KR20090090924A - Development of optimal eukaryotic expression vector - Google Patents

Abstract

An expression vector of eukaryote which has optimal condition in gene therapy and DNA vaccine is provided to highly induce the expression by inserting the interleukin-12 mutant gene. A recombinant vector pGX27 for expressing foreign gene comprises a CMV promoter (pCMV), intron (gIVS) of beta gloving gene of rabbit, nucleic acid cloning area (MCS) which encodes a target protein, polyadenylation signal(SV40 poly(A)) of SV40 virus, SV40 virus enhancer and antibiotics-resistant gene. The antibiotics-resistant gene is a kanamycin resistant gene (Kan). A composition for vaccine immunization or gene therapy comprises a recombinant vector pGX27 of expressing a foreign gene.

Description

Development of Optimal Eukaryotic Expression Vectors {Development of Optimal Eukaryotic Expression Vector}

The present invention relates to the development of vectors with optimal conditions for gene therapy and DNA vaccines, and more particularly to eukaryotic expression vectors developed by combining various components. The present invention also relates to a pGX27 vector and a pGX28 vector capable of inducing high expression of an interleukin-12 variant gene when an interleukin-12 variant gene is inserted into various eukaryotic expression vectors.

Many chemicals that have been developed and used as existing immunotherapeutics have limitations with their limited effects and many side effects, and protein preparations that are mostly cytokines or antibodies are in vitro. In addition to the enormous cost of mass production in China, the long-term safety testing of humans has been a major obstacle to development. Currently, chemotherapy and protein preparation are mainly used for the treatment of many refractory and infectious diseases, but it is difficult to cure them. For example, the recent surge in tuberculosis, which has been considered curable, is due to the development of new types of tuberculosis bacteria that are resistant to any type of chemotherapeutic agent.

New treatment methods that can overcome the disadvantages such as chemicals and protein preparations are DNA and gene therapy and prophylactic methods. These methods are not resistant, but also inexpensively and effectively treat infectious diseases, diseases caused by genetic defects. It shows potential for use in therapeutic and prophylactic vaccines. In particular, DNA vaccines used for immunotherapy using DNA can induce both antigen-specific humoral and cellular immune responses, and in particular, can produce strong Th1 and CTL responses, which can result in cellular immune responses for treatment. It can be applied to the treatment and prevention of various chronic diseases that are essential.

In general, the most important requirement in the immunization method using DNA is how efficiently the target gene to be expressed can be delivered into the cell. The DNA immunization method has received a spotlight in vector systems using viruses due to its low efficiency of in vivo delivery, but its use is currently limited due to its safety. On the other hand, attempts to use non-viral vectors, such as naked DNA, are safe, but their delivery efficiency in vivo is relatively low. Therefore, the key to DNA immunization method using such a non-viral vector is whether to enhance the expression of the target gene and maximize its stability through the development of the optimal vector.

Several factors have been reported to influence the efficiency of non-viral plasmid vectors used in DNA immunization methods. The most important factor is how stable the vector can express a large amount of the target protein in vivo. Factors that may affect the expression and stabilization of the target genes are, firstly, viral promoters and enhancers, which regulate their expression by controlling transcription initiation and enhancing transcriptional efficiency of the target genes in the vector, respectively. Can be improved. Second, intron and poly (A) (poly adenylation) sequences can enhance the expression of the gene of interest by inducing the stabilization of mRNA. High levels of target gene expression are known to induce potent immune responses (Barry et al., Vaccine 15: 788-91, 1997; Leitner et al., J. Immunol. 159: 6112-9, 1997), Genetic deficiencies are also essential in the treatment of diseases. Third, sequences that can enhance the expression of the target gene by enhancing the translation efficiency or the stability of the DNA itself can be a component of the vector. In addition, it is known that DNA motifs composed of unmethylated CpG dinucleotides present in the vector backbone can stimulate the immune system (Krieg et al., Nature, 374: 546-9, 1995). Such immunostimulatory sequence (ISS) can enhance the efficiency of DNA immunotherapy and the like by inducing a Th1 immune response (Roman et al., Nat. Med. 3: 849-54, 1997; Chu et al., J. Exp. Med. 186: 1623-31, 1997). Thus, optimal gene expression efficiency and immunotherapeutic efficiency will be obtained when the most appropriate combination of these various factors is achieved.

Against this background, the present inventors have diligently tried to develop vectors suitable for inducing an immune response in vivo by increasing the expression level of the gene of interest by combining various promoter / enhancer sequences, poly (A) sequences, intron sequences, and the like, known to date. Results: pGX27 vector consisting of pCMV, gIVS, MCS, SV40 poly (A), SV40 enhancer and antibiotic resistance genes and pGX28 vector consisting of pCMV, TPL, MCS, bGH40 poly (A) and antibiotic resistance genes were the optimal plasmid vectors. The present invention has been completed by confirming that it can be usefully used.

Accordingly, it is an object of the present invention to provide optimal vector components selected by comparing the effects of various plasmid vector components on enhancing the desired gene expression.

Specifically, an object of the present invention is pGX27 vector consisting of pCMV, gIVS, MCS, SV40 poly (A), SV40 enhancer and antibiotic resistance genes and pGX28 consisting of pCMV, TPL, MCS, bGH40 poly (A) and antibiotic resistance genes To provide a vector.

Another object of the present invention is to provide a host cell transformed with the recombinant vector.

Another object of the present invention is to provide a composition for vaccine immunization or gene therapy comprising the recombinant vector.

To achieve the above object, in one aspect, the present invention provides a pGX27 vector consisting of pCMV, gIVS, MCS, SV40 poly (A), SV40 enhancer and antibiotic resistance genes and pCMV, TPL, MCS, bGH40 poly (A) and It relates to a pGX28 vector consisting of antibiotic resistance genes.

As used herein, the term "pCMV" refers to a Cytomegalovirus (CMV) early promoter. pCMV has been known to be the most potent regulatory element and has activity in a variety of cells (Schmidt et al., Mol. Cell. Biol. 10: 4406-11, 1990). Thus, pCMV has been used in most mammalian cell expression vectors.

As used herein, the term "gIVS" is one of a kind of intron sequence, and refers to an intron of a rabbit Better globin gene. Intron sequences generally have a positive effect on gene expression, as well as in vitro ( Buchman and Berg , 1988, Mol . Cell . Bilol . 8, 4395-4405; Huang and Gorman , 1990, Nucleic Acid Res . 18, 937-947) have also been reported in transgenic mice ( Brinster et al .. 1988, Proc Natl . Acad . Sci . USA 85, 836-840 .

As used herein, the term "TPL" refers to a tripartite leader (TPL) sequence of adenovirus. TPL of adenoviruses is known to increase translation efficiency (Logan et al., Proc. Natl. Acad. Sci. USA. 81: 3655-9, 1984).

As used herein, the term "SV40 poly (A)" refers to a late polyadenyl signal of Simian virus 40 (SV40). SV40 late poly (A) is known to stabilize RNA five times more than the SV40 early poly (A) sequence ( Carswell et al ., Mol . Cell . Biol . 9: 4248-58, 1989 ).

As used herein, the term "bGH40 poly (A)" refers to a bovine growth hormone (bGH) poly adenyl signal. The bGH40 poly (A) sequence showed three times higher target gene expression than SV40 initial poly (A) or human collagen poly (A) ( Pfarr et al ., DNA 5: 115-22, 1986 ).

As used herein, the term "SV40 enhancer" refers to an enhancer of Simian virus 40 (SV40), and is known to increase transcription efficiency by increasing the number of RNA polymerase II binding to DNA. ( Treisman et al., Nature , 315: 73-5, 1985 ).

As used herein, the term "antibiotic resistance gene" is a gene having resistance to antibiotics, and since the cells having the same survive in the environment treated with the antibiotic, it is useful as a selection marker in the process of obtaining a large amount of plasmid from E. coli. . In the present invention, since the antibiotic resistance gene is not a factor that greatly affects the expression efficiency according to the optimal combination of vectors, which is the core technology of the present invention, antibiotic resistance genes generally used as selection markers can be used without limitation. As specific examples, resistance genes for ampicillin, tetracyclin, kanamycin, kanamycin, chloroamphenicol, streptomycin, or neomycin may be used. Can use kanamycin resistance gene (Kan). The kanamycin gene, which can be used in human clinic, has been widely used for the development of naked DNA vectors for treating human refractory diseases.

As used herein, the term "MCS" refers to a multiple cloning site encoding a protein of interest. The MCS is a site that is recognized and cleaved by a specific restriction enzyme, and the nucleic acid sequence encoding the protein of interest can be easily inserted into the site of cleavage.

Examples of medically useful target proteins that can be inserted into the cloning site of the vector include hormones, cytokines, enzymes, coagulation factors, transport proteins, receptors, regulatory proteins, structural proteins, transcription factors, antigens, antibodies, and the like.

The protein of interest is preferably a human interleukin-12 variant gene (hp35 / IRES / hp40-N222L; hIL-12m). The human interleukin-12 variant gene (hIL-12m) is a mutated Asn-222 amino acid that is a glycosylation portion of the human IL-12p40 subunit, and its expression increases the expression of the active IL-12p70 while increasing its expression. Secretion of -12p40 is known to reduce and is disclosed in Korean Patent No. 10-0399728.

In a specific embodiment of the present invention, a mouse interleukin-12 variant gene (mp35 / IRES / mp40-N220Q; mIL-12m) corresponding to the human interleukin-12 variant gene (hIL-12m) was used. mIL-12m is a mutated Asn-220 amino acid, a glycosylated moiety of the murine IL-12p40 subunit. The expression of this gene increases the expression of active IL-12p70 while decreasing the secretion of IL-12p40. Immunization with HCV E2 gene in mice, a known and small animal model, has been reported to induce an optimal cell-mediated immune response, especially after a long period of time ( Ha. et al ., Nat . Biotechnol . 20: 381-6, 2002 ).

In the present invention, the elements may be prepared by artificially synthetic or genetic recombination methods, and are provided by operatively connecting to a vector capable of expressing the same.

As used herein, the term "vector" refers to a gene construct that is capable of expressing a protein of interest in a suitable host cell, and includes a genetic construct comprising essential regulatory elements operably linked to express the gene insert. As used herein, the term "operably linked" refers to a functional linkage of a nucleic acid expression control sequence and a nucleic acid sequence encoding a protein of interest to perform a general function. For example, a promoter and a nucleic acid sequence encoding a protein may be operably linked to affect expression of the nucleic acid sequence encoding. Operational linkage with recombinant vectors can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation uses enzymes commonly known in the art and the like.

In the present invention, a combination of various promoters / enhancer sequences, poly (A) sequences, intron sequences, and the like, to increase the expression level of a gene of interest and develop vectors suitable for inducing an immune response in vivo, Based on this, the components of the plasmid vectors such as pCMV, gIVS, SV40 poly (A), SV40 enhancer, RSV enhancer, bGH poly (A), and Kan are selected, and various restriction enzymes are used as follows. Vectors were prepared (FIG. 1). The produced plasmid vectors are arranged in the order of the components.

pVAX1-mIL-12m: pCMV, mIL-12m, bGH poly (A), Kan

pGX23-mIL-12m: pCMV, mIL-12m, SV40 poly (A), Kan

pGX24-mIL-12m: pCMV, gIVS, mIL-12m, SV40 poly (A), Kan

pGX25-mIL-12m: pCMV, gIVS, mIL-12m, SV40 poly (A), RSV Enhancer, Kan

pGX26-mIL-12m: pCMV, gIVS, mIL-12m, bGH poly (A), Kan

pGX27-mIL-12m: pCMV, gIVS, mIL-12m, SV40 poly (A), SV40 enhancer, Kan

pGX10-mIL-12m: pCMV, TPL, mIL-12m, SV40 poly (A), SV40 Enhancer, Kan

pGX28-mIL-12m: pCMV, TPL, mIL-12m, bGH40 poly (A), Kan

In addition, in order to select plasmid vectors exhibiting high levels of target gene expression through an optimal combination of these plasmid vectors, the present invention inserts a mouse interleukin 12 variant gene into each of the developed plasmid vectors and inserts each vector into a mammalian cell. Phosphorus COS-7 cells and Hela cells were transformed and then ELISA assays for murine interleukin 12 p70 were performed using the cell supernatant obtained therefrom (FIG. 11).

Comparing the expression of interleukin 12 obtained in cell supernatants after transformation of expression plasmid vectors with SV40 poly (A), pGX23-mIL-12m exhibited the lowest level of interleukin 12 expression. PGX24-mIL-12m with more gIVS added to this expression plasmid vector showed higher expression levels than pGX23-mIL-12m, which can be expected to be an effect of gIVS. In addition, pGX25-mIL-12m with RSV enhancer at pGX24-mIL-12m showed similar expression levels as pGX24-mIL-12m, which would be expected that RSV40 enhancer did not affect expression level improvement in the composition of this combination. Can be. However, pGX27-mIL-12m substituted with SV40 enhancer instead of RSV enhancer in pGX25-mIL-12m showed higher expression levels compared to pGX24-mIL-12m and pGX25-mIL-12m, with the highest expression of the combination You can see the level. As another example demonstrating that the combination of pGX27 vector is optimal, pGX10-mIL-12m with TPL instead of gIVS in pGX27-mIL-12m showed significantly lower expression levels than pGX27-mIL-12m.

Comparing the expression of interleukin 12 obtained in cell supernatants after transformation of expression plasmid vectors with bGH poly (A), the expression of interleukin 12 with the lowest pVAX1-mIL-12m inserted into the currently available pVAX1 (invitrogen) vector Levels were indicated. PGX26-mIL-12m with more gIVS added to this expression plasmid vector showed higher expression levels than pVAX1-mIL-12m, which can be expected to be an effect of gIVS. Also, pGX28-mIL-12m, which substituted gIVS with TPL in pGX26-mIL-12m, showed higher expression levels than pGX26-mIL-12m, indicating that TPL, not gIVS, is the optimal combination for bGH poly (A). Can be.

Similar results can be obtained in ELISA using Hela cell supernatant. These results indicate that pGX27 vector among SV40 poly (A) vector and pGX28 vector among bGH poly (A) mice were interleukin 12. It can be seen that it is an optimal plasmid vector for expression of variant genes.

Thus, pGX27 and pGX28 vectors are not limited to, but are not limited to, antigenic genes in the prevention and treatment of diseases that can be cured by increased immunity in the human body, such as AIDS, hepatitis C and B, cancer, flu, tuberculosis, and malaria. It can be usefully used as a vector into which the immune regulatory factor gene can be inserted.

As another aspect, the present invention relates to a host cell transformed with the recombinant vector.

The cell transformed with the vector of the present invention is preferably an animal cell or a cell derived from an animal.

The method of introducing the vector of the present invention into the cell includes any method of introducing nucleic acid into the cell, and may be performed by selecting a suitable standard technique as known in the art. Electroporation, calcium phosphate co-precipitation, retroviral infection, microinjection, DEAE-dextran, cationic liposome Law, and the like.

As another aspect, the present invention relates to a vaccine immunization or gene therapy composition comprising the recombinant vector.

When the vector of the present invention is used as a DNA vaccine, a pharmaceutically acceptable carrier, for example, a phosphate buffer, is preferably used together with the DNA vaccine vector component of the present invention containing a therapeutically effective amount of a foreign gene. (PBS), saline, glucose, water, glycerol, ethanol or mixtures thereof. Preferably, the vector of the present invention can be used with phosphate buffer solution. In addition, adjuvant components known in the art may be further used.

DNA vaccine vectors and vaccine compositions comprising the same can be administered in a variety of ways including, but not limited to, oral, nasal, pulmonary, intramuscular, subcutaneous or intradermal routes. They may be administered to the subject as an injectable composition, for example as a sterile aqueous suspension, preferably isotonic solution.

The mode of application of the vaccine composition containing the vector of the present invention includes the size and species of the animal to be treated, the amount of the composition or composition of the DNA vaccine vector to be administered, the route of administration, the efficacy and dose of the adjuvant compound used, and the person skilled in the art. It will be appreciated that this may vary depending on other factors that are apparent to the person.

The present invention provides vectors with optimal conditions for gene therapy or DNA vaccines. In addition, when the interleukin-12 variant gene is inserted into the various eukaryotic cell expression vectors of the present invention, it is possible to induce high expression of the interleukin-12 variant gene among various eukaryotic cell expression vectors. In addition, the optimal vector of the present invention can be usefully used for gene therapy or DNA vaccine by inserting the gene of interest.

Hereinafter, the present invention will be described in more detail in the following examples. However, these examples are only illustrative of the present invention and do not limit the scope of the present invention.

Example  General Molecular Biology

Treatment of restriction enzymes (all restriction enzymes were from New England Biolab, Inc.), agarose gel electrophoresis, gel extraction kit (Solgent gel extraction kit), protein extraction with phenol / chloroform, ethanol precipitation Methods commonly used in molecular biology, such as DNA fragmentation and E. coli transformation, pure plasmid DNA purification, and polymerase chain reaction (all primers are synthesized from Genome), are described by Molecular Cloning of Sambrook et al. The method described in) was applied with minimal modification.

Example  2. Preparation of Various Plasmid Vectors

A. pGX26 Manufacture

PCR was carried out by PCR amplification method with primers using vector pcDNA6 / TR (Invitrogen) as a template, and the amplified product was cut with NheI and HindIII, and the DNA fragment (0.58kb) was obtained, and the vector pVAX1 (Invitrogen) was used as a restriction enzyme. PGX26 (3.56 kb) was prepared by insertion into the site cut with (Fig. 2).

5'-gIVS (NheI): 5'-gctggctagcgtgagtttggggacccttgat-3 '(SEQ ID NO: 1, sense)

3'-gIVS (HindIII): 5'-taccaagcttcaattcgccctatagtgagtc-3 '(SEQ ID NO: 2, antisense)

B. pGX28 Manufacture

Vector pGX10 ( Ha et al., Nat. Biotechnol. 20: 381-6, 2002 ) was cut with restriction enzymes NdeI and Asp718, and the one of the two fragments (0.86 kb) DNA fragment treated with the same restriction enzyme The vector pGX28 (3.42 kb) was obtained by binding to a large size (2.56 kb) DNA fragment among two fragments of pGX26 (FIGS. 3 and 4).

C. pGX27 Manufacture

The vector pGX27 (3.77 kb) was obtained by combining the DNA fragment (1 kb) generated by cleaving the vector pGX26 with restriction enzymes NdeI and Asp718 with the DNA fragment (2.78 kb) generated by treating the vector pGX10 with the same restriction enzyme (Fig. 5). And FIG. 6).

D. pGX25 Manufacture

After PCR using PCR amplification method with primers using vector pRc / RSV (Invitrogen) as a template, the amplified product was cut with ClaI and SalI, and the obtained DNA fragment (0.32kb) was treated with vector pGX27 with the same restriction enzyme. The resulting DNA fragment (3.53 kb) was combined to obtain a vector pGX25 (3.85 kb) (FIG. 7).

5'-RSV Enhancer (ClaI): 5'-atctatcgatacgatgtacgggccagatata-3 '(SEQ ID NO: 3, sense)

3'-RSV Enhancer (SalI): 5'-atctgtcgaccaattatctctgcaatgcgg-3 '(SEQ ID NO: 4, antisense)

E. pGX24 Manufacture

The vector pGX27 was cut with ClaI and SalI and treated with Klenow fragments to make blunt ends, followed by binding to obtain vector pGX24 (3.5kb) (FIG. 8).

F. pGX23 Manufacture

Vector pGX24 was cut with restriction enzymes NheI and HindIII, treated with Klenow fragments to make blunt ends, and bound to obtain vector pGX23 (2.9kb) (FIG. 9).

Example  3. Rat Interleukin 12 Variant  ( mIL -12m) Preparation of Gene Inserted Vector

The mIL-12m gene fragment (2.3 kb) resulting from cleavage of the vector pGX10-mIL-12m with XhoI was combined with the DNA fragment resulting from cleavage of each vector (pGX23, pGX24, pGX25, pGX26, pGX27 and pVAX1) with the same restriction enzyme. By this, a mouse interleukin 12 variant (mIL-12m) gene-inserted vector was obtained (FIG. 10).

Example  4. In vitro of plasmid vectors ( in in vitro Efficacy analysis

In the constructed vectors, murine interleukin 12 mutant genes were inserted and transformed into mammalian cells to examine the expression level of this gene in an in vitro cell culture system, and the expression level of murine interleukin 12 was examined by ELISA.

COS-7 cells (ATTC) and Hela cells (ATCC) incubated with DMEM medium (Dulbecco's modified Eagle's medium, GIBCO-BRL) containing 10% heat-inactivated FBS (fetal bovine serum, GIBCO-BRL) Transformation was performed using lipofectamine (Invitrogen). Two micrograms of each of the various plasmid vectors containing the mouse interleukin 12 variant gene the next day after incubating 5 × 10 ⁵ COS-7 cells and 1 × 10 ¹⁰ Hela cells (ATCC) in a 60 mm dish (SARSTED) the day before transformation, and 0.5 μg of pNEB-Enhanced Green Fluorescence Protein (pNEB-EGFP), which can serve as a control, was placed in 100 μl of DMEM medium without FBS, and then 10 μg of lipofectamine was added to DMEM medium without FBS. Put into the μl and mix the two solutions and left at room temperature for 30 minutes. The cells cultured yesterday were washed twice with DMEM medium containing no FBS, filled with medium, and placed in the incubator. After 30 minutes, the cell medium was aspirated, and then 2.8 ml of DMEM medium containing no FBS was added to a mixture of plasmid DNA and lipofectamine. The cells were added to the cells and incubated in the incubator for 4-6 hours. Subsequently, the cells were replaced with DMEM medium containing 10% of FBS, incubated for 24 to 48 hours, cells were harvested, centrifuged, and the supernatants were recovered. The pellets were transformed by comparing the degree of EGFP expression through FACS analysis. Measured for efficiency comparison. The level of murine interleukin 12 variant present in the supernatant was measured by ELISA capable of measuring murine interleukin 12 (Phamingen). Figure 11 shows the relative amount of expression when looking at the expression level of the mouse interleukin 12 in COS-7 cell culture transformed with pVAX1-mIL-12m measured by ELISA at 100%.

1 is a schematic of various eukaryotic cell expression vectors used in the present invention.

Figure 2 is a schematic diagram showing the manufacturing process of the vector pGX26 used as eukaryotic cell expression vector of the present invention.

Figure 3 is a schematic diagram showing the manufacturing process of the vector pGX28 used as eukaryotic cell expression vector of the present invention.

Figure 4 shows a cleavage map of the vector pGX28 used as eukaryotic cell expression vector of the present invention.

Figure 5 is a schematic diagram showing the manufacturing process of the vector pGX27 used as eukaryotic cell expression vector of the present invention.

Figure 6 shows a cleavage map of the vector pGX27 used as eukaryotic cell expression vector of the present invention.

Figure 7 is a schematic diagram showing the manufacturing process of the vector pGX25 used as eukaryotic cell expression vector of the present invention.

Figure 8 is a schematic diagram showing the manufacturing process of the vector pGX24 used as eukaryotic cell expression vector of the present invention.

Figure 9 is a schematic diagram showing the manufacturing process of the vector pGX23 used as eukaryotic cell expression vector of the present invention.

FIG. 10 is a schematic diagram of a vector inserted with a mouse interleukin-12 modified gene (mp35 / IRES / mp40-N220Q; mIL-12m) used as a target gene in the present invention. FIG.

FIG. 11 shows relatively ELISA results for interleukin-12 performed in a cell culture supernatant obtained after intracellular transformation of expression vectors containing murine interleukin-12 variant (mIL-12m) genes in the present invention. will be. The X axis represents the relative expression of murine interleukin-12 in cell culture supernatants (%) and the Y axis represents the various eukaryotic cell expression vectors.

<110> POSTECH FOUNDATION <120> Development of Optimal Eukaryotic Expression Vector <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> sense primer for amplification of gIVS <400> 1 gctggctagc gtgagtttgg ggacccttga t 31 <210> 2 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> antisense primer for amplification of gIVS <400> 2 taccaagctt caattcgccc tatagtgagt c 31 <210> 3 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> sense primer for amplification of RSV enhancer <400> 3 atctatcgat acgatgtacg ggccagatat a 31 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> antisense primer for amplification of RSV enhancer <400> 4 atctgtcgac caattatctc tgcaatgcgg 30  

Claims (11)

       CMV promoter (pCMV), intron of rabbit Better globin gene (gIVS), nucleic acid cloning region (MCS) encoding target protein, late polyadenylic signal of SV40 virus (SV40 poly (A)), enhancer of SV40 virus ( SV40 enhancer) and antibiotic resistance gene comprising a recombinant vector for foreign gene expression, in order from the 5 'end. The vector of claim 1, wherein the antibiotic resistance gene is Kanamycin resistance gene (Kan). The vector of claim 1, wherein the vector is pGX27 disclosed in FIG. 6. A recombinant vector in which an interleukin-12 variant gene is operably inserted into the MCS of the vector according to claim 1. pCMV, tripartite leader (TPL) of adenovirus, nucleic acid cloning region (MCS) encoding target protein, bovine growth hormone (bGH) poly (A) and antibiotic resistance genes in order from 5 'end, Recombinant vector for foreign gene expression. The vector of claim 5, wherein the antibiotic resistance gene is Kanamycin resistance gene (Kan). The vector of claim 5, wherein the vector is pGX28 disclosed in FIG. 4. A recombinant vector in which an interleukin-12 variant gene is operably inserted into the MCS of the vector according to claim 5. A host cell transformed with the recombinant vector of any one of claims 1 to 8. The host cell of claim 9, wherein the cell is an animal cell or a cell derived from an animal. A composition for vaccine immunization or gene therapy comprising the recombinant vector of any one of claims 1 to 8.

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