CN101805724B - Targeting WWOX (WW domain-containing oxidoreductase) gene recombinant adenovirus and preparation method and application thereof - Google Patents

Abstract

The invention discloses a targeting WWOX (WW domain-containing oxidoreductase) gene recombinant adenovirus and a preparation method and application thereof. The recombinant adenovirus comprises a WWOX gene expression box, wherein the WWOX gene expression box comprises a Survivin promoter, a WWOX gene and a terminator, and the expression of the WWOX gene is regulated and controlled by the Survivin promoter. The recombinant adenovirus can effectively and specifically induce apoptosis of tumor cells and inhibit the cloning of the tumor cells, can effectively inhibit the growth of the tumor cells, and can prolong the recombinant adenovirus also has the advantages of high transfection efficiency, good stability, wide oncotherapy spectrum, simple preparation method and the like, can be used for preparing various anti-tumor medicines, and has better development and application prospect in the tumor gene therapy field.

Description

Targeting WWOX (WW domain-containing oxidoreductase) gene recombinant adenovirus

Technical field

The present invention relates to a kind of recombinant adenovirus, particularly targeting WWOX (WW domain-containing oxidoreductase) gene recombinant adenovirus also relates to the preparation method and the application of this recombinant adenovirus.

Background technology

Tumour is the important diseases that threatens human health, and the oncotherapy Study on new method is the emphasis of medical science and life science always.It is the fourth-largest treat-ment after operation, radiotherapy and chemotherapy that tumor biotherapy is known as by world medical circle, wherein one of most active research field in the present especially tumor biotherapy of therapy of tumor.The principle of therapy of tumor is that goal gene is imported target cell with gene transfer technique, makes target cell express this gene and obtains particular functionality, carry out then or mediation to the killing and wounding and restraining effect of tumour, thereby reach the purpose of treatment.

Double-colored propylhomoserin territory (the WW domain-containing oxidoreductase of oxydo-reductase; WWOX) gene is a new cancer suppressor gene having crossed over whole common chromosomal fragile site FRA16D, in multiple human malignancies such as lung cancer, cancer of the stomach, expresses and reduces or disappearance.WWOX albumen comprises the SDR district (oxydo-reductase district) of two WW territories that two functional zone are N-terminal (double-colored propylhomoserin territory) and C-terminal, and the WW territory is prone to combine with the structure of proline rich, participates in the interaction between the albumen; The SDR district is relevant with the steroid hormone metabolism.Infer that according to the WWOX protein structure it maybe be through intervening tumor growth with other protein-interactings and participation hormone metabolism.

In therapy of tumor, in order to improve the specificity of killing tumor cell, it is very important to seek tumour cell and Normocellular difference.Wherein, the expression with exogenous tumor-specific promoters regulation and control goal gene is a kind of important techniques means.Survivin (Survivin) is the strongest survivin of finding up to now, has to suppress apoptosis and the dual-use function of keeping cell mitogen.Survivin expresses in normal last differentiated tissue eventually hardly, but in nearly all malignant tumor tissue high expression level, and closely related with progress, the prognosis of disease.Previously research is verified; In tumours such as lung cancer, cancer of the stomach, liver cancer, mammary cancer, ovarian cancer, glioma and melanoma; The Survivin promotor all has stronger ability of regulation and control, shows that the Survivin promotor is the tumor-specific promoters of a wide spectrum.

Summary of the invention

In view of this; One of the object of the invention is to provide a kind of targeting WWOX (WW domain-containing oxidoreductase) gene recombinant adenovirus; Expression through tumor-specific promoters control wwox gene; Wwox gene can optionally be expressed in the tumour cell, thereby accurate killing tumor cell reduce the damage of healthy tissues as far as possible; Two of purpose is to provide the preparation method of said targeting WWOX (WW domain-containing oxidoreductase) gene recombinant adenovirus; Three of purpose is to provide the application of said targeting WWOX (WW domain-containing oxidoreductase) gene recombinant adenovirus aspect medical.

For achieving the above object, the present invention adopts following technical scheme:

1, targeting WWOX (WW domain-containing oxidoreductase) gene recombinant adenovirus; Said recombinant adenovirus contains a wwox gene expression cassette; Said wwox gene expression cassette comprises Survivin promotor, wwox gene and terminator, and the expression of said wwox gene is by the Survivin promoter regulation.

Further, said recombinant adenovirus is the wwox gene expression cassette to be inserted in the replication defective 5 type adenoviral gene groups obtain.

2, the preparation method of said targeting WWOX (WW domain-containing oxidoreductase) gene recombinant adenovirus may further comprise the steps:

The clone of a, Survivin promotor: design and synthesize primer according to the segmental nucleotide sequence of Survivin gene promoter; With people's laryngocarcinoma Hep-2 cell genomic dna is template; The pcr amplification two ends have the Survivin promoter fragment of Xho I restriction enzyme site and Nco I restriction enzyme site respectively; Be cloned into the pGEM-T carrier, get recombinant vectors pGEM-T/SurP;

The clone of b, wwox gene: the nucleotide sequence according to wwox gene designs and synthesizes primer; With the total RNA rt of human embryonic kidney 293 cell is total cDNA; Be template with total cDNA again; The pcr amplification two ends have the wwox gene of Hind III restriction enzyme site and BamH I restriction enzyme site respectively, are cloned into the pMD18-T carrier, get recombinant vectors pMD18-T/WWOX; In the carrier pMD18-T/WWOX that recombinates, go out wwox gene again with restriction enzyme Hind III and BamH I double digestion; With under the effect of T4 dna ligase, be connected with the carrier for expression of eukaryon pcDNA3.1 (+) of BamH I double digestion through Hind III equally, recombinant vectors pcDNA3.1-WWOX;

The structure of c, Survivin promoter regulation wwox gene recombinant adenovirus shuttle vectors: go out the Survivin promoter fragment with Xho I and Nco I double digestion among the carrier pGEM-T/SurP that recombinates certainly; With under the effect of T4 dna ligase, be connected with the recombinant vectors pcDNA3.1-WWOX of Nco I double digestion through Xho I equally, recombinant vectors pcDNA3.1-SurP-WWOX; In the carrier pcDNA3.1-SurP-WWOX that recombinates, go out the SurP-WWOX fragment again with Xho I and BamH I double digestion; With under the effect of T4 dna ligase, be connected with the adenovirus shuttle vector pShuttle of BamH I double digestion through Xho I equally, recombinant adenovirus shuttle vectors pShuttle-SurP-WWOX;

The structure of d, Survivin promoter regulation wwox gene recombinant adenoviral vector: with recombinant adenovirus shuttle vectors pShuttle-SurP-WWOX with Pme I linearization for enzyme restriction after; Carry out homologous recombination in the born of the same parents with adenovirus skeleton carrier pAdEasy-1 cotransfection intestinal bacteria BJ5183 competent cell, get recombinant adenoviral vector pAd-SurP-Apoptin;

Packing, amplification and the purifying of e, Survivin promoter regulation wwox gene recombinant adenovirus: with liposome method with recombinant adenoviral vector pAd-SurP-Apoptin transfection human embryonic kidney 293 cell; When treating that obvious pathology appears in cell; Centrifugal collecting cell, multigelation makes lysis, centrifugal removal cell debris; Collection contains the supernatant of virus, promptly gets recombinant adenovirus Ad-SurP-Apoptin stoste; Virus stock solution used is infected 293 cells again with amplicon virus, and the supernatant that gained contains virus adopts the caesium chloride density gradient centrifugation purifying.

Further, primer sequence is described in the step a:

Upstream primer: 5 '-gcctcgagctggccatagaaccagagaagtga-3 ';

Downstream primer: 5 '-gcccatggccacctctgccaacgggtcccgcg-3 ';

Further, the cycling condition of PCR described in step a parameter is: 94 ℃ of preparatory sex change 3 minutes, and 1 minute, 58 ℃ annealing of 94 ℃ of sex change are 1 minute then, and 72 ℃ were extended 1.5 minutes, totally 30 circulations, last 72 ℃ were extended 7 minutes;

Further, primer sequence is described in the step b:

Upstream primer: 5 '-gggaagcttttggagcgggagtgag-3 ';

Downstream primer: 5 '-atgggatcccagcagttgttgaagtaca-3 ';

Further, the cycling condition of PCR described in step b parameter is: 94 ℃ of preparatory sex change 3 minutes, and 30 seconds, 65 ℃ annealing of 94 ℃ of sex change are 1 minute then, and 72 ℃ were extended 1 minute, totally 30 circulations, last 72 ℃ were extended 10 minutes.

3, the application of said targeting WWOX (WW domain-containing oxidoreductase) gene recombinant adenovirus in the preparation antitumor drug.

Beneficial effect of the present invention is: targeting WWOX (WW domain-containing oxidoreductase) gene recombinant adenovirus of the present invention has stronger selectivity and specificity; Expression through tumour-specific Survivin promotor control wwox gene; Wwox gene optionally is expressed in the tumour cell, thereby can in accurate killing tumor cell, reduces the damage of healthy tissues as far as possible; The inside and outside result of study shows; Recombinant adenovirus of the present invention can be effectively and the apoptosis of inducing tumor cell specifically; Can be effectively and suppress the clone of tumour cell specifically; Can effectively suppress the growth of tumour cell, can prolong the mean survival time (MST) of tumor bearing nude mice and improve the The average survival time rate simultaneously; In addition, this recombinant adenovirus also has the transfection efficiency height, good stability, and advantage such as oncotherapy spectrum is wide, and the preparation method is simple can be used for preparing antitumor drug, in field of tumor gene therapy the excellent development application prospect is arranged.

Description of drawings

In order to make the object of the invention, technical scheme and advantage clearer, will combine accompanying drawing that the present invention is made further detailed description below, wherein:

Fig. 1 is the agarose gel electrophoresis figure of Survivin promotor PCR product;

Fig. 2 is the agarose gel electrophoresis figure of wwox gene PCR product;

Fig. 3 is the tumour cell of infection recombinant adenovirus of the present invention and the apoptosis rate comparison diagram of normal tissue cell;

Fig. 4 is the tumour cell of infection recombinant adenovirus of the present invention and the cloning efficiency comparison diagram of normal tissue cell;

Fig. 5 is the tumor growth curve that gives the tumor bearing nude mice of recombinant adenovirus of the present invention or PBS;

Fig. 6 is the The average survival time rate that gives the tumor bearing nude mice of recombinant adenovirus of the present invention or PBS.

Embodiment

Below will carry out detailed description to the preferred embodiments of the present invention with reference to accompanying drawing.The experimental technique of unreceipted actual conditions in the preferred embodiment, usually according to normal condition, the molecular cloning experiment guide (third edition for example; J. work such as Sa nurse Brooker, Huang Peitang etc. translate, Science Press; 2002) described in condition, or the condition of advising according to manufacturer.

One, the preparation of targeting WWOX (WW domain-containing oxidoreductase) gene recombinant adenovirus

1, the clone of Survivin promotor

Adopt genome DNA extracting reagent kit to extract people's laryngocarcinoma Hep-2 cell genomic dna, operate according to the test kit specification sheets.With the gained cell genomic dna is template, with F1:5 '-gc CtcgagCtggccatagaacca-gagaagtga-3 ' (SEQ ID No.1, underscore partly are restriction enzyme Xho I restriction enzyme site) and R1:5 '-gc CcatggCcacctctgccaacgggtcccgcg-3 ' (SEQ ID No.2, underscore partly are restriction enzyme Nco I restriction enzyme site) is the upstream and downstream primer, adopts PCR test kit amplification Survivin promoter fragment, operates according to the test kit specification sheets.PCR cycling condition parameter is: 94 ℃ of preparatory sex change 3 minutes, and 1 minute, 58 ℃ annealing of 94 ℃ of sex change are 1 minute then, and 72 ℃ were extended 1.5 minutes, totally 30 circulations, last 72 ℃ were extended 7 minutes.The PCR product is after agarose gel electrophoresis evaluation, gel recovery test kit are cut glue recovery purifying; Be connected with the pGEM-T carrier; Connect product transformed into escherichia coli JM109 competent cell,, extract plasmid with the LB plate screening positive colony that contains penbritin; Order-checking is identified, with positive colony plasmid called after pGEM-T/SurP.

The agarose gel electrophoresis result shows that unique specific band (Fig. 1) appears in the PCR product at about 980bp place, conform to the purpose clip size; The insertion gene order (SEQ IDNo.3) that sequencing result shows the positive colony plasmid and GenBank accession number are that the Survivin promoter fragment sequence of U75285 is consistent.

2, the clone of wwox gene

Adopt Trizol reagent to extract human embryo kidney (HEK) 293 cell total rnas, operate according to the reagent specification sheets.With gained cell total rna rt is total cDNA, is template with total cDNA again, with F2:5 '-ggg- AagcttTtggagcgggagtgag-3 ' (SEQ ID No.4, underscore partly are restriction enzyme Hind III restriction enzyme site) and R2:5 '-atg GgatccCagcagttgttgaagtaca-3 ' (SEQ ID No.5, underscore partly are restriction enzyme BamH I restriction enzyme site) is the upstream and downstream primer, adopts PCR test kit amplification wwox gene, operates according to the test kit specification sheets.PCR cycling condition parameter is: 94 ℃ of preparatory sex change 3 minutes, and 30 seconds, 65 ℃ annealing of 94 ℃ of sex change are 1 minute then, and 72 ℃ were extended 1 minute, totally 30 circulations, last 72 ℃ were extended 10 minutes.The PCR product is after agarose gel electrophoresis evaluation, gel recovery test kit are cut glue recovery purifying; Be connected with the pMD18-T carrier; Connect product transformed into escherichia coli JM109 competent cell,, extract plasmid with the LB plate screening positive colony that contains penbritin; Order-checking is identified, with positive colony plasmid called after pMD18-T/WWOX.

The agarose gel electrophoresis result shows that unique specific band (Fig. 1) appears in the PCR product at about 1250bp place, conform to the purpose clip size; The insertion gene order (SEQ IDNo.6) that sequencing result shows the positive colony plasmid and GenBank accession number are that the wwox gene sequence of NP_057457 is consistent.

In positive colony plasmid pMD18-T/WWOX, go out wwox gene with Hind III and BamH I double digestion; With under the effect of T4DNA ligase enzyme, be connected with the carrier for expression of eukaryon pcDNA3.1 (+) of BamH I double digestion through Hind III equally; Connect product transformed into escherichia coli JM109 competent cell, with the LB plate screening positive colony that contains penbritin, extracting plasmid; Hind III and BamH I double digestion are identified and order-checking is identified, with positive colony plasmid called after pcDNA3.1-WWOX.

3, the structure of Survivin promoter regulation wwox gene recombinant adenovirus shuttle vectors

In positive colony plasmid pGEM-T/SurP, go out the Survivin promoter fragment with Xho I and Nco I double digestion; Be connected under the effect of T4 dna ligase through the positive colony plasmid pcDNA3.1-WWOX of Xho I and Nco I double digestion equally; Connect product transformed into escherichia coli JM109 competent cell, with the LB plate screening positive colony that contains penbritin, extracting plasmid; Xho I and Nco I double digestion are identified, with positive colony plasmid called after pcDNA3.1-SurP-WWOX.

In positive colony plasmid pcDNA3.1-SurP-WWOX, go out the SurP-WWOX fragment with Xho I and BamH I double digestion; With under the effect of T4 dna ligase, be connected with the adenovirus shuttle vector pShuttle of BamH I double digestion through Xho I equally; Connect product transformed into escherichia coli JM109 competent cell; With the LB plate screening positive colony that contains penbritin, the extracting plasmid, Xho I and BamH I double digestion are identified; Gained positive colony plasmid is Survivin promoter regulation wwox gene recombinant adenovirus shuttle vectors, with its called after pShuttle-SurP-WWOX.

4, the structure of Survivin promoter regulation wwox gene recombinant adenoviral vector

With recombinant adenovirus shuttle vectors pShuttle-SurP-WWOX with Pme I linearization for enzyme restriction after; Carry out homologous recombination in the born of the same parents with adenovirus skeleton carrier pAdEasy-1 electric transformed into escherichia coli BJ5183 competent cell of while; With the LB plate screening positive colony that contains kantlex, the extracting plasmid, Pac I enzyme is cut evaluation; Gained positive colony plasmid is Survivin promoter regulation wwox gene recombinant adenoviral vector, with its called after pAd-SurP-WWOX.

5, packing, amplification and the purifying of Survivin promoter regulation wwox gene recombinant adenovirus

The human embryonic kidney 293 cell is seeded in the T-25 culturing bottle with 40%～60% cell density; Treat that cell grows to 30%～50% and discards nutrient solution when merging, with Lipofectamine 2000 reagent with recombinant adenoviral vector pAd-SurP-WWOX rotaring redyeing 293 cell, through the microscope observing cell pathological change; Centrifugal collecting cell when waiting to occur obvious cytopathic effect; Resuspended with PBS, multigelation makes lysis 3 times, centrifugal removal cell debris; Collection contains the supernatant of virus, promptly gets Survivin promoter regulation wwox gene recombinant adenovirus Ad-SurP-WWOX stoste; Virus stock solution used is infected 293 cells again with amplicon virus, and the supernatant that will contain virus at last adopts the caesium chloride density gradient centrifugation purifying.

Two, the antitumor action of targeting WWOX (WW domain-containing oxidoreductase) gene recombinant adenovirus detects

1, extracorporeal anti-tumor function

(1) apoptosis rate detects

Respectively with human hepatoma HepG2 cell and America grivet kidney COS-7 cell inoculation in culturing bottle; Cultivating after 24 hours, is 100 cells infecteds with recombinant adenovirus Ad-SurP-WWOX by infection multiplicity (MOI), and corresponding control group (substituting recombinant adenovirus Ad-SurP-WWOX with PBS) is set simultaneously; Infect after 3 days; Collecting cell, after the PBS washing, the adjustment cell concn is 1 * 10 ⁵Individual/ml; Add concentration and be phospholipids incorporate albumen V (Annexin V) 5 μ l and propidium iodide (PI) the 5 μ l that concentration is 250 μ g/ml of the marked by fluorescein isothiocyanate of 250 μ g/ml; The ice bath lucifuge was hatched 10 minutes; Use the PBS washed cell, detect the apoptosis situation with FACS Calibur flow cytometer again: Annexin V and PI are all low, and the cell that dyes is normal viable cell; The AnnexinV height dyes and the low cell that dyes of PI is an apoptotic cell, and the cell that the equal height of Annexin V and PI dyes is a non-viable non-apoptotic cell.

The result sees Fig. 3; The apoptosis rate that infects the tumour cell HepG2 of recombinant adenovirus Ad-SurP-WWOX or PBS is respectively 46.8% and 5.2%; And the apoptosis rate that infects the normal tissue cell COS-7 of recombinant adenovirus Ad-SurP-WWOX or PBS is respectively 4.3% and 3.9%, shows that recombinant adenovirus Ad-SurP-WWOX of the present invention can be effectively and the apoptosis of inducing tumor cell specifically.

(2) the cell clone rate of formation detects

Respectively human hepatoma HepG2 cell and each 200 in America grivet kidney COS-7 cell are inoculated in 6 orifice plates; Cultivate after 24 hours; Is 100 cells infecteds with recombinant adenovirus Ad-SurP-WWOX by MOI; Corresponding control group (substituting recombinant adenovirus Ad-SurP-WWOX with PBS) is set simultaneously, after infecting 2 thoughtful cell colonies and forming, the row Giemsa staining; Mirror counting down calculates the cell clone rate of formation by following formula: cell clone rate of formation=clone's number/inoculating cell number * 100% greater than the colony of 50 cells.

The result sees Fig. 4; The cloning efficiency that infects the tumour cell HepG2 of recombinant adenovirus Ad-SurP-WWOX or PBS is respectively 13% and 87%; And the cloning efficiency that infects the normal tissue cell COS-7 of recombinant adenovirus Ad-SurP-WWOX or PBS is respectively 82% and 89%, shows that recombinant adenovirus Ad-SurP-WWOX of the present invention can be effectively and suppress the clone of tumour cell specifically.

2, anti-tumor in vivo effect

With 1 * 10 ⁵Individual HepG2 cell inoculation is in the subcutaneous tumor bearing nude mice of setting up of nude mice; Tumor bearing nude mice is divided into two groups at random: control group and experimental group; Control group tail vein injection PBS; Experimental group tail vein injection recombinant adenovirus Ad-SurP-WWOX observes survival rate and the gross tumor volume of two groups of mouse in 60 days, draws tumor growth curve.

The result: two groups of mouse tumor growth curves are seen Fig. 5, and in the time of the 30th day, the control group mice gross tumor volume reaches 5500mm ³, and the experimental mice tumor growth is slow, gross tumor volume is merely 1800mm ³Two groups of mouse The average survival time rates are seen Fig. 6, and control group mice is all dead in 30 days, and 60 days survival rate of experimental mice is 60%; Show that recombinant adenovirus Ad-SurP-WWOX of the present invention can effectively suppress the growth of tumour cell, can prolong the mean survival time (MST) of tumor bearing nude mice and improve the The average survival time rate simultaneously.

Explanation is at last; Above embodiment is only unrestricted in order to technical scheme of the present invention to be described; Although through invention has been described with reference to the preferred embodiments of the present invention; But those of ordinary skill in the art should be appreciated that and can make various changes to it in form with on the details, and the spirit and scope of the present invention that do not depart from appended claims and limited.

Sequence table

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Claims (1)

1. the preparation method of targeting WWOX (WW domain-containing oxidoreductase) gene recombinant adenovirus; It is characterized in that: said recombinant adenovirus contains a wwox gene expression cassette; Said wwox gene expression cassette comprises Survivin promotor, wwox gene and terminator, and the expression of said wwox gene is by the Survivin promoter regulation;

Its preparation method may further comprise the steps:

The clone of a, Survivin promotor: design and synthesize primer according to the segmental nucleotide sequence of Survivin gene promoter; With people's laryngocarcinoma Hep-2 cell genomic dna is template; The pcr amplification two ends have the Survivin promoter fragment of Xho I restriction enzyme site and Nco I restriction enzyme site respectively; Be cloned into the pGEM-T carrier, get recombinant vectors pGEM-T/SurP; Said primer sequence is: upstream primer: 5 '-gcctcgagctggccatagaaccagagaagtga-3 '; Downstream primer: 5 '-gcccatggccacctctgccaacgggtcccgcg-3 '; Said PCR cycling condition parameter is: 94 ℃ of preparatory sex change 3 minutes, and 1 minute, 58 ℃ annealing of 94 ℃ of sex change are 1 minute then, and 72 ℃ were extended 1.5 minutes, totally 30 circulations, last 72 ℃ were extended 7 minutes;

The clone of b, wwox gene: the nucleotide sequence according to wwox gene designs and synthesizes primer; With the total RNA rt of human embryonic kidney 293 cell is total cDNA; Be template with total cDNA again; The pcr amplification two ends have the wwox gene of Hind III restriction enzyme site and BamH I restriction enzyme site respectively, are cloned into the pMD18-T carrier, get recombinant vectors pMD18-T/WWOX; In the carrier pMD18-T/WWOX that recombinates, go out wwox gene again with restriction enzyme Hind III and BamH I double digestion; With under the effect of T4DNA ligase enzyme, be connected with the carrier for expression of eukaryon pcDNA3.1 (+) of BamH I double digestion through Hind III equally, recombinant vectors pcDNA3.1-WWOX; Said primer sequence is: upstream primer: 5 '-gggaagcttttggagcgggagtgag-3 '; Downstream primer: 5 '-atgggatcccagcagttgttgaagtaca-3 '; Said PCR cycling condition parameter is: 94 ℃ of preparatory sex change 3 minutes, and 30 seconds, 65 ℃ annealing of 94 ℃ of sex change are 1 minute then, and 72 ℃ were extended 1 minute, totally 30 circulations, last 72 ℃ were extended 10 minutes;

The structure of c, Survivin promoter regulation wwox gene recombinant adenovirus shuttle vectors: go out the Survivin promoter fragment with Xho I and Nco I double digestion among the carrier pGEM-T/SurP that recombinates certainly; With under the effect of T4DNA ligase enzyme, be connected with the recombinant vectors pcDNA3.1-WWOX of Nco I double digestion through Xho I equally, recombinant vectors pcDNA3.1-SurP-WWOX; In the carrier pcDNA3.1-SurP-WWOX that recombinates, go out the SurP-WWOX fragment again with Xho I and BamH I double digestion; With under the effect of T4DNA ligase enzyme, be connected with the adenovirus shuttle vector pShuttle of BamH I double digestion through Xho I equally, recombinant adenovirus shuttle vectors pShuttle-SurP-WWOX;

The structure of d, Survivin promoter regulation wwox gene recombinant adenoviral vector: with recombinant adenovirus shuttle vectors pShuttle-SurP-WWOX with Pme I linearization for enzyme restriction after; Carry out homologous recombination in the born of the same parents with adenovirus skeleton carrier pAdEasy-1 cotransfection intestinal bacteria BJ5183 competent cell, get recombinant adenoviral vector pAd-SurP-Apoptin;

Packing, amplification and the purifying of e, Survivin promoter regulation wwox gene recombinant adenovirus: with liposome method with recombinant adenoviral vector pAd-SurP-Apoptin transfection human embryonic kidney 293 cell; When treating that obvious pathology appears in cell; Centrifugal collecting cell, multigelation makes lysis, centrifugal removal cell debris; Collection contains the supernatant of virus, promptly gets recombinant adenovirus Ad-SurP-Apoptin stoste; Virus stock solution used is infected 293 cells again with amplicon virus, and the supernatant that gained contains virus adopts the caesium chloride density gradient centrifugation purifying.

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