KR20030079214A - Expression vector containing the restricted E1 gene of wild type adenoviurs serotype 5 and the animal cell line thereof - Google Patents

Abstract

PURPOSE: An expression vector containing the restricted E1 gene of wild type adenovirus serotype 5 and an animal cell line thereof are provided, thereby effectively inhibiting the formation of replication competent adenoviruses(RCA) which is usually formed in the cell line, and producing stable self-replication deficit recombinant adenovirus. CONSTITUTION: An expression vector pcDNA3-AdE1 (KCTC 10169BP) contains nucleotides 400 to 3532 of the restricted E1 gene sequence of wild type adenovirus serotype 5. A transgenic animal cell line (KCTC 10168BP) is produced by transforming an animal cell line with the expression vector pcDNA3-AdE1 (KCTC 10169BP), wherein the animal cell line is HeLa cell line; and the restricted E1 gene has the nucleotide length of 3.1kb.

Description

Expression vector containing the restricted early gene 1 of wild type adenovirus serotype 5 and animal cell line comprising the same {expression vector containing the restricted E1 gene of wild type adenoviurs serotype 5 and the animal cell line}

As the gene sequences of human beings are completed, the causes of intractable diseases such as cancer of the related art are being identified at the gene level, and gene therapy using the same has been applied in various diseases. Among the various virus mediators that deliver genes for therapeutic purposes to the target cells, adenoviruses are used a lot more. The adenovirus mediators have much higher gene transfer capacity than other types of virus mediators, and they are able to isolate high titers of adenovirus relatively easily. This is because infection with various tissue cells is possible, and in vivo delivery ability is superior (Brody, SL, and Crystal, RG, Ann NY Acad Sci., 716: 90-101, 1994).

The genome of the adenovirus is a 36Kb linear double-stranded DNA molecule with reverse repeating segments (ITR) at both ends and divided into capsidizing sequences (Psi), early genes and late genes. Early genes are divided into E1, E2, E3 and E4, among which E1 gene is essential for replication of adenoviruses (Tooze, J., DNA Tumor Viruses (revised), Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1981).

The E1 region of adenovirus is the place where expression occurs primarily after target cell infection, and consists of two transcription units, E1A and E1B genes. E1A protein is a transcriptional regulator that stimulates cell cycles, leads to endless growth, and activates transcription of E2, E3, and E4 genes. In addition, co-expression of E1B protein helps to proliferate the virus while inhibiting cell apoptosis and expression of host cell proteins (Jochemsen, AG et al., EMBO J., 6 (11): 3399-3405 , 1987; Telling, GC et al., J. Virol. 68 (1): 541-547, 1994).

By introducing an external gene at the position where the E1 gene is deleted, a non-replicable recombinant virus can be produced, and thus the viral vector can serve as a mediator of a specific gene into a target cell. Several steps are required to propagate the nonreplicable recombinant adenovirus that lacks the initial E1 region. First, the host packaging cell line is infected with the nonreplicable recombinant adenovirus and then transduced by the E1 protein expressed in the host packaging cell. Trans- complementation results in replication of the nonreplicable recombinant adenovirus. Subsequently, a sufficient amount is obtained through the cell crushing step and the purification process.

Host packaging cells can be produced by cells of mammalian species infected with human adenovirus, and can be made mainly from kidney cells, muscle cells, epithelial cells, fetal mesenchymal cells and the like. The most commonly used are human embryonic kidney cells comprising 293 cell lines including the left reverse repeat section, the encapsidated region, the E1 region, the pIX and the pIVa2 protein code region, which constitute the left end 12% of the entire serotype 5 adenovirus genome. (Graham, FL et al., J. Gen. Virol., 36: 59-74, 1977).

However, the problem related to the production technology of the present recombinant adenovirus is the homologous recombination between the unnecessary overlapping sequence consisting of 12% of the left end of the nucleotide sequence of the adenovirus genome in the cell and the non-replicating recombinant adenovirus genome sequence. Through replication competent adenoviruses (hereinafter referred to as RCA) (Imler, JL et al., Gene Ther, 3: 75-84, 1996; Graham, FL et al. J. Gen. Virol., 36: 59-74, 1977), which not only proliferates the virus in an uncontrolled manner, but also helps with unreplicable recombinant adenoviruses, leading to uncontrolled proliferation and eventually serious side effects in living tissues. (Lochmuller, H. et al., Hum. Gene Ther., 5: 1485-1492, 1994).

Accordingly, the problem to be solved by the present invention is to provide an expression vector comprising a limited minimum E1 gene fragment regulated by an inducible promoter in a cell line, and transduced with the provided expression vector, thereby replicating the host genome and a non-replicating recombinant adenovirus base. It is an object of the present invention to provide a transformed animal cell line which lowers the possibility of generating RCA by homologous recombination between sequences.

1 is a sequence map of the pCMV-AdE1 expression vector and adenovirus serotype 5 genome base sequence comparison photograph.

Figure 2 is an immunoblot picture showing the expression of E1A and E1B 55kDa and 21kDa protein in 293HEK, HeLa and H17 and H19 cells.

Figure 3 is a graph of the production comparison of the nonreplicable recombinant adenovirus-LacZ in 293HEK and transformed HeLa cell line.

Figure 4 is a photograph of the PCR with a non-replicating recombinant adenovirus-LacZ gene generated in each cell line to confirm the generation of replication competitive adenovirus (RCA) in 293HEK and transformed HeLa cell line.

In order to achieve the above object, the present invention provides an expression vector under the control of an inducible promoter with a 3.1 kb E1 gene fragment of a limited minimum adenovirus, and by transducing the provided expression vector, non-replicable recombination Provided is a transformed animal cell line that inhibits production of RCA by gene sequences overlapping with adenovirus.

Hereinafter, the present invention will be described in more detail.

An expression vector is provided characterized by the cloning of a sequence of 3532 at a limited minimum of nucleotides 400 having the functions necessary for viral replication from the wild type adenovirus serotype 5 genome.

Also provided is a transformed animal cell line characterized in that a wild type adenovirus E1 gene having a limited minimal nucleotide sequence is transduced with a cloned expression vector.

In addition, the animal cell line provides an animal cell line, characterized in that HeLa cell line using the adenovirus packaging cell line.

Hereinafter, the present invention will be described in detail with reference to Examples. The following examples are intended to specifically illustrate the present invention, but the present invention is not limited thereto.

Example 1 Construction of an Expression Vector, pcDNA3-AdE1, Containing a Limited E1 Gene of Wild-type Adenovirus Serotype 5

Adenoviruses have a wide range of serotypes, but the use of adenovirus 5 serotypes, which are relatively molecularly informative, can increase the reliability of the results.

To obtain an E1 region gene in the genome of wild type adenovirus serotype 5, after synthesizing primers for amplifying genes encoding the E1A and E1B gene regions, PCR of the wild type adenovirus serotype 5 was carried out with PCR as a template. . The base sequence of the 5 'side primer is 5'-GATCGAATTCTCAGGTGTTTTCCGCGT-3' (SEQ ID NO: 1), and the base sequence of the 3 'side primer is 5'-GATCCTCGAGCCACGCCCACACATTTC-3' (SEQ ID NO: 2). EcoRI and XhoI restriction enzyme recognition sites were inserted.

The template DNA was mixed with wild-type adenovirus serotype 5 culture medium and 10 mM Tris-Cl (pH.8) / 10 mM EDTA / 0.5% SDS buffer at 1:10, crushed, and treated with phenol, followed by 0.3M Sodium acetate / 100% ethanol. Precipitated and separated. PCR was carried out 30 times with 1 μg of template DNA and 100 pmol of primer, followed by preheating at 98 ° C. for 2 minutes, followed by 1 minute at 98 ° C., 1 minute at 55 ° C. and 2 minutes at 72 ° C. The reaction was terminated by a final extension reaction at 10 ° C. (DNA Engine gradient cycler PTC200, MJ Research, USA).

The 3.1 kb product obtained by PCR contained 3532 to nucleotides 400 encoding nucleotides E1A and E1B in the human adenovirus serotype 5 genome, double cleaved with EcoRI and XhoI restriction enzymes and then double cleaved to the same restriction site. The plasmid pcDNA3 (Invitro gen, USA) with a promoter was cloned to confirm the nucleotide sequence of the final wild type E1 DNA in the vector (Fig. 1). Hereinafter, it is named pcDNA3-AdE1. The expression vector pcDNA3-AdE1 was deposited on February 5, 2002 to the Gene Bank of Biotechnology Research Institute, affiliated with the Korea Institute of Science and Technology (Accession No .: KCTC 10169BP).

Example 2: Selection and Construction of New Cell Lines HeLa-H17 and H19

The construction of the cell line for the E1 region of adenovirus will be described. This cell line allows for the propagation of nonreplicable recombinant adenovirus lacking these regions. All transfections were performed by the SuperFect, Qiagen, USA system according to the manufacturer's protocol. More specifically, HeLa cells in a 5 cm diameter dish were transduced with pcDNA3-AdE1 for expression of the E1 protein induced by the CMV promoter of 5 µg to 10 µg.

The transduced cells were incubated for two days, and then the cells were detached with trypsin / EDTA, grown in an appropriate dish at an appropriate rate, and replaced with a culture medium containing G418 (GibcoBRL, USA) at a concentration of 800 µg / ml. If G418 is replaced every 3 days, selectable clones can be isolated after 3 weeks. When cell clones were large enough to be visually distinguished, they were individually implanted into Wells of 96-well culture plates.

If the number of cells is appropriately increased, infection is performed with mutant self-replicating recombinant adenovirus-LacZ at 1moi per cell number. After 5 days, the clones showing CPE (Cytopa thic effect) were selected to be strongly stained with X-gal and gradually amplified. X-gal staining was performed by removing the medium of cultured cells, washing with PBS, adding 0.5% glutaaldehyde (Sigma, USA) for cell fixation, fixing at 37 ° C. for 15 minutes, and then removing the fixed solution and PBS. After washing twice with X-gal solution and dyeing at 37 ℃ for 2 hours, the X-gal solution was removed and washed twice with PBS to proceed to the blue stained clone screening method. Each cell clone is then frozen and preserved in liquid nitrogen. In the present invention, the experiment was performed using two selected clones, of which HeLa-H19 clone was deposited on February 5, 2002, to the Gene Bank of Korea Institute of Science and Technology, Biotechnology Research Institute (Accession Number: KCTC 10168BP).

Example 3: Expression of Ad5 E1A and E1B Proteins in Modified HeLa-E1 Cells

Expression of Ad5 E1A and 21 kDa, 55 kDa E1B proteins in HeLa cells was confirmed by Western blotting using monoclonal antibody (mAb) (FIG. 2). M58 (PharMingen, San Diego, Calif.) Recognized the E1A protein, while 1G11 and 9C10 (Oncogene, USA) recognized 21kDa and 55kDa, respectively. First, total protein was extracted from 293, HeLa, and selected clonal cells used as a control using Lysisbuffer (0.1% Nonidet P-40 / 50mM Tris-Cl, pH.8 / 5mM EDTA / 250mM NaCl / 50mM NaF). 30 µg per well was added to the SDS-PAGE, followed by electrophoresis, and transferred to the nitrocellulose membrane. After blocking for 1 hour with 5% skim milk PBST (1 × PBS, 0.1% Tween 20), 1.5 μg / ml of M58, 1G11 and 9C10 were reacted at room temperature overnight. The immunoreactive protein was reacted with horseshoe-radish peroxidase-bound anti-mouse secondary antibody and anti-let secondary antibody (Promega, USA) in 5% skim milk PBST solution for 1 hour at room temperature. After washing with PBST it was confirmed by ECL detection kit (Amersham, England). As a result, the expression level of E1A protein and 55kDa, 21kDa protein can be confirmed in 293 cells used as a control (FIG. 2).

Example 4 Determination of Proliferative Capacity of E1 Deficient Adenovirus

It is the explanation that the selected cell clone contributes complementarity to the propagation of the nonreplicating recombinant adenovirus. Nonreplicated recombinant adenovirus-LacZ was measured by transduction over time.

Example 4-1 Propagation of Nonreplicable Recombinant Adenovirus

293 and HeLa cell lines were used as positive and negative controls, respectively, and selected cell lines were introduced into 10moi. After each cell was dispensed in an appropriate amount in a 10 cm diameter dish, the cells were transduced with a non-replicating recombinant adenovirus-LacZ at 10 moi per target cell for 24 hours, followed by 37 for 8, 24, 48, 72, 96 hours. All lysates including cells were collected after incubation in the incubator. Thereafter, three times of freezing and dissolution were repeated, and the supernatant was separated by centrifugation at 2,000 rpm.

Example 4-2: Import Rate Measurement

The titer of the nonreplicating recombinant adenovirus-LacZ virus present in the supernatant was measured by a gene transfer unit (GTU) by the beta-galactosidase gene. Untransformed HeLa cells in 6-well culture plates were dispensed with 4 × 10 ⁵ per well and washed with PBS the next day and then infected with the above-described timely separated supernatant at 37 ° C. for 1 hour. The supernatant was removed, washed with PBS, and then replaced with 2% FBS / DMEM. After 48 hours, the import rate of beta-galactosidase was measured. Cell culture medium was removed, washed with PBS, and lysed with 0.25M Tris-Cl (pH 7.8). After complete freeze / dissolution of the cells, the cells were centrifuged at 1000 rpm for 3 minutes in a 1.5 ml Eppendorf tube, and the supernatant of the cell extract was separated into a new tube. In welnae of a 96-well culture plates for color reaction 1mM MgCl ₂ / 45mM beta-Merced kepto ethanol / 67mM phosphate (pH 7.5) solution 204㎕ with 4mg / ㎖ the concentration of ONPG (2-nitrophenyl-β- D-galactopyranoside , Roche) solution 66μl and each cell extract 30μl was mixed and left for 2 hours at 37 ℃ and measured at 420nm wavelength by ELISA reader (Molecular dynamics), 3 days after infection HeLa-H19 cells The amount of virus in medium cultured during the same period showed the same result as that obtained from the positive control 293 cells (FIG. 3).

Example 5 Confirmation of RCA Generation

Dilute the virus medium taken after 3 days in infected cells of 293, H17 and H19 to 1:10 with distilled water, and add 1u / μl of DNAase I (Roche) to remove DNA from host cells. After incubation at 25 ° C. for 20 minutes, it was inactivated at 70 ° C. for 15 minutes. After boiling for 5 minutes for complete crushing of the virus (boiling) and left on ice. First, the base sequence of the 5'-side primer for amplifying the beta-galactosidase gene, which is a marker gene, is 5'-TGGTTATGCCGATCG-3 '(SEQ ID NO: 3), and the base sequence of the 3'-side primer is 5'-CTCGAATCAGCAACG- 3 '(SEQ ID NO: 4). The base sequence of the 5'-side primer for amplifying the E1 gene is 5'-AAGGTCCTGTGTCTGAACCT-3 '(SEQ ID NO: 5), and the base sequence of the 3' side primer is 5'-TCAGGAGGTGTGTTAGAAGG-3 '(SEQ ID NO: 6). PCR was performed 20 times with 1 μl of the diluted culture solution and 100 pmol of primer, followed by preheating for 2 minutes at 98 ° C., 30 seconds at 98 ° C., 30 seconds at 55 ° C., and 30 seconds at 72 ° C. The reaction was terminated by a final extension reaction at 72 ° C. for 10 minutes (DNA Engine gradient cycler PTC200, MJ Research, USA). The resulting PCR product was confirmed by electrophoresis (Fig. 4, M: marker, 1, 3, 5: when amplified 208bp using beta-galactosidase gene primer, 2, 4, 6: E1 gene primer When amplified using 175bp, 1, 2,: PCR in virus medium produced in 293 cells, 3, 4: PCR in virus medium produced in H17 cells, 5, 6: in H19 cells PCR in virus medium produced). As a result, unlike all of the 208 bp beta-galactosidase gene was identified, 175 bp E1 fragment was identified only in 293 culture solution (FIG. 4).

As described above, the present invention relates to the production of an animal vector transformed with an expression vector and a provided expression vector for selectively expressing only the E1 gene essential for self-proliferation of adenovirus to produce self-replicating recombinant adenovirus. By overcoming the RCA problem caused by the use of existing packaging cell lines, it is possible to produce a non-replicable recombinant adenovirus that can be used safely for treatment.

Although the present invention has been described in detail only with respect to the described embodiments, it will be apparent to those skilled in the art that various modifications and variations are possible within the technical scope of the present invention, and such modifications and modifications are within the scope of the appended claims.

Claims (3)

Expression vector pcDNA3-AdE1 (Accession No .: KCTC 10169BP), characterized in that the sequence of 3532 was cloned from E1 gene nucleotide 400 of wild-type adenovirus serotype 5. The animal cell line transformed with the expression vector pcDNA3-AdE1 cloned with the cloned E1 gene of wild type adenovirus serotype 5 of claim 1 (Accession No .: KCTC 10168BP). The method of claim 2, And wherein said animal cell line is a HeLa cell line.