the present invention
A kind of tumor diagnosis kit, wherein this tumor diagnosis kit comprises aforesaid another kind of recombinant herpes simplex virus.
The object of the invention to solve the technical problems is also applied to the following technical measures to achieve further.
Aforesaid tumor diagnosis kit, it is characterised in that the sample of this tumor diagnosis kit detection is complete selected from experimenter
Blood, blood plasma, Lymphocyte suspension, bone marrow, hydrothorax or seroperitoneum.
Aforesaid tumor diagnosis kit, it is characterised in that this tumor diagnosis kit also includes: RPMI-1640 cultivates
Base, pH are the erythrocyte cracked liquid of 7 and phosphate buffer that pH is 7.2～7.4, and wherein, described erythrocyte cracked liquid comprises
0.15M ammonium chloride, 10nM potassium bicarbonate and 1nM ethylenediaminetetraacetic acid；Or RPMI-1640 culture medium, proportion be 1.077～
The Ficoll-Hypaque of 0.001 kg/liter and the phosphate buffer that pH is 7.2～7.4.
The present invention compared with prior art has clear advantage and beneficial effect.By technique scheme, the present invention
Recombinant herpes simplex virus, its preparation method and application can reach suitable technological progress and practicality, and have in industry
Extensive value, it at least has the advantage that
(1) recombinant herpes simplex virus of the present invention is for existing herpesvirus, can more effectively selectivity
Ground growth and breeding in human tumor cells, and do not breed in human normal cell line, thus more effectively killing cancerous cell, protection is just
Often cell；
(2) the another kind of recombinant herpes simplex virus of the present invention, it is at former recombinant herpes simplex virus genome
In insert fluorescent protein expression box, it can be luminous in tumor cell, so can be more rapid, accurate, sensitive and wide spectrum ground is real
The early diagnosis of existing tumor and the diagnosis of neoplasm metastasis.
The Classification And Nomenclature of the recombinant herpes simplex virus that the present invention obtains is I herpes simplex virus type, and Latin name is
Herpes Simplex Virus Type 1, is preserved on August 14th, 2012 and is positioned at BeiChen West Road, Chaoyang District, BeiJing City 1
China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) of institute 3, deposit number is CGMCC
No.6397.The biomaterial oHSV1-hTERTp_ICP4 strain being preserved, the implication of its strain number is: oHSV1 refers to I type herpes simplex
Virus 17+ strain (HSV1)；HTERTp_ICP4 refers to replace ICP4 gene promoter with hTERT promoter hTERTp
Son.
Described above is only the general introduction of technical solution of the present invention, in order to better understand the technological means of the present invention,
And can be practiced according to the content of description, and in order to allow the above and other objects, features and advantages of the present invention can
Become apparent, below especially exemplified by preferred embodiment, and coordinate accompanying drawing, describe in detail as follows.
Accompanying drawing explanation
Fig. 1 is that the embodiment of the present invention 1 is by the ICP4 gene promoter in the genome of wild type herpes simplex virus 17+ strain
Son replaces with the method step schematic diagram of hTERT promoter hTERTp.
Fig. 2 shows recombinant herpes simplex virus oHSV1-hTERTp_ICP4 and wild type herpes simplex prepared by embodiment 1
Virus (17+) infects the contrast and experiment of normal cell (human fibroblasts).
Fig. 3 shows that recombinant herpes simplex virus oHSV1-d34.5-d47-hTERTp_ICP4 prepared by embodiment 2 is with existing
Technology only eliminates the ICP34.5 gene of wild type herpes simplex virus genome and the virus (oHSV1-of ICP47 gene
D34.5-d47) contrast and experiment of normal human leukocytes is infected
Fig. 4 A-Fig. 4 F display recombinant herpes simplex virus oHSV1-hTERTp_ICP4 is to hepatocarcinoma, pulmonary carcinoma, head and neck scale carcinoma, black
The lethal effect result of the test (MOI=1) of melanoma, colon cancer and carcinoma of prostate.
Detailed description of the invention
By further illustrating the technological means and effect that the present invention taked by reaching predetermined goal of the invention, below in conjunction with
Accompanying drawing and preferred embodiment, to recombinant herpes simplex virus, its preparation method proposed according to the present invention and apply it specifically real
Execute mode, structure, feature and effect thereof, after describing in detail such as.
The invention provides a kind of recombinant herpes simplex virus, wherein, this recombinant herpesvirus is by containing ICP4 gene
Herpes simplex virus genes group in ICP4 gene promoter replace with hTERT promoter hTERTp or its
Its tumor-specific promoters.Can should be wild type herpes simplex virus containing the herpes simplex virus of ICP4 gene, eliminate
The virus of any genetic fragment (except ICP4 gene and ICP4 gene promoter) in wild type herpes simplex virus genome, but
It is not limited to this, can be that those skilled in the art utilize routine techniques that it is changed any containing ICP4 gene of gained
Herpes simplex virus.
More specifically, the invention provides a kind of recombinant herpes simplex virus, its microbial preservation number is CGMCC
No.6397。
Preferably, this herpes simplex virus eliminates one or both in ICP34.5 gene and ICP47 gene, rejects
ICP34.5 gene (Nervous toxicity gene), can make oncolytic virus safer；And reject ICP47 gene, to promote immunne response and increasing
Strong oncolytic activity.Preferably reject two kinds of genes of ICP34.5 and ICP47.
Present invention also offers a kind of method preparing described recombinant herpes simplex virus, wherein the method includes following step
Rapid: to replace containing the ICP4 in the herpes simplex virus genes group of ICP4 gene with hTERT promoter hTERTp
Gene promoter, builds this recombinant herpes simplex virus HSV-hTERTp_ICP4:
(1) shuttle plasmid pICP4del-hTERTp_ICP4 and pICP4del-eGFP is built:
A. this herpes simplex virus containing ICP4 gene, and its genomic DNA of purification are cultivated with bhk cell；
B. amplification ICP4 upstream region of gene (US) flanking sequence (FlankingRegion is called for short FLR): with gained in step a
Virus genom DNA is template, with following ICP4USf forward primer and ICP4USr reverse primer:
ICP4USf forward primer: CCCTCCAGACGCACCGGAGTCGGGGG
ICP4USr reverse primer: AAGTCGACTCTAGAGGATCGATCTCTGACCTGAGATTGGCGGCACTGAGGTA
Amplify ICP4 upstream region of gene flanking sequence；
Amplification ICP4 downstream of gene (DS) flanking sequence: gained virus genom DNA is as template in step a, with following
ICP4DSf forward primer and ICP4USr reverse primer:
ICP4DSf forward primer: AAAAGTCGACCTGCAGGCATGCTAACGAGGAACGGGCAGGGGGC
ICP4DSr reverse primer: AAAAAAGCTTGCATGCCCACGTGCGCGGGGCCAGACGGGCT
Amplify ICP4 downstream of gene flanking sequence；
Upstream and downstream flanking sequence is cloned on pSP73 plasmid, builds pICP4del and pICP4del-eGFP plasmid: will
Upstream flanking sequence and the aforementioned of SalI/HindIII double digestion of the aforementioned ICP4 gene amplified of SalI enzyme action amplify
The downstream flanking sequence of ICP4 gene mix and be connected to the EcoRV/HindIII site of pSP73, obtain pICP4del；With
EcoRI/XhoI cuts, from pcDNA3.1-eGFP, the eGFP expression cassette that CMV promoter controls, through T4 archaeal dna polymerase filling-in end
After be inserted into the EcoRV site of pICP4del, obtain pICP4del-eGFP；
Three sections of sequences during PCR amplifies ICP4 gene the most in three times: first, use following primer:
ICP4-1stForward primer: TTTTTTGAATTCATGGCGTCGGAGAACAAGCAGCGCC
ICP4-1stReverse primer: TGGAGCCACCCCATGGCCTCCGCGT
ICP4-2ndForward primer: CGACGCCGCGCAGCAGTACGCCCTG
ICP4-2ndReverse primer: CGGCGGGGGCGGGCCCGGCGCACCG
ICP4-3rdForward primer: CCTCATGTTTGACCCGCGGGCCCTG
ICP4-3rdReverse primer: TTTTTTCTCGAGTTACAGCACCCCGTCCCCCTCGAAC
In step a, gained virus genom DNA is as template, amplifies three fragment gene fragments ICP4-1 respectivelyst、ICP4-
2ndAnd ICP4-3rd, following three kinds of matter are constructed in the EcoRV site that this three fragment genes fragment is inserted pSP73 plasmid the most respectively
Grain: pSP73-ICP4-1st、pSP73-ICP4-2nd、pSP73-ICP4-3rd, cut with EcoRI and BsrGI from these three kinds of plasmids
Cut out ICP4-1st, be cut out ICP4-2 with BsrGI and PvuIndAnd it is cut out ICP-3 with PvuI and XhoIrdStand-by；
D. from the plasmid containing hTERT promoter hTERTp, hTERTp sheet is cut with NruI and HindIII
Section, replaces the CMV promoter with NruI and HindIII excision from pcDNA3-NHN, obtains plasmid pcDNA3-NHN-
HTERTp, wherein, pcDNA3-NHN is to insert NheI-HapI-NheI restriction enzyme site sequence gained in the NheI site of pcDNA3；
E. by this ICP4-1 in step cst、ICP4-2ndAnd ICP4-3rdMix and be connected in step d should
EcoRI and XhoI site (these three kinds of genetic fragments ICP4-1 of pcDNA3-NHN-hTERTpst、ICP4-2ndAnd ICP4-3rd's
Connecting order is to be determined by the sequence of its end bit sequence Yu EcoRI and the XhoI site of this pcDNA3-NHN-hTERTp, its
Can automatically carry out coupling to connect), obtain plasmid pcDNA3-NHN-hTERTp_ICP4；
F. the plasmid pICP4del containing ICP4 gene upstream and downstream flanking sequence in SalI enzyme action step b, and filling-in end are used
Stand-by, this plasmid pcDNA3-NHN-hTERTp_ICP4 obtained from step e with PmeI and HpaI is cut out hTERTp_ICP4 table
Reach box fragment, and the pICP4del plasmid that it is stand-by with after this enzyme action is connected, construct plasmid pICP4del-hTERTp_
ICP4；
G. structure BHK-ICP4 auxiliary cell: this plasmid pcDNA3-NHN-obtained from step e with EcoRI and XhoI
In hTERTp_ICP4, enzyme action goes out ICP4 gene, and is cloned into downstream EcoRI and the XhoI site of CMV promoter in pcDNA3,
To pcDNA3-CMV-ICP4 plasmid；This pcDNA3-CMV-ICP4 plasmid is transfected bhk cell, this pcDNA3-CMV-ICP4 matter
Grain DNA can recombinate in bhk cell genome, makes some BHK reconstitution cell obtain the resistance to neomycin and express ICP4, uses
Antibiotics G418 kills the bhk cell do not recombinated, and screens through several sub-clones taken turns, and filters out expression ICP4 by RT-PCR method
BHK-ICP4 assist cell；
(2) reject in genome ICP4 gene promoter and insert reverse transcriptase of telomere promoter hTERTp promoter:
A. cultivate this herpes simplex virus containing ICP4 gene with bhk cell, and extract virus genom DNA；
B. this virus genom DNA in step A is entered step with this plasmid pICP4del-eGFP corotation in step (1) b
(1) in g, this BHK-ICP4 auxiliary is intracellular, through homologous recombination, and this plasmid pICP4del-eGFP Green fluorescent protein expression box
GFP has replaced the ICP4 gene of this herpes simplex virus HSV containing ICP4 gene so that the malicious speckle green-emitting of recombinant virus is glimmering
Light, through several plaque purifications taken turns, selects green fluorescence poison speckle, and (d4 represents rejecting to be just purified into recombinant virus HSV-d4GFP
ICP4 gene)；
This HSV-d4GFP virus in C, incubation step B, and extract genomic DNA；
D, by the genomic DNA of step C this recombinant virus HSV-d4GFP and step (1) f this plasmid pICP4del-
The DNA corotation of hTERTp_ICP4 enters this BHK-ICP4 and assists cell, and through homologous recombination, hTERTp_ICP4 expression cassette has replaced this
The egfp expression box GFP of recombinant virus HSV-d4GFP, makes the malicious speckle not fluoresced green of new recombinant virus, passes through
Several plaque purifications taken turns, select unstressed configuration poison speckle, just can be purified into this recombinant herpes simplex virus HSV-hTERTp_ICP4.
In like manner, the preparation method of described recombinant herpes simplex virus, wherein, can first reject this and contain the simple of ICP4 gene
One or both in the ICP34.5 gene of herpesvirus and ICP47 gene, then with hTERT promoter
HTERTp replaces ICP4 gene promoter；Or replacing ICP4 gene promoter with hTERT promoter hTERTp
After obtaining this recombinant herpes simplex virus HSV-hTERTp_ICP4, then reject this recombinant herpes simplex virus HSV-hTERTp_
One or both in the ICP34.5 gene of ICP4 and ICP47 gene.
The preparation method of described recombinant herpes simplex virus, wherein can substitute this people with other tumor-specific promoters
Reverse transcriptase of telomere promoter hTERTp, replaces the ICP4 gene promoter in this herpes simplex virus containing ICP4 gene
Son, completes gene recombinaton.
Preferably, the preparation method of above-mentioned recombinant herpes simplex virus also include the plasmid related to is checked order with
Confirm the step occurred without sudden change.Monitoring for plasmid sequence, it is ensured that the accuracy of whole preparation process.
It addition, in the preparation method of described recombinant herpes simplex virus, be not limited to only use egfp expression
Box, also can be by cyan fluorescent protein expression cassette, red fluorescent protein expression cassette, yellow fluorescence protein expression cassette and other instruction egg
In white expression cassette, any one is replaced.
Present invention also offers a kind of pharmaceutical composition, wherein, the aforesaid recombinant herpesvirus of this pharmaceutical composition, and
Pharmaceutically acceptable carrier or excipient.Preferably this pharmaceutical composition is injection, and this injection includes pharmaceutically acceptable
Carrier and aforesaid recombinant herpes simplex virus, containing 10 in every milliliter of described injection2～1010This recombinant herpes simplex individual
Virus.More preferably, wherein this pharmaceutically acceptable carrier be pH value be the phosphate buffer of 4.0～9.0.It addition, more preferably,
This injection is possibly together with protective agent and/or osmotic pressure regulator；On the basis of this injection, described protectant content is 0.01
～30% (weight), described protective agent is selected from one or more in inositol, sorbitol and sucrose；Every kilogram of this injection
Containing 200～700 milligrams of described osmotic pressure regulators, described osmotic pressure regulator is sodium chloride and/or potassium chloride.
Above-mentioned herpes simplex virus and pharmaceutical composition can be used for the medicine of preparation treatment tumor.
It addition, present invention also offers another kind of recombinant herpes simplex virus, it is at aforementioned recombinant herpes simplex virus
Inserting fluorescent protein expression box in genome, this fluorescent protein expression box can be egfp expression box, hanced cyan fluorescent egg
In white expression cassette, red fluorescent protein expression cassette, yellow fluorescence protein expression cassette and other fluorescent protein expression box any one；By
In this fluorescent protein expression box can in tumor cell luminous, it is possible to realize with realizing more rapid, accurate, sensitive and wide spectrum
The early diagnosis of tumor and the diagnosis of neoplasm metastasis.
Based on the above-mentioned another kind of restructuring inserting fluorescent protein expression box in aforementioned recombinant herpes simplex virus genome
Herpes simplex virus, the invention provides a kind of tumor diagnosis kit, and this tumor diagnosis kit includes aforementioned another kind of weight
Group herpes simplex virus, this tumor diagnosis kit detection sample selected from experimenter's whole blood, blood plasma, Lymphocyte suspension,
Bone marrow, hydrothorax and seroperitoneum, this tumor diagnosis kit also includes: RPMI-1640 culture medium, pH are the erythrocyte of 7
Lysate and the phosphate buffer that pH is 7.2～7.4, wherein, described erythrocyte cracked liquid comprises 0.15M ammonium chloride, 10nM carbon
Potassium hydrogen phthalate and 1nM ethylenediaminetetraacetic acid；Or the ficoll that RPMI-1640 culture medium, proportion are 1.077～0.001 kg/liter-
Cardiografin and the phosphate buffer that pH is 7.2～7.4.Promulgated by the State Council in can using for the using method of this tumor diagnosis kit
Method disclosed in bright patent CN102220292A (publication date: on October 19th, 2011).
The present invention is further illustrated below in conjunction with embodiment.
Following example selection I herpes simplex virus type strain working standard strain: 17+ strain (Genebank
JN555585.1), for buying from Britain HPA (Health Protection Agency) company.17+ strain complete genome sequence is
Know.If no special instructions, enzyme used and plasmid are purchase gained.
Embodiment 1
The present embodiment is to be replaced with by the ICP4 gene promoter in the genome of wild type herpes simplex virus 17+ strain
HTERT promoter hTERTp, refers to Fig. 1, and step is:
(1) shuttle plasmid pICP4del-hTERTp_ICP4 and pICP4del-eGFP is built:
A. cultivate this wild type herpes simplex virus 17+ strain with BHK (Ren Mus) cell (purchased from ATCC), and purification its
Genomic DNA.
B. amplification ICP4 upstream region of gene flanking sequence: the virus genom DNA of gained 17+ in step a, as template, is used
ICP4USf (forward primer: CCCTCCAGACGCACCGGAGTCGGGGG) and ICP4USr (reverse primer:
AAGTCGACTCTAGAGGATCGATCTCTGACCTGAGATTGGCGGCACTGAGGTA) ICP4 upstream region of gene flank sequence is amplified
Row；
Amplification ICP4 downstream of gene flanking sequence: the virus genom DNA of gained 17+ in step a, as template, is used
ICP4DSf (forward primer: AAAAGTCGACCTGCAGGCATGCTAACGAGGAACGGGCAGGGGGC) and ICP4DSr is (reversely
Primer: AAAAAAGCTTGCATGCCCACGTGCGCGGGGCCAGACGGGCT) amplify ICP4 downstream of gene flanking sequence；
Upstream and downstream flanking sequence is cloned into pSP73 plasmid (buying from Promega company), build pICP4del and
PICP4del-eGFP plasmid: by upstream flanking sequence and the SalI/ of the aforementioned ICP4 gene amplified of SalI enzyme action
The downstream flanking sequence of the aforementioned ICP4 gene amplified of HindIII double digestion mixes and is connected to the EcoRV/ of pSP73
HindIII site, obtains pICP4del.Sequence analysis confirms that this pICP4del plasmid is without sudden change.With EcoRI/XhoI from
PcDNA3.1-eGFP (buying from YRGENE, Beijing) cuts the eGFP that CMV promoter (cytomegalovirus early promoter) controls
Expression cassette, is inserted into the EcoRV site of pICP4del after T4DNA polymerase filling-in end, obtains pICP4del-eGFP；
Table 1
Three sections of sequences during PCR amplifies ICP4 gene the most in three times: first, use the primer shown in table 1, with in step a
Gained 17+ virus genom DNA is template, amplifies three fragment gene fragments ICP4-1 respectivelyst、ICP4-2ndAnd ICP4-3rd, and
Following three kinds of plasmid: pSP73-ICP4-1 are constructed in the rear EcoRV site that this three fragment genes fragment is inserted pSP73 respectivelyst、
pSP73-ICP4-2nd、pSP73-ICP4-3rd.After confirming that sequence is without sudden change, with EcoRI and BsrGI from these three kinds of plasmids
It is cut out ICP4-1st, be cut out ICP4-2 with BsrGI and PvuIndAnd it is cut out ICP-3 with PvuI and XhoIrdStand-by；
D. from the plasmid containing hTERT promoter hTERTp, hTERTp sheet is cut with NruI and HindIII
Section, from pcDNA3-NHN, (pcDNA3 buys from Invitrogen company, inserts NheI-in the NheI site of pcDNA3 in replacement
HapI-NheI restriction enzyme site sequence, is built into pcDNA3-NHN) upper NruI and HindIII excision CMV promoter, obtain
Plasmid pcDNA3-NHN-hTERTp；
E. by this ICP4-1 in step cst、ICP4-2ndAnd ICP4-3rdMix and be connected in step d should
EcoRI and the XhoI site of pcDNA3-NHN-hTERTp, obtains plasmid pcDNA3-NHN-hTERTp_ICP4；
F. contain the plasmid pICP4del of ICP4 gene upstream and downstream flanking sequence with SalI enzyme action, and filling-in end is stand-by, use
This plasmid pcDNA3-NHN-hTERTp_ICP4 that PmeI and HpaI obtains from step e is cut out hTERTp_ICP4 expression cassette sheet
Section, and the pICP4del carrier that it is stand-by with after this enzyme action is connected, construct plasmid pICP4del-hTERTp_ICP4.
G. structure BHK-ICP4 auxiliary cell: this plasmid pcDNA3-NHN-obtained from step e with EcoRI and XhoI
In hTERTp_ICP4, enzyme action goes out ICP4 gene, and is cloned into the downstream EcoRI and XhoI site that in pcDNA3, CMV starts, and obtains
PcDNA3-CMV-ICP4 plasmid；This pcDNA3-CMV-ICP4 plasmid is transfected bhk cell, this pcDNA3-CMV-ICP4 plasmid
DNA can recombinate in bhk cell genome, makes some BHK reconstitution cell obtain the resistance to neomycin (neomycin) and table
Reach ICP4.The bhk cell do not recombinated is killed with antibiotics G418.Screen through several sub-clones taken turns, screen by RT-PCR method
Cell is assisted to the BHK-ICP4 expressing ICP4.
(2) reject in genome ICP4 gene promoter and insert reverse transcriptase of telomere promoter hTERTp promoter:
A. cultivate this wild type herpes simplex virus 17+ with bhk cell, and extract virus genom DNA；
B. this virus genom DNA in step A is entered step with this plasmid pICP4del-eGFP corotation in step (1) b
(1) in g, this BHK-ICP4 auxiliary is intracellular, through homologous recombination, and this plasmid pICP4del-eGFP Green fluorescent protein expression box
Replace the ICP4 gene of this herpes simplex virus HSV containing ICP4 gene so that the malicious speckle fluoresced green of recombinant virus,
Through several plaque purifications taken turns, select green fluorescence poison speckle, just can be purified into recombinant virus oHSV1-d4GFP；
This oHSV1-d4GFP virus in C, incubation step B, and extract genomic DNA；
D, by the genomic DNA of step C this recombinant virus oHSV1-d4GFP and step f this plasmid pICP4del-
The DNA corotation of hTERTp_ICP4 enters BHK-ICP4 and assists cell, and through homologous recombination, it is heavy that hTERTp_ICP4 expression cassette has replaced this
The egfp expression box GFP of papova oHSV1-d4GFP, makes the malicious speckle not fluoresced green of new recombinant virus, passes through
Several plaque purifications taken turns, select unstressed configuration poison speckle, just can be purified into this recombinant herpes simplex virus oHSV1-hTERTp_ICP4.
All plasmids all confirm without sudden change through sequence analysis.
Fig. 2 is this recombinant herpes simplex virus oHSV1-hTERTp_ICP4 and the wild type herpes simplex of embodiment 1 preparation
Virus (17+) infects the contrast and experiment of normal cell (human fibroblasts), (by 1 virus under identical infectious condition
Infect 1 cell, i.e. MOI=1, infect 24 or 48 hours), by this two-strain, normal cell (human fibroblasts) is killed
The comparison of wound effect understands, and wild type herpes simplex virus 17+ causes serious cytopathy and kills normal cell (people's one-tenth
Fibrocyte), this recombinant herpes simplex virus oHSV1-hTERTp_ICP4 then on normal cell without impact.
Embodiment 2
The present embodiment is similar to Example 1, difference be the present embodiment be will to eliminate ICP47 gene and ICP34.5
ICP4 gene promoter in the genome of the wild type herpes simplex virus of gene replaces with human telomerase reverse transcriptase and starts
Sub-hTERTp, specifically comprises the following steps that
(1) genomic DNA of 17+ virus is extracted: cultivate 17+ with BHK (Ren Mus) cell viral and extract genome
DNA。
(2) ICP47 gene, construction of recombinant virus oHSV1-d47 are rejected
A. amplification ICP47 upstream region of gene flanking sequence: with gained 17+ virus genom DNA in step (1) as template, uses
ICP47USf (forward primer: AAAAGAATTCGATTGGGTTCGATTGGCAATGTTGTCTC) and ICP47USr (reverse primer:
AAAAACTAGTGATGTCCCGGGTACGACCATCACCCGAG) ICP47 US FLR is amplified.
B. amplify ICP47 downstream of gene FLR: with gained 17+ virus genom DNA in step (1) as template, use
ICP47DSf (forward primer: AAAAAAGCTTCACGACATGCTCCCCCCCGACGAGC) and ICP47DSr (reverse primer:
AAAACAGCTGACGCGGAACTAGCGCGGACCGGTCG) ICP47 DS FLR is amplified.
C. upstream and downstream flanking sequence is cloned into pBSK (buying from Stratagene company) plasmid, builds pdICP47
And pdICP47-eGFP plasmid: by the upstream flanking sequence of the aforementioned ICP47 gene amplified of EcoRI/SpeI double digestion,
The downstream flanking sequence of the aforementioned ICP47 gene amplified of HindIII/SalI double digestion and complementation there is Spel/
Catenation sequence (the Linker1 CTAGTGAATTCTAGTGGATCCCCCGGGCTGCAGGAATTCGA of HindIII double enzyme site
TATCA；Linker 2 AGCTTGATATCGAATTCCTGCAGCCCGGGGGATCCACTAGAATTCA) mix and connect
To the EcoRI/SalI site of pBSK, obtain pdICP47.With EcoRI/XhoI from pcDNA3.1-EGFP (buy from YRGENE,
Beijing) cut the eGFP expression cassette that CMV promoter controls, after T4 archaeal dna polymerase filling-in end, it is inserted into pdICP47's
EcoRV site, obtains pdICP47-eGFP.
D. by genomic DNA viral for gained 17+ in step (1) and pdICP47-eGFP cotransfection bhk cell.Homology weight
After group, through several plaque purifications taken turns, select green fluorescence poison speckle, just can obtain pure recombinant virus oHSV1-d47-GFP.With
Same method pdICP47 rejects GFP expression cassette in oHSV1-d47-GFP, obtains oHSV1-d47.
(3) ICP34.5 gene, construction of recombinant virus oHSV1-d34.5-d47 are rejected
A. the genomic DNA of 17+ virus is extracted: cultivate oHSV1-d47 virus with bhk cell and extract genomic DNA.
B. amplification ICP34.5 upstream region of gene flanking sequence: with step (1) gained 17+ virus genom DNA as template, uses
ICP34.5USf (forward primer: CTCTGACCTGAGATTGGCGGCACTG) and ICP34.5USr (reverse primer:
GCGGCCGCAGCGCTGCGGCCGCCGCGGGCGCGCTCCTGACCGCGGG) ICP34.5US FLR is amplified.
C. amplification ICP34.5 downstream of gene flanking sequence: with step (1) gained 17+ virus genom DNA as template, uses
ICP34.5DSf (forward primer: GCGGCCGCAGCGCTGCGGCCGCCAGCGCGG CGGGGCCCGGCCAACCA) and
ICP34.5DSr (reverse primer: TTCTTCCCTCTTCTCCCGCCCTCCA) amplifies ICP34.5 downstream flanking sequence.
D. upstream and downstream flanking sequence is cloned into pSP72 (buying from Promega) plasmid, build pdICP34.5 and
PdICP34.5-eGFP plasmid: connect ICP34.5 upstream and downstream flank sequence with Overlapping Polymerase Chain Reaction (overlapping PCR)
Row, and cut and the pSP72 carrier of filling-in is connected with BamHI/Xhol is double, obtain pdICP34.5.With EcoRI/XhoI from
PcDNA3.1-eGFP cuts eGFP expression cassette, is inserted into the AfeI position of pdICP34.5 after T4 archaeal dna polymerase filling-in end
Point, obtains pdICP34.5-eGFP.
E. by thin with step D gained pdICP34.5-eGFP cotransfection BHK for step (2) gained oHSV1-d47 genomic DNA
Born of the same parents.After homologous recombination, through several plaque purifications taken turns, select green fluorescence poison speckle, just can obtain pure recombinant virus oHSV1-
d47-d34.5-GFP.Reject GFP expression cassette in oHSV1-d47-d34.5-GFP with pdICP34.5 in the same way, obtain
oHSV1-d34.5-d47。
The method using embodiment 1 afterwards, replaces aforementioned obtaining with hTERT promoter hTERTp
The ICP4 gene promoter of oHSV1-d34.5-d47, builds recombinant herpes simplex virus oHSV1-d34.5-d47-hTERTp_
ICP4.Certainly, in the present embodiment, it is possible to use method well known in the prior art that ICP47 gene and ICP34.5 gene are entered
Row is rejected.
In the present embodiment, all plasmids all confirm without sudden change through sequence analysis.
In another embodiment, it is possible at embodiment 1 gained this recombinant herpes simplex virus oHSV1-hTERTp_ICP4
After, then its ICP47 gene and ICP34.5 gene are rejected.The method rejecting ICP47 gene and ICP34.5 gene can be
Method in embodiment 2, it is possible to use other method of the prior art.
Fig. 3 is that this recombinant herpes simplex virus oHSV1-d34.5-d47-hTERTp_ICP4 of embodiment 2 preparation is with existing
Technology only eliminates the ICP34.5 gene of wild type herpes simplex virus genome and the virus (oHSV1-of ICP47 gene
D34.5-d47) infect the contrast and experiment of normal human leukocytes, under identical infectious condition, (infect 1 carefully by 1 virus
Born of the same parents, i.e. MOI=1, infect 24 hours), flow cytometer detection finds to infect leukocyte by oHSV1-d34.5-d47, and (detected value is
0.8%), this recombinant herpes simplex virus oHSV1-d34.5-d47-hTERTp_ICP4 does not the most infect leukocyte (detected value is
0.0%), thus explanation the latter's Clinical practice is safer.
Fig. 4 A-Fig. 4 F is this recombinant herpes simplex virus oHSV1-hTERTp_ICP4 to hepatocarcinoma, pulmonary carcinoma, head and neck scale carcinoma, black
The lethal effect result of the test (MOI=1) of melanoma, colon cancer and carcinoma of prostate, wherein matched group is wild type herpes simplex
Virus.From this experimental result, the recombinant herpes simplex virus oHSV1-hTERTp_ICP4 of the present invention can kill multiple swollen
Oncocyte, has the antitumor action of wide spectrum.
Although both of the aforesaid embodiment is as a example by I herpes simplex virus type strain 17+ strain, but the present invention is not limited to 17
+ strain, can equally be well applied to I herpes simplex virus type strain F strain, KOS strain, JS1 strain and BLI strain etc., it addition, be equally applicable to II
Herpes simplex virus type, such as HG52 strain, is i.e. applicable to the arbitrary herpes simplex virus containing ICP4 gene.
Although it should be noted that previous embodiment is by the ICP4 gene promoter in the genome of herpes simplex virus
Replace with hTERT promoter hTERTp, but the scope of the present invention is not limited to human telomerase reverse transcriptase and starts
Sub-hTERTp, the ICP4 promoter of the herpes simplex virus genes group containing ICP4 gene can be replaced with people in the art by it
Other tumor-specific promoters that member is known, such as: survivin (survivin) promoter, prostate specific antigen
(PSA) promoter, hepatoma carcinoma cell distinctive alpha-fetoglobulin (AFP) promoter, the carcinoembryonic antigen (CEA) of epithelial cell tumor open
Mover etc..
The above, be only presently preferred embodiments of the present invention, and the present invention not makees any pro forma restriction, though
So the present invention is disclosed above with preferred embodiment, but is not limited to the present invention, any technology people being familiar with this specialty
Member, in the range of without departing from technical solution of the present invention, when the technology contents of available the disclosure above makes a little change or modification
For the Equivalent embodiments of equivalent variations, as long as being the content without departing from technical solution of the present invention, the technical spirit of the foundation present invention
Any simple modification, equivalent variations and the modification being made above example, all still falls within the range of technical solution of the present invention.
Claims (18)
1. a recombinant herpes simplex virus, it is characterised in that by the herpes simplex virus genes group containing ICP4 gene
ICP4 gene promoter replaces with hTERT promoter hTERTp, and the system of described recombinant herpes simplex virus
Preparation Method comprises the following steps:
Replace containing the ICP4 gene in the herpes simplex virus of ICP4 gene with hTERT promoter hTERTp
Promoter, builds this recombinant herpes simplex virus HSV-hTERTp_ICP4:
(1) shuttle plasmid pICP4del-hTERTp_ICP4 and pICP4del-eGFP is built:
A. this herpes simplex virus containing ICP4 gene, and its genomic DNA of purification are cultivated with bhk cell；
B. amplification ICP4 upstream region of gene flanking sequence: gained virus genom DNA is as template in step a, uses following ICP4USf
Forward primer and ICP4USr reverse primer:
ICP4USf forward primer: CCCTCCAGACGCACCGGAGTCGGGGG
ICP4USr reverse primer: AAGTCGACTCTAGAGGATCGATCTCTGACCTGAGATTGGCGGCACTGAGGTA
Amplify ICP4 upstream region of gene flanking sequence；
Amplification ICP4 downstream of gene flanking sequence: gained virus genom DNA is as template in step a, with following ICP4DSf just
To primer and ICP4USr reverse primer:
ICP4DSf forward primer: AAAAGTCGACCTGCAGGCATGCTAACGAGGAACGGGCAGGGGGC
ICP4DSr reverse primer: AAAAAAGCTTGCATGCCCACGTGCGCGGGGCCAGACGGGCT
Amplify ICP4 downstream of gene flanking sequence；
Upstream and downstream flanking sequence is cloned on pSP73 plasmid, builds pICP4del and pICP4del-eGFP plasmid: by SalI
The aforementioned upstream flanking sequence of the ICP4 gene amplified of enzyme action and the aforementioned of SalI/HindIII double digestion amplify
The downstream flanking sequence of ICP4 gene mixes and is connected to the EcoRV/HindIII site of pSP73, obtains pICP4del；With
EcoRI/XhoI cuts, from pcDNA3.1-eGFP, the eGFP expression cassette that CMV promoter controls, through T4 archaeal dna polymerase filling-in end
After be inserted into the EcoRV site of pICP4del, obtain pICP4del-eGFP；
Three sections of sequences during PCR amplifies ICP4 gene the most in three times: first, use following primer:
ICP4-1stForward primer: TTTTTTGAATTCATGGCGTCGGAGAACAAGCAGCGCC
ICP4-1stReverse primer: TGGAGCCACCCCATGGCCTCCGCGT
ICP4-2ndForward primer: CGACGCCGCGCAGCAGTACGCCCTG
ICP4-2ndReverse primer: CGGCGGGGGCGGGCCCGGCGCACCG
ICP4-3rdForward primer: CCTCATGTTTGACCCGCGGGCCCTG
ICP4-3rdReverse primer: TTTTTTCTCGAGTTACAGCACCCCGTCCCCCTCGAAC is with gained virus base in step a
Because group DNA is template, amplify three fragment gene fragments ICP4-1 respectivelyst、ICP4-2ndAnd ICP4-3rd, the most respectively by this three
Fragment gene fragment is inserted the EcoRV site of pSP73 plasmid and is constructed following three kinds of plasmid: pSP73-ICP4-1st、pSP73-
ICP4-2nd、pSP73-ICP4-3rd, from these three kinds of plasmids, it is cut out ICP4-1 with EcoRI and BsrGIst, with BsrGI and
PvuI is cut out ICP4-2ndAnd it is cut out ICP-3 with PvuI and XhoIrdStand-by；
D. from the plasmid containing hTERT promoter hTERTp, hTERTp fragment is cut with NruI and HindIII,
Replace the CMV promoter with NruI and HindIII excision from pcDNA3-NHN, obtain plasmid pcDNA3-NHN-hTERTp, its
In, pcDNA3-NHN is to insert NheI-HapI-NheI restriction enzyme site sequence gained in the NheI site of pcDNA3；
E. by this ICP4-1 in step cst、ICP4-2ndAnd ICP4-3rdThis pcDNA3-mixed and be connected in step d
EcoRI and the XhoI site of NHN-hTERTp, obtains plasmid pcDNA3-NHN-hTERTp_ICP4；
F. use the plasmid pICP4del containing ICP4 gene upstream and downstream flanking sequence in SalI enzyme action step b, and filling-in end be stand-by,
This plasmid pcDNA3-NHN-hTERTp_ICP4 obtained from step e with PmeI and HpaI is cut out hTERTp_ICP4 expression cassette
Fragment, and the pICP4del plasmid that it is stand-by with after this enzyme action is connected, construct plasmid pICP4del-hTERTp_ICP4；
G. structure BHK-ICP4 auxiliary cell: this plasmid pcDNA3-NHN-hTERTp_ obtained from step e with EcoRI and XhoI
In ICP4, enzyme action goes out ICP4 gene, and is cloned into downstream EcoRI and the XhoI site of CMV promoter in pcDNA3, obtains
PcDNA3-CMV-ICP4 plasmid；This pcDNA3-CMV-ICP4 plasmid is transfected bhk cell, this pcDNA3-CMV-ICP4 plasmid
DNA can recombinate in bhk cell genome, makes some BHK reconstitution cell obtain the resistance to neomycin and express ICP4, with anti-
Rhzomorph G418 kills the bhk cell do not recombinated, and screens through several sub-clones taken turns, and filters out by RT-PCR method and expresses ICP4's
BHK-ICP4 assists cell；
(2) reject in genome ICP4 gene promoter and insert reverse transcriptase of telomere promoter hTERTp promoter:
A. cultivate this herpes simplex virus HSV containing ICP4 gene with bhk cell, and extract virus genom DNA；
B. this virus genom DNA in step A is entered in step (1) g with this plasmid pICP4del-eGFP corotation in step (1) b
This BHK-ICP4 auxiliary is intracellular, and through homologous recombination, this plasmid pICP4del-eGFP Green fluorescent protein expression box GFP puts
Change the ICP4 gene of this herpes simplex virus HSV containing ICP4 gene so that the malicious speckle fluoresced green of recombinant virus, warp
Cross several plaque purification taken turns, select green fluorescence poison speckle, just can be purified into recombinant virus HSV-d4GFP；
This HSV-d4GFP virus in C, incubation step B, and extract genomic DNA；
D, by the genomic DNA of step C this recombinant virus HSV-d4GFP and step (1) f this plasmid pICP4del-hTERTp_
The DNA corotation of ICP4 enters this BHK-ICP4 and assists cell, and through homologous recombination, it is sick that hTERTp_ICP4 expression cassette has replaced this restructuring
The green fluorescent protein GFP expression cassette of poison HSV-d4GFP, makes the malicious speckle not fluoresced green of new recombinant virus, takes turns through several
Plaque purification, selects unstressed configuration poison speckle, just can be purified into this recombinant herpes simplex virus HSV-hTERTp_ICP4.
Recombinant herpes simplex virus the most according to claim 1, it is characterised in that its microbial preservation number is CGMCC
No.6397。
Recombinant herpes simplex virus the most according to claim 1, it is characterised in that this herpes simplex virus eliminates
One or both in ICP34.5 gene and ICP47 gene.
Recombinant herpes simplex virus the most according to claim 1, it is characterised in that first reject this list containing ICP4 gene
One or both in the ICP34.5 gene of pure herpesvirus and ICP47 gene, then with hTERT promoter
HTERTp replaces ICP4 gene promoter, or is replacing ICP4 gene promoter with hTERT promoter hTERTp
After obtaining this recombinant herpes simplex virus HSV-hTERTp_ICP4, then reject this recombinant herpes simplex virus HSV-hTERTp_
One or both in the ICP34.5 gene of ICP4 and ICP47 gene.
5. according to the recombinant herpes simplex virus described in claim 1 or 4, it is characterised in that with other tumor-specific promoters
Substitute this hTERT promoter hTERTp.
6. according to the recombinant herpes simplex virus described in claim 1 or 4, it is characterised in that the method also includes being related to
Plasmid carry out checking order confirming the step occurred without sudden change.
7. according to the recombinant herpes simplex virus described in claim 1 or 4, it is characterised in that this egfp expression box can
Expressed by cyan fluorescent protein expression cassette, red fluorescent protein expression cassette, yellow fluorescence protein expression cassette or other indicator protein
Box is replaced.
8. a pharmaceutical composition, it is characterised in that this pharmaceutical composition comprises the recombinant herpes simplex described in claim 1 or 2
Virus, and pharmaceutically acceptable carrier or excipient.
Pharmaceutical composition the most according to claim 8, it is characterised in that this pharmaceutical composition is injection, described injection
Including the recombinant herpes simplex virus described in pharmaceutically acceptable carrier and claims 1 to 3 any claim, every milli
Rise in described injection containing 102～1010This recombinant herpes simplex virus individual.
Pharmaceutical composition the most according to claim 9, it is characterised in that this pharmaceutically acceptable carrier is that pH value is
The phosphate buffer of 4.0～9.0.
11. pharmaceutical compositions according to claim 9, it is characterised in that this injection is possibly together with protective agent and/or infiltration
Pressure regulator；On the basis of this injection, described protectant content is 0.01～30% weight, and described protective agent is selected from flesh
One or more in alcohol, sorbitol and sucrose；Every kilogram of this injection contains 200～700 milligrams of described osmotic pressuries to be adjusted
Joint agent, described osmotic pressure regulator is sodium chloride and/or potassium chloride.
Recombinant herpes simplex virus described in 12. claims 1 to 3 any claim, or described in 8 to 11 any claim
Pharmaceutical composition preparation treatment tumor medicine in application.
13. 1 kinds of recombinant herpes simplex virus, it is characterised in that in the restructuring as described in claims 1 to 3 any claim
Herpes simplex virus genes group is inserted fluorescent protein expression box or other indicator protein expression cassette.
14. recombinant herpes simplex virus according to claim 13, it is characterised in that this fluorescent protein expression box is green
Fluorescent protein expression box, cyan fluorescent protein expression cassette, red fluorescent protein expression cassette, yellow fluorescence protein expression cassette and other
In indicator protein expression cassette any one.
The application in the medicine preparing diagnosing tumour of the recombinant herpes simplex virus described in 15. claim 13 or 14.
16. 1 kinds of tumor diagnosis kits, it is characterised in that this tumor diagnosis kit comprises as described in claim 13 or 14
Recombinant herpes simplex virus.
17. tumor diagnosis kits according to claim 16, it is characterised in that the sample of this tumor diagnosis kit detection
Product are selected from experimenter's whole blood, blood plasma, Lymphocyte suspension, bone marrow, hydrothorax or seroperitoneum.
18. tumor diagnosis kits according to claim 16, it is characterised in that this tumor diagnosis kit also includes:
RPMI-1640 culture medium, pH are the erythrocyte cracked liquid of 7 and phosphate buffer that pH is 7.2～7.4, wherein, described red
Cell pyrolysis liquid comprises 0.15M ammonium chloride, 10nM potassium bicarbonate and 1nM ethylenediaminetetraacetic acid；Or
RPMI-1640 culture medium, proportion are the Ficoll-Hypaque of 1.077～0.001 kg/liter and pH is 7.2～7.4
Phosphate buffer.
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| CN103436558B (en) * | 2013-08-14 | 2015-01-28 | 中国人民解放军第三军医大学第二附属医院 | Diagnostic agent for tracing tumor cells by magnetic resonance |
| CN106434573B (en) * | 2016-09-21 | 2022-08-23 | 重庆宇珩生物科技有限公司 | Recombinant herpes simplex virus HSV-hTERTp _ ICP4_ Hep-GFP and corresponding diagnostic kit |
| CN106318915B (en) * | 2016-09-21 | 2020-02-18 | 重庆宇珩生物科技有限公司 | Recombinant herpes simplex virus HSV-hTERTp _ ICP4_ LungCA-GFP and diagnostic kit |
| CN108265051B (en) * | 2016-12-30 | 2020-08-25 | 张婧 | Non-invasive method and kit for capturing and isolating fetal cells from a mother |
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| CN109182381A (en) * | 2018-08-09 | 2019-01-11 | 湖北科技学院 | A kind of the novel oncolytic virus and its construction method of selectively killing colon cancer cell |
| CN109161561A (en) * | 2018-08-09 | 2019-01-08 | 湖北科技学院 | A kind of the novel oncolytic virus and its construction method of selectively killing prostate gland cancer cell |
| CN109679928B (en) * | 2018-12-19 | 2019-12-10 | 重庆点检生物科技有限公司 | Recombinant herpes simplex virus, kit and application thereof |
| CN111676199B (en) * | 2020-06-24 | 2022-04-19 | 武汉波睿达生物科技有限公司 | CAR-T technology for presenting and activating HSV-1 type oncolytic virus and application thereof |
| CN111676245B (en) * | 2020-06-24 | 2022-09-13 | 武汉波睿达生物科技有限公司 | NFAT-Cre-CAR-T cell containing HSV-1 type oncolytic virus and application thereof |
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| CN102220292A (en) * | 2010-04-15 | 2011-10-19 | 中国医学科学院肿瘤研究所 | Recombined II-type herpes simplex virus, preparation method and application and tumour diagnostic reagent kit |
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| CN102220292A (en) * | 2010-04-15 | 2011-10-19 | 中国医学科学院肿瘤研究所 | Recombined II-type herpes simplex virus, preparation method and application and tumour diagnostic reagent kit |
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| 新型溶瘤病毒Ohsv2hGM-CSF的构建及其抗肿瘤作用;施桂兰等;《中华肿瘤杂志》;20120228;第34卷(第2期);89-96 * |
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| WO2014036807A1 (en) | 2014-03-13 |
| CN103205399A (en) | 2013-07-17 |
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