CN101168742A - Oncolytic virus and its preparing method - Google Patents
Oncolytic virus and its preparing method Download PDFInfo
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- CN101168742A CN101168742A CNA200710027080XA CN200710027080A CN101168742A CN 101168742 A CN101168742 A CN 101168742A CN A200710027080X A CNA200710027080X A CN A200710027080XA CN 200710027080 A CN200710027080 A CN 200710027080A CN 101168742 A CN101168742 A CN 101168742A
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Abstract
The invention discloses specific TRAIL oncolytic adenovirus (Ad/TRAIL-E1) of a recombinant tumor. The main components comprise tumor necrosis factor-related apoptosis inducing ligand (TRAIL), human telomerase reverse transcriptase gene promoter (hTERT), adenovirus vector (Ad) and cytomegalo virus (CMV-E). The construction form of the specific TRAIL oncolytic adenovirus (Ad/TRAIL-E1) of the recombinant tumor is shown in the figure. Simultaneously, the invention also discloses the preparation method of the oncolytic adenovirus.
Description
Technical field
The present invention relates to oncolytic virus and preparation method thereof, relate in particular to special tumor necrosin relative death inducing ligand oncolytic virus of a kind of recombinant tumor and preparation method thereof.
Background technology
Malignant tumour is one of important diseases of present serious threat human life health.Although traditional treatment means such as operation, radiation and chemotherapy have had bigger development, most late tumor patient is still died from cancer itself.Though conventional chemotherapy has certain curative effect to malignant tumour, most tumors can occur because of chemotherapy resistance being continued development fatal in many courses of treatment after the chemotherapy.So seeking new treatment means and strategy will be very necessary and urgent to the treatment level that improves malignant tumour.
(tumor necrosis factor-relatedapoptosis-inducing ligand TRAIL) is a new tnf family cytokines part of just finding in recent years to tumor necrosin relative death inducing ligand.It becomes one of focus in the present oncotherapy because of the characteristics that powerful antitumor action arranged and do not have a multidrug resistance in the test in vivo and in vitro.Preclinical research is verified, TRAIL can induce the apoptosis of kinds of tumor cells, intratumor injection can suppress growth of tumor and prolong the lifetime of animal tumor model, and good synergy is all arranged with multiple chemotherapeutics such as Zorubicin, cis-platinum, CPT-11 and radiotherapy, even the tumour of anti-chemoradiotherapy still had curative effect preferably, pointing out it is a kind of very promising cancer therapy drug.The result of study of we and other shows, by being carrier with adenovirus, can well trail dna being transfected into kinds of tumor cells and expressing the TRAIL that symphysis connects and cause its apoptosis.And because of it produces that TRAIL that symphysis connects has bystander effect (bystander effect) thereby the anti-tumor activity that obviously strengthens TRAIL, its antitumour activity even be better than the TRAIL of solubility.
Studies confirm that TRAIL cultivates external former be commissioned to train foster human liver cell and people's cerebral tissue serious toxicity, thereby has caused the worry that TRAIL is applied to the genotoxic potential of clinical cancer therapy.How to reduce TRAIL and will clear away important obstacle for TRAIL enters human clinical's research the toxicity of these organs.
Summary of the invention
Shortcoming at prior art the purpose of this invention is to provide a kind of oncolytic virus and preparation method thereof, solves the xicity related problem of tumor necrosin relative death inducing ligand gene therapy.
To achieve these goals, the technical scheme of oncolytic virus of the present invention is: a kind of oncolytic virus, its moiety comprise tumor necrosin relative death inducing ligand (TRAIL), human telomerase reverse transcriptase's promotor (hTERT), adenovirus carrier (Ad) and cytomegalovirus (CMV-E).
The structure form of described oncolytic virus is:
The method that the present invention prepares described oncolytic virus comprises the steps:
(1) amplification of trail dna, clone and order-checking: according to the sequence and the bibliographical information design synthetic primer of the trail dna of GenBank report, carry out pcr amplification, the PCR product reclaims test kit with glue and reclaims purifying, and the product routine behind the purifying is carried out enzyme and cut evaluation and dna sequence analysis;
(2) structure of Ad/gTRAIL expression vector: hTERT is targeted on the trail dna.With the trail dna PCR product fragment of purifying, insert in the hTERT carrier by ligase enzyme is directed, be built into the adenovirus carrier of Ad/gTRAIL, strengthen the expression of the GFP/TRAIL fusion gene of hTERT mediation again by the regulator of GAL4;
(3) reorganization of Ad/gTRAIL expression vector and oncolytic virus carrier: on the adenovirus carrier basis of Ad/gTRAIL, recombinate with the oncolytic virus carrier, form a new virus, be referred to as the special TRAIL oncolytic virus (Ad/TRAIL-E1) of recombinant tumor; After Ad/TRAIL-E1 carries out further amplification purification, measure purity and quantitative with ultraviolet spectrophotometer.
Compared with prior art, the present invention has successfully made up the adenovirus carrier (being called for short Ad/gTRAIL) by the specific expressed trail protein of hTERT promotor.Confirm that in preliminary preclinical research it is effective that Ad/gTRAIL has extensive powerful antitumor action to comprise the human tumor of anti-chemotherapy, specifically to the external former foster human liver cell of being commissioned to train, normal cell and animal nontoxicity.And be the TRAIL of promotor with the CMV or the PGK-GV-16 of non-tumour-specific, then produced serious liver toxicity.Illustrate that hTERT can make the TRAIL targeted expression at tumour cell, thereby avoided its hepatotoxicity.Recombinate with the oncolytic virus carrier on the Ad/gTRAIL basis, form a new virus, we are referred to as the special TRAIL oncolytic virus (Ad/TRAIL-E1) of recombinant tumor.New A d/TRAIL-E1 has overcome trail dna and has carried out the more weak characteristics of antineoplaston effectiveness, can in tumour cell, carry out self-replacation, increase gene at the copy number of tumour cell or the expression level in tumour cell, thereby strengthen the antitumor action of Ad/TRAIL-E1.
Description of drawings
Fig. 1 is the strong apoptosis-induced effect synoptic diagram of Ad/gTRAIL to people's various tumor cell strains; In Fig. 1, H460: people's non-small cell lung cancer cell strain; A549, H1299: human lung adenocarcinoma cell line; DLD-1: human colon cancer cell strain; Lovo: human large intestine cancer cell strain; PBS: phosphate buffered saline buffer; GFP: green fluorescent protein; The G-TRAIL:GFP-TRAIL fusion gene; The GT-TRAIL:GFP-hTERT-TRAIL fusion gene.
Fig. 2 is the good resistance tumor promotion synoptic diagram of Ad/gTRAIL to the drug-fast MDA-MB-231 of Zorubicin; In Fig. 2, MDA-231: human breast cancer cell strain; MDA-231/ADR: mammary cancer Zorubicin drug-resistant cell strain.
Fig. 3 is the dose-effect relationship synoptic diagram of Ad/TRAIL-E1 virus dosage and active cells; In Fig. 3, A549, H1299: human lung adenocarcinoma cell line; H460: people's non-small cell lung cancer cell strain; DLD-1: human colon cancer cell strain; DLD-1/TRAIL-R: colorectal carcinoma TRAIL drug-resistant cell strain; NHFB: the normal endothelium fibrocyte of people; Ad/CMV-GFP: adenovirus/cytomegalovirus carrier; Ad/GFP-E1: adenovirus carrier; The adenovirus mediated trail dna that Ad/TRAIL-RGD:RGD modifies; Ad/TRAIL-E1: recombinant tumor specificity TRAIL oncolytic virus; MOI: infection multiplicity.
Fig. 4 be Ad/TRAIL-E1 do not have obvious apoptosis-induced effect synoptic diagram to the lethal effect of kinds of tumor cells and to people's normal fiber cell; In Fig. 4, H460: people's non-small cell lung cancer cell strain; H1299: human lung adenocarcinoma cell line; DLD1: human colon cancer cell strain; DLD1/TRAIL-R: colorectal carcinoma TRAIL drug-resistant cell strain; NHFB: the normal endothelium fibrocyte of people; PBS: phosphate buffered saline buffer; Ad/CMV-GFP: adenovirus/cytomegalovirus carrier; Ad/GFP-E1: adenovirus carrier; The adenovirus mediated trail dna that Ad/TRAIL-RGD:RGD modifies; Ad/TRAIL-E1: recombinant tumor specificity TRAIL oncolytic virus.
Fig. 5 is that Ad/TRAIL-E1 can carry out self-replacation and there is no in NHFB duplicating the effect synoptic diagram in various kinds of cell; In Fig. 5, DLD1: human colon cancer cell strain; DLD1/TRAIL-R: colorectal carcinoma TRAIL drug-resistant cell strain; Ad/GFP-E1: adenovirus carrier; Ad/TRAIL-E1: recombinant tumor specificity TRAIL oncolytic virus; H1299: human lung adenocarcinoma cell line; NHFB: the normal endothelium fibrocyte of people; Ad/GFP-E1: adenovirus carrier; Ad/TRAIL-E1: recombinant tumor specificity TRAIL oncolytic virus.
Fig. 6 is that Ad/gTRAIL is at the intravital antitumor action synoptic diagram of mammary cancer mice with tumor; In Fig. 6, MDA-MB-231: human breast cancer cell strain; PBS: phosphate buffered saline buffer; Ad/CMV-GFP: adenovirus/cytomegalovirus carrier; Ad/gTRAIL: adenovirus mediated trail dna.
Fig. 7 is the existence situation synoptic diagram of mammary cancer mice with tumor; In Fig. 7, MDA-MB-231: human breast cancer cell strain; PBS: phosphate buffered saline buffer; Ad/CMV-GFP: adenovirus/cytomegalovirus carrier; Ad/gTRAIL: adenovirus mediated trail dna.
Fig. 8 is that Ad/gTRAIL is at the intravital antitumor action synoptic diagram of anti-ADR mammary cancer mice with tumor; In Fig. 8,231/ADR: mammary cancer Zorubicin drug-resistant cell strain; Ad/CMV-GFP: adenovirus/cytomegalovirus carrier; Ad/gTRAIL: adenovirus mediated trail dna.
Fig. 9 is the existence situation synoptic diagram of anti-ADR mammary cancer mice with tumor; In Fig. 9,231/ADR: mammary cancer Zorubicin drug-resistant cell strain; Ad/CMV-GFP: adenovirus/cytomegalovirus carrier; Ad/gTRAIL: adenovirus mediated trail dna.
Figure 10 is the situation synoptic diagram of Ad/gTRAIL associating paclitaxel treatment pulmonary metastases; In Figure 10, Ad/GFP: adenovirus carrier; Paclitaxel: taxol; Ad/gTRAIL: adenovirus mediated trail dna.
Figure 11 is that Ad/gTRAIL-E1 suppresses growth of tumor synoptic diagram in the nude mouse; In Figure 11, PBS: phosphate buffered saline buffer; Ad/CMV-GFP: adenovirus/cytomegalovirus carrier; Ad/GFP-E1: recombinant adenoviral vector; The adenovirus mediated trail dna that Ad/TRAIL-RGD:RGD modifies; Ad/TRAIL-E1: recombinant tumor specificity TRAIL oncolytic virus.
Figure 12 is the existence situation synoptic diagram of mice with tumor between different groups; In Figure 12, PBS: phosphate buffered saline buffer; Ad/CMV-GFP: adenovirus/cytomegalovirus carrier; Ad/GFP: adenovirus carrier; Ad/GFP-E1: recombinant adenoviral vector; The adenovirus mediated trail dna that Ad/TRAIL-RGD:RGD modifies; Ad/TRAIL-E1: recombinant tumor specificity TRAIL oncolytic virus.
Embodiment
The present inventor confirms that through research trial repeatedly prevention TRAIL to Normocellular toxicity, needs the correct target tumor of TRAIL.Confirmer's reverse transcriptase of telomere promotor (hTERT) can be used to the short apoptogene of target tumor at present, and hTERT expresses in the human tumor more than 85%, and remains static in most of somatocyte.Therefore, hTERT is targeted on the trail dna, can solves the xicity related problem of trail dna treatment.In view of the above, the composition of the special TRAIL oncolytic virus (Ad/TRAIL-E1) of recombinant tumor of the present invention comprising: tumor necrosin relative death inducing ligand (TRAIL), human telomerase reverse transcriptase's promotor (hTERT), adenovirus carrier (Ad) and cytomegalovirus (CMV-E).The structure form of the TRAIL oncolytic virus (Ad/TRAIL-E1) that this recombinant tumor is special is:
Simultaneously, the method for preparing above-mentioned oncolytic virus comprises the steps:
(1) amplification of trail dna, clone and order-checking: according to the sequence and the bibliographical information design synthetic primer of the trail dna of GenBank report, carry out pcr amplification, the PCR product reclaims test kit with glue and reclaims purifying, and the product routine behind the ability purifying is carried out enzyme and cut evaluation and dna sequence analysis.
(2) structure of Ad/gTRAIL expression vector: TRAIL is directly imported tumour cell by adenovirus mediated gene therapy method, can the induced tumor apoptosis and suppress tumor growth, and the tumour cell that is not imported into goal gene also can be killed by the bypass effect.Because the main drawback of TRAIL is that its treatment is xicity related, limited the application of TRAIL in oncotherapy, we can express by the tumor-specific promoters target gene and overcome this shortcoming at present, hTERT is targeted on the trail dna, has solved the xicity related problem of trail dna treatment.With the trail dna PCR product fragment of purifying, insert in the hTERT carrier by ligase enzyme is directed, be built into the adenovirus carrier of Ad/gTRAIL, strengthen the expression of the GFP/TRAIL fusion gene of hTERT mediation again by the regulator of GAL4; GAL4 gene regulating system can strengthen the transgene expression of hTERT promotor mediation, and don't can reduce the specificity of target.The GAL4 gene regulatory system can make the transgene expression of CEA promotor mediation strengthen 20-100 doubly, and does not reduce the specificity of its expression at all.
(3) reorganization of Ad/gTRAIL expression vector and oncolytic virus carrier: on the adenovirus carrier basis of Ad/gTRAIL, recombinate with the oncolytic virus carrier, form a new virus, be referred to as the special TRAIL oncolytic virus (Ad/TRAIL-E1) of recombinant tumor; After Ad/TRAIL-E1 carries out further amplification purification, measure purity and quantitative with ultraviolet spectrophotometer.The TRAIL oncolytic virus has overcome TRAIL and has carried out the more weak characteristics of antineoplaston effectiveness, Ad/TRAIL-E1 can rush in capable self-replacation at tumour cell, increase gene at the copy number of tumour cell or the expression level in tumour cell, thereby strengthen the antitumor action of Ad/TRAIL-E1.
Further confirm effect of the present invention below in conjunction with each concrete testing data.
The extracorporeal anti-tumor function of example 1:Ad/TRAIL
See also Fig. 1, the Ad/gTRAIL in vitro tests confirms that tumor cell lines such as human lung carcinoma cell H460 and A549, human colon cancer cell DLD1 and Lovo are all had favorable anti-tumor effect; And to normal human liver cell and human bronchial endotheliocyte free of toxic effects.See also Fig. 2, Ad/gTRAIL also has very strong tumor inhibition effect to the human breast cancer cell M231 of adriamycin-resistant, and Ad/gTRAIL and paclitaxel and vinorebine have good synergy at the drug-fast MDA-MB-231 cell strain of Zorubicin.
The extracorporeal anti-tumor function of example 2:Ad/TRAIL-E1
See also Fig. 3 and Fig. 4, the Ad/TRAIL-E1 in vitro tests confirms: Ad/TRAIL-E1 is to human lung carcinoma cell line H1299, A549, H460 with to the human colon cancer cell strain DLD1 of TRAIL sensitivity with to the drug-fast human colon cancer cell strain of TRAIL DLD1/TRAIL-R favorable anti-tumor effect, but the normal endothelium fibrocyte of people (NHFB) is not had tangible toxic action.
The virus replication in tumour cell of example 3:Ad/TRAIL-E1
See also Fig. 5, Ad/TRAIL-E 1 can carry out self-replacation in tumour cell H1299, DLD1 and DLD1/TRAIL-R cell, and having the regular hour dependency, experimental data there is no duplicating of Ad/TRAIL-E1 virus in the normal fibrocyte of confirmer (NHFB) simultaneously.
The effect of example 4:Ad/gTRAIL anti-tumor in vivo
See also Fig. 6, it is subcutaneous that human breast cancer cell strain MDA-MB-231 is inoculated in the nude mice in age in 6-8 week.When diameter of tumor reaches 0.4-0.5cm, intratumor injection Ad/gTRAIL or control vector.Intratumor injection 9 * 10
10Particle number, injection in 5 days 1 time, totally 3 times.Take a blood sample and have or not liver toxicity.Compare with control group, Ad/gTRAIL significantly suppresses tumor growth (P＜0.01).And 60% animal, disappearing fully appears in tumour, and after May, these animals still do not have knurl existence.See also Fig. 7, with the animal that Ad/CMV-GFP handles, the tumour size is the same with the PBS group, and this 2 treated animal is all dead in March.2,12,30 days ALT and AST are in normal range the 1st treatment back.Zorubicin drug-resistant cell strain transplanted tumor in nude mice is also observed equifinality.The nude mice of inoculation 231/ADR is divided into Ad/gTRAIL group and Ad/CMV-GFP group, 10 every group.See also Fig. 8, Ad/gTRAIL significantly suppresses tumor growth (P＜0.05).See also Fig. 9, the Ad/gTRAIL group has 4 still not have knurl existence after May, and control group is all dead.See also Figure 10, the result of study in the drug-fast 231/ADR pulmonary metastases of Zorubicin model shows that be compared to negative control and Ad/CMV-GFP blank group, Ad/gTRAIL or taxol can significantly suppress the intravital lung of mouse knurl tubercle.And, can make the metastatic lesion of lung in the mouse body with Ad/gTRAIL connection and paclitaxel treatment, be suppressed with respect to other animal is more significant.Particularly, the tumor nodule of this test group lung obviously is less than other group (P＜0.05).
The anti-tumor in vivo effect of example 5:Ad/TRAIL-E1
See also Figure 11 and Figure 12, it is subcutaneous that human lung carcinoma cell line H1299 is inoculated in the nude mice in 6 ages in week, when diameter of tumor reaches 0.3-0.5cm, intratumor injection Ad/gTRAIL-E1 or control vector, Ad/TRAIL-E1 can obviously suppress growth of tumor, and cause the intravital tumour of animal to disappear fully, and there is not knurl existence for a long time.
Claims (3)
1. oncolytic virus, the main component of its composition comprises tumor necrosin relative death inducing ligand TRAIL, human telomerase reverse transcriptase's promotor hTERT, adenovirus carrier Ad and cytomegalovirus CMV-E.
2. oncolytic virus as claimed in claim 1 is characterized in that, its structure form is:
3. a method for preparing the described oncolytic virus of claim 1 comprises the steps:
(1) amplification of trail dna, clone and order-checking: according to the sequence and the bibliographical information design synthetic primer of the trail dna of GenBank report, carry out pcr amplification, the PCR product reclaims test kit with glue and reclaims purifying, and the product routine behind the purifying is carried out enzyme and cut evaluation and dna sequence analysis;
(2) structure of Ad/gTRAIL expression vector: hTERT is targeted on the trail dna.With the trail dna PCR product fragment of purifying, insert in the hTERT carrier by ligase enzyme is directed, be built into the adenovirus carrier of Ad/gTRAIL, strengthen the expression of the GFP/TRAIL fusion gene of hTERT mediation again by the regulator of GAL4;
(3) reorganization of Ad/gTRAIL expression vector and oncolytic virus carrier: on the adenovirus carrier basis of Ad/gTRAIL, recombinate with the oncolytic virus carrier, form a new virus, be referred to as the special TRAIL oncolytic virus (Ad/TRAIL-E1) of recombinant tumor; After Ad/TRAIL-E1 carries out further amplification purification, measure purity and quantitative with ultraviolet spectrophotometer.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102958940A (en) * | 2010-06-25 | 2013-03-06 | 阿达梅德公司 | Anticancer fusion protein |
| CN111471715A (en) * | 2020-04-07 | 2020-07-31 | 南昌大学第一附属医院 | Adenovirus vector and construction method and application thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102958940A (en) * | 2010-06-25 | 2013-03-06 | 阿达梅德公司 | Anticancer fusion protein |
| CN102958940B (en) * | 2010-06-25 | 2015-12-02 | 阿达梅德公司 | Anticancer fusion protein |
| CN111471715A (en) * | 2020-04-07 | 2020-07-31 | 南昌大学第一附属医院 | Adenovirus vector and construction method and application thereof |
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Open date: 20080430 |