KR100760320B1 - ANTITUMOR COMPOSITION COMPRISING EXPRESSION VECTOR ENCODING IMPORTIN α GENE AND RECOMBINAT p53 GENE - Google Patents

Abstract

The present invention relates to an anti-cancer composition comprising a vector expressing an importin α gene and a recombinant p53 gene or proteins thereof.

Cancer detection method using a mutant of the impotin α gene according to the present invention is capable of searching for cancer cells that are not mutated p53, anticancer comprising a vector or a protein thereof that expresses the import gene and recombinant p53 gene together The composition may be effective in chemotherapy because it induces apoptosis of cancer cells by normalizing or activating the function of p53.

p53, importin α, VP16, cancer cell killing, gene therapy

Description

Anticancer composition comprising a recombinant vector expressing an impotin α gene and a recombinant gene 35. {ANTITUMOR COMPOSITION COMPRISING EXPRESSION VECTOR ENCODING IMPORTIN α GENE AND RECOMBINAT p53 GENE}

1 is a photograph (400-fold) showing the results of confirming the position of p53 protein by immunohistochemistry in HBL-100 and ZR-75-1 cells.

Figure 2 is a photograph showing the results of electrophoresis after RT-PCR of the importin α gene in ZR-75-1 breast cancer cells.

Figure 3 is a schematic diagram showing the structure of the wild type (truncated-mutant type) importin α.

Figure 4 is a schematic diagram and photograph showing the experimental results proved by the yeast two-hybrid assay that the binding of the importin α with p53 and interaction.

Figure 5 is a schematic diagram showing the recombinant gene structure of the normal and missing type impartin α fused with Green fluorescence protein (GFP).

Figure 6 is a photograph taken by fluorescence and optical microscopy after transforming CHO cells with pIα / GFP and pIαNLSb / GFP vectors containing recombinant genes of normal and missing impartin α fused with GFP.

Figure 7 is a photograph showing the results of analysis of immunohistochemical staining of the position of the normal p53 protein in each cell after transforming the CHO-K1 cells with the genes of normal importin α and missing impartin α.

8 is a result of Northern blotting analysis of the expression level of p21 / WAF1, which is a major downstream mediator of p53 after transforming CHO cells with the normal type impotin α gene.

Figure 9 is a photograph of Western blot analysis of the stability of p53 protein itself after transforming CHO cells with the normal type impotin α gene.

10 is a graph of cell cycles analyzed by FACS analysis after transforming CHO cells with a normal type of importin α gene.

11 is a gene cleavage map of the pVP16 / p53-Impα expression vector.

12a transforms a human hepatocellular cell line Hep3B lacking p53 (homozygous deletion, null type) into a normal p53 gene, a normal importin α gene, a recombinant p53 gene, or a combination of a recombinant p53 gene and a normal importin alpha gene After the survival rate of the cells analyzed by the MTT assay is a graph showing the results.

12B shows the survival rate of cells after transforming HeLa, a uterine cancer cell having p53 normal, into a normal p53 gene, a normal import protein α gene, a recombinant p53 gene, or a combination of a recombinant p53 gene and a normal import protein α gene. It is a graph showing the result analyzed by the assay.

12C shows the survival rate of cells after transforming the human liver cancer cell line HepG2 having a normal p53 into a normal p53 gene, a normal importin α gene, a recombinant p53 gene, or a combination of a recombinant p53 gene and a normal importin α gene. The graph shows the result of analysis.

12D shows the survival rate of cells after transforming a human breast cancer cell line JAR having a normal p53 to a normal p53 gene, a normal import protein α gene, a recombinant p53 gene, or a combination of a recombinant p53 gene and a normal import protein α gene. It is a graph showing the results of the analysis.

12e shows the survival rate of cells after transforming CHO-K1, an animal ovarian cancer cell with normal p53, into a normal p53 gene, a normal import protein α gene, a recombinant p53 gene, or a combination of a recombinant p53 gene and a normal import protein α gene. A graph showing the results analyzed by the MTT assay.

Figure 12f shows the survival rate of cells after transforming human lung cancer cell line A549 with normal p53 into normal p53 gene, normal import protein α gene, recombinant p53 gene, or recombinant p53 gene and normal import protein α gene. It is a graph showing the results of the analysis.

Figure 12g shows the survival rate of cells after transforming human lung cancer cell line NCI-H322 with a mutation in p53 into a normal p53 gene, a normal importin α gene, a recombinant p53 gene, or a combination of a recombinant p53 gene and a normal importin α gene. Is a graph showing the results of the analysis by the MTT assay.

Figure 12h shows that the survival rate of cells after transforming NIH-3T3, a fibroblast with normal p53, into a normal p53 gene, a normal importin α gene, a recombinant p53 gene, or a combination of a recombinant p53 gene and a normal importin α gene It is a graph showing the result analyzed by the assay.

The present invention relates to an anticancer composition comprising an importin α gene and a recombinant p53 gene. More specifically, the present invention relates to an anticancer composition comprising a vector or proteins thereof simultaneously expressing the importin α gene and the recombinant p53 gene. It is about a method.

P53, a well-known tumor suppressor gene, activates genes such as p21 / WAF1 to inhibit cell proliferation, and is known to induce cell death (apoptosis) by being involved in the formation of boxes and superoxide radicals. (Ko, LJ, and Prives C, 1996 Genes Dev. 10: 1054-1072), which have recently been shown to have the same potent antiangiogenic function as angiostatin, which has been spotlighted as an anticancer drug (Clayman GL et al. , 1995, Cancer Res. 55: 1-6). In addition, the p53 gene mutation is the most frequently used anti-cancer gene for gene therapy of various human cancers because it is most frequently found in various tumors (Hollstein MD et al., 1991, Science, 253: 49-53; Hall S et al., Amer. J. Hum. Gen. 1997, 61: 785-9.).

Most cancer suppressor genes act as clear transcription factors, p53 is one of them. That is, since mRNA of the genes is synthesized by transcription in the nucleus, they are exported out of the nucleus so that the protein is synthesized and imported into the nucleus in order to act as a transcription factor. Even if a problem occurs, it will not function properly.

However, in breast cancer, about 37% of normal p53 was found in the cytoplasm of the tissue, and although it is a protein with normal function, it is a normal transcription factor because there is a problem in the smooth nuclear transport process required to function as a transcription factor. (Moll UM et al. Proc Natl acd Sci USA (1992) 89: 7262-6; Kim, IS, et al., J. Biol. Chem. 275 (30): 23139-23145).

The major factor of inactivation of the p53 gene is known to be mediated by depletion or nuclear exclusion by hDM2 (Mamand J et al. Cell (992). 69: 1237-45; Finlay Ca. Mol Cell biol. (1993). 13; 301-6; Haupt YR et al. Nature (1997). 387: 296-9). As described above, the p53 protein synthesized in the cytoplasm may move to the nucleus and then function in the nucleus through transport into the nucleus, and the protein that has completed its function in the nucleus is generally transported out of the nucleus through normal decomposition. . However, it is difficult to play a role if there is a problem with transport into the nucleus or if it is released out of the nucleus prematurely before it can function. Therefore, although the p53 gene is an ideal gene therapy material, it is difficult to expect sufficient activity of the p53 protein in cancer cells with abnormalities in transporting the p53 gene product into or out of the nucleus. Therefore, in order to maintain p53 activity in cancer cells with mutations in proteins related to intranuclear or extranuclear transport of these p53 proteins, it is necessary to modify the p53 gene according to the purpose. Therefore, genetic engineering techniques can be used to inhibit pDM hDM2 damage and increase the stability of p53, or to co-express the p53 gene in the nucleus transport protein to facilitate the transport of p53 in the nucleus to facilitate tumor cell growth. DNA vaccines for treating cancer that maintain tumor suppressor function that inhibits or induces apoptosis can be prepared. However, there are no recombinant p53 genes that have increased the stability of p53 by causing inhibition of degradation by hDM2, and the cause of abnormality in transport of the normal p53 gene into the nucleus has not been determined yet.

Therefore, the present inventors have increased the intracellular stability of p53 used in gene therapy therapy to enhance the function as a tumor suppressor gene and also conducted extensive studies on the causes of abnormalities in the nuclear transport mechanism of p53. It was confirmed that the recombinant p53 gene prepared to reduce the increase in the intracellular stability of p53, and recently found that the inventors are involved in the transport of p53 into the nucleus (Kim, IS, et al., J. Biol. Chem. 275 (30), 23139-23145) It was confirmed that the importin α was mutated in breast cancer cells in which a normal p53 gene was found, and the present invention was completed based on this.

Accordingly, an object of the present invention is to provide an anticancer agent capable of increasing the stability of p53, a tumor suppressor protein in cancer cells, and smoothing transport into the nucleus to normalize or activate the ability of p53 to kill cancer cells.

To achieve the above object, the anticancer agent of the present invention comprises a normal type impotin α gene and a protein thereof represented by SEQ ID NOs: 1 and 2, a recombinant p53 gene represented by SEQ ID NOs: 4 and 5, a protein thereof, and / or SEQ ID NO: 4 And a recombinant p53 gene and a normal type of importin alpha gene represented by SEQ ID NO: 1 and a protein thereof.

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

In the present invention, first, to determine the reason why the p53 activity does not appear in the cancer cell line in which the normal p53 gene is present.

Immunohistochemical staining was performed using anti-p53 antibody DO-1 to determine the intracellular location of p53 protein in cancer cell lines in which the normal p53 gene is present. As a result, p53 was located in the nucleus in normal cells, but a significant amount of p53 was located in the cytoplasm in ZR-75-1, which is a breast cancer cell. Expected to be abnormal.

Therefore, in order to confirm whether the importer α, which plays an important role in the nuclear transport of p53 protein in cancer cells, has been mutated in the cancer cells, the implanter α gene was cloned in breast cancer cells. The cloned importin α gene had a nucleotide sequence represented by SEQ ID NO: 3 and compared with the normal form represented by SEQ ID NO: 1 to bind with nuclear localization signals (hereinafter abbreviated as "NLS"). It was confirmed that the part was missing (see Figs. 2 and 3). Next, as a result of confirming the binding ability of the defective impartin α to p53, it could be seen that the improper binding (see Fig. 4), the intracellular local analysis of the defective impintin α using GFP, normal importin α Compared with the majority present in the nucleus, it was confirmed that a significant amount of the missing type impartin α exists in the cytoplasm and could not migrate into the nucleus (see FIGS. 5 and 6). In addition, as a result of confirming the position of the p53 protein by immunoassay using the anti-p53 antibody Pab 246 in the animal cell line transformed with the defective impintin α (Fig. 7) it was confirmed that only in the cytoplasm. Based on the foregoing, it was found that the cause of the lack of p53 activity in the cancer cell line in which the normal p53 gene is present was due to the deletion of the NLS binding portion of the importin α.

Subsequently, the normal impotin α was transfected into CHO cells, and then, to confirm the stability of p53, whether the expression level of p21 RNA whose transcription was regulated by p53 was increased was analyzed by Northern blotting. It was confirmed that the amount was significantly increased (see FIG. 8), and at the same time, the normal impotin α was transduced into the CHO cells, and Western blotting was performed to confirm the stability of p53. The monoclonal antibody PAb240 against p53 (or As a result of confirming the expression level of p53 using DO-1), it was confirmed that the expression level of p53 was high (see FIG. 9). Fluorescence activated cell sorting (FACS) was also used to measure the DNA content of CHO cells transformed with defective or normal importin α, but 24% of the cells were killed when normal importing α was added. Higher values than when normal p53 was delivered (see FIG. 10).

In addition to the above-described problems in the nuclear transport process of p53, the activity of normal p53 can also be degraded by protein degradation.

Human double minute2 (hDM2) protein, which plays an important role in the intracellular stability of p53, is often overexpressed in cancer cells, and hDM2 binds to the transactivation domain of p53 to inhibit p53 activity. Known. Accordingly, in the present invention, the recombinant p53 gene VP16 / p53 was prepared by replacing the binding site (h1 -75 amino acid) with hDM2 present in p53 cDNA by VP16 active domain (amino acids 412-490) and inserted into pIRESneo (Clontech). PVP16 / p53 vector was obtained. The recombinant p53 gene VP16 / p53 is represented by SEQ ID NO: 4 and the amino acid sequence of recombinant p53 based on the nucleotide sequence is represented by SEQ ID NO: 5. In addition, the pVP16 / p53 vector was transformed into E. coli strain TOP10 and the transformed E. coli strain Escherichia coli / pVP16 / p53 was deposited on January 22, 2001 with the accession number KFCC-11258 to the Korean microbial conservation center affiliated with the Korean spawn association.

Meanwhile, an expression vector pVP16 / p53-Impα capable of expressing two genes together was further prepared by inserting a normal impartin α into the pVP16 / p53 vector prepared above, and the vector was introduced into E. coli strain DH5α to prepare a transformant. The introduction method may be introduced through a known technique, that is, a thermal shock method, an electroporation method, and the like, and in addition to the E. coli strain, various transformation E. coli may be used. The Escherichia coli strain Escherichia coli / pVP16 / p53-Impα was deposited as Accession No. KFCC-11260 at the Korean Microbial Conservation Center attached to the Korean spawn association on January 22, 2001. The expression vector pVP16 / p53-Impα includes a combined form of the recombinant p53 gene represented by SEQ ID NO: 4 and the normal type impotin α gene represented by SEQ ID NO: 1.

The pVP16 / p53-Impα not only prevents hDM2 from inhibiting p53 activity by removing the binding site with hDM2, but also facilitates intracellular transport of p53 protein according to the expression of normal importin α, thereby promoting p53 activity. Normalize or increase.

In order to confirm the anticancer effects of the pVP16 / p53-Impα, normal type importin α, and pVP16 / p53, various viable cell lines and cancer cell lines were treated to determine cell viability through MTT assay. As a result, it was confirmed that the necrosis rate in cancer cells is high, and the anticancer effect is excellent.

The above results are excellent because the expression vector pVP16 / p53-Impα expressing the recombinant p53 gene VP16 / p53 and normal type importin α produced in the present invention significantly increases the anticancer activity of p53 gene regardless of the state of p53 of target cancer cells. It can be used as an anticancer agent, and the recombinant p53 gene and the normal type impotin α gene can also be used alone as anticancer agents because they exhibit excellent anticancer effects. In addition, the administration of the importin alpha gene, the recombinant p53 gene, and the combined administration of the recombinant p53 gene and the importin alpha gene in the present invention have a synergistic effect by combining anticancer chemicals or radiation therapy.

In addition to the specific parent vector used for the production of the expression vector in the gene insertion process of all the above-described process, various expression vectors are possible, so that other expression vectors can be easily used according to the purpose of expression, and also the foreign gene insertion site of the expression vector. The size, nucleotide sequence, etc. of the gene to be inserted into can also be variously changed by known techniques.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the scope of the present invention is not limited thereto.

Example 1

Intracellular Location of p53 Protein in Breast Cancer Cell Lines

Known and not specifically described methods in all the examples below follow the method of Sambrook (Sambrook J. et al., Molecular cloning, 2nd edition, Cold Spring Harbor Laboratory Press (1989)).

To determine the intracellular location of the p53 protein in normal and breast cancer cell lines, first place a sterile cover slip at the bottom of the plate and place HBL-100 in McCoy's 5a medium (Life Technologies) containing 10% Fetal Bovine serum (FBS). Human mammary epithelial cells (ATCC) cells and human breast cancer cell ZR-75-1 cell lines (ATCC) were suspended and cultured until cell monolayers were formed. After incubation, the cells were washed with PBS (Phosphate buffered saline) buffer, fixed with 4% paraformaldehyde for 30 minutes, blocked with 10% normal goat serum, and then p53 antibody (anti-p53 antibody; DO-1, Santa Cruz Co.) was treated for 1 hour and washed three times with PBS buffer. Next, the anti-mouse secondary antibody (Fast red-conjugated secondary anti-mouse antibody; Zymed laboratories Inc.) was treated for 1 hour, washed with PBS buffer and observed under an optical microscope (see Fig. 1).

As a result, p53 was located in the nucleus in normal cells HBL-100, but a significant amount of p53 was located in the cytoplasm in ZR-75-1, a breast cancer cell. Therefore, it was confirmed that the mechanism involved in the influx of p53 into the nucleus of the breast cancer cell line was confirmed.

Example 2

Impotin α Gene Isolation

In order to purely isolate the impotin α gene, which is an intranuclear transporter and is known to be involved in the transport of p53 into the nucleus, and to determine whether the gene is abnormal in breast cancer cells, RT-PCR and Sequencing was performed. HBL100 cell lines were cultured in McCoy's 5a medium with 10% FBS, and ZR-75-1 was cultured by manufacturer's method in RPMI medium (GIBCO BRL.) With 10% FBS.

Using a RNA purification kit (Catrimox TM-14 RNA Isolation Kit; Takara Shazo Co., Shiga, Japan), total RNA was isolated from HBL100 cells by the manufacturer's protocol, and cDNA was prepared using the isolated RNA as a template. RT-PCR for the synthesis was performed. RT-PCR was performed using an RNA LA PCR kit (Takara Shazo Co., Shiga, Japan). First, 4 μl of 25 mM MgCl ₂ , 10 × RNA PCR buffer (100 mM Tris-HCl, pH 8.3) were added to the PCR tube. ), 2 μl 500 mM KCl), 2 μl each 10 mM dNTP mixture, 0.5 μl 40 units / μl RNase inhibitor, 1 μl 5 unit / μL AMV reverse transcriptase XL, Oligo dT-Adaptor Primer 1 μl, 400 ng of RNA was added, and the total reaction amount was 20 μl with distilled water, and the mixture was mixed well. 30 ° C. for 10 minutes at 60 ° C. and 60 ° C. at PTC-100 heat cycler It was performed once under the conditions of min, 99 ° C. for 5 minutes and 5 ° C. for 5 minutes to obtain a total cDNA of HBL100 cells.

20 μl of the finished cDNA, 6 μl of 25 mM MgCl _2, 8 μl of 10 × LA PCR buffer II, 65.1 μl of sterile water, and polymerase (TaKaRa LA Taq). 0.5 μl of polymerase) was added, and 2 μl (10 pmol) of the forward primer represented by SEQ ID NO: 6 and the reverse primer represented by SEQ ID NO: 7 specific to the importin α were mixed well, followed by a total of 100 μl of the reaction. The solution was amplified once at 94 ° C for 2 minutes, and then amplified at 30 cycles under conditions of 94 ° C 30 seconds, 56 ° C 30 seconds, and 72 ° C 1 minute 30 seconds. Electrophoresis of the amplified PCR product on a 9% agarose gel confirmed that the DNA fragment of about 1.6 kb was amplified in the HBL-100 cell line, and in addition to the normal importin α transcript in the ZR-75-1 breast cancer cell line. It was confirmed that the truncated transcripts were amplified (FIG. 2).

Known Southern blotting assay confirmed that the amplified transcript was an importin αDNA, and the imported DNA fragment (387bp) for cloning the identified DNA fragment into the pRIP-V vector (Bioneer). Was electrophoresed on a 1.0% agarose gel to cut only the desired bands, followed by Vent DNA polymerase (New England Biolabs, Inc., Beverly, MA, USA) and the forward primer and SEQ ID NO: PCR was amplified by the same method as above using the reverse primer represented by 9. After phenol extraction of the PCR product, T4 DNA kinase was cloned into the pRIP-V vector by a known ligation method to obtain a pRIP-V / Imp vector (Sambrook J. et al., Molecular cloning, 2nd edition, Cold Spring Harbor Laboratory Press (1989).

Ligation Product is CaCl ₂ To examine whether the desired DNA fragment was cloned and introduced into the TOP10F 'cells, a competent E. coli strain prepared by treatment with a solution (50 mM CaCl ₂ , 10 mM Tris-HCl, pH 8.0), was cloned. The plasmid DNA of colonies generated by incubating for 12 hours was isolated by a known method. The resulting plasmid DNA was confirmed by electrophoresis on 1.0% agarose gel after double deletion with EcoRI and Hind III restriction enzymes (FIG. 3). Sequencing of the importin αDNA in the pRIP-V / Imp vector was performed using the TopTM DNA Sequencing Kit (Bionia).

Sequence analysis confirmed that the site of binding to NLS in the normal type impotin α gene is missing (see FIG. 3).

Example 3

Analysis of the Interaction of Importin α with p53

The yeast two-hybrid assay was used to determine the binding and interaction of the impotin α with p53. The yeast strain YRG-2 was combined with pBD-impotin α gene (Impα) and pAD-p53 from Stratagene, followed by a filter lift assay (Bartel, P., and Field, S. 1995, Methods Enzymol.) Β-galactosidase activity was assayed.

As a result, it was confirmed that p53 and the importin α bind to each other (see FIG. 4). However, in the case of the mutant impartin α lacking the NLS-binding domain, it was confirmed that it could not bind to p53. Thus, the truncated impintin α-expressing plasmid (pBD-IαΔNLSb) was used as the import-in β-expressing plasmid Β-galactosidase is normally expressed upon introduction into yeast with -Iβ).

These results indicate that the missing part of the defective type of importin α plays a key role in the binding to p53.

Example 4

Preparation of Recombinant Importin α Vector and Location of Recombinant Protein in Cells

In order to confirm the intracellular positions of the normal and mutant importin α, the genes of the normal and mutated importin α obtained in Example 2 are known to pEGFP-N1 (Clontech) expression vector containing GFP (green fluorescent protein), respectively. PEGFP-Imp and pEGFP-mImp expression vectors were obtained by inserting the same method (see FIG. 5).

2 μg of the two expression vectors prepared above were introduced into CHO cells using the protocol of lipofectamine (Life Science Inc.) and the manufacturer. First, the CHO cells were cultured on a cover slip, and then treated with DNA-liposome complexes for about 8 hours to introduce respective expression vectors into the cells. After 48 hours, cells were washed with PBS buffer and fixed for 30 minutes with 4% paraformaldehyde. CHO cells expressing GFP-impotin α or GFP-impotin α mutant fusion proteins were observed under fluorescence microscopy (see FIG. 6), and DAPI (4 ′, 6′-diamidine-2′-phenylindole) for staining the nucleus. dihydrochloride) staining was performed by known methods.

As a result, it was confirmed that the defective impintin α exists in the cytoplasm while the normal impartin α was observed to be present more in the nucleus. Therefore, it is expected that the defective impartin α cannot move smoothly into the nucleus, so that the normal intranuclear migration of the proteins carried by the importin α into the nucleus cannot be achieved.

Example 5

Intracellular Location of p53 Protein in Cell Lines Expressing Defective Impotin α

In a breast cancer cell line expressing a deficiency mutation of the impartin α, a sterile cover slip was first placed at the bottom of the plate and the RPMI containing 10% FBS was added to confirm the location of the p53 protein that is transported into the nucleus. CHO cells were suspended in Gibco BRL) medium and cultured until cell monolayers were formed. The cultured cells were washed with PBS buffer and fixed for 30 minutes with 4% paraformaldehyde. Next, after blocking with 10% normal goat serum, anti-p53 antibody (Pab 246, Santa Cruz Co.) was treated for 1 hour and washed three times with PBS buffer. Fast red-conjugated secondary anti-mouse antibody (Zymed laboratories Inc.) was treated for 1 hour, washed three times with PBS buffer, and CHO cells were observed by fluorescence microscopy (see FIG. 7).

As a result, it was confirmed that p53 protein was mainly present in the nucleus in CHO cells expressing the normal type importin α, but not in the nucleus but in the cytoplasm in the CHO cells expressing the defective importin α.

Example 6

Northern blotting analysis confirmed whether p53 increased transcriptional activity

In order to determine whether p53 transcriptional activity was increased due to an increase in p53 safety in the cell line into which importin α was introduced, Northern blot analysis was performed to determine whether the expression level of p21 gene regulated by p53 was increased. Total RNA was extracted using TRIzol reagent (Life Technologies, Inc.) in CHO-K1 cells transduced with importin α. The extracted RNA was used for electrophoresis on 1% agarose-formaldehyde gel and then transferred to a nylon membrane (Hybond-N + nylon membrane; Amersham). Northern blotting was performed using p21waf1 / cip1 labeled with ³² P to determine the expression level of p21 gene using the transferred nylon membrane (see FIG. 8).

As a result, it was confirmed that the RNA expression level of p21 gene was significantly increased in the cells into which importin α was introduced.

Example 7

Western blotting analysis confirmed the stability of p53

In order to determine the stability of p53 in the CHO-K1 cell line into which the importin α was introduced, the whole protein of the cells transfected with the importin α was extracted by a known method. Next, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed using the extracted protein, and then transferred to a nitrocellulose membrane (Hybond-C extra membrane; Amersham). The transferred membrane was treated with PAb240 or DO-1 monoclonal antibody (manufactured by Santa Cruz Biotechnology) capable of detecting p53, followed by treatment with a secondary antibody (horseradish peroxidase-conjugated secondary antibody), followed by an ECL kit (Amersham). Color) to confirm the expression level (see FIG. 9).

As a result, it was observed that the expression level of p53 was high in the cells into which the importin α was introduced, and it was confirmed that the stability of p53 was increased.

Example 8

Cell cycle analysis

CHO-K1 cells (ATCC) were inoculated at 60 mm dishes at 2 × 10 ⁵ cells / dish and the pRIP-V / Imp and pRIP-V / mImp expression vectors of Example 2 were transduced into CHO-K1 cells. Incubate for 48 hours. After incubation, cells were harvested, suspended in PBS solution at a cell concentration of 10 ⁶ cells / ml, stained with propiodium iodide (produced by sigma), and DNA content was measured by FACS (see FIG. 10). ).

As a result, the percentage of apoptosis cells in the cell group transfected with the normal importin α gene was high as 24%, even higher than when the normal p53 gene was delivered.

Example 9

Recombinant p53 Gene Preparation

For the preparation of the recombinant p53 gene, first, the site of p53 active domain (amino acids 1-75) was replaced with VP16 active domain to block the depletion by hDM2. The VP16 active domain (amino acids 412-490) was subjected to PCR in the same manner as in the gene isolation of the importin α in Example 2 using the forward primer represented by SEQ ID NO: 10 and the reverse primer represented by SEQ ID NO: 11. . Next, the 250 bp VP16 active domain DNA fragment obtained by PCR was digested with EcoRI and BamHI, and then cloned into pIRESneo (Clontech) vector digested with the same restriction enzyme to obtain a pSneo / VP16 vector. It was confirmed.

After cleaving the vector prepared above with BamHI, p53 (amino acids 75-393) was used as a template of the pCMV53 vector in which p53 was cloned, and the forward primer represented by SEQ ID NO: 12 and the reverse primer represented by SEQ ID NO: 13 were used. In this example, PCR was performed under the same conditions as above to obtain a 1.5 kb p53 DNA fragment. The obtained DNA fragment was treated with BamHI, electrophoresed in a 0.9% agarose gel, separated from the gel, and then inserted into a pSneo / VP16 vector treated with BamHI by a known method to complete the pVP16-p53 vector. Next, the sequence was performed using the vector.

Sequencing analysis confirmed that the recombinant p53 gene of the present invention has a sequence represented by SEQ ID NO: 4.

Example 10

Recombinant p53 and Importin α Simultaneous Expression Vector Preparation

In order to simultaneously express the VP16-p53 gene and the normal import phosphorin α gene, a forward primer represented by SEQ ID NO: 14 and a SEQ ID NO: 15 using a pRIP vector cloned with the normal import phosphorin α gene as described above are used as a template. In the same manner as in Example 9 using the reverse primer shown in the amplification of the importin α and then cleaved with XbaI. On the other hand, double-cutting the pVP16-p53 vector with SmaI and XbaI and then inserted the above-described importin α DNA to complete the pVP16 / p53-Impα vector expressing the recombinant p53 and importin α at the same time (Fig. 11). The base sequence of the pVP16 / p53-Impα vector was confirmed by a known method.

As a result, it was confirmed that cloning was properly performed on the pVP16 / p53-Impα vector.

Example 11

Induction of pVP16 / p53-Impα Vectors into Cancer Cells and Death of Cancer Cells

Normal cell lines CHO-K1 (chinese hamster; ATCC), NIIH-3T3 (NIH swiss mouse; ATCC) and cancer cell lines Hep3B (hepatocellular carcinoma; ATCC), HeLa (epithelioid carcinoma; ATCC), HepG2 (hepatocellular carcinoma (ATCC), The cell death effect by pVP16 / p53-Impα vector was confirmed by MTT assay using JAR (choriocarcinoma; ATCC), A549 (lung carcinoma; ATCC), and NCI-H358 (lung bronchoalveolar; Korea Cell Line Bank).

First, each cell line was inoculated in a 96-well plate at a concentration of 10 ⁴ per well, and then cultured overnight in DMEM medium containing fetal calf serum and penicillin, and then washed with the same medium. Next, 5 μl (1 μg / 5 μl) of pVP16 / p53-Impα vector was added to a 1.5 mL microtube, and 0.375 μl (1 mg / ml) of poly-L-lysine was added thereto and left for 10 minutes. Thereafter, 4 μl of DCK / DOPE was slowly mixed and left for 20 minutes, 1 ml of DMEM medium was added to prepare a medium composition solution, and 100 μl of each well was added thereto. 24 hours after the addition of the medium, the degree of cell proliferation was measured using a CellTiter 96TM Non-Radioactive Cell Proliferation Assay kit (Promega). A dye solution ([3- (4,5-dimethylthiaxol-2yl) -2,5-diphenyltetrazolium bromide (MTT)) was added to each vector-introduced cell line and incubated for 4 hours at 37 ° C. in a CO ₂ incubator. After that, the reaction solution was added and left at 37 ° C. for 1 hour. After completion of the above reaction, the 96-well plate was transferred to an enzyme enhanced immunosorbent assay (ELISA) reader, and the OD value was measured at 570 nm (see FIGS. 12A, B, C, D, E, F, G, H). ).

As a result, in the cell line transformed with pVP16 / p53 / Impα, it was observed that all cancer cells were killed, and the effect was excellent.

12A, b, c, d, e, f, g and h, pVP16 / p53- even when 5 μl (1 μg / 5 μl) of pVP16 / p53 and normal-type importin α were administered, respectively. Although less effective than Impα, anti-cancer effects were shown.

As seen in the above examples, the pVP16 / p53-Impα, a vector expressing the recombinant p53 gene VP16 / p53 or VP16 / p53 gene and the normal type importin α produced by the present invention, has the stability and transport capacity of p53 into the nucleus. By increasing the concentration of the target cancer cells, regardless of the state of p53 when administered to cancer cells can be effectively used for gene therapy of various cancers. In addition, administration of the VP16 / p53 gene, the importin α gene, and the VP16 / p53-impotin α gene according to the present invention may exhibit a synergistic effect by performing anticancer chemistry or radiation therapy.

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Claims (10)

delete delete delete delete delete delete An expression vector pVP16 / p53-Impα expressing a recombinant p53 gene represented by SEQ ID NO: 4 and a normal type impotin α gene represented by SEQ ID NO: 1. The transformant transformed with the expression vector pVP16 / p53-Impα of claim 7 (Accession No .: KFCC-11260). An anticancer composition consisting of a vector expressing a recombinant p53 gene represented by SEQ ID NO: 4 and an impotin α gene represented by SEQ ID NO: 1. An anticancer composition comprising a recombinant p53 protein represented by SEQ ID NO: 5 and an impotin α protein represented by SEQ ID NO: 2.

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