CN100415298C - Adenovirus vector for idiopathy liver genetherapy and using method - Google Patents

Abstract

The present invention relates to an adenovirus carrier used for the gene therapy of a primary liver cancer, and a using method thereof; a method and a compound for treating a primary liver cancer on the basis of the oncolytic virus therapyare provided. The adenovirus carrier builds gene recombination adenovirus, wherein the gene recombination adenovirus comprises an alpha-fetoprotein promotor gene and a theoretical gene. The research shows that the recombination adenovirus can be used for specifically killing primary liver cancer cells used for expressing alpha-fetoprotein. The gene recombination adenovirus can be used for the gene therapy of the primary liver cancer.

Description

The adenovirus vector and the using method that are used for the primary hepatocarcinoma gene therapy

Technical field

The present invention relates to genetic treatment of tumor and viral therapy field, be specifically related to, carry out the technology that virus replication and external source are inserted gene expression under given conditions by tumor-specific promoters regulating and controlling adenovirus carrier, promptly utilize afp promoter regulating and controlling adenovirus carrier, in the primary hepatic carcinoma cell of expressing alpha-fetoprotein, carry out virus replication and external source specifically and insert gene expression, thereby in the hepatoma carcinoma cell of expressing alpha-fetoprotein, insert gene expression because of being subjected to afp promoter control to carry out virus replication and external source specifically, thereby kill the technology of hepatoma carcinoma cell.

Background technology

Malignant tumor serious threat human health is the mankind No. second " killer " that are only second to cardiovascular disease, and traditional tumor therapeuticing method has suitable limitation, for example, puts, chemotherapy kill tumor cell the time, and is extremely strong to the toxic and side effects of tumor patient health.

Molecular biology and other subject development in recent years, people to the function of range gene and and disease between relation understand gradually, the Biotherapeutics of tumor develops rapidly, wherein mainly comprise aspects such as immunotherapy of tumors and gene therapy, the former comprises the bringing out of activation, cytokine of anticarcinogenic effect cell, the screening of anticancrin (being targeted therapy), the methods such as development of new generation vaccine, and the latter comprises the methods such as treatment of viral vector and non-virus carrier.

With virus is the gene therapy of the malignant tumor of carrier, and carrier can enter tumor cell efficiently, specifically, and expresses entrained treatment exogenous gene, or duplicates in tumor cell specifically, breeds, and then the kill tumor cell.At present common gene therapy viral vector has retroviral vector, adenovirus vector, slow virus carrier and gland relevant viral vector, and wherein the maximum of research are adenovirus vectors.

People's adenovirus is divided into 50 serotypes according to the difference of particle surface epi-position, returns A-F6 subgenus respectively.Crossing over mutually between the member of each subgenus, but between different subgenus, can not recombinate mutually.People's 5 type Adenovirus adenovirus hominis C subgenus.Adenovirus is a kind of double-stranded DNA virus, and its genome length is about 36kb, divides early transcription district (E district) and late transcription district (L district), and the early transcription district can be divided into 6 distinct area: E1A, E1B, E2 (E2A and E2B), E3, E4 in detail.

Deleted the adenovirus vector in E1 district and E3 district, be commonly called first generation adenovirus vector, this carrier can insert and express the exogenous gene that length is no more than 8kb, this adenovirus vector not reproducible itself produces progeny virus, and have following characteristics: can infect the polytype cell, comprise nondividing cell resting stage; Genome not with the genome generation random integration of host cell, safer; Be easy to large-scale production, can obtain the virus of higher titre.

How which kind of gene therapy vector no matter improves and improves its specificity in tumor cell, be this carrier can Secure Application in clinical key.For reaching this purpose, people pass through gene engineering method, constantly transform adenovirus vector: perhaps make improved virus in tumor cell, duplicate propagation by specificity, and in normal cell reproducible propagation not, become a kind of so-called oncolytic virus, as the Onyx-015 virus of Onyx company, the tumor cell of specific killing p53 gene mutation; Perhaps, by transforming adenoviral gene group itself (Lieber A et al.), modification (Curiel D et al.) by the adenovirus ciliary structures, and by using duplicate (the Yu DC et al.) of external source tumor-specific promoters control recombinant adenovirus, realize the purpose of tumour-specific carrier, as the CN706 virus of Calydon company, by the effect of tumor of prostate specificity promoter SPA, the specific killing prostate gland cancer cell.

An important difference of primary hepatic carcinoma cell and normal hepatocytes cancerous cell is, surpass 70% primary hepatic carcinoma cellular expression alpha-fetoprotein, normal liver cell is not then expressed, has only the expression that alpha-fetoprotein is arranged in the embryonic liver cell, this is a kind of embodiment of cancerous cell primitivization, in clinical diagnosis, the detection of alpha-fetoprotein is one of important means of primary hepatic carcinoma diagnosis.We are with self promoter gene deletion in the adenovirus vector genome, replace to afp promoter, make the adenovirus vector that is controlled by afp promoter after the reorganization, can only be under the situation of expressing alpha-fetoprotein, duplicate and express because of promoter is activated, in normal hepatocyte, because of there not being the existence of alpha-fetoprotein, adenovirus vector is reproducible and expression not, has reached the purpose to the hepatocyte selective killing thus.

Using afp promoter control adenovirus and produce the liver cancer tissue specificity, is common anti-hepatocarcinoma therapy of tumor research method.But because the afp promoter complex structure, different parts effect difference has difference in functionality, makes people produce the liver cancer-specific of afp promoter and doubts very greatly.We improve afp promoter commonly used, use a kind of afp promoter with special construction, it contains enhancer (Enhancer), silencer (Silencer) and promoter core space all functional elements such as (Core promoter).In addition, in order to strengthen the specificity of this promoter, the specific genes controlling element has been inserted in the upstream of our afp promoter gene order in the genome of recombinant adenoviral vector---and separaant (Insulator) makes it reach very high specificity.

As the carrier of therapy of tumor, require to have tumour-specific on the one hand; On the other hand, should have the antitumor characteristic, promptly under tumor-specific promoters controlled condition,, express at specifically inside tumor cell with entrained relevant Antioncogene, thus killing tumor cell effectively, and normal cell is not had lethal effect.At this, apoptosis induction ligand (the TNF-related apoptosis-inducing ligand that we are relevant with tumor necrosis factor with the E1a gene of Ad5 type adenovirus, abbreviation TRAIL) gene carries out gene recombinaton by genetic engineering means with the adenoviral gene group, can realize above-mentioned target.

Human adenovirus 5 type E1a genes are positioned at the genomic left end of adenovirus linear dsdna, have 84% conservative.This gene produces two kinds of main mRNA, and size is respectively 13S and 12S, and 289 and 243 amino acid whose two albumen of encoding respectively, these two albumen impel duplicate (Shenk T, 1996) of virus by activating the expression of viral gene.These two albumen are the initial expressed proteins of adenovirus, have the function that activates adenovirus other gene, particularly E2 district and E4 district gene, are the keys of adenoviral replication.The albumen of E1a gene code has 3 to transform necessary zone: non-conserved amino acid end (aa2-24); Conserved region 1 (CR1; Aa40-80); Conserved region 2 (CR2; Aa120-140).The proteic difference in functionality zone of E1a gene code combines with intracellular two albuminoids, thereby make the activation of E1a encoded protein, bring into play its function: transcribe coactivator p300/CBP family, combine with the protein N terminal and the CR1 of E1a gene, Rb family combines with CR1 and CR2, these reactions are from the different growth regulating approach of functionally influencing, thus the promotion differentiation.Though cell experiment shows that E1a is under the synergism of E1b and ras gene outcome, can make normal embryo fibroblast is vicious transformation, becomes tumor (Yu D when being inoculated in nude mice; HungMC, 1998), but E1a gene itself can not make cell line vicious transformation (Chang JY et al., 1996), up to now, there is not evidence to show that the E1a gene is an oncogene, opposite, discovered in recent years E1a is the gene with cancer suppressing action, and it can suppress the formation and the transfer of tumor by number of ways.

The inhibition of E1a gene pairs tumor realizes by the following aspects: suppress tumor by the mechanism that relies on p53 1..When cell DNA damages, but p53 gene inducing cell advances the G1 phase, suppresses cell proliferation and obtains repairing up to DNA damage, if the DNA of damage can not get repairing, the gene that p53 can activate cell death inducing makes cell generation apoptosis.And the E1a gene can be kept p53 wild type conformation, makes p53 albumen be in higher level.This may be the protein bound result of P300 in E1a gene protein and the cell.2. the mechanism by non-dependence p53 suppresses tumor, may be relevant with the activation or the inactivation of some genes in the cell, thus the apoptosis of promotion cell, mechanism it be unclear that (Ricardo SP et al., 1996) at present.3.E1a gene suppresses tumor by the immunoreation of regulate tumor cell.The E1a gene can obviously increase the NIH3T3 cell to the Cytotoxic sensitivity of TNF, influences the susceptibility (Chen MJ et al., 1987) of target cell to the molten cell mechanism of non-dependence TNF by NK cell and activating macrophage.4.E1a gene can suppress different human tumor cells' pernicious behavior (Yu D by tumor cell being changed into the epithelial cell phenotype; Hung MC, 1998).5.E1a gene suppresses tumor invasion by the Profilin enzyme gene expression, as (Yu D ﹠amp such as IV Collagen Type VI enzyme, plasminogen activator, interstitial collagenase, urokinase and stromelysins; Hung MC, 1998, RicardoSP et al., 1996).6.E1a gene can shift (Steeg PS et al., 1988) by the expression inhibiting that strengthens nm-23 nm23.

In addition, the E1a gene can make tumor cell that radiotherapy and chemotherapeutical sensitivity are increased.Its action pathway: 1. by blocking the activity of NB-.Transcription factor NB-is relevant with chemotherapeutical negation to radiotherapy with tumor cell, ray and some chemotherapeutic agents can activate the activity of NF-κ B, make tumor cell produce negation to radiotherapy and chemotherapy, and the E1a gene can be blocked the activity of NF-κ B, thereby improve tumor cell to radiotherapy and chemotherapeutical sensitivity (Shao RP et al., 1997).2. by changing the activity of some gene in the cell.Descend as the erbB2 expression of gene, the wild type p53 level increases.(Ricardo SP et al. such as Ricardo, 1996) studies show that the E1a gene improves tumor cell to radiotherapy and chemotherapeutical sensitivity, do not rely on the change of state, H-ras and other oncogenes of p53, even in the cancerous cell of HPV-E6 gene high expression, the E1a expression of gene can make tumor cell that the sensitivity of DNA damage class medicine and lonizing radiation is improved 4～10 times.

Therefore, people begin the inhibitory action of broad research E1a gene pairs tumor: in the nude mice tumor model of setting up with human ovarian cancer cell's strain, by the liposome-mediated E1a gene of local injection, growth of tumor obviously is suppressed (Yu D et al., 1995) with sending out than matched group; In the pulmonary carcinoma nude mice model,, obtained Biotherapeutics effect (Chang JY et al., 1996) preferably in the human trachea by the liposome-mediated E1a gene of tail vein injection; Liposome-mediated E1a gene therapy metastatic breast cancer, epithelial ovarian cancer show that tumor growth is suppressed (Ueno NT et al., 1998); The treatment of the correct neck neoplasms of E1a gene obtained the FDA approval and is used for clinical research in 1996, the head and neck cancer patient that can not excise and recur with E1a gene therapy 7 examples, carry out the injection of tumor body with four groups of different dosage (15,30,60 and 120 μ g DNA/cm3 tumor), do not find tangible toxic action; Among the 6 routine patients of assessment, 4 routine tumors are controlled, and 1 example part is dwindled, and 1 example has less change (Yoo GH et al., 1998).E1a not only intravenous injection has obtained better therapeutic effect in animal model, do not see obvious toxic and side effects, and clinical research also shows sure curative effect, by number of ways, acts on different genes, and E1a should have bright prospect in oncotherapy.

Among the present invention, when we have selected for use the E1a gene with function of tumor inhibition to treat with one of insertion gene as external source, selected for use comparatively noticeable recently gene participating in apoptosis to treat with inserting gene as the another one external source, the purpose of the two combination is, improves the oncotherapy effect.

Apoptosis is the process of cell suicide, and it makes multicellular organism control certain cell number in tissue and eliminates indivedual cells that threaten biological existence.Cell apoptosis after serious DNA damage starts failure and causes the generation (Evan G et al., 1998) of malignant tumor.The unique pick off of some cell surface tool, be called as death receptor, it can survey extracellular dead signal (apoptosis activity factor), and can start inherent apoptotic process (the Ashkenazi A et Dixit VM of cell rapidly, 1998), recent research has been found a kind of apoptosis activity factor-TRAIL (TNF-related apoptosis-inducing ligand) albumen, is an II type memebrane protein.The total length memebrane protein TRAIL of eukaryotic expression and pmol soluble TRAIL can be induced kinds of tumor cells apoptosis (Wiley SR et al., 1995 rapidly; Pitti RM et al., 1996).

TRAIL has expression at many tissues, and its receptor does not all combine (Ashkenazi A et Dixit VM, 1998) with other cell toxicant parts such as TNF-α.Normal and tumor tissues is all expressed DR5, the DR4 receptor of TRAIL, and the apoptotic effect (Pan G et al., 1997) of mediation TRAIL, and the DcR1 of TRAIL and DcR2 receptor, high expressed in normal structure (containing hepatic tissue), the low expression in embryo's (containing the tire liver) and tumor tissues.Particularly importantly, DcR1 and DcR2 receptor can not transmit dead signal, are " bait " receptor of tool protective effect, make that normal cell avoids being killed and wounded.Transfection experiment has confirmed this protective effect (Sheridan JP et al., 1997; Pan G et al., 1997).Infer according to TRAIL and multiple receptor expression thereof, normal structure is to insensitive (the Lars EF et al. of its apoptosis-induced effect, 2000), simultaneously, TRAIL kills and wounds oncocyte and the same p53 of not relying on of other factor of its family, can more effectively kill and wound the cell (Rohn TA et al., 2001) that some p53 have undergone mutation like this.

Genetic treatment of tumor, key issue are the selection specificity of tumor cell and kill and wound two aspects of effectiveness.The present invention inserts viral vector by using afp promoter, the control external source is inserted expression of gene, feature with specific killing hepatoma carcinoma cell, simultaneously, the treatment gene that external source is inserted is selected E1a gene that enters clinical research and the trail dna with apoptosis activation for use, by the regulating and controlling effect of internal ribosome entry site IRES gene, express the albumen of these two kinds of gene codes simultaneously respectively, strengthened effectiveness the tumor cytotoxicity effect.Promptly, E1 district promoter deletion with 5 type adenoviruss replaces afp promoter, the tumour-specific of research afp promoter and separaant, and, obtain the Ad.HS4.AFP.E1a/TRAIL recombinant adenoviral vector and make up at the downstream of their gene orders connection E1a and TRAIL.Studies show that, this recombinant adenoviral vector, have in the primary hepatic carcinoma cell because of the expression specificity of alpha-fetoprotein and regulated and control the characteristics that afp promoter carries out adenoviral replication, make that two kinds of different treatments under this promoter control are expressed with gene, effectively strengthened under the specified conditions, be in the primary hepatic carcinoma cell, this viral vector is to the lethal effect of tumor cell.

The present invention also provides the viral vector and the available pharmaceutical carrier that contain this tumour-specific to use jointly.

The present invention also provides the viral vector of this tumour-specific being used for the drug regimen therapeutic use of oncotherapy.

The present invention also provides the viral vector of similar tumour-specific being used for the purposes of oncotherapy.

The present invention also provides the viral vector of similar tumour-specific being used for the combined therapy purposes of oncotherapy with other medicines and/or available pharmaceutical carrier.

Description of drawings

Fig. 1. recombinant adenovirus Ad.HS4.AFP.E1a/TRAIL structural representation.Wherein, HS4 is the betaglobulin separaant gene of chicken, AFP is the afp promoter gene, constitute by 2 enhancers, a 6-8 silencer and promoter core gene respectively, E1a is 5 type adenovirus early gene E1a district genes, IRES is the internal ribosome entry site gene, and TRAIL is the relevant apoptosis induction ligand of tumor necrosis factor, and Δ E3 represents 5 type adenovirus early gene E3 district's gene elminations.

Fig. 2. when being 100 (TCID50) by living cells colorimetric detection experimental study multiplicity of infection MOI, cell toxicant specificity in AFP feminine gender/positive cell.HepG2, Hep3B, SMMC-7721, BEL-7404 is respectively hepatoma cell line, and L-02 is people's a normal liver cell, and LS174T is a human colon cancer cell, and Hek293 is a HEKC.When above-mentioned cell is cultured to 104 cells in every hole in 96 orifice plates, add AdHS4.AFP.E1a/TRAIL respectively, dl1520, or culture fluid is in contrast, after 6 days, carries out the MTS method and detects the 490nm light absorption value, and the result is three different tests reproducible results averages.

Fig. 3. recombinant adenovirus AdHS4.AFP.E1a/TRAIL is to the dosage dependent cells toxic action of different hepatoma cell line under the different TCID50 multiplicity of infection MOI conditions.Hep3B, HepG2 is an AFP high expressing cell system, BEL-7404, SMMC-7721 are the low express cell of AFP systems, when above-mentioned cell is cultured to 104 cells in every hole in 96 orifice plates, use MOI1000 respectively, 100,10,1 AdHS4.AFP.E1A/TRAIL infects, infect the back and carried out MTS method detection 490nm light absorption value in continuous 6 days, the result is three different tests reproducible results averages.

Fig. 4 .E 1A, the RT-PCR that TRAIL and Caspase-3 express analyzes.BEL-7404, HepG2 and L02 cell are Ad.HS4.AFP.E1A/TRAIL (as shown in the figure) infection of 100TCID50 or do not infect (in contrast) 48h that through MOI the quantitative analysis that E1A, TRAIL and Caspase-3 transcribe is an internal contrast with β-actin.

Fig. 5. the apoptotic effect of the colorimetric detection research TRAIL by the Caspase-3 enzymatic activity.HepG2, Hep3B, SMMC-7721, BEL-7404 is respectively hepatoma cell line, when above-mentioned cell is cultured to 2 * 106 cells in every hole in 6 orifice plates, obstruction by low Nutrient medium cultivation and aphidocolin, make the cell growth be tending towards synchronous, change cell culture fluid then, carry out MOI simultaneously respectively and be 100 AdHS4.AFP.E1a/TRAIL, dl1520 infects and culture fluid contrasts, and infects after 30 hours, collecting cell, cell lysis detects the activity that the colorimetric reagent box detects Caspase with CaspaceTM, detects through the 405nm light absorption value and obtains the result, apoptotic effect is that the free pNA thing of pmol/mg/min represents that the result is a secondary different tests reproducible results average by vertical coordinate unit.

Fig. 6. the replication capacity of recombinant adenovirus Ad.HS4.AFP.E1A/TRAIL in BEL-7404 transplanted tumor and the comparison of Ad.HS4.AFP.E1A/TRAIL and dl1520E1A expression.Transplanted tumor to the athymic nude mice of lotus BEL-7404 tumor is carried out Ad.HS4.AFP.E1A/TRAIL (1.0 * 107TCID50/mm3, C and F), dl1520 (1.0 * 107TCID50/mm3 respectively, B and E), perhaps PBS (A and D, contrast as a setting) injection was injected 5 days continuously.Mice with tumor is condemned to death the 7th day the time behind the drug administration by injection the last time and carries out histopathological analysis, and the slice of tumor is with anti-E1A antibody (brown) dyeing, and reuse hematoxylin (blueness) is redyed other parts.E1A quantitative analysis (G) shows, Ad.HS4.AFP.E1A/TRAIL group and dl1520 group and PBS group on adding up notable difference (p＜0.01, ^*), simultaneously, the Ad.HS4.AFP.E1A/TRAIL group is organized the difference that also has on the statistical significance with quantitative dl1520.Concrete section as shown in the figure.

Fig. 7. recombinant adenovirus AdHS4.AFP.E1a/TRAIL is Antitumor Effects in the lotus human liver cancer cell BEL-7404 tumor nude mice in vivo.PBS, (1.0 * 107TCID50/mm3), dl1520 (1.0 * 107TCID50/mm3) for AdHS4.AFP.E1a/TRAIL, or DDP is injected directly in the low BEL-7404 transplanted tumor tumor of expressing of AFP, volume injected is 50 μ l, injects continuously 5 days, and DDP injected 7 days continuously, gross tumor volume calculates by the detection of diameter of tumor, measure weekly 2 times, the gross tumor volume of injection day is set at 100%, compares with negative control PBS group, other 3 class mean all have remarkable tumor-inhibiting action (p＜0.01, AVONA).The Ad.HS4.AFP.E1A/TRAIL group is compared with the dl1520 group, and also there were significant differences for its tumor-inhibiting action (p＜0.05, student t test).

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Summary of the invention

Constructed recombinant adenoviral vector is 5 type adenoviruss through transforming among the present invention.The genome of 5 type adenoviruss contains 35935 nucleotide, is a kind of virus of studying at present, mainly causes people's respiratory tract infection on the epidemiology, and has the virus of obvious self-healing property.Adenovirus is applied to the human body history of existing decades as vaccine, transforms human normal cell line and the generation of carcinogenic phenomenon from finding no.

The constructed recombinant adenoviral vector AdHS4.AFP.E1a/TRAIL of the present invention as shown in fig. 1, the E1a promoter region of adenovirus vector self is deleted, the substitute is afp promoter (AFP Promoter), and insert separaant (Insulator) at its upstream, be the betaglobulin gene order HS4 of one section chicken, be used to control and the specificity of strengthening whole recombinant adenoviral vector duplicates specific expressed with related gene.The downstream of afp promoter is an adenovirus early gene E1a gene, and by internal ribosome entry site (internal ribosome entry site, IRES) the relevant apoptosis induction ligand trail dna of tumor necrosis factor of gene connection, E1a gene and trail dna are under the control of specificity afp promoter, regulating and controlling effect through IRES, express simultaneously respectively, strengthened effective lethal effect hepatoma carcinoma cell.

The constructed recombinant adenoviral vector AdHS4.AFP.E1a/TRAIL of the present invention submitted Chinese typical culture collection center preservation on 9th in JIUYUE in 2003, and deposit number is: V 2003010.

The afp promoter complex structure, sequence reaches 8kb.Use different piece, its liver cancer-specific has very big difference.The present invention adopts gene engineering method, the human a-fetoprotein promoter structure is optimized, particularly, promptly adopt 2 multiple enhancers (Enhancer), 6 multiple silencers (Silencer), and afp promoter core space (Core promoter), common form one brand-new, efficiently, 5 type adenoviral gene groups are placed under this promoter control, and, form a specificity and in hepatoma carcinoma cell, duplicate the recombinant adenovirus of expression in conjunction with controlling elements such as separaants.

Advantage of the present invention: the present invention has killing tumor cells high specificity and the high advantage of effectiveness.

The specificity aspect:

At first select to use alpha-fetoprotein AFP promoter among the present invention, making the entrained external source of recombinant adenoviral vector insert gene duplicates in can expressing the primary hepatocarcinoma cell of alpha-fetoprotein and expresses, and, employed afp promoter is through research and the afp promoter of the reorganization after improving, on its performance, its tumour-specific is strong than other bibliographical information, and simultaneously, it starts to have a bit on the function and optimizes.

On this basis, insert separaant, further strengthened the specificity of this promoter in the promoter upstream.That select here is the betaglobulin gene order HS4 of chicken, and other similar separaant all can be used for strengthening the specific separaant of promoter and uses, and makes adenovirus vector duplicating with expression specificity in tumor cell strengthen thus.

The effectiveness aspect:

The adenovirus early gene E1a gene of at first selecting in this aspect to have entered clinical research with effective tumor-inhibiting action that also has been proved to be is expressed with gene as treatment, simultaneously, under the special important regulating and controlling effect of internal ribosome entry site IRES gene, can use under the situation of same transcript unit, express two kinds of different genes encoded protein simultaneously respectively, select to activate relevant TRAIL, strengthen lethal effect tumor cell with apoptosis.

Apoptosis is the process of cell suicide, and it makes multicellular organism control certain cell number in tissue and eliminates indivedual cells that threaten biological existence.Cell apoptosis after serious DNA damage starts failure and causes the generation of malignant tumor.TRAIL, promptly tumor necrosin relative death inducing ligand is an II type memebrane protein, studies show that soluble TRAIL can be induced the kinds of tumor cells apoptosis rapidly.Therefore, the co expression of TRAIL and E1a is with the fragmentation effect that strengthens tumor cell.

The present invention adapts to the tumor-specific promoters of different targeting equally, specific treatment at different tumors, select different tumor CDKN2s simultaneously for use and/or induce co expression such as activated gene, gene involved in immunity, to strengthen effective lethal effect tumor.

Definition

Unless different definition is arranged in addition, as used herein common understand the same of those skilled in the art in the field under the implication of all technology and scientific terminology and the present invention.Although any similar or identical any method and material of describing therewith all can be used for enforcement of the present invention or test.Only preferable methods and material are described at this.The present invention is defined with regard to following term for this reason:

" separaant " this term as used herein is meant any betaglobulin separaant gene order that has with chicken of coding, i.e. HS4 strengthens the exogenous gene sequence of promoter specific function equally.

" exogenous gene " this term as used herein, be meant and insert any interested, the proteic gene order that biological function is arranged of the coding in the used adenovirus vector among the present invention, this inserts the coded albumen of exogenous gene, can have medicinal or further feature.The upper limit of the length of the exogenous gene that is inserted depends on the packing limit of adenovirus vector.Exogenous gene can pass through conventional method, promptly synthesizes, is prepared by methods such as natural origin extraction, gene clones.

These two terms of " liver cancer-specific gene expression " and " liver cancer-specific " are meant adenovirus vector duplicating and expressing in the primary hepatic carcinoma cell as used herein.Though this carrier also has expression in normal cell, expression is very low, makes carrier duplicate and packs and can't effectively carry out, to such an extent as to low like this copy package level can be ignored.

" first generation adenovirus vector " or " first generation recombinant adenoviral vector " this term is meant the adenovirus vector of having deleted early gene E1 district and E3 district as used herein, and such adenovirus vector is reproducible not.

The specific embodiment

The structure of example I recombinant adenovirus AdHS4.AFP.E1a/TRAIL

Recombinant adenovirus AdHS4.AFP.E1a/TRAIL described below, be the 5 type adenovirus vectors that have afp promoter, and, has HS4 separaant gene in this promoter upstream, it is that the relevant early stage E1a gene of adenovirus of tumor-inhibiting action activates the relevant apoptosis induction ligand trail dna of relevant tumor necrosis factor with apoptosis that external source insertion gene is carried in the promoter downstream, and the two connects by internal ribosome entry site IRES, the same transcript unit of common use expresses respectively.

Unless otherwise indicated, the technology used in the present invention all is this area routine techniquess, and as molecule clone technology, microbiological technique, cytobiology technology, these technology all are fully explained in the literature.As " molecular cloning laboratory manual " second edition (1989) of Sambrook etc. etc.

The structure of plasmid vector:

People's afp promoter gene order derives from plasmid pXZAFP (Kaneko S et al., 1995), people's tumor necrosin relative death inducing ligand hTRAIL gene order derives from plasmid pORF.TRAIL (Invivogen), internal ribosome entry site IRES derives from plasmid pIRES2-EGFP (Clonetech Labotorary), the betaglobulin separaant gene HS4 of chicken derives from plasmid pSLJCa (Steinwaerder DS and Lieber A, 2000), adenovirus early gene E1a gene order derives from the PCR product of plasmid pFG140 (Microbix), and its amplimer is 5 '-GGGACTGAAAATGAGAC-3 ' and 5 '-CGCCATGCAAGTTAAACA-3 '.Plasmid pShuttle that all adenovirus constructions are used and rescue AdEasy1 carrier are AdEasy TM adenovirus system test kit (Stratagene), and the bacterial strain that is used for the antibacterial conversion is BJ5183 and XL10-gold.

With plasmid pORF-TRAIL restricted enzyme SalI and NheI hydrolysis, obtain the dna fragmentation of TRAIL,, mend plain film section end through the effect of T4 polymerase, insert AfiII hydrolysis, the T4 polymerase of pShuttle-pA plasmid and mend flat dna fragmentation, obtain plasmid pShuttle-TRAIL-pA.Plasmid pIRES2-EGFP through XhoI and enzyme hydrolysis of BstXI restriction enzyme and the flat processing of T4 polymerase benefit, is connected to BglII enzyme action, the benefit prosposition point of plasmid pShuttle-TRAIL-pA, obtains plasmid pShuttle-IRES-TRAIL-pA.The EcoRVI flush end hydrolysis site that to insert plasmid pShuttle-IRES-TRAIL-pA with the plasmid pFG140 flush end E1a gene that to be template obtain through pcr amplification obtains plasmid pShuttle-E1A-IRES-TRAIL-pA.Come from AFP promoter gene plasmid pXZAFP, that have enhancer, silencer and core promoter zone, after SalI and AflII hydrolysis, be connected to the corresponding site of plasmid pShuttle-E1A-IRES-TRAIL-pA, obtain plasmid pShuttle-AFP-E1A-IRES-TRAIL-pA.The HS4 gene that will come from the XbaI enzyme cutting hydrolysis site of plasmid pSLJca then through mending the XhoI benefit prosposition point that plasmid pShuttle-AFP-E1A-IRES-TRAIL-pA is inserted in flat back, obtains final plasmid pShuttle-HS4-AFP-E1A-IRES-TRAIL-pA.

The structure of recombinant adenovirus AdHS4.AFP.E1a/TRAIL:

With the further enzyme action hydrolysis linearisation of plasmid pShuttle-HS4-AFP-E1A-IRES-TRAIL-pA through the PmeI restricted enzyme, AdEasy1 carries out homologous recombination in escherichia coli BJ5183 with the rescue plasmid, and the correct clone who obtains by analysis will be transformed among the escherichia coli XL10-Gold and further increase and purification.Purification gained plasmid is behind the PacI linearization for enzyme restriction, transfection is in HEKC, through transcribing, translate and packing, the adenovirus vector that obtains recombinating after further restriction analysis is identified, further increases constructed correct recombinant adenovirus AdHS4.AFP.E1a/TRAIL in the AE25 cell, through the CsCl gradient centrifugation, dialysis and titre are demarcated (TCID50/ml), and packing is stored in-80 ℃ (Dirk SS et al., 2000).

The specific effect research of example II recombinant adenovirus AdHS4.AFP.E1a/TRAIL in hepatoma carcinoma cell

The MTS method detects recombinant adenovirus AdHS4.AFp.E1a/TRAIL to the influence in the different tumor cells:

For the tomour specific that detects recombinant adenovirus AdHS4.AFP.E1a/TRAIL lethal, by using MTS (Promega) test kit to detect living cells when multiplicity of infection MOI is 100 (TCID50), cell toxicant specificity that should virus in AFP feminine gender/positive cell.HepG2, Hep3B, SMMC-7721, BEL-7404 is respectively hepatoma cell line, and L-02 is people's a normal liver cell, and LS174T is a human colon cancer cell, and Hek293 is a HEKC.Above-mentioned cell is seeded to 104 cells in every hole in 96 orifice plates, behind the 24hr, use AdHS4.AFP.E1a/TRAIL respectively, dl1520 infects, or culture fluid in contrast, adds MTS reagent in different time points, and 37 ℃ of insulations are after one hour, carry out the 490nm light absorption value and detect (BIO-RAD), the results are shown in Figure 2.

The MTS method detects the dosage dependence specificity influence of recombinant adenovirus AdHS4.AFP.E1a/TRAIL to different hepatoma carcinoma cell

Recombinant adenovirus AdHS4.AFP.E1a/TRAIL is to the dosage dependent cells toxic action of different hepatoma cell line under different TCID50 multiplicity of infection MOI conditions.Hep3B, HepG2 is an AFP high expressing cell system, BEL-7404, SMMC-7721 are the low express cell of AFP systems, when above-mentioned cell is cultured to 104 cells in every hole in 96 orifice plates, use MOI1000 respectively, 100,10,1 AdHS4.AFP.E1a/TRAIL infects, infect the back and carried out MTS method detection 490nm light absorption value in continuous 6 days, the results are shown in Figure 3.

The CaspaceTM method detects the apoptotic effect research of expressing the hepatoma carcinoma cell that causes among the recombinant adenovirus AdHS4.AFP.E1a/TRAIL because of TRAIL

In order to detect the activity of TRAIL encoded protein, respectively with hepatoma cell line HepG2, Hep3B, SMMC-7721, BEL-7404 is seeded in when being cultured to 2 * 106 cells in every hole in 6 orifice plates, by low Nutrient medium, make the cell growth be tending towards synchronous, 0.5%FBS MEM cultivates 12hr, change 10%FBS MEM culture fluid and continue to cultivate 9hr, add Aphidicolin (4ug/ml) and cultivate 12hr, change 0.5%FBS MEM culture fluid and cultivate 6hr, change the normal cell culture fluid then, add MOI simultaneously respectively and be 100 AdHS4.AFP.E1a/TRAIL, dl1520 and culture fluid contrast, infect after 30 hours collecting cell, cell lysis (1%NP40,20mM Hepes, PH7.4,2mM EDTA, protease inhibitor cocktail (Roche)), 4 ℃ of its supernatants of centrifugal back carry out protein content and detect (Pierce Biotechnology), 30-50 μ g is used for CaspaceTM and detects the activity that the colorimetric reagent box detects Caspase, and substrate is Ac-DEVD-pNA (Promega), detects through the 405nm light absorption value and obtains the result, see Fig. 4, apoptotic effect is that the free pNA thing of pmol/mg/min is represented by vertical coordinate unit.

The antitumor action research of EXAMPLE III recombinant adenovirus AdHS4.AFP.E1a/TRAIL in lotus hepatocarcinoma transplanted tumor nude mice

Recombinant adenovirus AdHS4.AFP.E1a/TRAIL is Antitumor Effects in the lotus human liver cancer cell BEL-7404 tumor nude mice in vivo: the BALB/C nude mice in 6 ages in week, inoculation 1 * 107BEL-7404 cell suspending liquid 100 μ l.After becoming tumor, when transplanted tumor is grown to 80-200mm3, carry out random packet, every group of 7-12 nude mice, PBS, AdHS4.AFP.E1a/TRAIL (1.0 * 107 TCID50/mm3), dl1520 (1.0 * 107 TCID50/mm3), or DDP is injected directly in the transplanted tumor tumor, and volume injected is 50 μ l, injects continuously 5 days, DDP injected 7 days continuously, gross tumor volume calculates by the detection of diameter of tumor, measures weekly 2 times, puts to death all tumor bearing nude mices in the time of the 31st day, the gross tumor volume of injection day is set at 100%, compare with negative control group, other 3 groups all have remarkable tumor-inhibiting action, the results are shown in Figure 5.

Although the present invention for the ease of clearly understanding by way of example mode describe in detail in some aspects, it is feasible obviously carrying out certain variation and modification within the scope of the claims.

Claims (12)

1. recombinant adenoviral vector, it is by the adenovirus vector construct of having deleted E1 and/or E3 district gene, E1 district promoter is replaced as exogenous promoter, this exogenous promoter sequence upstream has separaant, in order to strengthen the specificity of exogenous promoter, described recombinant adenoviral vector carry can specificity duplicates and expresses in tumor the insertion gene, described insertion gene comprises and is subjected to the internal ribosome entry site adenovirus E 1 a gene apoptosis induction ligand gene relevant with tumor necrosis factor regulation and control, that can express respectively simultaneously.

2. adenovirus vector described in the claim 1, form by following elements:

A) terminal repeat is inverted in the adenovirus left side,

B) adenovirus packaging sequence is positioned at the downstream 3 ' end that terminal repeat is inverted in described a) left side,

C) separaant sequence is positioned at described b) downstream of adenovirus packaging sequence 3 ' end,

D) exogenous promoter and regulating and controlling sequence thereof are positioned at described c) downstream of separaant sequence 3 ' end,

E) E1a gene order is positioned at described d) the downstream 3 ' end of exogenous promoter and regulating and controlling sequence thereof, and regulated and control by this exogenous promoter,

F) the internal ribosome entry site gene order of external source is positioned at described e) downstream of E1a gene order 3 ' end, be used to control when gene order is inserted in its downstream and express respectively,

G) the relevant apoptosis induction ligand gene order of tumor necrosis factor is positioned at described f) the downstream 3 ' end of internal ribosome entry site, and controlled by this controlling gene, with described e) the E1a gene order expresses respectively simultaneously,

H) insert gene order and express termination site poly adenosine sequence, be positioned at described g) the downstream 3 ' end of the apoptosis induction ligand gene order that tumor necrosis factor is relevant,

I) one or more adenoviral gene sequences, hold in the downstream 3 ' that is positioned at above-mentioned poly adenosine sequence,

J) terminal repeat is inverted on the adenovirus right side, is positioned at 3 ' end of recombinant adenovirus virus gene.

3. adenovirus vector described in the claim 1, its exogenous promoter that has are the relevant specificity promoters of tumor.

4. adenovirus vector described in the claim 1, its exogenous promoter that has is an afp promoter.

5. adenovirus vector described in the claim 4, the external source afp promoter sequence that it has is made of enhancer sequence, silencer sequence and three componentries of promoter core space sequence.

6. adenovirus vector described in the claim 5, enhancer sequence repeats 2 times in the external source afp promoter sequence that it has, and silencer sequence repeats 6 to 8 times.

7. any described adenovirus vector among the claim 1-6, the betaglobulin separaant gene order that wherein said separaant sequence is a chicken.

8. the adenovirus vector described in the claim 7, it is that to be deposited in Chinese typical culture collection center deposit number be the adenovirus vector of V2003010.

9. method of selectivity kill tumor cell in vitro, this method comprises by any described recombinant adenoviral vector among the aforesaid right requirement 1-8 is contacted with the cell colony that carries of tumor cell, produces a kind of cell colony of infected tumor cell.

10. pharmaceutical composition that is used for the kill tumor cell, it contains any described recombinant adenoviral vector among the aforesaid right requirement 1-8.

11. the pharmaceutical composition described in the claim 10, wherein said tumor cell comprises:

A) tumor cell that contacts with immunosuppressant;

B) tumor cell that contacts with the radiation treatment thing; Or

C) tumor cell that contacts with chemotherapy compound.

12. any described recombinant adenoviral vector is used for the treatment of purposes in the pharmaceutical composition of tumor in preparation among the claim 1-8.

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