CN1327000C

CN1327000C - Shuttle vector of repeatable adenovirus of targeting melanoma and adenovirus

Info

Publication number: CN1327000C
Application number: CNB021293619A
Authority: CN
Inventors: 陆应麟; 谢庆军
Original assignee: Institute of Basic Medical Sciences of AMMS
Current assignee: Institute of Basic Medical Sciences of AMMS
Priority date: 2002-09-06
Filing date: 2002-09-06
Publication date: 2007-07-18
Anticipated expiration: 2022-09-06
Also published as: CN1480534A

Abstract

The invention relates to a replicable adenovirus shuttle vector and an adenovirus containing the vector. The replicable adenovirus targeting human melanoma can selectively infect human melanoma cells, replicate autonomously in the cells, and achieve the purpose of specifically killing cancer cells, and has broad application prospects in the treatment of tumors.

Description

The shuttle vector of repeatable adenovirus of of targeting melanoma and adenovirus

Technical field

The invention belongs to the genetically engineered field, relate to a kind of repeatable adenovirus carrier of targeting melanoma, also relate to this construction of carrier and purposes.

Background technology

Idea with the viral therapy tumour is of long duration.Before more than 100 year, the tumour patient that physicians just notice the temporary phenomenon that disappears of tumour can occur after infecting certain virus, and begins to seek the virus that can kill tumour cell.To 1970, people found that 38 kinds have the virus of anti-tumor activity in animal or human's body body, and begin to carry out clinical trial in the tumour patient late.

In the last thirty years, because virusology, the develop rapidly of molecular biology and oncology, people have finished multiple viral full gene examining order, and the structure and the function of gene carried out comparatively detailed research, and can carry out structure of modification to virogene effectively, make improved virus can be in tumour cell proliferated specifically, and in normal cell, do not breed, can be with this virus infection tumour cell at time multiplexed cell system propagation and cracking tumour cell, the virion that discharges after the lysis is other tumour cell of subinfection again, up to whole tumour cells are killed.Because this virus can not be in the normal cell internal breeding, so little to normal primary cellular defect, this virus is called as the tumour-specific virus of proliferation.Meanwhile, the purifying of various viruses and the scheme that concentrates are also reached its maturity, thereby can prepare high purity in a large number, the virus that height is tired is used for oncotherapy.At present, existing multiple virus of proliferation enters clinical trial, wherein adenovirus mediated cancer cells cracking strategy is the most effective, doctor has tried out the adenoviral mutants ONYX-015 of the tumour cell that selectivity attacks the P53 defective on one's body and has united conventional chemotherapy in 30 patients that suffer from the incidence cancer the Fa Deluohu, 25 patients' tumour is dwindled, wherein 8 patients' tumour completely dissolve.(A controlled trial of intratumoral ONYX-015, a selectively-replicatingadenovirus, in combination with cisplatin and 5-fluorouracil in patients withrecurrent head and neck cancer.Nature Medicine 2000.6 (8): 879) this is to obtain the result that a large amount of tumours disappears and do not recur first in the phase ii clinical trial that the more patient of quantity participates in.

Tumour is the collaborative and multistage morbidity of polygene, the mechanism complexity, only be difficult to prove effective with the gene therapy scheme at single pathogenic factors such as cytokine or cancer suppressor gene, some directly kill the method for cancer cells, and then effect is remarkable, as suicide gene therapy etc.Select the replicability adenovirus too, it is also killed the cancer cells cracking in amplification, and can seek target cell on one's own initiative.But adenoviral mutants ONYX-015 still has its limitation, what be that it is primarily aimed at is the tumour cell of P53 defective, and in all tumor types, the tumour of P53 functional defect only accounts for about 50%, it to other 50% powerless, therefore be necessary to develop the selection replicability adenovirus of other type.

At present, the method that obtains recombinant adenovirus has two classes, and the one, the adenovirus expression carrier that has foreign gene is cut by enzyme, it is directly connected on the adenovirus DNA.At first the expression of exogenous gene box (is comprised promotor, exogenous gene sequence and tailing signal) be inserted into the below of adenovirus left end genome (0-1.3mu), this zone has comprised viral left end ITR, the assembling signal, the enhanser of E1A downcuts destination gene expression sequence and adenoviral sequence then, is connected into adenovirus 2.6-100mu sequence, rotaring redyeing 293 cell, packing obtains to have infective recombinant virus therein.Another group of methods is intracellular homologous recombination method, the shuttle vectors and the replication-defective virus DNA that are about to adenovirus change in 293 cells jointly, in 293 cells, pack then by homologous recombination (wanting homologous sequence to be no less than 1Kb at least), obtain recombinant adenovirus.

Foreign gene inserts the carrier in district, and promptly adenovirus shuttle vector generally all is designed to the efficient protein expression carrier, inserts the common same strong promoter of gene and links to each other, and transcribes to impel.Conventional construction process is to insert an expression cassette in the E1 district, by efficient promoter, and foreign gene, and poly (A) tailing signal of mRNA3 ' end constitutes.The direction of expression cassette may be not too important for expressing, but the promotor of dextrad can produce unusual mRNA.

People are for killing tumor cell specifically, and according to the biological characteristics of tumour cell, the singularity of genetic expression in the distribution of the acceptor molecule of cell surface and the born of the same parents has designed the method for some targets, mainly contains two kinds.The one, the outer target of born of the same parents mainly is the part or the antibody of the specific combination with it that designs at some special acceptors of tumor cell surface or antigen, arrives target site in order to guide various treatments unit; The one, target in the born of the same parents mainly is to utilize tumour-specific controlling element (promotor or enhanser) to be expressed in specific target cell to drive therapeutic gene, and this method has been used for the experimental study of multiple solid tumor, and has obtained expected effect.But various tissue-specific promoters generally be upstream regulatory sequence with gene coding region in the genomic dna after the Function Identification of organizing specific expression, directly use it for the target that instructs genetic expression.This natural gene expression regulating and controlling sequence is not to aim at gene therapy and be provided with, and their promoter activity is generally lower, therefore can't adapt to the high degree of specificity that therapy of tumor proposes, and the requirement of high expression level is so need be improved.Recently, Siders etc. (Transcriptional targetingof recombinant adenoviruses to human and murine melanoma cells.Cancer Res.56:5638) have obtained the tyrosine oxidase promotor of people and mouse and the dna fragmentation of enhanser respectively with PCR clone's method, then each fragment is carried out artificial recombination, and therefrom filter out the melanoma high specificity, the reorganization regulating and controlling sequence that expression level is high, its transcriptional activity is equally matched with at present the strongest CMV promotor, and its array mode is two placed in-line enhansers and adds a promotor.Under the guiding of the special regulating and controlling sequence of melanoma, can guarantee the optionally melanoma cell of infected person of the engineering adenovirus that makes up, reach the purpose of specific killing cancer cells.Park etc. utilize this reorganization regulating and controlling sequence to instruct the expression of suicide gene PNP, reached target kill the purpose of melanoma cell (Augmentation ofMelanoma-Specific Expression Using a Tandem Melanocyte-Specific EnhancerResults in Increased Cytotoxicity of the Purine Nucleoside Phosphorylase Gene inMelanoma.Hum Gene Ther.1999,10:889).But the adenovirus carrier of structures such as Siders is not reproducible.

Summary of the invention

For a kind of complete repeatable adenovirus carrier and adenovirus with melanoma target are provided, the present inventor has done following improvement on the basis of existing technology: the one, add controlling element at melanocyte, with the Humanmachine tumour is target, has the specific target tropism; The 2nd, make up the repeatable adenovirus carrier, utilize virus direct cracking of self-replacation in oncocyte to kill and wound oncocyte, have the high-level efficiency of gene transfection, to reach the purpose of more effectively treating malignant tumour.

The present invention is when making up the expression vector of adenovirus, and the adenovirus shuttle vector pACSR (-) that selects for use left-hand to express is to avoid producing unusual mRNA.Because the adenoviral gene group is big, the restriction enzyme site complexity, the difficulty of directly recombinating is so selected the method for homologous recombination for use.

Adenovirus carrier of the present invention and adenovirus can obtain by the following technical programs.

At first design two primers, the essential adenoviral replication promotor gene E1 district of amplification adenovirus propagation from recombinant plasmid pAd5XhoIC, then it is cloned into the T carrier, enzyme is cut and is identified and definite its direction of insertion, then with E1 district gene subclone to adenovirus shuttle vector pACSR (-), make up recombinant vectors pAC-AdE1.The design primer is template with murine melanoma B16 cell genomic dna with containing mouse tyrosine oxidase core enhanser plasmid phsTyr (0.9) PCAT respectively, the promotor and the enhanser gene of amplification mouse tyrosine oxidase, promotor and enhanser are cloned into the T carrier respectively, and enzyme is cut evaluation and is carried out sequencing.Other designs two pairs of enhanser primers, insert restriction enzyme site respectively, so that product connects, and is template with plasmid pGEM-enh, carry out pcr amplification respectively with newly-designed two pairs of primers, obtain two enhanser fragments that have specific restriction enzyme site, then the external connection of two fragments is made up enhanser dimer dihtenh, be cloned into the T carrier, carry out PCR and enzyme and cut and identify and check order, then it is connected with promotor, makes up the regulating and controlling sequence dihtenh-prom of target Humanmachine tumour.With the downstream of AdE1 gene regions subclone to regulating and controlling sequence, connect into dihtenh-prom-AdE1, enzyme is cut evaluation and is inserted restriction enzyme site by PCR, so that follow-up clone.Displace CMVAdE1 fragment among the pAC-AdE1, establishing target adenovirus shuttle vector pAdhepE1 with its entire segment then.With itself and adenovirus recombinant plasmid pJM17 cotransfection 293 cells, through homologous recombination, PCR identifies, obtain the selection replicability adenovirus AdhepE1 (preservation date: on 08 30th, 2002 of target Humanmachine tumour, depositary institution: the common micro-organisms center C GMCC of China Committee for Culture Collection of Microorganisms, deposit number: CGMCC No.0794), identify correct through PCR.

The selection replicability adenovirus AdhepE1 of target Humanmachine tumour of the present invention, preservation date is on 08 30th, 2002, depositary institution is China Committee for Culture Collection of Microorganisms common micro-organisms center, and unit abbreviates CGMCC as, and deposit number is CGMCC No.0794.

Description of drawings

Fig. 1 (a) is the pcr amplification figure that shows adenoviral replication promotor gene district.Wherein 1 is AdE1 PCR product (about 3000bp), and 2 is Lamda/HindIII DNA Marker

Fig. 1 (b) is that demonstration AdE1 gene clone is cut the figure of evaluation to the enzyme that the T vector construction transforms recon pGEM-AdE1, and 1 is Lamda/HindIII DNA Marker among the figure, and 2 are

PGEM-AdE1 BamHI+SpeI, 3 is pGEM-AdE1/BamHI+ApaI

Fig. 1 (c) shows pGEM-AdE1 plasmid synoptic diagram.

Fig. 1 (d) shows that AdE1 district gene subclone to adenovirus shuttle vector, makes up recombinant vectors pAC-AdE1 synoptic diagram

Fig. 1 (e) is that the enzyme that shows recombinant plasmid pAC-AdE1 is cut the figure of evaluation.5 are among the figure

Lamda/HindIII DNA Marker, 1,2,3,4,6,7,8,9 1-8# cloned plasmids/KpnI

Fig. 2 (a) is the figure that shows the pcr amplification of mouse tyrosine oxidase promotor enhanser.1 is mouse tyrosine oxidase enhanser PCR product among the figure, and 2 is mouse tyrosine oxidase promotor PCR product, and 3 is Φ X 174/HincIIDNA Marker.

Fig. 2 (b) is that the enzyme that shows mouse tyrosine oxidase promotor is cut the figure of evaluation.Wherein left Fig. 1,2,3,4 is that 1,2,3,4 clone BamHI enzymes are cut the result, and 5 is Lamda/HindIII DNA Marker, and 6,7,8,9 is 1,2,3,4 clones' EcoRI result.Right Fig. 1,2,3,4 is that 1,2,3, the 4 HindIII enzymes of cloning are cut the result, and 5 is Lamda/HindIII DNA Marker, and 6,7,8,9 is 1,2,3,4 clones' XbaI enzyme cutting results.

Fig. 2 (c) is the figure that shows the double digestion evaluation of mouse tyrosine oxidase promotor.1 is Lamda/HindIII DNA Marker among the figure, and 2 is pGEM-prom/EcoRV, and 3,4,5 is that 1,2,3, the 4 BamHI+EcoRI enzymes of cloning are cut the result, and 6 is mouse tyrosine oxidase promotor PCR product.

Fig. 2 (d) is the PCR evaluation figure that shows mouse tyrosine oxidase enhanser.1 is Φ X 174/HincIIDNA Marker among the figure, and 2,3,4 is 1,2,3 clone PCR products.

Fig. 3 (a) is the PCR amplification figure again that shows mouse tyrosine oxidase enhanser.1 is Φ X 174/HaeIII DNA Marker among the figure, and 2 is enhA PCR product, and 3 is enhB PCR product, and 4 is the enhPCR product.

Fig. 3 (b) is the external interface chart that shows mouse tyrosine oxidase enhanser.1 is enh PCR product (207bp) among the figure, and 2 is enhA, the external connection product of B, and 3 is Neo PCR product (433bp).

Fig. 3 (c) shows the dimeric PCR evaluation of mouse tyrosine oxidase enhanser figure.

Fig. 3 (d) shows the dimeric PCR structure of mouse tyrosine oxidase enhanser evaluation figure.1 is enh PCR product (207bp) among the figure, and 2,3 is A4, A8 clone's PCR product, and 4 is enh+Neo PCR product.

Fig. 3 (e) is that the dimeric enzyme of demonstration mouse tyrosine oxidase enhanser is cut evaluation figure.Wherein 1 is enh PCR product (207bp), and 2,3 is A4, and A8 clone's HindIII enzyme is cut the result, and 4,5 is A4, and A8 clone's EcoRI enzyme is cut the result, and 6 is middle Φ X 174/HaeIII DNA Marker.

Fig. 3 (f) is that display target makes up synoptic diagram to the transcription regulating nucleotide sequence dihtenh-prom of Humanmachine tumour.

Fig. 3 (g) is the PCR evaluation figure that shows dihtenh-prom.1 is dihtenh-prom PCR product among the figure, and 2 is Lamda/EcoT 14 I DNA Marker

Fig. 4 (a) shows that dihtenh-prom-AdE1 makes up synoptic diagram.

Fig. 4 (b) is that the enzyme that shows dihtenh-prom-AdE1 is cut evaluation figure.1 cuts the result for the SalI enzyme of pGEM-dhepE1 among the figure, and 2 cut the result for the HindIII enzyme of pGEM-dhepE1, and 3 is Lamda/EcoT 14 I DNA Marker.

Fig. 4 (c) is the pcr amplification figure that shows SpeI-T7-dhepE1-SP6.1 is the SpeI-T7-dhepE1-SP6LAPCR product among the figure, and 2 is Lamda/HindIII DNA Marker.

Fig. 4 (d) shows that pAdhepE1 makes up synoptic diagram.

Fig. 4 (e) is that the enzyme that shows pAdhepE1 is cut evaluation figure.1,2 for just among the figure, oppositely inserts clone's single endonuclease digestion, and 3 is oppositely to insert clone BamHI+XhoI, and 4 for forward inserts clone BamHI+XhoI, and 5 is Lamda/EcoT 14 I DNA Marker.

Fig. 5 (a) shows the influence figure of AdhepE1 to the tumour cell form.

Fig. 5 (b) shows the specific killing statistical graph of AdhepE I to Humanmachine tumour.1 is SK-Mel-1 group (does not infect on a left side, the right infection) among the figure, and 2 are HepG2 group (does not infect on a left side, the right infection).

Fig. 5 (c) shows that RT PCR detects the figure of the specifically expressing of gene.1 is HepG2 cell β-actin RT PCR among the figure, and 2 is SK-Mel-1 cell β-actin RT PCR, and 3 is Φ X 174/HaeIII DNAMarker, and 4 is HepG2 cell E1A RT-PCR, and 5 is SK-Mel-1 cell E1A RT PCR.

Embodiment

Below in conjunction with, with embodiment the present invention is described in further detail.

The clone of embodiment one, adenoviral replication promotor gene district AdE1

And the structure of adenovirus shuttle vector pAC-AdE 1

One. material

1. bacterial strain and plasmid

JM109 host bacterium, preserve this chamber; PAd5XhoIC contains Ad5E1 district 80～5788bp, Dutch Lai Deng university, and Van der professor Eb is so kind as to give; PACSR (-) recombinant adenovirus shuttle vectors, doctor Yang Qicheng of U.S.West Coast biotech company is so kind as to give; PIRES 1.neo eucaryon co-expression carrier is clontech company product; The pAC-GFP plasmid, this chamber makes up; PGFP-IRES 1 neo, this chamber makes up.

2. virus and cell strain

Ad-LacZ expresses the replication-defective adenoviral of beta galactose former times enzyme, and one Room doctor Chen Qian of this institute is so kind as to give; 95-D (PLA-801D) human cytomegalovirus lung cancer cell line, preserve this chamber; Hela, human cervical carcinoma cell, preserve this chamber; Hela-GFP, the human cervical carcinoma cell of stably express green fluorescent protein, preserve this chamber.

Two. methods and results

1. find the complete sequence in adenoviral gene group E1 district from Genbank, visible adenoviral gene group E1 district is by E1A and two genomic constitutions of E1B.

2. design the PCR primer in adenoviral replication promotor gene district

At 5 of upstream primer ' end, add the BamHI restriction enzyme site, be convenient to later subclone.

Upstream primer 5 ' AAAATGAGACATATTATC3 ' downstream primer 5 ' TCAATCTGTATCTTCATCGC 3 '

3. the pcr amplification in adenoviral replication promotor gene district

To contain Ad5E1 district 80～5788bp plasmid pAd5XhoIC is template, carries out pcr amplification with the Pfu high-fidelity DNA polymerase, obtains the amplified band of about 3000bp, shown in Fig. 1 (a).

With the PCR product cloning in adenoviral replication promotor gene district to the T carrier

PCR product in E1 district directly is connected with the pGEM-T carrier, transforms JM109 host bacterium, be laid on and contain X-gal, IPTG, in the agar plate of Amp, the result grows many bluenesss and white colony, shown in Fig. 1 (b).

5. enzyme is cut and is identified and definite its direction of insertion

Choose blueness, each one of white clone, the 2ml that all increases, little upgrading grain, restriction enzyme digestion and electrophoresis is identified, the blue cloned plasmids size of result is about 3000bp, is empty carrier, and white cloned plasmids size is about 6000bp, the AdE1 fragment has been inserted in preliminary affirmation, and then use BamHI+SpeI, and BamIIII+ApaI carries out double digestion, and the result is shown in Fig. 1 (c).

Because of BamHI is that E1 district head inserts the site, SpeI and ApaI are respectively the T carrier and insert fragment two ends restriction enzyme site, therefore can judge the direction of its insertion, shown in Fig. 1 (d).

6. E1 district gene subclone to adenovirus shuttle vector is made up collection of illustrative plates such as Fig. 1 (e).

Enzyme is cut evaluation, selects the KpnI restriction endonuclease for use, and because of shuttle vectors itself contains single KpnI site, the E1 fragment also has a KpnI site, so a restriction endonuclease can be judged recombinant chou, shown in Fig. 1 (f):

Use the SpeI restriction endonuclease again, this has single SpeI site in CMV promotor upstream because of shuttle vectors, the E1 fragment is also brought a SpeI site at afterbody from the T carrier, so can judging, a restriction endonuclease inserts segmental direction in the recombinant chou, the result shows that direction of insertion is correct, and recombinant vectors pAC-AdE1 successfully constructs.

The clone of embodiment two, mouse tyrosine oxidase promotor, enhanser gene

One. material

PhsTyr (0.9) PCAT contains mouse tyrosine oxidase core enhanser, and Gtinther professor Schtitz of Heidelberg, Germany DKFZ is so kind as to give; The B16 cell, murine melanoma, preserve this chamber; All the other materials are with embodiment one.

Two. methods and results

1. from Genbank, find the complete sequence of mouse tyrosine oxidase promotor, enhanser.

2. design the PCR primer of mouse tyrosine oxidase promotor, enhanser.

Mouse tyrosine oxidase promoter primer

Upstream primer 5 ' ATCCAGTAAGTCCATTAC3 '

Downstream primer 5 '

TGTCACAGACTTCTTTTCC3 '

Insert the EcoRI site at its upstream in the primer, be convenient to insert subsequently enhancer sequence, insert the BamHI site in the downstream primer, be beneficial to link to each other with the head in E1 district.

Mouse tyrosine oxidase enhanser primer

Upstream primer 5 ' CAAGGTCATAGTTCCTGCC3 '

Downstream primer 5 ' TATTGTGGTTTGCCAGGACC3 '

3. the pcr amplification of mouse tyrosine oxidase promotor, enhanser

With murine melanoma B16 cell genomic dna is template, carries out pcr amplification with designed primer, and electrophoresis observation is shown in Fig. 2 (a).As seen the amplified band of about 780bp and 207bp, and be primary product, consistent with expected result.

With the PCR product cloning of mice tyrosine enzyme promotor, enhanser to the T carrier

Downcut required fragment from sepharose, glass milk reclaims purifying, links to each other with the T carrier, transforms JM109 host bacterium, be laid on to contain X-gal, and IPTG, in the agar plate of Amp, the result grows many bluenesss and white colony.

5. enzyme is cut and is identified mouse tyrosine oxidase promotor

Choose white and clone several, the 2ml that all increases, little upgrading grain is identified promotor with BamHI, EcoRI, HindIII and XbaI enzyme cutting electrophoresis, the result is shown in Fig. 2 (b).Have among 4 clones 3 positive, and then carry out double digestion with BamHI+EcoRI, be further analyzed, the result is shown in Fig. 2-3:

Above result shows that the restriction enzyme site of the mouse tyrosine oxidase promotor of being cloned and size are correct, can send company's order-checking, to do last evaluation.

6.PCR identify mouse tyrosine oxidase enhanser

With G ü nther Sch ü professor tz of Heidelberg, Germany DKFZ be so kind as to give to contain mouse tyrosine oxidase core enhanser plasmid phsTyr (0.9) PCAT be template, carry out pcr amplification, directly be connected with the T carrier, transform, choose the clone, identify that with PCR the result is shown in Fig. 2 (d) again.As seen three big or small homogeneous of clone should be required enhanser fragment.

7. order-checking is identified

The promotor and the enhanser clone of preliminary evaluation are served the order-checking of sea living worker biotech company, the enhanser complete sequence has been surveyed the consistent (Lowings with bibliographical information of part with promotor as a result, P, Yavuzer, U., and Gading, C.R.Positive and negative elements gegulate a melanocyte-specificpromoter.Mol.Cell.Biol.1992,12 (8): 3653-3662; Nylarder, K.Bourdon, J.C.Bray, J.E., et al.Transcriptional activator of tyrosinase and TRP-1 by p53 linksUV irradiation to the protective tanning response.J.Pathol.2000,190 (1), 39-46), with two plasmid called after pGEM-mTyr-prom, pGEM-mTyr-enh, be abbreviated as pGEM-prom, pGEM-enh.

The transcription regulating nucleotide sequence of embodiment three, structure target Humanmachine tumour

One material

PGEM-mTyr-prom, this chamber makes up; PGEM-mTyr-enh, this chamber makes up; PGEM-Teasy (Promega company); Restriction enzyme EcoRI, HlndIII etc. (precious biotech firm); T4 dna ligase (magnificent company, Promega company); Taq archaeal dna polymerase (worker bio-engineering corporation is given birth in Shanghai); Pfu high-fidelity DNA polymerase (match Parkson company); Φ X174/HaeIII (precious biotech firm); λ/EcoT 14I DNA marker (precious biotech firm); Fool-PCR test kit (match Parkson company) MILLIPORE Ultrafree-DA recovery tube (MILLIPORE company); All the other are with embodiment one.

Two. methods and results

1. design the PCR primer of two pairs of mouse tyrosine oxidase enhansers

According to the sequencing result of pGEM-enh, design two couples of primer enhA and enhB again.In enhancer sequence, with transcription factor bonded one end is head, the other end then is a tail, in the upstream primer of enhA, kept the SpeI site of enhanser head among the pGEM-enh, add a SphI site, so that subsequently connected head-to-tail enhanser dimer is cloned into the upstream of promotor among the pGEM-prom; In the downstream primer of enhA, then inserted the HindIII site.In the upstream primer of enhB, insert a HindIII site, so that be connected with the downstream of enhA; In the downstream primer of enhB, then inserted the ECoRI site, so that link to each other with the upstream of promotor.Two primer sequences are as follows:

enhA

Upstream primer 5 ' CT C

ACTAGT GATTCAAGGTCATA3 '

SphI SpeI

Downstream primer 5 ' CTCAAGCT TATTGTGGTTTGCCAGGACC3 '

HindIII

enhB

Upstream primer 5 ' CTCAAGCTTCAAGGTCATAGTTCCTGCC3 '

HindIII

Downstream primer 5 ' CTCGAATTCTATTGTGGTTTGCCAGGACC3 '

EcoRI

According to the T7 of pGEM-T carrier multiple clone site both sides, the SP6 promoter sequence designs a pair of primer in addition, so that increase its inner cloned sequence, sequence is as follows:

Upstream primer 5 ' GACTCACTATAGGGCGAA3 '

Downstream primer 5 ' GGTGACACTATAGAATAC3 '

2. the PCR of mouse tyrosine oxidase enhanser increases again

With plasmid pGEM-enh is template, and with newly-designed two couples of primer enhA, enhB carries out pcr amplification respectively, obtains two enhanser fragments that have specific restriction enzyme site, shown in Fig. 3 (a).

3. external connection

With enhA, enhB PCR product ethanol sedimentation reclaims, and all carries out enzyme with the HindIII restriction enzyme and cuts, and repurity is reclaimed, and carries out external connection, and with Modified TAE electrophoretic buffer electrophoresis, the result is shown in Fig. 3 (b).

4. mice tyrosine enzyme enhanser dimer is cloned into the pGEM-Teasy carrier

The enhanser dimer connection product of bp more than 400 is downcut from sepharose, MilliporeUltrafree-DA manages recovery, the Taq archaeal dna polymerase adds the A tail, be connected with the pGEM-Teasy carrier again, transform JM 109 host bacterium, be laid on and contain X-gal, IPTG, in the agar plate of Amp, the result grows 38 bacterium colonies, and great majority are white.

5.PCR identify

Choose 32 of white clones, each 2ml that increases is divided into four groups of A, B, C, D, and thermo-cracking system pcr template is selected the T7 of pGEM-Teasy carrier multiple clone site both sides for use, the SP6 primer, and usefulness fool-PCR test kit Rapid identification, wherein one group result is shown in Fig. 3 (c).

A4 wherein, A8, B1, B3, D3 clone's size meets the requirements, and therefore selects A4, A8 to do further structural analysis, selects a pair of primer of enh for use, carries out fool-pcr amplification, and the result is shown in Fig. 3 (d).Distinctive two bands of the dimer of connecting end to end because of expanding, A4, A8 are the connected head-to-tail dimer of enhanser really.

6. enzyme is cut evaluation

The restriction enzyme site that inserts in order to confirm, and the needs of being convenient to follow-up clone are selected A4 for use, the A8 clone, and amplification, little upgrading grain carries out enzyme with HindIII and EcoRI and cuts, and the result is shown in Fig. 3 (e).Illustrate and inserted the HindIII site in the dimer really, respectively there is an EcoRI site at the dimer two ends.

7. order-checking is identified

Cut on the basis of evaluation at PCR and enzyme, the host bacterium that will contain recombinant plasmid pGEM-T-easy-dihtenh is sent the order-checking of the logical company of the six directions, consistent (the Lowings of sequencing result with bibliographical information, P., Yavuzer, U., andGading, C.R.Positive and negative elements gegulate a melanocyte-specificpromoter.Mol.Cell.Biol.1992,12 (8): 3653-3662; Nylarder, K.Bourdon, J.C.Bray, J.E., et al.Transcriptional activator of tyrosinase and TRP-1 by p53 linksUV irradiation to the protective tanning response.J.Pathol.2000,190 (1), 39-46).

8. make up the transcription regulating nucleotide sequence dihtenh-prom of target Humanmachine tumour

With plasmid pGEM-Teasy-dihtenh is template, T7, SP6 is a primer, uses the Pfu high-fidelity DNA polymerase, and PCR expands and the T7-dhtenh-SP6 fragment, ethanol sedimentation reclaims, after the ECORI enzyme is cut, and cut through the EcoRI enzyme, the linearized vector pGEM-prom of CIAP dephosphorization acid links to each other, make up the transcription regulating nucleotide sequence dihtenh-prom of target Humanmachine tumour, shown in Fig. 3 (f).

Connect product and transform JM 109 competence host bacterium, the result grows 6 bacterium colonies, selected clone, amplification, thermo-cracking system pcr template is with T7 and the pairing of promotor downstream primer, with fool-PCR test kit Rapid identification, the result has 5 clones positive, and the about 1200bp of its PCR product size is shown in Fig. 3 (g).

The structure of embodiment four, adenovirus shuttle vector pAdhepE1

One. material

1. bacterial strain and plasmid

PGEM-dihtenh-prom, this chamber makes up; PAC-AdE1, this chamber makes up; PJM17, the adenovirus recombinant plasmid, doctor Yang Qicheng of U.S.West Coast biotech company is so kind as to give;

2. cell

293 cells, two the Wu ancestral pool academicians in the court are so kind as to give; The SK-Mel-1 cell, human melanoma cell is available from preclinical medicine institute of consonance medical university cell bank.

3. reagent and test kit

Poly-l-lysine (Sigma company); Lipofectamine 2000 lipofectamine (Invitragen company); LA PCR test kit (precious biotech firm); PureFection transfection level plasmid purification test kit (Promega company)

Two. methods and results

AdE1 gene regions subclone to the dihtenh-prom downstream, being connected into dihtenh-prom-AdE1, construction strategy such as Fig. 4 (a). enzyme is cut and is identified correctly, shown in Fig. 4 (b).

Because of dihtenh, prom respectively has a HindIII site in the AdE1 fragment, and therefore list can cut out three bands with HindIII, and is consistent with expection.Obtain recombinant plasmid pGEM-dihtenh-prom-AdE1, abbreviate pGEM-dhepE1 as.

Design a primer again, with SP6 primer pairing, expand and whole dihtenh-prom-AdE1 fragment, and insert the SpeI site, be convenient to follow-up clone.

Upstream primer 5 ' CTC

GACTCACTATAGGGCGAA 3 '

SpeI T7

Obtain fragment SpeI-T7-dhepE1-SP6 through LA PCR, the result is shown in Fig. 4 (c):

With its whole CMV-AdE1 fragment that displaces among the pAC-AdE1, be built into target adenovirus shuttle vector pAdhepE1 again.Construction strategy such as Fig. 4 (d).

Enzyme is cut and is identified correctly, shown in Fig. 4 (e).

The preparation of embodiment five, target Humanmachine tumour recombinant adenovirus

With adenovirus shuttle vector pAdhepE1 and pJM17 cotransfection 293 cells, through homologous recombination, PCR identifies, obtains the selection replicability adenovirus AdhepE1 of target Humanmachine tumour.

One, material

PJM17, the adenovirus recombinant plasmid, doctor Yang Qicheng of U.S.West Coast biotech company is so kind as to give; 293 cells, two the Wu ancestral pool academicians in the court are so kind as to give; The SK-Mel-1 cell, human melanoma cell is available from preclinical medicine institute of consonance medical university cell bank; Poly-l-lysine (Sigma company); Lipofectamine 2000 lipofectamine (Invitragen company); PureFection transfection level plasmid purification test kit (Promega company).Other material is with front embodiment.

Two, methods and results

1. cell cultures

293 cells are in containing the high sugared DMEM nutrient solution (pH 7.2) of 10% foetal calf serum, 50U/ml penicillin, 501g/ml Streptomycin sulphate, in 37 ℃, 5%CO ₂Cultivate under the condition.Cell is an adherent growth, covers with the back and goes down to posterity with 0.25% tryptic digestion in 2～3 days.

The SK-Mel-1 cell is containing the MEM nutrient solution seal H 7.2 of 10% foetal calf serum, 50U/ml penicillin, 50ug/ml Streptomycin sulphate) in, in 37 ℃, 5%CO ₂Cultivate under the condition.Cell is a suspension growth, covers with the back and divides bottle to go down to posterity in 3～5 days.

2. the poly-lysine bag is by 6 orifice plates

Poly-lysine mother liquor (20 ℃ frozen) with tri-distilled water preparation 0.1mg/ml; Every hole adds 0.5ml, and room temperature is placed and spent the night; Liquid is removed in suction, washes once with tri-distilled water, dries up in the super clean bench, is used for 293 cell bed boards subsequently.

3.Lipofectamine2000 mediation cotransfection 293 cells.

Plasmid pAdhepE1 and pJM17 are increased in a large number, and with PureFection test kit plasmid purification, with Lipofectamine2000 mediation cotransfection 293 cells, with pAC-GFP and pJM17 cotransfection indication transfection efficiency, operation is carried out according to the test kit explanation.Observe by the expression of green fluorescent protein in 293 cells, it is higher to see transfection efficiency, and about about 30～50%.

4. the CPE method is surveyed virus titer fast

With 293 cell inoculations in 6 well culture plates, 2 * 10 ⁵Cells/well was cultivated after 24 hours, and cell grows to 90% converging state, and the nutrient solution sucking-off with in each hole adds 10 respectively ²～10 ⁶The venom of doubling dilution adsorbed 1～2 hour, and with the sucking-off from each hole of viral liquid, every hole adds the DMEM nutrient solution that 2ml contains 10%FBS, cultivates and observes CPE after 48 hours, selects the extension rate that 100%CPE takes place, and calculates virus titer with following formula:

5. after the transfection the 7th day, pathology appearred in visible cell, and lesion region enlarges gradually afterwards, until complete pathology.Cell is reclaimed, freeze thawing three times, releasing virus is inoculated in the SK-Mel-1 cell, and complete pathology appears in cell after 2 days, cell is reclaimed, freeze thawing three times, releasing virus, centrifuging and taking supernatant, extract the recombinant adenovirus genomic dna as template, carry out PCR with mouse tyrosine oxidase promoter primer and identify.Mouse tyrosine oxidase promoter fragment is peculiar by mouse, with people and adenovirus genomic dna without any homology, therefore can conclude that the recombinant adenovirus that is obtained is AdhepE1.CPE method mensuration recombinant adenovirus AdhepE1 titre is about 2 * 10 fast ⁶Pfu/ml.

Embodiment six, targeting melanoma repeatable adenovirus are crossed the specific killing effect of tumour

After homologous recombination goes out gene engineering adenovirus AdhepE1, can carry out vitro detection, see whether it can carry out specific killing to Humanmachine tumour.

One. material

AdhepE1, the gene engineering adenovirus of the target Humanmachine tumour that this chamber makes up; HepG ₂, human hepatocellular carcinoma, preserve this chamber; All the other materials are with front embodiment

Two. methods and results

1.PCR primer

β-Actin primer

Upstream primer: 5 ' GTGGG GCGCC CCAGG CACCA3 '

Downstream primer: 5 ' CTTCC TTAAT GTCAC GCACG ATTTC3 '

The E1A primer

Upstream primer: 5 AAA ATGAG ACATA TTATC3 '

Downstream primer: 5 ' TTATG GCCTG GGGCG TTTAC3 '

2.AdhepE1 specific killing to Humanmachine tumour

With 5 * 10 ⁴Human melanoma cell is that SK-Mel-1 and human hepatocellular carcinoma cell line HepG2 are laid on six orifice plates, with 1 * 10 ⁴Pfu low dosage AdhepE1 attacks respectively, the result, and big death of SK-Mel-1 cell after 6 days, the HepG2 cell is then uninfluenced, and growth is normal, the results are shown in Figure 5 (a).Through viable count, statistical results show, there is marked difference in the two, sees Fig. 5 (b).

3.RT-PCR detect the differential expression of E1A

With 1 * 10 ⁶Pfu AdhepE1 infects SK-Mel-1 and HepG2 respectively, and after 3 hours, collecting cell extracts cell total rna.With the expression of RT PCR detection E1A, AdhepE1 has expressed E1A in SK-Mel-1 as a result, and does not express in HepG2, sees Fig. 5 (c).Prompting AdhepE1 to the specific killing of Humanmachine tumour owing to this virus copy choice in human melanoma cell causes.

Claims

1. reproducibility recombinant adenovirus AdhepE1, preserving number is CGMCC No.0794.

2. the purposes of the described reproducibility recombinant adenovirus of claim 1 AdhepE1 in preparation melanoma medicine.