CN105755043A - Double-copy human p53 gene recombinant adenovirus and preparation method thereof - Google Patents

Double-copy human p53 gene recombinant adenovirus and preparation method thereof Download PDF

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CN105755043A
CN105755043A CN201610224992.5A CN201610224992A CN105755043A CN 105755043 A CN105755043 A CN 105755043A CN 201610224992 A CN201610224992 A CN 201610224992A CN 105755043 A CN105755043 A CN 105755043A
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高贵
光炜
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Sanuosheng Shenzhen Gene Industry Development Co ltd
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Abstract

The invention discloses double-copy human p53 gene recombinant adenovirus and a preparation method thereof. A commercialized 5-type recombinant replication-deficient adenovirus construction system (AdEasy) is inserted into a double-copy human p53 tumor inhibition gene eukaryotic expression box as shown in SEQ ID NO.1 to construct a p53 tumor inhibition gene recombinant adenovirus expression carrier system, and recombinant replication-deficient adenovirus granules for expressing double-copy human p53 tumor inhibition gene are further obtained. Experiment shows that after being injected by tumor cells, the double-copy human p53 gene recombinant adenovirus can efficiently express p53 tumor inhibition genes carried by the virus. As the double-copy human p53 tumor inhibition gene eukaryotic expression box is integrated, the p53 tumor inhibition gene expression amount can be greatly increased, meanwhile the virus amount can be reduced, and a relatively good gene treatment effect can be achieved. The double-copy human p53 gene recombinant adenovirus is good in specificity and wide in spectrum, directly aims at gene mutation of tumor cells, and can be applied to malignant tumor of various tissue types at the early stage, the middle stage and the late stage.

Description

A kind of double; two copy Human p53 gene recombinant adenovirus and preparation method thereof
Technical field
The invention belongs to biomedicine field, be specifically related to a kind of double; two copy Human p53 gene recombinant adenovirus and preparation method thereof.
Background technology
Normal tissue cell develops into malignant cell due to the gene mutation of long-term accumulated, and malignant cell is characterized by the growth of uncontrollable unlimitedness, destroys the organizational structure of local and metastasis site.The final purpose for the treatment of tumor is to kill completely or remove tumor tissues.The method of traditional treatment tumor includes operation, chemotherapy and radiation three big main method and some complementary therapies, and these Therapeutic Method have significant side effect and limitation.Gene therapy is the new antitumour treatments that grows up at present, is directed to the genetic disorder of tumor cell and controls root.
P53 gene is most important antioncogene.After cytogene body sustains damage, the p53 albumen of p53 gene expression strengthens, and plays cell growth inhibiting and causes apoptotic function.P53 plays the effect for the treatment of malignant tumor by following mechanism: the tumor cell G1 phase suppresses and makes tumor cell stop growing, inducing tumor cell is committed suiside or apoptosis, Antineoplastic angiogenesis, raising chemotherapeutic efficacy, raising radiotherapy effect, bystander effect, and namely the antitumor action of P53 can be diffused into the tumor cell of surrounding.
Publication number has been the patent disclosure of CN1471977A a kind of " adenovirus vector for the treatment of proliferative disease and the gene recombinaton medicine of p53 gene ", and (commodity are called its antineoplastic gene prod recombinant human adenovirus p 53 injection ) be a kind of through genetic engineering modified, there is restructuring Human p53 gene adenovirus particles (rAd-p53) of infection activity, it is made up of the replication defective mankind 5 type adenovirus and normal p53 tumor suppressor gene two parts, replication defective adenovirus particles brings tumor cell as carrier into p53 gene, and p53 gene is p53 protein exhibits antineoplastic biological function at expression in tumor cells.(authentication code: the quasi-word S20040004 of traditional Chinese medicines) is ratified by state food and drug administration (SFDA) in 2004, it is used for the clinical treatment of nasopharyngeal carcinoma through local intratumor injection mode, is the therapy of tumor medicine of first listing in the world.
Five type adenoviruss are conventional genophore, and target gene is imported in target cell by carrier, is expressed as biological activity protein and plays a role.The advantage of adenovirus vector is no matter cell is in trophophase or resting stage, can high-efficiency transfection, transfer gene；And unconformity viral DNA to host cell gene group therefore does not produce to insert inactivation or mutation；Adenovirus vector safety is good；Titre is high, mass industrialized production technical maturity.The E1 district coding viral replication protein of adenovirus, after removing E1 district, adenovirus becomes replication-defective adenoviral, loses the ability at time multiplexed cell, so safety increases；The albumen of E3 gene expression can resist the antiviral defense system of host, and the removal in E3 district does not affect virus replication and can reduce the vivo immunization reaction of host.
Summary of the invention
The present invention solves that above-mentioned technical problem is adopted and gives disclosed technical scheme is, application molecule clone technology, merge having the double; two copy Human p53 gene recombinant adenovirus expression cassette sequences for various oncotherapy effects with the 5 type recombinant adenoviral vectors having in transfection importing target cell, it is thus achieved that gene recombinaton medicine.It is characterized in that this genomic medicine is for a kind of double; two copy Human p53 gene recombinant adenoviruss, namely as the high expressed human P 53 tumor suppressor gene recombinant adenovirus of gene therapy.Constructing technology path is to select commercial 5 types recombinant replication-defective sexual gland virus constructive system (AdEasySystem), insert double; two copy human P 53 tumor suppressor gene eukaryotic expression boxes of synthesis, build p53 tumor suppressor gene recombinant adenoviral expressing vector, obtain the recombinant replication-defective adenoviral granule of high expressed double; two copy human P 53 tumor suppressor gene further.This adenovirus construction system relates to two plasmid vectors: the shuttle plasmid (pShuttle) of clone's genes of interest and replication defect type 5 type adenovirus basic framework recombinant vector (pAdEasy), this carrier system can be inserted into the alien gene carrying 7.5kb.
1. shuttle plasmid (pShuttle) is except containing multiple clone site, to insert outside genes of interest fragment, possibly together with 5 type adenovirus promoters, adenoviral packaging signal sequence and (vector DNA sequence is referring to NCBI gene bank, AF334399 for the adenovirus both sides homologous sequence of homologous recombination；GI:12698892).
2. replication-defective adenoviral basic framework recombinant vector (pAdEasy) be then delete virus E1, E3 district and left distal end inverted repeat (invertedterminalrepeats,ITR), (vector DNA sequence is referring to NCBI gene bank, AY370909.2 for 5 type adenovirus full gene sequence carriers after viral packaging signal sequence；GI:46371999).
3. after two plasmids of step 1 and 2, in cell, homologous recombination occur, then novel constructs is containing genes of interest, and deletes the plasmid vector of the adenoviral gene full sequence of E1 and E3 sequence.Because E1 sequence is required for virus packaging, therefore only this recombination adenovirus construction carrier being transferred to HEK293 incasing cells, the latter provides adenovirus packaging required E1 viral gene products, thus producing the adenovirus particles containing genes of interest.Because other host cells are not provided that E1 viral gene products, thus this adenovirus can only infect once these host cells, but can not copy package again, therefore be called replication-defective adenoviral.
Concrete technical scheme is as follows:
The present invention first open a kind of double; two copy human P 53 tumor suppressor gene eukaryotic expression structure box fragments, described nucleic acid molecules is following 1) or 2) DNA molecular:
1) nucleotides sequence is classified as the DNA molecular shown in SEQIDNO.1；
2) with 1) DNA sequence that limits at least has 90%, at least has 95%, at least has 96%, at least has 97%, at least has 98% or at least have the DNA molecular of 99% homology coded protein.
Described double; two copy human P 53 tumor suppressor gene eukaryotic expression structure box fragment can be obtained by chemical synthesis in conjunction with polymerase chain reaction (PCR) and implement.Because this expression structure box fragment involves starting up son, the synthesis of genes of interest open reading frame and multiple fragments such as poly-A tail and double; two copy expression cassettes, its construction method of disclosure is: the disposable Jinmen cloning assembling multiple DNA fragmentation (including identical repeated fragment) in application molecular cloning, design synthesis 6 is to primer such as table 1, PCR expands Human p53 gene, CMV promoter and poly adenine cauda section respectively.Purification reclaims each pcr amplified fragment, sequence verification.Then through endonuclease digestion, connect, it is thus achieved that pCMV-p53-polyA-pCMV-p53-polyA double; two copy p53 expression casette fragment.
Another aspect of the present invention is in that openly a kind of recombinant vector utilizing described double; two copy human P 53 tumor suppressor gene eukaryotic expression structure box fragments to obtain, and described recombinant vector is the adenovirus shuttle plasmid pShuttle of one of adenovirus construction system carrier (AdEasy) and adenovirus construction system carrier.
Another aspect of the present invention is in that open one double; two copy Human p53 gene is recombinated 5 type adenovirus vector plasmids, its preparation method is, by in double; two copy Human p53 gene expression cassette sequence clones to adenovirus shuttle plasmid pShuttle, the pShuttle plasmid purified with HindIII and KpnI enzyme action respectively and sequence are the double; two copy human P 53 tumor suppressor gene eukaryotic expression structure box fragments shown in SEQIDNO.1, connecting, clone obtains containing double; two copies Human p53 gene expression cassette adenovirus shuttle plasmid (pShuttle-du-p53)；Utilize containing double; two copies Human p53 gene expression cassette adenovirus shuttle plasmid (pShuttle-du-p53), by the restructuring 5 type adenovirus vector containing double; two copy human P 53 tumor suppressor gene eukaryotic expression structure box fragments that homologous recombination obtains.
Another aspect of the present invention is in that openly a kind of restructuring 5 type adenovirus vector described above, its preparation method is, with PmeI enzyme action containing double; two copies Human p53 gene expression cassette adenovirus shuttle plasmid (pShuttle-du-p53), by digestion products and adenovirus basic framework recombinant vector pAdEasy, electroporated method cotransfection E.coli BJ5183, through homologous recombination, screening positive clone (prAd5-Du-p53), extracting plasmid, enzyme action is identified.
Another aspect of the present invention is in that open one double; two copy Human p53 gene recombinant adenovirus, and its preparation method is, with the restructuring 5 type adenovirus vector plasmid prAd5-Du-p53 described in PacI enzyme action so that it is linearisation, then uses CsCl2Purification, through LipofecAMINE2000 method transfected HEK 293,5%CO2, 37 DEG C hatch cultivation 14 days after, scraping cells, centrifugal segregation culture supernatants, stay 2mL culture fluid, concussion mixing, add ethanol bath method with dry ice freezing, 37 DEG C of water-baths are dissolved, and repeat freezing, dissolving, concussion cell lysis, it is thus achieved that virion.
The genomic medicine that double; two copy Human p53 gene recombinant adenovirus of the present invention is constituted, by the adenovirus carrier as gene delivery, after infected tumor's cell, its p53 tumor suppressor gene carried of high efficient expression.High expressed, double; two copy human P 53 tumor suppressor gene eukaryotic expression box because of this viral integrase, therefore can reduce viral dose reaches better gene therapy effect simultaneously to be greatly increased p53 tumor suppressor gene expression.Described double; two copy Human p53 gene recombinant adenovirus can also play synergism with traditional antitumour treatments use in conjunction.
Beneficial effect:
1. being inserted into double; two copy p53 gene at replication defect type five type adenovirus makes functional p53 expressing quantity increase；
2.p53 gene eucaryon expression box selects CMV strong promoter and enhancer to make functional p53 expressing quantity increase further and speed, double; two copy each architectonicals of expression cassette used；Thus double; two copy Human p53 gene recombinant adenoviruss improve 3-5 times than single copy rAd-p53 anti-tumor activity.
3. deleting more hiv region (E1 and E3 district), increasing Insert Fragment capacity be double; two copy expression casettes, thus increasing gene expression amount and minimizing virus makes consumption, curative effect and safety all increase.
4. pair copy Human p53 gene recombinant adenovirus can also play synergism with traditional antitumour treatments use in conjunction.
5. pair copy Human p53 gene recombinant adenovirus is Antioncogene medicine, compares safety height with traditional antitumour treatments such as chemotherapy or radiotherapy, significantly improves the quality of life of tumor patient；
6. high specificity, is directed to the gene mutation of tumor cell；
7. wide spectrum, the effect data in embodiment shows the application in preparation tumor suppression medicine of double; two copy Human p53 gene recombinant adenovirus.In the inhibiting tumour cells to hepatocarcinoma, pulmonary carcinoma, squamous cell carcinoma of the head and neck, cervical cancer and breast carcinoma, specifically there is good effect, indicate that it can be used for the phase morning, noon and afternoon malignant tumor of various organization type.
Accompanying drawing explanation
Fig. 1: double; two copy human P 53 tumor suppressor gene recombination adenovirus construction schematic diagrams, in figure, labelling is mainly: multiple clone site (MCS), wherein KpnI and HindIII is double; two copy p53 expression casette insertion points, plasmid replication starting point (Ori), bacterial resistance gene, two carrier differences, the respectively anti-kanamycin gene of Kan, the anti-ammonia benzyl of Amp because of.RightArm and LeftArm represents respectively for homologous recombination, is positioned at the adenovirus homologous sequence of genes of interest left and right sides.PmeI is sitting between the homologous sequence of left and right in pShuttle plasmid, therefore for homologous recombination linearized prior pShuttle plasmid.PacI is positioned at the both sides of the adenoviral gene deleting E1 and E3 after restructuring containing genes of interest, builds virion linearized prior plasmid for rotaring redyeing 293 cell and removes bacterial component.
Fig. 2: double; two copy Human p53 gene recombinant adenoviruss are for the killing tumor cells effect of animal model for tumour, it is illustrated that result proves that double; two copy Human p53 gene recombinant adenovirus includes pulmonary carcinoma, hepatocarcinoma, head-neck malignant tumor, breast carcinoma and cervical cancer etc. for various tumors and has significant fragmentation effect.Intratumor injection double; two copy Human p53 gene recombinant adenovirus is after 2 weeks, and various tumors significantly reduce, and almost arrive and are wholly absent.Illustrate that double; two copy Human p53 gene recombinant adenovirus has the antitumous effect of wide spectrum.
Detailed description of the invention
Following non-limiting example can make those of ordinary skill in the art more fully understand the present invention, but does not limit the present invention in any way.
PShuttle plasmid selected in pEGFP-C1 plasmid, adenovirus construction system and pAdEasy plasmid are all from commercial prod, and it is constituted and sequence can referring to NCBI gene bank (pEGFP-C1, U55763, GI:1377914；PShuttle, AF334399, GI:12698892；pAdEasy,AY370909.2,GI:46371999).
PrAd5-Du-p53 sequence illustrates:
1.5 type adenovirus (ad5) recombinant vectors delete the adenoviral gene group sequence of E1 and E3 and the left and right arms sequence for homologous recombination all refers to 5 type adenoviral gene group complete sequence (GI:56160529；AC_000008.1)；In constructive system carrier, the left and right arms sequence of adenovirus basic framework sequence and homologous recombination both is from 5 type adenoviruss, AC000008；GI:56160529 is the sequence registration number of NCBI gene bank 5 type adenoviral gene group.5 types adenoviral gene group complete sequence (35938bp) are registered as source with this, adenovirus basic framework carrier deletes E1 (5 ' ends lack) and E3 from 1 to 3532bp (from 28138 to 30819,2681bp disappearance altogether), in shuttle plasmid, homologous sequence right arm is from 3534 to 5774, altogether long 2241bp；Homologous sequence left arm is 34952 to 35937, long 986bp.
2. couple copy human P 53 tumor suppressor gene eukaryotic expression structure box fragment SEQIDNO.1: wherein:
1) human cytomegalovirus (CMV) early promoter sequence: copy 1:7～581；Copy 2:2023～2576；
2) human P 53 tumor suppressor gene coded sequence: copy 1:586～1784；Copy 2:2580～3795；
3) 3 ' end non-coding sequence and poly-A tail (polyA) sequence: copy 1:1789～2017；Copy 2:3798～4032；
4) sticky end of design when 4 lower cases are Jinmen clone in series, from 5 ' to 3 ' are followed successively by: attc (585), tctt (1183), taac (2027), gatc (2575), tatc (3798).
5) HindIII and 3 are introduced at 5 ' ends of double; two copy Human p53 gene whole sequences of expression structure box ' hold the KpnI endonuclease digestion site introduced to be selected from the restriction endonuclease in shuttle plasmid pShuttle multiple clone site, and be not present in extension increasing sequence.
Embodiment 1
1. build double; two copy human P 53 tumor suppressor gene eukaryotic expression structure box:
nullFor Jinmen clone technology (goldengatecloning) of disposable assembling polymolecular fragment in application novel molecular clone technology，Design synthesis double; two copy p53 gene expression structure box，Namely eukaryotic cell expression promoter (what use in the embodiment of the present invention is cytomegalovirus CMV promoter) is included，P53 tumor suppressor gene exploitation reading frame (ORF) and poly-A tail (polyA): be built into human cytomegalovirus (CMV) virus early promoter--Kozak sequence P53 gene--3 ' end non-coding sequence poly-A tail (polyA) sequence human cytomegalovirus (CMV) virus early promoter--Kozak sequence P53 gene--3 ' end non-coding sequence poly-A tail (polyA) sequences.
Jinmen clone technology utilizes II S type restricted enzyme exactly, and its DNA identifies what binding site and shearing point were spatially separated, produces the characteristic of a sticky end after enzyme action at be positioned at binding site the 3 ' specific sites held.Principle accordingly, 5 ' the ends expanding corresponding distinguished sequence primer in design introduce BsmBI restriction endonuclease (without this restriction enzyme site in amplified fragments) and different specified viscosity ends (referring to table 1, structure double; two copy human P 53 tumor suppressor gene eukaryotic expression box the primer sequence, Tm value and clip size) respectively with normal person cDNA for template, amplification generates p53 open reading frame；CMV promoter and polyA sequence is obtained with pEGFP-C1 plasmid for template amplification.Normal person's cDNA template comes from normal person's blood leukocytes, extracts RNA according to corresponding reagent box guide, and reverse transcription synthesis cDNA, as amplification p53 gene template.For ease of the follow-up shuttle plasmid that is cloned into, in design of primers, the 5 ' ends in first CMV promoter introduce HindIII restriction enzyme site, and the 3 ' ends of last polyA introduce KpnI restriction enzyme site.PCR reaction adopts high-fidelity Taq polymerase, 6 PCR specific fragments (, referring to table 1, Product Sequence is referring to SEQIDNO.1 for primer sequence, Tm value and PCR primer size) that 6 pairs of primer amplifications produce.Agarose gel purification PCR primer (confirmation that can check order at this moment PCR primer), the 6 of purification PCR fragment are put into same test tube with BsmBI restriction endonuclease and T4 ligase, because namely restriction enzyme site disappears after a resection, therefore form single digestion-coupled reaction.Final 5 ' the ends that obtain are containing HindIII enzyme action point, 3 ' ends are containing KpnI enzyme action point, without BsmBI site, the single PCR junction fragment of about 4035bp, check order after glue purification to determine that fragment sequence is correct, i.e. double; two copies human P 53 tumor suppressor gene eukaryotic expression structure box fragment (SEQIDNO.1).
Table 1, structure double; two copy human P 53 tumor suppressor gene eukaryotic expression box the primer sequence, Tm value and clip size
2. clone's double; two copy human P 53 tumor suppressor gene sequence is to adenovirus shuttle plasmid (pShuttle):
The adenovirus shuttle plasmid (pShuttle) in 5 type adenovirus expression carrier systems is selected to clone p53 Gene Double copy sequence.Double; two copy p53 tumor suppressor gene expression cassette fragments that the pShuttle plasmid purified with HindIII and KpnI enzyme action respectively is synthesized with step 1, glue purification digestion products, connect through T4 ligase, then DH5 α cell is converted, after kanamycin screening and culturing, choose 5～10 clones, extract plasmid, after enzyme action is accredited as expection normal colonic, determine that adenovirus shuttle plasmid contains correct double; two copies Human p53 gene expressed sequence (pShuttle-du-p53) then through order-checking.Simultaneously again by expression plasmid transfecting eukaryotic cells (such as HEK293 cell) correct for sequence, cultivate, extract cell lysis liquid, through WesternBlot, determine p53 expression product with special anti-p53 antibody.
3. clone's double; two copy Human p53 gene recombinant adenoviral vector (prAd5-du-p53)
With PmeI enzyme action pShuttle-du-p53, by linearizing, two ends are pShuttle-du-p53 plasmid and replication-defective adenoviral basic framework recombinant vector (pAdeasy) of left and right sides homologous sequence respectively, electroporated method cotransfection E.coli BJ5183 (cell with high homologous recombination efficiency of the special structure of these escherichia coli), then through kanamycin screening and culturing, select 10～20 little clones, transfer in the LB culture fluid containing kanamycin, 37 DEG C of cultivations overnight, extracting plasmid in a small amount, PacI endonuclease digestion is identified.Identify that correct clone is then for recombinating 5 type adenovirus vectors (prAd5-du-p53) containing double; two copy human P 53 tumor suppressor genes.Meanwhile, by identifying that correct plasmid vector is transfected in DH10B escherichia coli again, cultivate and extract substantial amounts of plasmid, and freezen protective part bacterium solution, preserve for a long time.(because of carrier instability, easy recombination mutation in BJ5183 cell).
4. obtain double; two copy Human p53 gene recombinant adenovirus granule:
The prAd5-du-p53 plasmid purified with PacI enzyme action so that it is linearisation, removes carrier bacteria plasmid redundance simultaneously.By linearizing positive plasmid CsCl2Purification, through LipofecAMINE2000 method transfected HEK 293,5%CO2, hatch cultivation for 37 DEG C.After about 14 days, scraping cells, it is transferred to 15mL centrifuge tube, 1000rpm, 10 minutes, remove culture supernatants, stay 2mL culture fluid, concussion mixing, add ethanol bath method with dry ice freezing, 37 DEG C of water-baths are dissolved, and repeat " freezing-to dissolve-concussion " 4 times, with cell lysis, discharge virion.Infect HEK293 cell with this virus mixed liquor again, replicate amplification and obtain more, the recombinant adenovirus granule that titre is higher.Expand obtained virion through CsCl2 ultracentrifugation separation purification of Recombinant virus, to dialyse then through SpectraMW6000, filtration sterilization, last subpackage every time.-80 DEG C of preservations, and make plaque test experiment and virion assay.The virion of initial purification, titre is up to 107Left and right, after 2～3 repeat amplification protcols, finally can obtain up to 1012Titer.
Embodiment 2
(1) experiment of double; two plaque tests and virion assay
Infect monolayer HEK293 cell with a series of purified viruses (double; two copy Human p53 gene recombinant adenovirus) diluent, by the virus of purification in the ratio of 1:10 successively serial dilutions, select 106To 1013Dilution virus (double; two copy Human p53 gene recombinant adenovirus) suspension is separately added in the monolayer HEK293 Tissue Culture Flask of densification, makes viruses adsorption, and 37 DEG C of cultivations suck viral suspension after 1 hour, then covers the agar of a melting layer, 37 DEG C of cultivations.Each dilution gradient at least 2 parts.Neutral red staining after 24 hours, checks result.Virus (double; two copy Human p53 gene recombinant adenovirus), after HEK293 time multiplexed cell system, can produce the focus of infection of a limitation, i.e. plaque.Contaminate living cells by dimethyl diaminophenazine chloride, it is seen that do not catch the plaque of color, calculate virus concentration and titre titer according to plaque number (two parts of averages) and dilution factor.Purified double; two copy Human p53 gene recombinant adenoviruss can reach 1012Titration titer.
(2) double; two copy Human p53 gene recombinant adenoviruss are for cultivating the lethal effect of various tumor cell
The tumor cell (MCF-7-ADR) (10 of hepatocarcinoma (MHCC97 cell strain), pulmonary carcinoma (H1299, non-small cell lung cancer cell strain), squamous cell carcinoma of the head and neck (UM-SCC-22A cell strain), cervical cancer (Hela cell strain) and breast carcinoma is cultivated at 96 orifice plates5-6Cell), these cell strains are all bought in the U.S. ATCC (AmericanTissuCultureCollection, Manassas, VA20108, USA；Phone 001-703365-2700).
Matched group defective five type adenovirus empty carrier (Shenzhen is matched hundred promise gene technology company limiteies and provided), test one group with rAd-du-p53, test two groups with rAd-p53 (Shenzhen is matched hundred promise gene technology company limiteies and provided), (107Virion) infect these tumor cells, measure cytoactive by MTT method.Each experiment repeats 3 times, calculates average cell growth inhibition ratio (see table 2 below).
Table 2 result proves that rAd-du-p53 has the function of anti-various tumors very by force, and its antitumous effect is 3 to 5 times of rAd-p53.…
(3) double; two copy Human p53 gene recombinant adenoviruss are for the killing tumor cells effect of animal model for tumour
(American National institute of oncology is derived from BALB/cnu/nu nude mice, NationalInstituteofHealth, Bethesda, Maryland, USA) tumor cell of subcutaneous implantation hepatocarcinoma, pulmonary carcinoma, squamous cell carcinoma of the head and neck, cervical cancer and breast carcinoma forms animal model for tumour, after planting 2 weeks, gross tumor volume is 2-4cm3.At intratumor injection rAd-du-p53 once (1x1011Virion is diluted to 1mL normal saline), adopt multi-level multi-direction injection, make virus be uniformly distributed in tumor tissues.Within every two days, measure gross tumor volume size.The size variation of tumor is shown in Fig. 2；Graphical results proves that double; two copy Human p53 gene recombinant adenovirus includes pulmonary carcinoma, hepatocarcinoma, head-neck malignant tumor, breast carcinoma and cervical cancer etc. for various tumors and has significant fragmentation effect.Intratumor injection double; two copy Human p53 gene recombinant adenovirus is after 2 weeks, and various tumors significantly reduce, and almost arrive and are wholly absent.Illustrate that double; two copy Human p53 gene recombinant adenovirus has the antitumous effect of wide spectrum.

Claims (5)

1. a double; two copy human P 53 tumor suppressor gene eukaryotic expression structure box fragment, it is characterised in that: described nucleic acid molecules is following 1) or 2) DNA molecular:
1) nucleotides sequence is classified as the DNA fragmentation shown in SEQIDNO.1；
2) with 1) DNA sequence that limits at least has 90%, at least has 95%, at least has 96%, at least has 97%, at least has 98% or at least have the DNA molecular of 99% homology coded protein.
2. utilize the recombinant vector that the double; two copy human P 53 tumor suppressor gene eukaryotic expression structure box fragments described in claim 1 obtain, it is characterised in that: described carrier is adenovirus shuttle plasmid pShuttle.
3. double; two copy Human p53 genes are recombinated 5 type adenovirus vector plasmids, it is characterized in that: its preparation method is, by double; two copy Human p53 gene expression cassette sequence clones to adenovirus shuttle plasmid pShuttle, the pShuttle plasmid purified with HindIII and KpnI enzyme action respectively is the DNA fragmentation shown in SEQIDNO.1 with sequence in claim 1, positive colony is used PmeI enzyme action, by digestion products and adenovirus basic framework recombinant vector pAdEasy, electroporated method cotransfection E.coli BJ5183, carry out homologous recombination, after screening positive clone, extract plasmid.
4. a double; two copy Human p53 gene recombinant adenovirus, it is characterised in that: its preparation method is, recombinates 5 type adenovirus vector plasmids with the double; two copy Human p53 genes described in PacI enzyme action claim 3 so that it is linearisation, then uses CsCl2Purification, through LipofecAMINE2000 method transfected HEK 293,5%CO2, 37 DEG C hatch cultivation 10～14 days after, scraping cells, centrifugal segregation culture supernatants, stay 2mL culture fluid, concussion mixing, add ethanol bath method with dry ice freezing, 37 DEG C of water-baths are dissolved, and repeat freezing, dissolving, concussion cell lysis, it is thus achieved that virion.
5. the double; two copy Human p53 gene recombinant adenovirus as claimed in claim 4 application in preparation tumor suppression medicine.
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