CN101892261A

CN101892261A - Recombinant adenovirus carrier and application thereof

Info

Publication number: CN101892261A
Application number: CN2009100845774A
Authority: CN
Inventors: 付国斌; 马大龙; 程书钧; 高燕宁; 郑宏伟; 石太平; 郭金海
Original assignee: Sinogenomax Co Ltd; Cancer Hospital and Institute of CAMS and PUMC
Current assignee: Sinogenomax Co Ltd; Cancer Hospital and Institute of CAMS and PUMC
Priority date: 2009-05-18
Filing date: 2009-05-18
Publication date: 2010-11-24

Abstract

The invention discloses a recombinant adenovirus carrier and application thereof. A plasmid of the recombinant adenovirus carrier is a pAdxsi plasmid carrying a DNA fragment (a) or (b): (a) a DNA fragment of a coding gene which contains a CMV promoter and DENND2D protein from the upstream to the downstream, and (b) a DNA fragment of a coding gene which contains a tetracycline reactive factor, a promoter, and a DENND2D protein from the upstream to the downstream. Two recombinant adenoviruses both have inhibiting effects on various tumor cell lines in vitro and in vivo and have a certain genetic therapy value.

Description

Recombinant adenoviral vector and application thereof

Technical field

The present invention relates to recombinant adenoviral vector and application thereof.

Background technology

Malignant tumour is the primary lethal factor of China at present, the serious harm people ' s health.Although the state of the art of medical science diagnosis and treatment in recent years is significantly improved, comprise that radiotherapy and chemotherapy means have all obtained rapid progress, but most solid tumors are prognosis mala still, the malignant tumor patient five year survival rate is still very low, as carcinoma of the pancreas (4%), liver cancer (7%), lung cancer (15%), press for new treatment means and remove the patient suffering, improve prognosis (Cross, D.and J.K.Burmester, Gene therapy for cancer treatment:past, present andfuture.Clin Med Res, 2006.4 (3): p.218-27.).Generation development to malignant tumour has in the last thirty years had comparatively deep understanding, present most scholar thinks that canceration is the process that takes place in the following multistage of comprehensive action of extraneous factor and inherited genetic factors, inactivation (the Vogelstein that comprises a plurality of oncogene active and cancer suppressor gene, B.and K.W.Kinzler, Cancer genes and the pathways they control.Nat Med, 2004.10 (8): p.789-99.), based on this understanding, the expression that recovers cancer suppressor gene by certain means is the most popular field of present malignant tumour gene therapy, existing a plurality of clinical experiments at cancer suppressor gene have obtained inspirer result, comprise p53, PTEN, FHIT (Moon, C., Y.Oh, and J.A.Roth, Current status of gene therapy for lung cancer and head and neck cancer.Clin Cancer Res, 2003.9 (14): p.5055-67.) etc.

In the gene therapy bio-carrier, replication-defective adenoviral vector has the advantage that high efficiency of infection, replicative phase and resting cell all can infect, unconformability is gone into the host cell gene group, security is high than the other biological carrier, therefore become the selection (Shirakawa of present most clinical experiments, T., The current status ofadenovirus-based cancer gene therapy.Mol Cells, 2008.25 (4): p.462-6.).Especially what deserves to be mentioned is China Shenzhen match the recombinant adenoviral vector of the p53 of hundred promise biotech companies ratified the listing (trade name: Gendicine), it is first therapy of tumor medicine in the world, median survival interval and the five year survival rate of using p53 recombinant adenovirus treatment group obviously are better than control group, benefited (the Peng of very many tumour patients has been arranged at present, Z., Current status of gendicine in China:recombinant humanAd-p53 agent for treatment of cancers.Hum Gene Ther, 2005.16 (9): p.1016-27.) (Kim, S., Z.Peng, and Y.Kaneda, Current status of gene therapin Asia.Mol Ther, 2008.16 (2): p.237-43.).

DENND2D is the lung cancer relative new gene that clone from lung squamous cancer differential expression cDNA library in laboratory, contriver place in 2006, it is positioned at karyomit(e) 1p13.1, the amino acid that 468 residues of encoding are formed, bioinformation predicts that it has conservative DENN structural domain, no clear signal peptide and membrane-spanning domain (Zheng Hongwei, evaluation and the functional study of new candidate tumor suppressor gene DENND2D. the doctorate paper, 2006.p.20-26).

Summary of the invention

The purpose of this invention is to provide recombinant adenoviral vector and application thereof.

Recombinant adenoviral vector plasmid provided by the invention is to insert following (a) or (b) recombinant plasmid that obtains of described dna fragmentation in the multiple clone site of pAdxsi plasmid (Fig. 2):

(a) contain the dna fragmentation of CMV promotor and the proteic encoding gene of DENND2D successively to the downstream from the upstream;

(b) dna fragmentation of tsiklomitsin response factor (TRE), promotor and the proteic encoding gene of DENND2D is contained in upstream to downstream successively.

Described DENND2D albumen can be following 1) or 2) protein:

1) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 2;

2) with the aminoacid sequence of sequence 2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have identical function by sequence 2 deutero-protein.

The proteic encoding gene of described DENND2D can be following 3) or 4) or 5) dna molecular:

3) its nucleotide sequence is the dna molecular shown in the sequence 1 in the sequence table;

4) under stringent condition with 3) the dna sequence dna hybridization and the coding identical function protein DNA molecule that limit;

5) with sequence table in the dna sequence dna that limits of sequence 1 have 90% above homology, and the identical function protein DNA molecule of encoding;

In the described dna fragmentation (b), described tsiklomitsin response factor (TRE) can be following 6) or 7) or 8) dna molecular:

6) its nucleotide sequence is the dna molecular shown in the sequence 3 in the sequence table;

7) under stringent condition with 6) dna sequence dna hybridization that limits and dna molecular with identical function;

8) with sequence table in the dna sequence dna that limits of sequence 3 have 90% above homology, and have the dna molecular of identical function.

In the described dna fragmentation (b), described promotor can be following 9) or 10) or 11) dna molecular:

9) its nucleotide sequence is the dna molecular shown in the sequence 4 in the sequence table;

10) under stringent condition with 9) dna sequence dna hybridization that limits and dna molecular with identical function;

11) with sequence table in the dna sequence dna that limits of sequence 4 have 90% above homology, and have the dna molecular of identical function.

The present invention also protects the recombinant adenovirus for preparing with described recombinant adenoviral vector plasmid.Described recombinant adenovirus can be by obtaining with described recombinant adenoviral vector transfection mammalian cell.Wherein, in the recombinant adenovirus that obtains with the pAdxsi plasmid transfection mammalian cell that carries (b) described dna fragmentation, the proteic encoding gene of described DENND2D can abduction delivering.

The present invention also protects a kind of recombinant adenovirus abduction delivering system, comprises Ad-teton plasmid and described recombinant adenoviral vector plasmid; Described recombinant adenoviral vector plasmid is the pAdxsi plasmid that carries (b) described dna fragmentation.With the common transfection mammalian cell of described recombinant adenovirus abduction delivering system, can induce the expression of the proteic encoding gene of DENND2D by tsiklomitsin or doxycycline.

Described mammalian cell specifically can be human embryonic kidney cell line HEK293 cell.

The medicine that described recombinant adenoviral vector plasmid, described recombinant adenovirus, described recombinant adenovirus expression system all can be applicable to prepare the treatment tumour and/or suppress tumor cell proliferation and/or inducing apoptosis of tumour cell.

The present invention also protects and a kind ofly treats tumour and/or suppress tumor cell proliferation and/or the medicine of inducing apoptosis of tumour cell, and its activeconstituents is at least a in the following material: described recombinant adenoviral vector plasmid, described recombinant adenovirus and described recombinant adenovirus expression system.

Described tumour can be common solid tumor, as the squama cancer, and common pathology somatotype such as gland cancer.Described squama cancer specifically can be lung squamous cancer.Described gland cancer specifically can be adenocarcinoma of lung.

Described tumour cell specifically can be H1299 cell, H520 cell or A549 cell.

The present invention has made up two kinds of recombinant adenoviral vectors of DENND2D.Two kinds of recombinant adenovirus all show restraining effect to kinds of tumor cells system in vivo and in vitro, have certain genetic therapy value.

Description of drawings

Fig. 1 is the structural representation of pShuttle-CMV shuttle vectors.

Fig. 2 is the structural representation of pAdxsi adenovirus skeleton carrier.

Fig. 3 cuts qualification result for the enzyme of pAdxsi-DENND2D.

Fig. 4 is that the Western blot of 48 hours total proteins of Ad-DENND2D cells infected detects.

Fig. 5 is the apoptosis-induced effect detected result of Ad-DENND2D.

Fig. 6 is the general form of each treatment group nude mice among the embodiment 3.

Fig. 7 is the tumor growth curve of each treatment group among the embodiment 3.

Fig. 8 is for respectively handling the main organs HE sections observation of treated animal among the embodiment 4.

Fig. 9 is the structural representation of pShuttle-TRE-T shuttle vectors.

Figure 10 cuts qualification result for the enzyme of pAdxsi-TRET-DENND2D.

Figure 11 is among the embodiment 5, and the Western blot of 72 hours total proteins of Ad-TRET-DENND2D cells infected detects.

Figure 12 is among the embodiment 6, and the phenotype of H1299 cell changes after 48 hours.

Figure 13 is among the embodiment 6, Western blot detected result.

Figure 14 is Flow cytometry apoptosis result (48 hours) among the embodiment 6.

Figure 15 is Flow cytometry apoptosis result (72 hours) among the embodiment 6.

Figure 16 is among the embodiment 6, apoptosis-induced time dose-dependent effect.

Figure 17 is among the embodiment 6, Flow cytometry H520 apoptosis result (48 hours).

Figure 18 is among the embodiment 6, Flow cytometry A549 apoptosis result (48 hours).

Figure 19 is among the embodiment 6, and Western blot detects the result of 3 kinds of clone endogenous expression DENND2D.

Embodiment

Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment if no special instructions, is ordinary method.Used test materials among the following embodiment if no special instructions, is to buy from routine biochemistry reagent shop and obtains.

The preparation of embodiment 1, Ad-DENND2D recombinant adenovirus

PShuttle-CMV shuttle vectors (see figure 1): Nuo Sai genome company.

PAdxsi adenovirus skeleton carrier (see figure 2): Nuo Sai genome company.

One, pAdxsi-DENND2D construction of recombinant plasmid

1, the structure of DENND2D recombinant shuttle vector

The primer that designs the DENND2D gene that has the SfiI restriction endonuclease sites is as follows:

DENND2D?Sfi?I?up：5’-AAAAAAGGCCGCTGCGGCCCACTCCAGGGGCCATGGATG-3’；

DENND2D?Sfi?I?dw：5’-AAAAAAGGCCTGTTTGGCCGTCATTCTTATTCACCACAGCTC-3’。

1. obtain the full length coding region of purpose DENND2D gene with above-mentioned primer PCR from human lung tissue cDNA library (available from Clontech company), after checking order correctly, cut the PCR product 3-4 hour with 37 ℃ of enzymes of restriction enzyme SfiI (NEB company).Reclaim the fragment of 1.4kb.

2. cut the pShuttle-CMV shuttle vectors 3-4 hour with 37 ℃ of enzymes of restriction enzyme SfiI, use calf intestinal alkaline phosphatase (CIP, Promega company) dephosphorylation to handle carrier 0.5 hour then.Reclaim the fragment of 3.4kb.

3. the 3.4kb fragment that 2. 1.4kb fragment and the step that 1. step is reclaimed reclaim is connected with the T4DNA ligase enzyme.

4. with step connection product transformed into escherichia coli DH5 α competence 3., that mycin solid medium flat board (the pShuttle-CMV shuttle vectors carries kalamycin resistance gene) of card-coating.

5. select positive colony and carry out the recombinant plasmid amplification purification, obtain recombinant plasmid I (DENND2D recombinant shuttle vector).

2, the structure of pAdxsi-DENND2D

1. utilize restriction enzyme I-CeuI and I-SceI (NEB company) double digestion recombinant plasmid I, reclaim the fragment of 2.5kb.

2. utilize restriction enzyme I-CeuI and I-SceI (NEB company) double digestion pAdxsi adenovirus skeleton carrier, reclaim the fragment of 30kb.

3. the 30kb fragment that 2. 2.5kb fragment and the step that 1. step is reclaimed reclaim is connected with the T4DNA ligase enzyme.

4. step connection product is 3. transformed DH5 α competence, be coated with penbritin solid medium flat board (pAdxsi adenovirus skeleton carrier carries ampicillin resistance gene).

5. select the positive colony amplification purification.

Positive colony is carried out enzyme cut evaluation.After purpose recombinant plasmid XhoI enzyme is cut following big or small fragment should be arranged: 14kb, 11.8kb, 2.66kb, 2.47kb, 2.4kb, 1.45kb, 0.6kb.What the enzyme of positive colony was cut evaluation the results are shown in Figure 3.Among Fig. 3, M:Marker (1kb DNA ladder) from top to bottom is followed successively by: 8kb, 7kb, 6kb, 5kb, 4kb, 3kb, 2kb, 1.6kb, 1kb, 517bp, 396bp, 230bp; 1-4: positive colony.Wherein, 14kb and 11.8kb fragment are too big, and electrophoresis is not easily distinguishable, and 2.47kb and 2.4kb clip size are too near also not being easily distinguishable.The result shows and has obtained the purpose recombinant plasmid, with its called after pAdxsi-DENND2D.

PAdxsi-DENND2D increases pAdxsi-DENND2D and purifying in a large number for the recombinant replication-defective type Ad5 carrier that the CMV promotor starts.

Two, the preparation of Ad-DENND2D recombinant adenovirus

1, virus packing

Select human embryonic kidney cell line's HEK293 cell (containing the required E1 of adenoviral replication district gene) (available from ATCC) as package cell line.

Get 5 μ g pAdxsi-DENND2D, after the linearizing of PacI restriction enzyme, utilize Lipofectamine2000 lipofectamine (Invitrogen company) transfection about 40 * 10 ⁴HEK293 cell (35mm plate); Change liquid after 6 hours; Test is provided with two repetitions.

Every day, observation of cell went out malicious sign.Going out malicious phenomenon is: cell becomes the big circle that becomes, be botryoidalis, and begin to occur obvious plaque, this be cytopathic effect (cytopathic effect, CPE).Treat the most of pathology of cell and when the bottom comes off, receive poison.Collect the cell and the nutrient solution of two plates and put into-80 ℃ of refrigerators.Cellular products is through dry ice alcohol and 37 ℃ of water-bath multigelations three times, smudge cells, releasing virus.Centrifugal 5 minutes of 3000rpm collects the supernatant liquor that contains virus, is the first-generation seed culture of viruses (P1) of Ad-DENND2D recombinant adenovirus.

2, virus amplification

Get 2ml P1 for viral supernatant, infect a 75cm ²The HEK293 cell of culturing bottle (cell density＞90%) treats that all cells bottom surface (after about 48 hours) that comes off can receive poison, collecting cell and substratum, and 2000rpm discarded supernatant in centrifugal 5 minutes.Add 1ml ST buffer (perfect medium+2.5% glycerine), be stored in-80 ℃ of refrigerators behind the mixing.Next generation's amplification is carried out in freeze thawing three times, centrifuging and taking supernatant or in-80 ℃ of preservations, this is the s-generation seed culture of viruses (P2) of Ad-DENND2D recombinant adenovirus.

Remain 50 μ l and preserve, get remaining whole P2 and infect 6 75cm for viral supernatant ²The cell of culturing bottle, cell detachment after about 48 hours, collecting cell adds 6ml ST buffer (perfect medium+2.5% glycerine), receives poison (process is the same) behind the mixing.With the viral supernatant of this batch collection, infect 40 75cm ²The cell of culturing bottle, it is the same to receive malicious method.

3, viral purification

Adopt cesium chloride (CsCl) density gradient centrifugation purified virus, concrete steps are as follows:

(1) the centrifugal removal cell contamination of discontinuous density gradient thing

(53g CsCl is dissolved in 87ml PH8.010mmol/L Tris-Cl to get 8ml CsCl 1.4 solution, filtration sterilization) adds in the centrifuge tube, (26.8g CsCl is dissolved in 92ml PH8.010mmol/L Tris-Cl to soft slowly adding 10ml CsCl 1.2 solution, filtration sterilization), top slowly adds the viral solution (10mMTris-Cl polishing) that the step 2 of about 20ml obtains.4 ℃ of 100000g are centrifugal 90 minutes.After the centrifugal end, the visible vaporific band of pearl opal, puncture sucking-off virus solution, with 10mM Tris-Cl solution dilution to 8ml.

(2) centrifugal differentiation infective virus of continuous density gradient and defective virus

Get 12ml CsCl 1.4 solution and add in the centrifuge tube, soft 14ml CsCl 1.2 solution that slowly add, its top adds 4 ℃ of centrifugal 16-20 of viral solution, 100000g hours that about 8ml step (1) obtains very slowly.The blue leukovirus band of centrifugal end back puncture sucking-off.

(3) CsCl is removed in dialysis

Step (2) gained virus liquid with 4 ℃ of dialysis of dialysis card (available from Pierce company) 16 hours, is changed liquid 3 times at interval, and dialysis buffer is: 10mM PH8.0Tris-Hcl, 2mM Mgcl ₂, 4%Sucrose.With the viral liquid that obtains as virus stock solution used.

4, virus titer is measured

The OD260/OD280 of the viral liquid that ultraviolet spectrophotometry step 3 obtains, the purity of this ratio reaction virus, ratio is 1.24, between normal range 1.2-1.3.Get virus stock solution used and establish 8 10 times dilution gradients, infect the HEK293 cell of cultivating in 96 orifice plates respectively, observation of cell pathology effect.The viral dilution degree of 100% pathology effect to occur, bring formula into and obtain the virus infection titre.

The viral volume (mL) of virus titer (pfu/ml)=(inoculating cell number * extent of dilution * 10)/adding, supposition has 10 viruses can cause cell complete pathology to occur at virus infection in the formula in each cell after 48 hours.

The titre that finally records Ad-DENND2D is 4 * 10 ¹¹Pfu/ml.

Three, contrast the preparation of viral I (Adv)

1, the preparation of empty carrier control plasmid I

1. utilize restriction enzyme I-CeuI and I-SceI (NEB company) double digestion pShuttle-CMV shuttle vectors, reclaim the fragment of 1.1kb.

3. the 30kb fragment that 2. 1.1kb fragment and the step that 1. step is reclaimed reclaim is connected with the T4DNA ligase enzyme.

5. select the positive colony amplification purification.

Obtain empty carrier control plasmid I.

2, the preparation of the viral I of contrast

Replace the pAdxsi-DENND2D plasmid with empty carrier control plasmid I, other same step 2 contrasts viral packing, amplification, purifying and titer determination.

Four, the evaluation of Ad-DENND2D recombinant adenovirus

If 3 Ad-DENND2D virus infection concentration (Moi value (multiplicity of infection) is respectively: moi50, moi100, moi200), with Ad-DENND2D (or Adv of moi100) infected person lung cancer cell line H1299 cell, extract the clone total protein after 48 hours and carry out Western blot detection.Get 40 μ g total proteins, change film behind the SDS-PAGE electrophoresis, detect the expression of DENND2D with the special antibody of DENND2D (the rabbit source polyclonal antibody that obtains with DENND2D immunity new zealand rabbit, 1: 1000).The results are shown in Figure 4.The result shows, infects the cell background of Adv and does not express, and the cell that infects Ad-DENND2D detects the expression of DENND2D after 48 hours, and with the increase of moi value, the expression enhancing of DENND2D.

The external evoked apoptotic effect of embodiment 2, Ad-DENND2D recombinant adenovirus

According to the dosage of the dosage of moi 100 or moi 200 infected person lung cancer cell line H1299 cell respectively, collecting cell carries out Annexin V-FITC and PI is two dyes after 48 hours with Ad-DENND2D (or Adv), and a situation arises for the Flow cytometry apoptosis.Test is provided with three repetitions, results averaged.

The results are shown in Figure 5 in (shown in the figure for wherein a result of experiment).After the dosage infection with moi100, Ad-DENND2D group apoptosis rate is 17.09 ± 3.22%, is significantly higher than Adv group 11.36 ± 1.25 (P＜0.05).After the dosage infection with moi200, Ad-DENND2D group apoptosis rate is 21.76 ± 1.99%, is significantly higher than Adv group 14.06 (P＜0.05).For the cell that infects Ad-DENND2D, the apoptosis rate of moi200 dosage group is higher than moi100 dosage group, has significant difference (P＜0.05), and there is the dose-effect relationship of positive in the apoptosis-induced effect of prompting Ad-DENND2D.

Tumor-inhibiting action in the body of embodiment 3, Ad-DENND2D recombinant adenovirus

1, selects 24 of the female BALB/c nude mices (available from dimension tonneau China company) in 3～4 ages in week; Collect the H1299 cell (available from ATCC) of logarithmic phase, adjusting cell concn is 2.5 * 10 ⁷/ ml.

2, it is subcutaneous behind the right armpit of nude mice to get 200 μ l cell suspension inoculations, treats about 10 days that tumor growth to 5 * 5mm is divided into four groups during size, 6 every group, carries out following processing respectively:

Physiological saline injection group (NS): in the injecting normal saline, per three days knurls multi-point injection once, each 50 μ l, totally three times.

Adv virus group (Adv): adjusting the Adv virus titer is 2 * 10 ¹⁰Pfu/ml, in per three days knurls multi-point injection once, each 50 μ l, totally three times, accumulative total virus injection amount is 3 * 10 ⁹Pfu.

P53 virus group (Ad-p53): adjusting Ad-p53 virus (available from promise match genome company limited) titre is 2 * 10 ¹⁰Pfu/ml, in per three days knurls multi-point injection once, each 50 μ l, totally three times, accumulative total virus injection amount is 3 * 10 ⁹Pfu.

Ad-DENND2D virus group (Ad-DENND2D): adjusting the Ad-DENND2D virus titer is 2 * 10 ¹⁰Pfu/ml, in per three days knurls multi-point injection once, each 50 μ l, totally three times, accumulative total virus injection amount is 3 * 10 ⁹Pfu.

Since the intratumor injection first time, measure transplanted tumor every other day once, record tumour major diameter (a) and minor axis (b) are with reference to formula gross tumor volume V=a * b ²/ 2, calculate the tumour size, draw the growth of xenografted curve.Since the intratumor injection first time, after injecting (on February 26th, 2009) 24 days for the first time, finish experiment, each is organized the form of nude mice and sees Fig. 6.Each is organized the tumor growth curve of nude mice and sees Fig. 7.From growth curve as can be seen the tumor growth of Ad-p53 group and Ad-DENND2D group nude mice lag behind the NS group and Adv organizes.

4 weeks back execution nude mice is got the subcutaneous transplantation knurl, weighs, and calculates the tumour inhibiting rate of injecting virus, and calculation formula is heavy for (it is heavy that NS organizes the average knurl of average knurl weight-experimental group)/NS organizes average knurl.It is 0.20 ± 0.14g heavily that Ad-DENND2D organizes average knurl, and tumour inhibiting rate is about 52.76%, with Ad-p53 group (average knurl is 0.19 ± 0.13g heavily, and tumour inhibiting rate is about 55.91%) no difference of science of statistics (P＞0.05).And Ad-p53 has applied clinically as gene therapy medicament, and prompting Ad-DENND2D recombinant adenovirus has a good application prospect.

Safety evaluation in the body of embodiment 4, Ad-DENND2D recombinant adenovirus

Get 64 female BALB/c nude mices in age in week (available from dimension tonneau China), be divided into two groups, 3 every group.

Physiological saline group (NS): every through tail vein injection saline 100 μ l;

Ad-DENND2D organizes (Ad-DENND2D): virus titer is adjusted into 5 * 10 ¹⁰Pfu/ml, (every injected dose is 5 * 10 through every injection of tail vein 100 μ l reorganization Ad-DENND2D ⁹Pfu).

Observe reaction of animals every day.Finish experiment after 7 days, get the embedding of whole body major organs and fix, system HE section, pathological analysis the results are shown in Figure 8.

Growth of animal shows no obvious abnormalities behind the virus injection, and the whole body main organs is not found pathological changes such as obvious necrosis, inflammatory infiltration; Prompting Ad-DENND2D has certain biological safety.

The structure of embodiment 5, Ad-TRET-DENND2D

Tsiklomitsin abduction delivering system (tet-on) is made up of two portions: the destination gene expression and the antisense tetracycline active element rtTA of tsiklomitsin response element TRE control.(doxycycline, when Dox) existing, rtTA activates and is bonded to the TRE element, starts downstream gene and transcribes when the tsiklomitsin or derivatives thereof.

The pShuttle-TRE-T shuttle vectors (see figure 9) that has tsiklomitsin response element TRE: Nuo Sai genome company.

The Ad-teton recombinant adenovirus plasmid of antisence tsiklomitsin active element rtTA: Nuo Sai genome company.

One, pAdxsi-TRET-DENND2D construction of recombinant plasmid

1, the structure of DENND2D reorganization pShuttle-TRE-T shuttle vectors (recombinant plasmid I I)

1. designing the upstream has Kpn I downstream to have the primer of Xba I restriction enzyme site as follows, with the normal lung tissue cDNA library available from Clontech company is template, pcr amplification obtains purpose DENND2D fragment, after checking order correctly, with restriction enzyme Kpn I and Xba I double digestion 37 ℃, 4 hours, reclaim the fragment of 1.4kb.

DENND2D?Kpn?I?up：5’CGGGGTACCCACTCCAGGGGCCATGGATG；

DENND2D?Xba?I?dw：5’TGCTCTAGAGTCATTCTTATTCACCACAGCTC。

2. use restriction enzyme KpnI and XbaI double digestion pShuttle-TRE-T shuttle vectors (37 ℃, 4 hours).Reclaim the fragment of 2.9kb.

3. the 2.9kb fragment that 2. 1.4kb fragment and the step that 1. step is reclaimed reclaim is connected with the T4DNA ligase enzyme.

4. with step connection product transformed into escherichia coli DH5 α competence 3., that mycin solid medium flat board (the pShuttle-TRET carrier carries kalamycin resistance gene) of card-coating.

5. select positive colony and carry out the recombinant plasmid amplification purification, obtain recombinant plasmid I I (DENND2D reorganization pShuttle-TRET shuttle vectors).

2, the structure of pAdxsi-TRET-DENND2D

1. utilize restriction enzyme I-CeuI and I-SceI (NEB company) double digestion recombinant plasmid I I, reclaim the fragment of 2kb.

3. the 30kb fragment that 2. 2kb fragment and the step that 1. step is reclaimed reclaim is connected with the T4DNA ligase enzyme.

5. select the positive colony amplification purification.

Positive colony is carried out enzyme cut evaluation.After purpose recombinant plasmid XhoI enzyme is cut following big or small fragment should be arranged: 14kb, 11.8kb, 2.5kb, 2.47kb, 1.9kb, 1.45kb, 0.6kb.The enzyme of positive colony is cut the Figure 10 that the results are shown in of evaluation.Among Figure 10, M:Marker (1kb DNA ladder) from top to bottom is followed successively by: 8kb, 7kb, 6kb, 5kb, 4kb, 3kb, 2kb, 1.6kb, 1kb, 517bp, 396bp, 230bp; 1-4: positive colony.Wherein, 14kb and 11.8kb fragment are too big, and electrophoresis is not easily distinguishable, and 2.5kb and 2.47kb clip size are too near also not being easily distinguishable.4 positive colonies are the purpose recombinant plasmid, with its called after pAdxsi-TRET-DENND2D.

PAdxsi-TRET-DENND2D is the recombinant replication-defective type Ad5 carrier of tsiklomitsin abduction delivering, and pAdxsi-TRET-DENND2D is increased and purifying in a large number.

Two, the preparation of Ad-TRET-DENND2D recombinant adenovirus

1, virus packing

Select human embryonic kidney cell line's HEK293 cell (containing the required E1 of adenoviral replication district gene) as package cell line.

Get 5 μ g pAdxsi-TRET-DENND2D plasmids, after the linearizing of PacI restriction enzyme, utilize Lipofectamine2000 lipofectamine (Invitrogen company) transfection about 40 * 10 ⁴HEK293 cell (35mm plate); Change liquid after 6 hours; Test is provided with two repetitions.

Every day, observation of cell went out malicious sign.Going out malicious phenomenon is: cell becomes the big circle that becomes, be botryoidalis, and begin to occur obvious plaque, this be cytopathic effect (cytopathic effect, CPE).Treat the most of pathology of cell and when the bottom comes off, receive poison.Collect the cell and the nutrient solution of two plates and put into-80 ℃ of refrigerators.Cellular products is through dry ice alcohol and 37 ℃ of water-bath multigelations three times, smudge cells, releasing virus.Centrifugal 5 minutes of 3000rpm collects the supernatant liquor that contains virus, is the first-generation seed culture of viruses (P1) of Ad-TRET-DENND2D recombinant adenovirus.

2, virus amplification

Get 2ml P1 for viral supernatant, infect a 75cm ²The HEK293 cell of culturing bottle (cell density＞90%) treats that all cells bottom surface (after about 48 hours) that comes off can receive poison, collecting cell and substratum, and 2000rpm discarded supernatant in centrifugal 5 minutes.Add 1ml ST buffer (perfect medium+2.5% glycerine), be stored in-80 ℃ of refrigerators behind the mixing.Next generation's amplification is carried out in freeze thawing three times, centrifuging and taking supernatant or in-80 ℃ of preservations, this is the s-generation seed culture of viruses (P2) of Ad-TRET-DENND2D recombinant adenovirus.

Remain 50 μ l and preserve, get remaining whole P2 and infect 6 75cm for viral supernatant ²The cell of culturing bottle, cell detachment after about 48 hours, collecting cell adds 6ml ST buffer (perfect medium+2.5% glycerine), receives poison (process is the same) behind the mixing.With the viral liquid that obtains as virus stock solution used.

3, virus titer is measured

The OD260/OD280 of the viral liquid that ultraviolet spectrophotometry step 3 obtains, the purity of this ratio reaction virus, ratio is 1.27, between normal range 1.2-1.3.Get virus stock solution used and establish 8 10 times dilution gradients, infect the HEK293 cell of cultivating in 96 orifice plates respectively, observation of cell pathology effect.The viral dilution degree of 100% pathology effect to occur, bring formula into and obtain the virus infection titre.

The viral volume (mL) of virus titer (pfu/ml)=(inoculating cell number * extent of dilution * 10)/adding.

Supposition has 10 viruses can cause cell complete pathology to occur at virus infection in the formula in each cell after 48 hours.

The titre of Ad-TRET-DENND2D is 1.5 * 10 ¹⁰Pfu/ml.

Three, the preparation of the viral II of contrast

1, the preparation of empty carrier control plasmid II

1. utilize restriction enzyme I-CeuI and I-SceI (NEB company) double digestion pShuttle-TRE-T shuttle vectors, reclaim the fragment of 0.6kb.

3. the 30kb fragment that 2. 0.6kb fragment and the step that 1. step is reclaimed reclaim is connected with the T4DNA ligase enzyme.

5. select the positive colony amplification purification.

Obtained empty carrier control plasmid II.

2, the preparation of the viral II of contrast

Replace the pAdxsi-TRET-DENND2D plasmid with empty carrier control plasmid II, other same step 2 contrasts viral packing, amplification and titer determination.

Four, the evaluation of Ad-TRET-DENND2D recombinant adenovirus

Tsiklomitsin abduction delivering adenovirus system need infect two kinds of viruses (Ad-teton and Ad-TRET-DENND2D) simultaneously, getting two kinds of viral ratios is 1: 1, if different total infective doses, with Ad-teton and Ad-TRET-DENND2D coinfection human lung cancer cell line H1299 cell together, add Dox after 4 hours in the substratum (available from Sima company, final concentration is 1 μ g/ml) abduction delivering, extract the clone total protein and carry out Western blot detection after 48 hours.Get 40 μ g total proteins, change film behind the SDS-PAGE electrophoresis, detect the expression of DENND2D with the special antibody of DENND2D (the rabbit source polyclonal antibody that obtains with the DENND2D immune rabbit, 1: 1000).

The results are shown in Figure 11.Among Figure 11,1-5: do not add the processing of DOX; 1: the cell that does not infect two kinds of viruses; 2: the cell (total infective dose is moi10) that infects two kinds of viruses; 3: the cell (total infective dose is moi20) that infects two kinds of viruses; 4: the cell (total infective dose is moi50) that infects two kinds of viruses; 5: the cell (total infective dose is moi100) that infects two kinds of viruses; 6-10: add the processing of DOX; 6: the cell that does not infect two kinds of viruses; 7: the cell (total infective dose is moi10) that infects two kinds of viruses; 8: the cell (total infective dose is moi20) that infects two kinds of viruses; 9: the cell (total infective dose is moi50) that infects two kinds of viruses; 10: the cell (total infective dose is moi100) that infects two kinds of viruses.The result shows: do not add the treatment group of Dox, the personal comments toxic agent amount of catching an illness increases and low-down leakage expression occurs; Add the treatment group of Dox, the expression of DENND2D obviously strengthens behind the infective virus, has played the effect of switch regulation and control, and its expression raises with viral dosage increase.

The apoptosis-induced effect of embodiment 6, Ad-TRET-DENND2D

One, to the effect of H1299 induced apoptosis

With Ad-teton and Ad-TRET-DENND2D (or contrasting viral II) according to 1: 1 ratio coinfection human lung cancer cell line H1299 cell, total moi value is 50, if Dox activity from low to high (0～1 μ g/ml), observation of cell changes after 48 hours, and Figure 12 is seen in the variation of the phenotype of H1299 cell after 48 hours.Among Figure 12, A: the H1299 cell that does not carry out any processing; B: the H1299 cell (concentration of Dox is 1 μ g/ml) that infects Ad-teton and the viral II of contrast; C: the H1299 cell (concentration of Dox is 0 μ g/ml) that infects Ad-teton and Ad-TRET-DENND2D; D: the H1299 cell (concentration of Dox is 0.01 μ g/ml) that infects Ad-teton and Ad-TRET-DENND2D; E: the H1299 cell (concentration of Dox is 0.1 μ g/ml) that infects Ad-teton and Ad-TRET-DENND2D; F: the H1299 cell (concentration of Dox is 1 μ g/ml) that infects Ad-teton and Ad-TRET-DENND2D.The result shows: follow the Dox activity to increase the H1299 cell and occur becoming circle, come off, and the necrocytosis ratio raises, and contrast virus adds high density Dox group and do not have similar phenotype.

Caspase3 is the apoptotic crucial molecule of carrying out.Usually the unactivated state of Caspase is the zymogen forms of total length, Caspase3 was cut by homology activation or allos activation and removes N end propetide when apoptosis took place, its active condition is generally two big subunits (about 20kD) and two small subunits (about 10kD) are formed, so the minimizing of full-length proteins ratio represents that generally Caspase transfers activation to by tranquillization, can shear downstream substrate PARP after the Caspase3 activation, PARP is cut into 85kD and two fragments of 29kD by 116kD.Extraction infection recombinant virus clone total protein after 72 hours, Western blot detects DENND2D, Caspase3 (1: 1000, Cell Signaling company) and PARP (Cell Signaling company).Western blot detected result is seen Figure 13.Among Figure 13, A: the H1299 cell that does not carry out any processing; B: the H1299 cell (concentration of Dox is 1 μ g/ml) that infects Ad-teton and the viral II of contrast; C: the H1299 cell (concentration of Dox is 0 μ g/ml) that infects Ad-teton and Ad-TRET-DENND2D; D: the H1299 cell (concentration of Dox is 0.01 μ g/ml) that infects Ad-teton and Ad-TRET-DENND2D; E: the H1299 cell (concentration of Dox is 0.1 μ g/ml) that infects Ad-teton and Ad-TRET-DENND2D; F: the H1299 cell (concentration of Dox is 1 μ g/ml) that infects Ad-teton and Ad-TRET-DENND2D.The result shows: the protein expression of DENND2D strengthens with the induced concentration rising of Dox, and the expression of the full-length proteins of Caspase3 weakens, the PARP shearing action significantly strengthens along with the abduction delivering of DENND2D simultaneously, the crucial activation of carrying out molecule Caspase3 of prompting apoptosis.

Add that Dox induced back 48 hours and 72 hours collecting cells carry out Annexin V-FITC and PI pair and dye, a situation arises for the Flow cytometry apoptosis.Test is provided with three repetitions, results averaged.48 hours detected result is seen Figure 14 (shown in the figure for wherein a result of experiment).72 hours detected result is seen Figure 15 (shown in the figure for wherein a result of experiment).Among Figure 14 and Figure 15, A: the H1299 cell that does not carry out any processing; B: the H1299 cell (concentration of Dox is 1 μ g/ml) that infects Ad-teton and the viral II of contrast; C: the H1299 cell (concentration of Dox is 0 μ g/ml) that infects Ad-teton and Ad-TRET-DENND2D; D: the H1299 cell (concentration of Dox is 0.01 μ g/ml) that infects Ad-teton and Ad-TRET-DENND2D; E: the H1299 cell (concentration of Dox is 0.1 μ g/ml) that infects Ad-teton and Ad-TRET-DENND2D; F: the H1299 cell (concentration of Dox is 1 μ g/ml) that infects Ad-teton and Ad-TRET-DENND2D.The result shows: transfection virus group H1299 apoptosis rate raises at 48 hours induced concentrations with Dox and rises to 18.78 ± 1.74% by 3.09 ± 1.31%, 72 hour cell apoptosis rates rise to 44.91 ± 1.73% with the induced concentration rising of Dox by 9.89 ± 1.03%, are significantly higher than 48 hours apoptosis incidences (P＜0.05).

According to the result of Flow cytometry, the apoptosis rate of different treatment is drawn, see Figure 16.Among Figure 16, A: the H1299 cell that does not carry out any processing; B: the H1299 cell (concentration of Dox is 1 μ g/ml) that infects Ad-teton and the viral II of contrast; C: the H1299 cell (concentration of Dox is 0 μ g/ml) that infects Ad-teton and Ad-TRET-DENND2D; D: the H1299 cell (concentration of Dox is 0.01 μ g/ml) that infects Ad-teton and Ad-TRET-DENND2D; E: the H1299 cell (concentration of Dox is 0.1 μ g/ml) that infects Ad-teton and Ad-TRET-DENND2D; F: the H1299 cell (concentration of Dox is 1 μ g/ml) that infects Ad-teton and Ad-TRET-DENND2D.The result shows: the DENND2D cell death inducing, and have the time dose-dependent effect of positive.

Two, to other induced apoptosis effect

Having selected lung squamous cancer and lung adenocarcinoma cell respectively is H520 clone (available from ATCC) and A549 clone (available from ATCC), observes the apoptosis-induced effect of Ad-TRET-DENND2D, the same step 1 of method.

H520 clone the results are shown in Figure 17 (total moi 200).A among Figure 17: the H520 cell that does not carry out any processing; B: the H520 cell (concentration of Dox is 1 μ g/ml) that infects Ad-teton and the viral II of contrast; C: the H520 cell (concentration of Dox is 0 μ g/ml) that infects Ad-teton and Ad-TRET-DENND2D; F: the H520 cell (concentration of Dox is 1 μ g/ml) that infects Ad-teton and Ad-TRET-DENND2D.The result shows: the apoptosis incidence is upgraded to 46.27 ± 21.99% of abduction delivering group (Dox 1 μ g/ml) by 9.09 ± 4.11% of the viral II of contrast.

A549 clone the results are shown in Figure 18 (total moi 100).A among Figure 18: the A549 cell that does not carry out any processing; B: the A549 cell (concentration of Dox is 1 μ g/ml) that infects Ad-teton and the viral II of contrast; C: the A549 cell (concentration of Dox is 0 μ g/ml) that infects Ad-teton and Ad-TRET-DENND2D; F: the A549 cell (concentration of Dox is 1 μ g/ml) that infects Ad-teton and Ad-TRET-DENND2D.The result shows: the apoptosis incidence is upgraded to 29.38 ± 9.93% of abduction delivering group (Dox 1 μ g/ml) by 14.80 ± 5.92% of the viral II of contrast; All has significant difference (P＜0.05).

Three, the detection that DENND2D expresses in the different cells

Detect in H1299 cell, H520 cell and the A549 cell the whether expression of DENND2D by Western blot.Detect confidential reference items GAPDH level with GAPDH antibody (1: 5000, health becomes biotech firm) simultaneously.

The results are shown in Figure 19.The result shows: the H520 cell has the expression of DENND2D, and H1299 and A549 cell do not detect the expression of DENND2D, points out the influence that inducing action is not subjected to DENND2D background expression of transferring to of Ad-TRET-DENND2D.

Above result shows that all it is that apoptosis takes place that Ad-TRET-DENND2D can induce multiple solid tumor cell, has the candidate gene medicine and is worth.

Sequence table

＜110〉Sinogenomax Co., Ltd. of Tumour Inst., Chinese Medical Academy

＜120〉recombinant adenoviral vector and application thereof

<130>CGGNARY92283

<160>4

<210>1

<211>1407

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>1

atggatgggc?tcggccgccg?ccttcgagcc?agtctgagac?tgaagcgcgg?ccatggggga 60

ccaccccagg?acaattcagg?ggaagcttta?aaggaaccag?aaagggccca?ggagcactct 120

ttgcccaact?ttgctggggg?gcagcacttc?tttgaatacc?ttcttgtggt?ttctctcaaa 180

aagaagcgtt?cagaggatga?ttacgagcct?ataatcacct?accaatttcc?caagcgggag 240

aacctgcttc?ggggtcagca?ggaggaggag?gagcggctgc?tcaaagctat?ccccttgttc 300

tgcttcccag?atgggaatga?gtgggcatca?ctcaccgagt?atcccaggga?gaccttctcc 360

ttcgttctga?ccaatgtgga?tgggagcaga?aagattggat?actgcaggcg?cctcttgcct 420

gccggccctg?gccctcgcct?tcccaaagtg?tactgcatca?tcagctgcat?cggctgcttc 480

ggcttgttct?ccaagatcct?ggatgaagtg?gagaagagac?atcagatctc?catggctgtc 540

atctacccgt?tcatgcaggg?cctccgagag?gcagccttcc?ctgctcctgg?gaagactgtc 600

actctcaaga?gcttcatccc?cgactcaggc?actgagttca?tttcactgac?acggcccctg 660

gactcccacc?tagaacatgt?ggattttagt?tctctattgc?actgtctcag?ttttgaacag 720

atacttcaga?tctttgcctc?tgccgtgctg?gagagaaaaa?tcatcttcct?ggcggaaggt 780

ctcagcacct?tgtctcagtg?catccatgct?gctgccgcac?tgctctaccc?cttcagctgg 840

gcgcacacct?acatccctgt?tgtccctgag?agccttctgg?ccaccgtctg?ctgccccacc 900

cccttcatgg?ttggagtaca?aatgcgcttc?cagcaggagg?tcatggacag?ccctatggaa 960

gaggtcctgc?tggtcaatct?ttgtgaagga?accttcttaa?tgtcggttgg?tgatgaaaaa 1020

gacatcctgc?caccgaagct?tcaggatgac?atcttagact?ctcttggtca?ggggatcaat 1080

gagttaaaga?ctgcagaaca?aatcaacgag?catgtttcag?gcccctttgt?gcagttcttt 1140

gtcaagattg?tgggccatta?tgcttcctat?atcaagcggg?aggcaaatgg?gcaaggccac 1200

ttccaagaaa?gatccttctg?taaggctctg?acctccaaga?ccaaccgccg?atttgtgaag 1260

aagtttgtga?agacacagct?cttctcactt?ttcatccagg?aagccgagaa?gagcaagaat 1320

cctcctgcag?gctatttcca?acagaaaata?cttgaatatg?aggaacagaa?gaaacagaag 1380

aaaccaaggg?aaaaaactgt?gaaataa 1407

<210>2

<211>468

<212>PRT

＜213〉artificial sequence

<220>

<223>

<400>2

Met?Asp?Gly?Leu?Gly?Arg?Arg?Leu?Arg?Ala?Ser?Leu?Arg?Leu?Lys?Arg

1 5 10 15

Gly?His?Gly?Gly?Pro?Pro?Gln?Asp?Asn?Ser?Gly?Glu?Ala?Leu?Lys?Glu

20 25 30

Pro?Glu?Arg?Ala?Gln?Glu?His?Ser?Leu?Pro?Asn?Phe?Ala?Gly?Gly?Gln

35 40 45

His?Phe?Phe?Glu?Tyr?Leu?Leu?Val?Val?Ser?Leu?Lys?Lys?Lys?Arg?Ser

50 55 60

Glu?Asp?Asp?Tyr?Glu?Pro?Ile?Ile?Thr?Tyr?Gln?Phe?Pro?Lys?Arg?Glu

65 70 75 80

Asn?Leu?Leu?Arg?Gly?Gln?Gln?Glu?Glu?Glu?Glu?Arg?Leu?Leu?Lys?Ala

85 90 95

Ile?Pro?Leu?Phe?Cys?Phe?Pro?Asp?Gly?Asn?Glu?Trp?Ala?Ser?Leu?Thr

100 105 110

Glu?Tyr?Pro?Arg?Glu?Thr?Phe?Ser?Phe?Val?Leu?Thr?Asn?Val?Asp?Gly

115 120 125

Ser?Arg?Lys?Ile?Gly?Tyr?Cys?Arg?Arg?Leu?Leu?Pro?Ala?Gly?Pro?Gly

130 135 140

Pro?Arg?Leu?Pro?Lys?Val?Tyr?Cys?Ile?Ile?Ser?Cys?Ile?Gly?Cys?Phe

145 150 155 160

Gly?Leu?Phe?Ser?Lys?Ile?Leu?Asp?Glu?Val?Glu?Lys?Arg?His?Gln?Ile

165 170 175

Ser?Met?Ala?Val?Ile?His?Pro?Phe?Met?Gln?Gly?Leu?Arg?Glu?Ala?Ala

180 185 190

Phe?Pro?Ala?Pro?Gly?Lys?Thr?Val?Thr?Leu?Lys?Ser?Phe?Ile?Pro?Asp

195 200 205

Ser?Gly?Thr?Glu?Phe?Ile?Ser?Leu?Thr?Arg?Pro?Leu?Asp?Ser?His?Leu

210 215 220

Glu?His?Val?Asp?Phe?Ser?Ser?Leu?Leu?His?Cys?Leu?Ser?Phe?Glu?Gln

225 230 235 240

Ile?Leu?Gln?Ile?Phe?Ala?Ser?Ala?Val?Leu?Glu?Arg?Lys?Ile?Ile?Phe

245 250 255

Leu?Ala?Glu?Gly?Leu?Ser?Thr?Leu?Ser?Gln?Cys?Ile?His?Ala?Ala?Ala

260 265 270

Ala?Leu?Leu?Tyr?Pro?Phe?Ser?Trp?Ala?His?Thr?Tyr?Ile?Pro?Val?Val

275 280 285

Pro?Glu?Ser?Leu?Leu?Ala?Thr?Val?Cys?Cys?Pro?Thr?Pro?Phe?Met?Val

290 295 300

Gly?Val?Gln?Met?Arg?Phe?Gln?Gln?Glu?Val?Met?Asp?Ser?Pro?Met?Glu

305 310 315 320

Glu?Val?Leu?Leu?Val?Asn?Leu?Cys?Glu?Gly?Thr?Phe?Leu?Met?Ser?Val

325 330 335

Gly?Asp?Glu?Lys?Asp?Ile?Leu?Pro?Pro?Lys?Leu?Gln?Asp?Asp?Ile?Leu

340 345 350

Asp?Ser?Leu?Gly?Gln?Gly?Ile?Asn?Glu?Leu?Lys?Thr?Ala?Glu?Gln?Ile

355 360 365

Asn?Glu?His?Val?Ser?Gly?Pro?Phe?Val?Gln?Phe?Phe?Val?Lys?Ile?Val

370 375 380

Gly?His?Tyr?Ala?Ser?Tyr?Ile?Lys?Arg?Glu?Ala?Asn?Gly?Gln?Gly?His

385 390 395 400

Phe?Gln?Glu?Arg?Ser?Phe?Cys?Lys?Ala?Leu?Thr?Ser?Lys?Thr?Asn?Arg

405 410 415

Arg?Phe?Val?Lys?Lys?Phe?Val?Lys?Thr?Gln?Leu?Phe?Ser?Leu?Phe?Ile

420 425 430

Gln?Glu?Ala?Glu?Lys?Ser?Lys?Asn?Pro?Pro?Ala?Gly?Tyr?Phe?Gln?Gln

435 440 445

Lys?Ile?Leu?Glu?Tyr?Glu?Glu?Gln?Lys?Lys?Gln?Lys?Lys?Pro?Arg?Glu

450 455 460

Lys?Thr?Val?Lys

465

<210>3

<211>249

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>3

gagtttactc?cctatcagtg?atagagaacg?tatgtcgagt?ttactcccta?tcagtgatag 60

agaacgatgt?cgagtttact?ccctatcagt?gatagagaac?gtatgtcgag?tttactccct 120

atcagtgata?gagaacgtat?gtcgagttta?ctccctatca?gtgatagaga?acgtatgtcg 180

agtttatccc?tatcagtgat?agagaacgta?tgtcgagttt?actccctatc?agtgatagag 240

aacgtatgt 249

<210>4

<211>60

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>4

taggcgtgta?cggtgggagg?cctatataag?cagagctcgt?ttagtgaacc?gtcagatcgc 60

Claims

1. recombinant adenoviral vector plasmid is to insert following (a) or (b) recombinant plasmid that obtains of described dna fragmentation in the multiple clone site of pAdxsi plasmid:

(b) dna fragmentation of tsiklomitsin response factor, promotor and the proteic encoding gene of DENND2D is contained in upstream to downstream successively.

2. recombinant adenoviral vector plasmid as claimed in claim 1 is characterized in that: described DENND2D albumen is following 1) or 2) protein:

3. recombinant adenoviral vector plasmid as claimed in claim 2 is characterized in that: the proteic encoding gene of described DENND2D is following 3) or 4) or 5) dna molecular:

5) with sequence table in the dna sequence dna that limits of sequence 1 have 90% above homology, and the identical function protein DNA molecule of encoding.

4. as arbitrary described recombinant adenoviral vector plasmid in the claim 1 to 3, it is characterized in that: in the described dna fragmentation (b), described tsiklomitsin response factor is following 6) or 7) or 8) dna molecular:

8) with sequence table in the dna sequence dna that limits of sequence 3 have 90% above homology, and have the dna molecular of identical function;

Described promotor is following 9) or 10) or 11) dna molecular:

5. recombinant adenovirus abduction delivering system comprises arbitrary described recombinant adenoviral vector plasmid and Ad-teton plasmid in the claim 1 to 4; Described recombinant adenoviral vector plasmid is the pAdxsi plasmid that carries (b) described dna fragmentation.

6. application rights requires the recombinant adenovirus that arbitrary described recombinant adenoviral vector plasmid prepares in 1 to 4.

7. at least a application in the medicine of preparation treatment tumour and/or inhibition tumor cell proliferation and/or inducing apoptosis of tumour cell in arbitrary described recombinant adenoviral vector plasmid, the described recombinant adenovirus expression system of claim 5 and the described recombinant adenovirus of claim 6 in the claim 1 to 4.

8. application as claimed in claim 7 is characterized in that: described tumour is a solid tumor; Described tumour cell is H1299 cell, H520 cell or A549 cell.

9. treat tumour and/or suppress tumor cell proliferation and/or the medicine of inducing apoptosis of tumour cell for one kind, its activeconstituents is at least a in the following material: arbitrary described recombinant adenoviral vector plasmid, the described recombinant adenovirus expression system of claim 5 and the described recombinant adenovirus of claim 6 in the claim 1 to 4.

10. medicine as claimed in claim 9 is characterized in that: described tumour is a solid tumor; Described tumour cell is H1299 cell, H520 cell or A549 cell.