JPH11332560A - Recombinant adenovirus, cosmid vector containing the recombinant virus genom and animal cell infected with the recombinant virus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To produce a cosmid vector enabling easy operations such as the modification and alteration of the gene of adenovirus and the introduction of a foreign gene and useful for the treatment of genetic diseases, etc., by including a specific adenovirus-like genom in the vector. SOLUTION: The cosmid vector contains an adenovirus-like genom having defects at E1 and E3 regions and completely containing the inverted terminal repeat sequences on both terminals or the vector has a restriction enzyme recognition site to isolate only the adenovirus-like genom part from the cosmid vector at both sides of the adenovirus-like genom part. Preferably, the cosmid vector is cut with a restriction enzyme to prepare a recombinant adenovirus wherein the expression of a gene subjected to the control of a tetracycline promoter is controllable by tetracycline.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a recombinant adenovirus, a cosmid vector having the recombinant virus gene, and an animal cell infected with the recombinant virus. It can be applied to the treatment of genetic diseases.

[0002]

2. Description of the Related Art Conventionally, in order to prepare a recombinant adenovirus, a basic adenovirus (usually a wild type) is grown in large amounts in cultured cells, and then the virus is purified. It requires a process to extract DNA from the virus. It also requires a step of causing homologous recombination between the purified viral DNA and the plasmid DNA grown in E. coli inside the cultured cells.
Therefore, when it was necessary to introduce or modify a foreign gene in multiple places of the recombinant adenovirus genome, an intermediate virus (a virus at the stage before the virus having the desired structure) was prepared. Later, the virus DNA must be purified, requiring a lot of time and effort.

[0003]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a method for growing and purifying a wild-type or intermediate adenovirus, which is indispensable in conventional techniques for preparing recombinant adenovirus. The object of the present invention is to eliminate the need for a DNA extraction step and to eliminate the need for a homologous recombination operation between purified virus genomic DNA and plasmid DNA grown in E. coli.

[0004]

SUMMARY OF THE INVENTION According to the present invention, the above-mentioned object is achieved by reversing the terminal repeat sequence at both ends.
(ITR: inverted terminal repeat), and the cosmid vector is characterized in that it has an adenovirus-like genome internally deficient in the E1 and E3 regions. Is achieved.

[0005] The cosmid vector according to the present invention has, on both sides of the adenovirus-like genome portion, restriction enzyme recognition sites for releasing only the adenovirus-like genome portion from the cosmid vector. In the cosmid vector according to the present invention, another restriction enzyme recognition site that cleaves the vector is present in each of the E1 and E3 deficient regions of the adenovirus-like genome, thereby enabling introduction of a foreign gene. Is advantageous. The cosmid vector according to the present invention further has another restriction enzyme recognition site in a portion on the right side of the adenovirus-like genome left inverted terminal repeat sequence or on a portion on the left side of the right inverted terminal repeat sequence, Advantageously, manipulations of modifying or altering the adenovirus-like genome have been made possible.

[0006] In the cosmid vector according to the present invention,
For example, an expression unit of a tetracycline transactivator and an expression unit of a gene controlled by a tetracycline promoter can be introduced into the E1- and E3-deficient regions. If this cosmid vector is cleaved with a restriction enzyme to give a structure in which both ends are inverted terminal repeat sequences, the DNA having this structure is introduced into an animal cell in which a recombinant adenovirus can proliferate. And the recombinant adenovirus can be used to infect animal cells.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention utilizes a cosmid vector because an adenovirus has a size of about 36000 base pairs in the case of a wild type, and a recombinant adenovirus vector has a size almost equivalent thereto. Is what you do.
The technique used to stably maintain and propagate the adenovirus-like genome inside E. coli using a cosmid vector is disclosed in JP-A-7-298877.
A method for preparing the recombinant adenovirus is also disclosed in the publication. Therefore, the present inventors stably maintain the adenovirus-like gene completely containing the ITRs at both ends with a different cosmid vector, and simultaneously treat the cosmid vector with a restriction enzyme to remove the adenovirus from the cosmid vector. It was possible to release only the genome. The recombinant adenovirus can be easily obtained by introducing the adenovirus-like genome completely containing both released ends into animal cells capable of growing the recombinant adenovirus, for example, 293 cells. Further, the cosmid vector according to the present invention is constructed to have a plurality of restriction enzyme recognition sites in order to facilitate introduction of a foreign gene or modification of the basic skeleton of the recombinant adenovirus.

Next, the structure of the cosmid vector according to the present invention will be described. Structure of Adenovirus-Like Genomic Portion The cosmid vector according to the present invention is composed of several fragments as described below, and Ax1cw derived from Ax1c (or Ax1w) disclosed in JP-A-7-298877.
(Ax1cw is available from RIKEN Genebank). This newly created cosmid vector is hereinafter referred to as “pacad1A”. An overview of this cosmid vector, pacad1A, is shown in FIG. pacad1A is composed of six fragments, with fragment 1 as the basic backbone, and fragments 2-6 linked to 1
In the form of two plasmids. This pacad1A is divided into four parts for genetic manipulation when it is used for producing a recombinant adenovirus. That is, the first part is the left part of the adenovirus-like genome (the region flanked by Bs-Bs in FIG. 1), which includes the complete left ITR, the packaging sequence and the E1 gene region (from 1.3% to 9.3%). % Deletion, and a recognition cleavage site by ClaI has been created at that site for the purpose of introducing a foreign gene). No. 2
The right part of the adenovirus-like genome (the region between E and E in FIG. 1) is the complete right IT
It contains R, and mainly contains the E3 gene region (removed from 79.6% to 84.8%, and a recognition cleavage site by SwaI has been created at that site for the purpose of introducing a foreign gene) and the E4 gene region. The third part is the middle part of the adenovirus-like genome, and is a part of the viral gene other than the above left and right parts. The final fourth part is a part other than the adenovirus-like genome, and in this case, is a part of one of the cosmid vectors, charomid. Each of these parts is composed of several fragments or parts of fragments. The origin of each fragment is clarified in Table 1 below, and pacad1A is directly derived from Ax1cw and the AxRSV-LacZ virus [H. Kojima et al.
m. Biophys. Res. Commun. Vol. 207, pages 8-12, (1
995)] as a template and a fragment amplified and synthesized by PCR [polymerase chain reaction] using primers, which are synthetic DNAs.

[0009]

[Table 1]

[0010] In order to solve the problem concerning the structure of the skeletal portion required for propagation in Escherichia coli as a charomid partial cosmid vector or packaging in λ phage, the present inventors have proposed p
acad1A has charomid 9-1 disclosed by Saito et al.
1 [I. Saito et al., "Proc. Natl. Acad. Sci. USA", Vol.
83, pages 8664-8668, (1986)] from which the polylinker portion (recognition and cleavage portion with EcoRI and HindIII of restriction enzymes) was removed.

Structure of Boundary Portion of Each Fragment The base structure at the boundary portion of each fragment constituting pacad1A shown in FIG. 1 is shown in FIG. 2 to FIG.
As shown in Fig. 2, the boundary between charomid 9-11 from which the polylinker portion was removed and the adenovirus-like genome was HindIII-Bst11 from the charomid side on the left side.
Restriction enzyme recognition sites are arranged in the order of 07I-PacI, followed by the left ITR of the adenovirus-like genome.
As shown in Figure 7, the right ITR portion of the adenovirus-like genome followed by the PacI-EcoRI
The restriction enzyme recognition sites are arranged in this order, and then reach the charomid portion. Used in the present invention
Since such a restriction enzyme recognition site is located in pacad1A, by treating with the restriction enzyme PacI, only the adenovirus-like genome
It is possible to release from the haromid part. In addition, Bs
By using t1107I or EcoRI, it is possible to selectively release the left end and the right end of the adenovirus-like genome, and therefore, it is easy to perform a processing operation when modifying or changing the adenovirus-like site.

For the purpose of inserting a foreign DNA fragment into the recombinant cosmid vector pacad1A or replacing a part of pacad1A with foreign DNA, p
processed acad1A (for some purposes, Klenowfragment
After addition of DNA for insertion or substitution)
A ligation reaction is performed with the fragments. Continue to Giga
Using a pack in vitro packaging kit (Stratagene), recombinant phages are prepared by packaging cosmid DNA into λ phage. The recombinant phage is used to infect a suitable E. coli (for example, DH5α) and incubated at 37 ° C. overnight on LB agar medium containing ampicillin. The colonies that appeared were individually collected and placed in 1.5 ml of LB medium containing ampicillin at 37 ° C overnight.
And shake culture. A cosmid vector-derived DNA is extracted from E. coli contained in the LB medium (miniprep operation), and the extracted DNA is treated with an appropriate restriction enzyme to isolate a clone having a desired structure.

Release and purification of adenovirus-like gene In order to purify a large amount of DNA of a pacad1A derivative clone having the desired structure, first, DNA obtained by extracting from 1.5 ml of LB medium is again used as a Gigapack invitro packagi.
Recombinant phage is made by packaging into λ-phage using ng kit. Infect the appropriate E. coli (DH5α) with the recombinant phage and overnight in an appropriate volume (50-200 ml) of LB medium containing ampicillin.
Perform shaking culture at 37 ℃. From this LB medium, use QIAGEN
DNA is extracted and purified using a column (QIAGEN) or a concentration density gradient ultracentrifugation method using CsCl. An appropriate amount of the obtained DNA was collected, and PacI (New Englan
d BioLabs). After the treatment with PacI, phenol / chloroform treatment and centrifugation are performed for the purpose of deproteinization. After a precipitation treatment with ethanol for the purpose of further purifying the DNA contained in the obtained supernatant, the precipitate (DNA) is further rinsed once with 70% ethanol. The rinsed DNA is dissolved in sterile distilled water, the DNA concentration is measured, and then frozen and stored until gene transfer to 293 animal cells is performed.

293 cells of adenovirus-like genomic DNA
The adenovirus virus-like genomic DNA obtained in the introduction above process to
600 μl Opti-MEM solution (Gibco) containing 10 μg
Mix with 80 µl of Lipofectamine reagent (Gibco) and 600 µl of Opti-MEM solution, and then stand at room temperature for 30 minutes. Then add another 7ml to this mixture.
Add Opti-MEM solution. This solution was prepared in advance
Replace with the culture solution of 293 cells [obtained from the ATCC (one flask with an area of 75 cm ² coated with collagen)] at 37 ° C, 5% CO ₂ (in a CO ₂ incubator)
Incubate at The DNA-containing mixture was then completely replaced with fresh cell culture and incubated overnight.
Incubate at ℃, 5% CO ₂ environment (in a CO ₂ incubator). On the day following the day of the above-described gene transfer procedure, 293 cells are released from the flask to form a suspension, and seeded on a 96-well plate coated with collagen, and the appearance and growth of the recombinant virus are awaited.

Isolation of recombinant adenovirus Normally, if 293 cells are maintained for 14 days after the gene transfer operation, newly generated recombinant adenovirus will
Since a cytopathic effect that proliferates in 293 cells and kills 293 cells is observed, it is possible to discriminate wells that have produced virus. That
Cryopreserve the dead cells containing the virus and culture medium from the wells. A portion of this stock solution infects fresh 293 cells and extracts genomic DNA from dead cells. After treating the obtained DNA with several restriction enzymes,
The cleavage pattern is confirmed by electrophoresis on an agarose gel. After a large amount of virus clones having the desired structure are propagated by the above-described operation, purification is performed.

Characteristics of the present invention Compared with the conventional method, the characteristics of the present invention are clarified. (1) Normally, the production of recombinant adenovirus is performed between two different DNA molecules in 293 cells. Although this step is unnecessary in the present invention,
(2) The recombinant adenovirus produced and emerging is extremely unlikely to have a structure other than the intended one, and (3)
In the conventional method, one of two different DNA molecules
Since DNA is extracted from virus particles grown and purified in large quantities, its preparation requires a relatively large amount of time and labor and specific facilities and equipment.In the present invention, pacad1A and its Since the derivative is a cosmid vector, it is only a step of growing in Escherichia coli.Therefore, large-scale preparation of a recombinant adenovirus can be performed easily and easily. (4) pacad1A has the form of a cosmid vector. As a result, the adenovirus-like genome can be stably maintained by λ phage and E. coli, and (5) complex operations required for modifying the genome are required for the adenovirus-like genome inside pacad1A. Various restriction enzymes (ClaI, Bst1107I,
XbaI, BamHI, EcoRI, SwaI and KpnI-see Figure 1)
Thus, it can be used to construct a new generation of recombinant adenovirus vector or a recombinant adenovirus vector having a complicated function.

[0017]

EXAMPLES Next, the present invention will be described in more detail and concretely with reference to examples and test examples, but it should be noted that this example is merely an example of application of the present invention. Example (Construction of recombinant adenovirus capable of regulating gene expression by tetracycline with a single vector) The construction of this type of recombinant adenovirus can be roughly divided into four steps, An overview is shown in Figure 8. Of these, steps 1 and 2 involve the recombination procedure for insertion of the expression unit into the cosmid vector, and step 3 involves adenovirus-like genomic DNA that completely contains the ITR portions at both ends (see Figure 1). For the release and purification of
Step 4 involves the operation of introducing the DNA into 293 cells and the operation of isolating emerging viruses.

[0018] Of the above steps, steps 1 and 2 each include the following steps. (a) treatment of a cosmid vector with a restriction enzyme and purification of the obtained DNA fragment, (b) purification of the DNA fragment to be introduced or replaced, (c) ligation reaction of both DNA fragments, (d) in In vitro packaging reaction and a step of infecting Escherichia coli (DH5α) with the obtained recombinant λ phage,
(e) extracting cosmid DNA from ampicillin-resistant Escherichia coli and confirming the structure of the DNA, and (f) adenovirus-like genomic DNA derived from a clone having the desired structure
A large amount of is prepared.

Steps 1 and 2 (Step of inserting expression unit into pacad1A): First, a tetracycline transactivator gene containing a nuclear translocation signal derived from the large T protein of SV40 virus (disclosed in JP-A-10-80274) NtTA gene) is placed downstream of a CAG promoter (disclosed in JP-A-3-168087) and a tetracycline transactivator expression unit (p disclosed in JP-A-10-80274).
(Derived from the CANtTA plasmid) was cut out with ClaI (Takara Shuzo Co., Ltd.) and purified. Next, after treating pacad1A with ClaI, a ligation reaction was performed with the NtTA gene expression unit, and GigapackIII Gold kit (Stratage
ne company) and in vitro packaging to λ phage. Escherichia coli (DH5α) was infected with this λ phage, seeded on LB agar medium containing ampicillin, and incubated at 37 ° C overnight. The next day, DNA was extracted from the newly emerged colonies, and the structure was confirmed through restriction treatment with Bst1107I and SpeI to obtain a clone (pacadCANT) having the desired structure. In the obtained pacadCANT, the NtTA expression unit is introduced rightward).

Next, using the Gigapack III Gold kit
E. coli with pacadCANT described above by in vitro packaging
(DH5α). The λ phage-infected Escherichia coli was cultured overnight in an LB medium containing ampicillin and grown.
Mass purification of pacadCANTDNA from this culture was performed using a QIAGEN column. A part of the obtained DNA was collected, subjected to restriction treatment with SwaI (Behringer-Mannheim), and the DNA fragment generated thereby was purified. This includes the tetracycline promoter [M. Gossen et al.
"Proc. Natl. Acad. Sci. USA", Vol. 89, pages
5547-5551 (1992)] downstream of the Picker Gene Promoter Vector 2 plasmid (Toyo Ink Co., Ltd.)
A ligation reaction was carried out by adding a DNA fragment containing a luciferase expression unit in which a firefly luciferase gene derived from the yeast and a late polyadenylation signal sequence derived from SV40 were arranged. About this ligation solution
Lambda phage was in vitro packaged using GigapackIII Gold kit (Stratagene). Escherichia coli (DH5α) was infected with this λ phage, seeded on LB agar medium containing ampicillin, and incubated at 37 ° C overnight. The next day, DNA was extracted from newly emerging colonies to confirm the structure. Desired structure (Luciferase expression unit controlled by tetracycline promoter
(leftward at the site of cleavage of pacadCANT by waI).
The DNA was named "TT-luc", and its DNA was prepared and purified in large quantities using a QIAGEN column. This DNA
It was dissolved in a TE solution [10 mM Tris HCl (pH 8.0) + 1 mM EDTA solution], and the DNA concentration was measured and then stored frozen (the above operations are Step 2).

Step 3 (Release and purification of adenovirus-like genomic DNA): 100 μg of pacadCANTT-luc DNA was added to 4
The cells were treated with 0 μl of PacI [4000 U / ml (New England BioLabs)] for 2 hours. Thereafter, phenol / chloroform treatment was performed once and chloroform treatment was performed twice, followed by precipitation treatment with ethanol. Precipitated DNA
Was rinsed with 70% ethanol and dissolved in 210 μl of sterile distilled water. Take 10 μl of this DNA solution and add D
The NA concentration was measured, and the remainder was stored frozen.

Step 4 (Transfection of adenovirus-like genomic DNA into 293 cells): The DNA obtained in Step 3 and having a known concentration and containing 10 μg of DNA was collected from the frozen stock. 600 μl of Opti
Dissolve in -MEM solution and contain 80μl Lipofectamine
It was mixed with 600 μl of Opti-MEM solution. Then, after leaving still at room temperature for 30 minutes, 7 ml of Opti-MEM solution was added and DNA-Lipofec for introduction of adenovirus-like genome was added.
A tamine mixture was used. 293 cell culture solution previously prepared in a 75 cm ² flask coated with collagen
(Cell confluence: 80%) from culture solution (DMEM containing 10% FBS)
The culture was removed, and the cells were lightly rinsed with 5 ml of Opti-MEM solution. Then, remove the Opti-MEM rinse solution and replace it with the above-mentioned mixture for genome introduction, at 37 ° C, 5% CO ₂
The cells were incubated for 4 hours in a CO ₂ incubator under the following conditions. Next, DNA for genome introduction
-Lipofectamine mixed solution with fresh culture solution (containing 10% FBS)
(DMEM solution) and further incubated overnight under the same conditions as above. The next day, the transfected 293
The cells were released from the flask, and used as a cell suspension using DMEM containing 10% FBS. This cell suspension was seeded on a 96-well plate coated with collagen and incubated at 37 ° C and 5% CO2.
The condition of ₂ was maintained.

After step 4 (Isolation and structure confirmation of recombinant adenovirus): As a result of the gene transfer operation performed twice independently, 16 to 14 days after the gene transfer treatment,
The cytotoxic effect of the virus was observed in 21 wells (gene transfer examples A and
B). We observed cytotoxic effect by virus
Both cells and culture medium were separately collected in sterile tubes and cryopreserved. After maintaining the cells for 14 days after the gene transfer treatment, the cells (dead cells + culture medium) from all wells with cytotoxic effects were observed using dry ice / ethanol and a 37 ° C water bath. After five times of rapid freezing and thawing, centrifugation was performed to collect the virus-containing supernatant.
A part of the supernatant was used to infect fresh 293 cells (inoculated on a 24-well plate), and when the cytotoxic effect was completely developed, dead cells were collected and DNA was extracted from the cells. This D
The structure was confirmed by treating NA with some restriction enzymes. Fig. 9 shows an example in which DNA derived from the virus obtained in the case of transfection example A was treated with SphI (16 cases).
It is shown. All viruses obtained are restriction enzymes
When treated with SphI, it is expected that DNA fragments of the size shown in Fig. 10 will be released, but in the case of transfection example A, all of the obtained 16-well viruses are identical. And it was found to have the expected structure (the newly obtained recombinant adenovirus was named "AcNTT-luc"). This proved that in the process of preparing the recombinant adenovirus according to the present invention, the probability of occurrence of unwanted viruses such as E1 positive virus was extremely low as compared with the conventional method. PacadCANTT-lu not treated with PacI
For c, transfection was performed under the same conditions as for pacadCANTT-luc treated with PacI (transfection examples C, D, E and F in Table 2 below), but no inhibitory effect on 293 cells was observed. Therefore, pacadC
Treatment of ANTT-luc with PacI revealed that it was necessary to release adenovirus-like genomic DNA completely containing ITRs at both ends.

[0024]

[Table 2] In Table 2, "Number of positive wells" refers to the number of wells in which the cytotoxic effect of the virus was observed on 293 cells.
Is the sum of the numbers.

Test Example (Evaluation on Function of AcNTT-luc Recombinant Adenovirus Vector) The AcNTT-luc virus vector according to the present invention has different gene expression units at different positions (E1-deficient site and E3-deficient site) on its genome. Have. In order for the luciferase gene under the control of the tetracycline promoter introduced into the E3 deficient site to be expressed, the expression of the NtTA gene introduced into the E1 deficient site is necessary. It is designed so that it can be inhibited by adding. Therefore, CHO-K1 cells (provided by the ATCC) were used to determine whether the genes introduced at different sites
Infect the NTT-luc virus and select [Multiplici
ty of infection): 1)], the expression of luciferase gene was examined by changing the concentration of tetracycline in the culture solution. The results are as shown in Table 3 below,
In the absence of tetracycline, high luciferase activity is observed, whereas in the presence of tetracycline, the activity decreases as the concentration increases, and in the absence of 1 μg / ml tetracycline, the activity decreases by 1000 μm.
Activity was reduced by a factor of one. From these results, the recombinant adenovirus according to the present invention infects animal cells,
It was confirmed that the virus had a function of expressing a target gene product (protein) from the virus invading the inside of the cell, that is, from different gene expression units present at different places on the genome. This means that in the case of AcNTT-luc, E3
Expression of a foreign gene under the control of the tetracycline promoter introduced into the deletion site is induced by expression of the NtTA gene introduced into the E1 deletion site, and the induction can be inhibited by adding tetracycline to the culture system. There is no other evidence that it has an adjustment function.

[0026]

[Table 3] In Table 3, ^* : average ± standard deviation

The recombinant adenovirus vector according to the present invention
Comparison of a recombinant adenovirus with a conventional recombinant adenovirus. The characteristics of the recombinant adenovirus according to the present invention, i.e.
The expression of a foreign gene (under the control of a promoter) is transferred to another site in a single adenovirus-like genome.
The characteristics of a type of recombinant adenovirus that is induced by the expression of the A gene and has an expression regulating function that the induction can be inhibited by adding tetracycline to the culture system are clarified in comparison with the conventional recombinant adenovirus. It is as follows. When a foreign gene is introduced into animal cells using a conventional recombinant adenovirus, the expression of the foreign gene is usually constitutive, and external control is often impossible, and the expression can be regulated. In such cases, the usefulness is low because the expression level of the target transgene itself is poor or the degree of induction or suppression thereof is poor. In contrast, Goss
en etc. ["Proc. Natl. Acad. Sci. USA", Vo
l. 89, pages 5547-5551 (1992)], improved by Hamada et al. (JP-A-10-80274), a system that can control the expression level of a transgene with tetracycline is an extremely useful system. is there. Although this system has already been applied to recombinant adenovirus vectors, the expression sites of the tetracycline transactivator and the tetracycline promoter are controlled at separate sites (E1 and E3 deletion sites) in a single adenovirus vector. This is the first time that the present invention is a recombinant adenovirus having an expression unit for a foreign transgene placed below. By introducing both the expression unit of the tetracycline transactivator and the expression unit of the foreign transgene under the control of the tetracycline promoter into a single adenovirus, a plurality of viruses are prepared in the present invention, In addition to saving the trouble of handling, if one virus invades animal cells, it has the advantage that the expression of the desired foreign transgene can be regulated. Furthermore, as shown in Table 3 of the test examples, the use of this type of recombinant adenovirus can induce (or suppress) gene expression at least 1000-fold. The recombinant adenovirus of the present invention having such an expression regulating function is extremely useful when used for the purpose of gene therapy or when applied to analysis on the function of a physiologically active substance possessed by cells or living bodies. Means.

[0028]

According to the present invention, the use of the cosmid vector having an adenovirus-like genome according to the present invention makes it possible to modify or change an adenovirus gene, introduce a foreign gene, etc., which have conventionally required complicated operation processes at a plurality of sites. The operation can be performed easily. In this case, it is not necessary to prepare a large amount of adenovirus or its genomic DNA, so that time and labor can be greatly reduced.

[Brief description of the drawings]

FIG. 1 is a diagram showing an outline of the structure of pacad1A, which is a recombinant cosmid vector used in the present invention. In the figure, E1 deletion site: early expression region of adenovirus (early
region) 1 deletion site, E3 deletion site: adenovirus early expression region 3 deletion site, COS: an essential part for packaging this cosmid vector into λ phage, Ori: in E. coli a plasmid origin of replication, parts performing a function of the cosmid vector is amplified in E. coli, Amp ^r: gene portion to confer resistance to ampicillin, an antibiotic in E. coli transformed with the cosmid vector , ITR: Inverted terminal repeats located at both ends of the adenovirus genome, Bs: Recognition and cleavage site by restriction enzyme Bst1107I, P: Recognition and cleavage site by restriction enzyme PacI, C: by restriction enzyme ClaI Recognition cleavage site, X: Recognition and cleavage site by restriction enzyme XbaI, B: Recognition and cleavage site by restriction enzyme BamHI, E: Restriction enzyme EcoRI Recognition cleavage site by, S: recognition cleavage site by a restriction enzyme SwaI, K: restriction enzyme KpnI by means recognized cleavage site is a restriction enzyme (C which is abbreviated in italics, X, B and
S) means that these restriction enzymes recognize and cut this cosmid vector at only one site.

FIG. 2 paca, a cosmid vector shown in FIG.
The figure which shows the base sequence of the boundary A part of the fragment 1 and 2 which comprise d1A, and the restriction enzyme which recognizes the base sequence in this boundary part and can cut | disconnect a vector.

FIG. 3 pcosm, a cosmid vector shown in FIG.
The figure which shows the base sequence of the boundary B part of the fragment 2 and 3 which comprise d1A, and the restriction enzyme which recognizes the base sequence in this boundary part and can cut | disconnect a vector.

FIG. 4 shows the cosmid vector paca shown in FIG.
The figure which shows the base sequence of the boundary C part of the fragment 3 and 4 which comprise d1A, and the restriction enzyme which recognizes the base sequence in the said boundary part and can cut | disconnect a vector.

FIG. 5: paca, a cosmid vector shown in FIG.
The figure which shows the base sequence of the boundary D part of the fragment 4 and 5 which comprise d1A, and the restriction enzyme which recognizes the base sequence in this boundary part and can cut | disconnect a vector.

FIG. 6 shows the cosmid vector paca shown in FIG.
The figure which shows the base sequence of the boundary E part of the fragment 5 and 6 which comprise d1A, and the restriction enzyme which recognizes the base sequence in this boundary part and can cut | disconnect a vector.

FIG. 7: paca, a cosmid vector shown in FIG.
The figure which shows the base sequence of the boundary F part of the fragment 6 and 7 which comprise d1A, and the restriction enzyme which recognizes the base sequence in this boundary part and can cut | disconnect a vector.

FIG. 8 is a diagram showing a recombinant adenovirus capable of regulating gene expression by tetracycline with a single vector, infection of animal cells with the virus, and expression of the virus by culturing the animal cells.

FIG. 9 shows the results obtained in Example A for gene transfer in Examples.
16 wells of recombinant adenovirus-derived DNA was treated with the restriction enzyme SphI and subjected to electrophoresis on an agarose gel to show the cleavage pattern.M indicates the marker lane, and the left side of the figure shows the marker The size (number of base pairs) of the DNA indicated by is indicated by a number.

[FIG. 10] Recombinant adenovirus gene DNA by SphI assumed to be represented by the target AcNTT-luc
The numbers indicate the size (number of base pairs) of DNA fragments released by digestion with SphI.

──────────────────────────────────────────────────の Continued on front page (51) Int.Cl. ⁶ Identification code FI (C12N 15/09 ZNA C12R 1:92)

Claims (8)

[Claims] 1. An adenovirus-like genome completely containing up to the inverted terminal repeats at both ends and having deletions in the E1 and E3 regions.
Cosmid vector. 2. On both sides of the adenovirus-like genome portion,
The cosmid vector according to claim 1, wherein the cosmid vector has a restriction enzyme recognition site that releases only the adenovirus-like genome portion from the cosmid vector. 3. The cosmid vector according to claim 1, wherein another restriction enzyme recognition site that cleaves the cosmid vector is present in the E1- and E3-deficient regions of the adenovirus-like genome, respectively. . 4. The adenovirus-like genome further has another restriction enzyme recognition site in a portion on the right side of the left inverted terminal repeat sequence or on a portion on the left side of the right inverted terminal repeat sequence. The modification or alteration operation of the virus-like genome is easily performed,
The cosmid vector according to any one of claims 1 to 3. 5. The method according to claim 3, wherein the E1 and E3 deficient regions have an expression unit of a tetracycline transactivator and an expression unit of a foreign gene controlled by a tetracycline promoter, respectively.
The cosmid vector according to 1. 6. A recombinant adenovirus, which is prepared by cutting the cosmid vector according to claim 1 with a restriction enzyme. 7. The recombinant adenovirus prepared from the cosmid vector according to claim 5, wherein the expression of a gene under the control of the tetracycline promoter can be regulated by tetracycline. 8. An animal cell which is infected with the recombinant adenovirus according to claim 6 or 7.