Description
Title of Invention: NOVEL HYBRID PROMOTER AND RE¬
COMBINANT VECTOR COMPRISING THE SAME
Technical Field
[1] The present invention relates to a hybrid promoter, in which a whole or a part of a
CMV enhancer, a whole or a part of a b-actin promoter, a whole or a part of a CMV
promoter, and a whole or a part of a b-actin intron are operably linked to each other, a
recombinant vector comprising the same, a transformant transformed with the r e
combinant vector, a pharmaceutical composition comprising the recombinant vector or
the transformant, and a method for preparing a target protein using the recombinant
vector or the transformant.
[2]
Background Art
[3] In order to express a target gene in a host cell, an expression vector and a gene
transfer technique for carrying a structural gene of interest and expressing the same
within the cells are required. In this regard, the expression vector capable of expressing
a DNA fragment inserted therein generally includes regulatory elements, such as a
promoter or an enhancer. Such regulatory elements facilitate the expression of a target
gene operably linked thereto. The expression vectors can be selected depending on
host cell type, target gene expression level, type of expression desired and the like, and
a variety of expression vectors have been developed to satisfy the desired purposes.
[4]
Disclosure of Invention
Technical Problem
[5] Accordingly, the present inventors have made an effort to develop an expression
vector suitable for increasing an expression level of a target protein, and found that a
hybrid promoter, in which a whole or a part of a CMV enhancer, a whole or a part of a
b-actin promoter, a whole or a part of a CMV promoter, and a whole or a part of a b-
actin intron are operably linked, is able to remarkably increase the expression level of
the target protein, thereby completing the present invention.
[6]
Solution to Problem
[7] An object of the present invention is to provide a hybrid promoter, in which a whole
or a part of a CMV enhancer, a whole or a part of a b-actin promoter, a whole or a part
of a CMV promoter, and a whole or a part of a b-actin intron are operably linked to
each other.
[8] Another object of the present invention is to provide a recombinant vector,
comprising the hybrid promoter and a target protein-encoding gene operably linked
thereto.
[9] Still another object of the present invention is to provide a transformant into which
the recombinant vector is introduced.
[10] Still another object of the present invention is to provide a pharmaceutical com
position comprising the recombinant vector or the transformant.
[11] Still another object of the present invention is to provide a method for preparing a
target protein, comprising the steps of:
[12] 1) culturing the transformant of the present invention;
[13] 2) inducing the expression of a target protein from the transformant; and
[14] 3) harvesting the expressed target protein from the transformant or the culture
solution thereof.
Advantageous Effects of Invention
[15] The present invention relates a novel hybrid promoter that is optimized for the
production of an antibody or a DNA vaccine. When a variety of target genes are
inserted into a recombinant vector including the hybrid promoter, transcription and ex
pression of the target genes can be improved. Therefore, the recombinant vector
including the hybrid promoter of the present invention can be effectively used for the
development of an antibody or the production of a DNA vaccine.
[16]
Brief Description of Drawings
[17] FIG. 1 shows the structure of a pGL3-Basic vector, which is used as a starting vector
in the present invention;
[18] FIG. 2 shows the structure of a pGL3-Promoter(SV40) vector, in which a SV40
promoter is introduced into a pGL3-Basic vector;
[19] FIG. 3 shows the structure of a pGL3-BA vector, in which a b-actin promoter is in
troduced into a pGL3-Basic vector;
[20] FIG. 4 shows a TATA box region of a CMV promoter of a pcDNA3.1 vector used in
the present invention;
[21] FIG. 5 shows the structure of a pGL3-B/C TA vector, in which a hybrid promoter
comprising a b-actin promoter (1.9 kb) and a TATA box region of a CMV promoter
(130 bp) is introduced into a pGL3-Basic vector;
[22] FIG. 6 shows the structure of a pGL3-B/C TA/B vector, in which a hybrid promoter
comprising a b-actin promoter (1.9 kb), a TATA box region of a CMV promoter (130
bp) and a b-actin intron region is introduced into a pGL3-Basic vector;
[23] FIG. 7 shows the structure of a pGL3-U/C TA Bi vector, in which a hybrid promoter
comprising a b-actin promoter (150 bp), a TATA box region of a CMV promoter (130
bp) and a b-actin intron region is introduced into a pGL3-Basic vector;
[24] FIG. 8 shows the structure of a pGL3-C e /U/CTA/B vector, in which a hybrid
promoter comprising a b-actin promoter (150 bp), a TATA box region of a CMV
promoter (130 bp), a b-actin intron region and a CMV enhancer region is introduced
into apGL3-Basic vector; and
[25] FIG. 9 shows the results of comparing luciferase expression levels in CHO cells
transformed with each of the vectors described above.
[26]
Best Mode for Carrying out the Invention
[27] In one embodiment, the present invention provides a hybrid promoter, in which a
whole or a part of a CMV enhancer, a whole or a part of a b-actin promoter, a whole or
a part of a CMV promoter, and a whole or a part of a b-actin intron are operably linked
to each other.
[28]
[29] As used herein, the term "b-actin" exists in most cell types as a major component of
the cytoskeleton and is a highly conserved protein that is involved in cell motility,
structure and integrity. The gene encoding b-actin serves as a housekeeping gene, and
can maintain a certain level of expression regardless of environmental conditions.
[30] As used herein, the term "promoter" refers to a polynucleotide sequence that allows
transcription of a target gene operably linked thereto and regulates expression thereof.
The promoter includes sequences that are recognized by a RNA polymerase and a tran
scription initiation site. In order to express a target protein in a particular cell type or a
host cell, a suitable functional promoter must be chosen carefully. For example, the
promoter sequences have been deposited in data banks such as GenBank, and may be
obtained as a separate element or elements cloned within a polynucleotide sequence
from commercial or individual sources.
[31] As used herein, the term "b-actin promoter" refers to a structural gene that is
involved in the regulation of transcriptional activity of the housekeeping gene, b-actin.
In case of influencing on the expression of a coding sequence under transcriptional
regulation of a promoter, the b-actin promoter is operably linked to a coding sequence.
The coding sequence may be operably linked to a nucleotide sequence regulating the
transcription in a forward or reverse direction.
[32] With respect to the objects of the present invention, the b-actin promoter of the
present invention can be composed of one or more DNA fragments selected from the
group consisting of the following:
[33] i) a DNA fragment having a nucleotide sequence represented by SEQ ID NO: 9,
[34] ii) a DNA fragment having a nucleotide sequence represented by SEQ ID NO: 10, or
[35] iii) a DNA fragment having a deletion, a substitution or an insertion of one or more
nucleotides in the nucleotide sequences of the DNA fragments i) and ii), and having a
promoter activity and an activity of regulating the expression of a target gene operably
linked downstream of the promoter.
[36] The present invention may include DNA fragments whose nucleotide sequences have
at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% homology to those of the DNA
fragments described above.
[37] The b-actin promoter of the present invention can be amplified by PCR using
forward and reverse primers represented by SEQ ID NOs: 1 and 2 and a whole or a
part of the b-actin promoter sequence as a template. The resulting b-actin promoter can
be a DNA fragment having a size of approximately 1.9 kb or 150 bp.
[38] 5 -BA l_F(NheI): 5 -CAG CTA GCG GGA CCA AGA CAG AAC CAT AA-3 (SEQ
ID NO: 1)
[39] 3 -BA 4_R(HindIII): 5'-GTA AGC TTC GGC GAA CTA TAT CAG GGC A-3 (SEQ
ID NO: 2)
[40]
[41] Any type of the b-actin promoters known in the art can be used as a b-actin promoter
of the present invention without limitation, and the preferred b-actin promoter can be a
b-actin promoter of CHO (Chinese Hamster Ovary) cell.
[42] The DNA fragment of SEQ ID NO: 9 comprises nucleotide sequences of 1930 bp,
which code for the full-length of a b-actin promoter derived from CHO cells, and the
DNA fragment of SEQ ID NO: 10 comprises nucleotide sequences of 154 bp, which
code for a U201 14 fragment having a b-actin promoter activity.
[43]
[44] As used herein, the term "b-actin intron" refers to a sequence regulating the tran
scription of a gene that is present inside the b-actin gene or a transcript thereof and that
is not included in a final RNA product of the gene. The nucleotide sequence of an
intron does not have information about an amino acid sequence.
[45] With respect to the objects of the present invention, the b-actin intron of the present
invention can be composed of a DNA fragment of the following:
[46] i) a DNA fragment having a nucleotide sequence represented by SEQ ID NO: 12, or
[47] ii) a DNA fragment having a deletion, a substitution or an insertion of one or more
nucleotides in the nucleotide sequence of the DNA fragment i), and having a promoter
activity and an activity of regulating the expression of a target gene operably linked
downstream of the promoter.
[48] The present invention may include DNA fragments, whose DNA sequences have at
least 80%, 90%, 95%, 96%, 97%, 98%, or 99% homology to those of the DNA
fragments described above.
[49] The b-actin intron of the present invention can be amplified by PCR using forward
and reverse primers represented by SEQ ID NOs: 6 and 2 and a whole or a part of the
b-actin intron sequence as a template. The resulting b-actin intron can be a DNA
fragment having a size of approximately 1 kb.
[50] 5 -CBint _F(NheI): 5'-CAA GCT AGC GAG CAC AGG CCT TTC-3'(SEQ ID NO:
6)
[51] 3 -BA 4_R(HindIII): 5'-GTA AGC TTC GGC GAA CTA TAT CAG GGC A-3'
(SEQ ID NO: 2)
[52]
[53] As used herein, the term "CMV (cytomegalovirus)" belongs to a viral genus of the
viral group known as Herpesviridae. The species that infects humans is commonly
known as human CMV (HCMV) or human hgerpesvirus-5 (HHV-5). It is classified
into the alpha-herpesvirus family and gamma-herpesvirus family, and all herpesviruses
share a characterictic ability to remain latent within the body over long periods.
[54] As used herein, the term "CMV promoter (pCMV)" refers to a cytomegalovirus
(CMV) early promoter. pCMV has been known as a powerful regulatory element, and
shows its activity in various cells.
[55] As used herein, the term "TATA box" refers to a region consisting of the nucleotide
sequence of TATAAA, which is included in many eukaryotic promoters. The TATA
box is typically located very close to a transcription initiation site (within 50 base
pairs), and a TATA binding protein binds to this region to assist the formation of a
RNA polymerase transcriptional complex.
[56] With respect to the objects of the present invention, the TATA box region of the
CMV promoter according to the present invention can be composed of a DNA
fragment of the following:
[57] i) a DNA fragment having a nucleotide sequence represented by SEQ ID NO: 11, or
[58] ii) a DNA fragment having a deletion, a substitution or an insertion of one or more
nucleotides in the nucleotide sequence of the DNA fragment i), and having a promoter
activity and an activity of regulating the expression of a target gene operably linked
downstream of the promoter.
[59] The present invention may include DNA fragments whose DNA sequences have at
least 80%, 90%, 95%, 96%, 97%, 98%, or 99% homology to those of the DNA
fragments described above.
[60] As used herein, the term "TATA box" refers to a sequence of alternating thymine (T)
and adenine (A) of a transcription initiation site present in the promoter region, and is a
highly conserved region common in most organisms.
[61] The TATA box region of the CMV promoter can be amplified by PCR using forward
and reverse primers represented by SEQ ID NOs: 4 and 5 and a whole or a part of the
TATA box region of the CMV promoter as a template. The resulting TATA box region
can be a DNA fragment having a size of approximately 130 bp.
[62] 5'-CMV TA_F(SalI): 5'-CAG TCG ACT AGG CGT GTA CGG TGG GAG-3' (SEQ
ID NO: 4)
[63] 3'-BGH reverse priming site: 5'-TAG AAG GCA CAG TCG AGG-3' (SEQ ID NO:
5)
[64]
[65] As used herein, the term "CMV enhancer" refers to a sequence that binds to other
protein of a transcription initiation complex and enhances the transcription initiation
regulated by the related promoter.
[66] With respect to the objects of the present invention, the CMV enhancer of the present
invention can be composed of a DNA fragment of the following:
[67] i) a DNA fragment having a nucleotide sequence represented by SEQ ID NO: 13, or
[68] ii) a DNA fragment having a deletion, a substitution or an insertion of one or more
nucleotides in the nucleotide sequence of the DNA fragment i), and having a promoter
activity and an activity of regulating the expression of a target gene operably linked
downstream of the promoter.
[69] The present invention may include DNA fragments whose DNA sequences have at
least 80%, 90%, 95%, 96%, 97%, 98%, or 99% homology to those of the DNA
fragments described above.
[70] The CMV enhancer of the present invention can be amplified by PCR using forward
and reverse primers represented by SEQ ID NOs: 7 and 8 and a whole or a part of the
CMV enhancer as a template. The resulting CMV enhancer can be a DNA fragment
having a size of approximately 530 bp.
[71] 5'-CMV En_F(MluI): 5'- CAG ACG CGT TGA CAT TGA TTA TTG ACT-3' (SEQ
ID NO: 7)
[72] 3'-CMV En_R(NheI): 5'-CAG GCT AGC AGT TGT TAC GAC ATT TTG-3' (SEQ
ID NO: 8)
[73] As used herein, the term "PCR (Polymerase Chain Reaction)" means a scientific
technique in molecular biology to amplify a single or a few copies of a piece of DNA
across several orders of magnitude, generating thousands to millions of copies of a
particular DNA sequence. The method relies on thermal cycling consisting of repeated
cycles as follows:
[74] 1) denaturation step: heating a DNA template, yielding single-stranded DNA
molecules,
[75] 2) annealing step: annealing primers to the single-stranded DNA template, and
binding DNA polymerase to the primer-template hybrid, and
[76] 3) extension/elongation step: synthesizing a new DNA strand complementary to the
DNA template strand, leading to exponential (geometric) amplification of the target
DNA.
[77] PCR provides a means to detect the presence of a target molecule under quantitative
or semi-quantitative conditions and to determine the relative amount thereof within the
starting pool of nucleic acids.
[78] As used herein, the term "downstream" refers to a nucleotide sequence that is located
3' to a reference nucleotide sequence. In particular, downstream nucleotide sequences
generally relate to sequences followed by a transcription initiation site. For example, a
translation initiation codon of a gene is located downstream of the transcription
initiation site.
[79] As used herein, the term "operably linked" refers to a functional linkage between a
promoter and a second sequence, wherein the promoter sequence initiates and mediates
transcription of the DNA corresponding to the second sequence. In particular, the term
operably linked means that the expression (operation) of a target gene sequence is
located under the control of a transcription regulation sequence (e.g. promoter,
enhancer or the like) or a translation regulation sequence.
[80] In a preferred embodiment of the present invention, a hybrid promoter has been con
structed, in which a whole or a part of a CMV enhancer, a whole or a part of a b-actin
promoter, a whole or a part of a CMV promoter, and a whole or a part of a b-actin
intron are operably linked to each other. It has been found that the hybrid promoter of
the present invention can significantly improve the transcription of a target gene and
the expression of a target protein, compared to conventional promoters known in the
art (see FIG. 9).
[81] In a more specific embodiment, the hybrid promoter of the present invention can be a
promoter comprising:
[82] a CMV enhancer represented by SEQ ID NO: 13,
[83] a TATA box region of a CMV promoter represented by SEQ ID NO: 11,
[84] a b-actin promoter represented by SEQ ID NO: 9, and
[85] a b-actin intron region represented by SEQ ID NO: 12,
[86] wherein the CMV promoter, TATA box, b-actin promoter and b-actin intron are
operably linked to each other.
[87] Further, the hybrid promoter of the present invention can be a promoter comprising:
[88] a CMV enhancer represented by SEQ ID NO: 13,
[89] a TATA box region of a CMV promoter represented by SEQ ID NO: 11,
[90] a b-actin promoter represented by SEQ ID NO: 10, and
[91] a b-actin intron region represented by SEQ ID NO: 12,
[92] wherein the CMV promoter, TATA box, b-actin promoter and b-actin intron are
operably linked to each other.
[93] The hybrid promoter of the present invention may include substitution, insertion and
deletion variants of one or more nucleotides, and combinations thereof. The sub
stitution variant as used herein may be a variant, in which at least one base is removed
and replaced with other base in the nucleotide sequence. The insertion variant as used
herein may be a variant, in which one or more bases are introduced into a prede
termined region within the nucleotide sequence. The deletion variant as used herein
may be a variant, in which one or more bases are removed from the nucleotide
sequence. In this regard, any combination of the substitution, deletion and insertion
may be made to remain function of the components intact.
[94] The hybrid promoter of the present invention may include DNA fragments, of which
DNA sequences have at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99%
homology to those of the DNA fragment represented by SEQ ID NO: 13, the DNA
fragment represented by SEQ ID NO: 11, the DNA fragment represented by SEQ ID
NO: 9 or 10, and the DNA fragment represented by SEQ ID NO: 12.
[95] As used herein, the term "homology" in relation to a sequence (e.g., a nucleic acid
sequence, an amino acid sequence, etc.) refers to the proportion of identity between
two or more gene sequences. Therefore, the greater the homology between two given
genes, the greater the identity or similarity between their sequences. Whether or not
two genes have homology is determined by comparing their sequences directly or by a
hybridization method under stringent conditions. When two gene sequences are
directly compared with each other, these genes have homology if the DNA sequences
of the genes have representatively at least 50% identity, preferably at least 70%
identity, more preferably at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity
with each other.
[96] As used herein, the term "homologous" in all its grammatical forms and spelling
variations refers to the relationship between proteins that possess a "common evo
lutionary origin," including superfamily-derived proteins (e.g., immunoglobulin superfamily)
and homologous proteins derived from different species (e.g., myosin light
chain)(Reeck et al., Cell 50: 667, 1987). Such proteins (and their encoding genes) have
sequence homology, as reflected by their high degree of sequence similarity. However,
in common usage and in the context of the present invention, the term "homologous"
being modified with an adverb such as "highly" can refer to sequence similarity and
not a common evolutionary origin.
[97] As used herein, the term "sequence similarity" refers to the degree of identity or cor
respondence between nucleic acid or amino acid sequences of proteins that may or
may not share a common evolutionary origin. In a specific embodiment, two DNA
sequences are "substantially homologous" or "substantially similar" when at least approximately
21% (preferably at least approximately 50%, and most preferably, ap
proximately 75%, 90%, 95%, 96%, 97%, 98% or 99%) of the nucleotides match over
the defined length of the DNA sequences.
[98] As used herein, the term "substantially similar" refers to nucleic acid fragments
wherein changes in one or more nucleotide bases result in substitution of one or more
amino acids, but do not affect the functional properties of the protein encoded thereby.
"Substantially similar" also refers to nucleic acid fragments wherein changes in one or
more nucleotide bases mediate alteration of gene expression by antisense or cosuppression
technology without influencing the functional properties thereof. "Sub
stantially similar" also refers to modifications of the nucleic acid fragments such as
deletion or insertion of one or more nucleotide bases that do not substantially affect the
functional properties of the resulting transcript. It is therefore understood that the
present invention encompasses more than the specific exemplary sequences. Each of
the proposed modifications is well known to those skilled in the art, as is the deter
mination of retention of biological activities of the encoded products.
[99] The similarity, identity and homology of amino acid sequences and base sequences
are herein compared using FASTA with the default parameters. Alternatively, an
identity search may be conducted, for example, using NCBI's BLAST 2.2.9 (published
May 12, 2004). As used herein, the value of identity usually refers to the value as a
result of alignment with the BLAST as described above using the default parameters. If
the change of parameters results in higher values, then the highest value is employed
herein as the value of the identity. When a plurality of regions is evaluated for identity,
the highest value is employed herein as the value of the identity.
[100]
[101] In another embodiment, the present invention provides a recombinant vector
comprising the hybrid promoter and a target protein-encoding gene operably linked
thereto.
[102] As used herein, the term "recombinant vector" refers to a vector transferring a
polynucleotide sequence of interest to a target cell. Such a vector is capable of selfreplication
or incorporation into a chromosome in a host cell (e.g., a prokaryotic cell,
yeast, an animal cell, a plant cell, an insect cell, an individual animal, and an individual
plant, etc.), and contains a promoter at a site suitable for transcription of a polynu
cleotide of the present invention. The recombinant vector may comprise a structural
gene and a promoter for regulating expression thereof, and in addition, various
regulatory elements in a state that allows them to operate within host cells. It is well
known in the art that a type of recombinant vector of a living organism such as an
animal and a species of a regulatory element used may vary depending on the type of
host cell used.
[104] In a specific embodiment, the recombinant vector of the present invention may
include the hybrid promoter. More specifically, the present invention provides a r e
combinant vector comprising the hybrid promoter, in which a whole or a part of a
CMV enhancer, a whole or a part of a b-actin promoter, a whole or a part of a CMV
promoter, and a whole or a part of a b-actin intron are operably linked to each other.
[105] More preferably, the recombinant vector of the present invention may include the
hybrid promoter, in which a CMV enhancer represented by SEQ ID NO: 13, a TATA
box region of a CMV promoter represented by SEQ ID NO: 11, a b-actin promoter
represented by SEQ ID NO: 9 or 10, and a b-actin intron region represented by SEQ
ID NO: 12 are operably linked to each other. Most preferably, the recombinant vector
of the present invention may be a pGL3-C e /U/C TA B vector having a cleavage map as
shown in FIG. 8. It has been found that the recombinant vector of the present invention
can induce the transcription of a target gene and the expression of a target protein with
excellent efficiency (see FIG. 9).
[106]
[107] The recombinant vector of the present invention may further include one or more
regulatory elements such as a replication origin, selectable markers, terminators and
the like.
[108] As used herein, the term "selectable marker" refers to a gene which functions as
guidance for selecting a host cell comprising a nucleic acid construct or a vector. The
selectable markers may include, but are not limited to: fluorescent markers, lu
minescent markers and drug selectable markers, and the like. The fluorescent markers
may include, but are not limited to, genes encoding fluorescence proteins such as green
fluorescent protein (GFP), cyan fluorescent protein (CFP), yellow fluorescent protein
(YFP), red fluorescent protein (dsRFP) and the like. The luminescent markers may
include, but are not limited to, genes encoding luminescent proteins such as luciferases.
The drug selectable markers suitable in the present invention may include,
but are not limited to, resistance genes to antibiotics, such as ampicillin, streptomycin,
gentamicin, kanamycin, hygromycin, tetracycline, chloramphenicol, and neomycin.
[109] As used herein, the term "terminator" refers to a sequence which is located
downstream of a protein-encoding region of a gene and which is involved in the ter
mination of transcription when DNA is transcribed into mRNA, and the addition of a
poly-A sequence. It is known that a terminator contributes to the stability of mRNA,
and has an influence on the amount of gene expression. Terminators include, but are
not limited to, a sequence including AATAAA.
[HO]
[111] To develop a hybrid promoter suitable for induction of immune responses in vivo
with increasing an expression level of a target gene, the present invention has prepared
the following recombinant vectors by combinations of various promoter/enhancer
sequences, poly (A) sequences and intron sequences:
[112] pGL3-Basic: f1 ori, synthetic poly (A)/transcriptional pause site, multiple cloning
site (MCS), liciferase reporter gene (luc+), SV40 late polyadenylation signal, Amp (see
FIG. 1)
[113] pGL3-Promoter(SV40): f1 ori, synthetic poly (A)/transcriptional pause site, MCS,
SV40 promoter, luciferase gene (luc+), SV40 late polyadenylation signal, ampicillin r e
sistance gene (Amp ) (see FIG. 2)
[114] pGL3-BA: f l ori, synthetic poly (A)/transcriptional pause site, MCS, b-actin
promoter, luc+, SV40 late polyadenylation signal, Amp (see FIG. 3)
[115] pGL3-B/CTA: f l ori, synthetic poly (A)/transcriptional pause site, MCS, hybrid
promoter of b-actin promoter (1.9 kb) and TATA box region (130 bp) of CMV
promoter, luc+, SV40 late polyadenylation signal, Amp (see FIG. 5)
[116] pGL3-B/CTA/B : f1 ori, synthetic poly (A)/transcriptional pause site, MCS, hybrid
promoter of b-actin promoter (1.9 kb) and TATA box region (130 bp) of CMV
promoter, b-actin intron, luc+, SV40 late polyadenylation signal, Amp (see FIG. 6)
[117] pGL3-U/CTA/B : f1 ori, synthetic poly (A)/transcriptional pause site, MCS, hybrid
promoter of U201 14 region (150 bp) of b-actin promoter and TATA box region (130
bp) of CMV promoter, b-actin intron, luc+, SV40 late polyadenylation signal, Amp
(see FIG. 7)
[118] pGL3-Ce /U/CTA/Bi : f l ori, synthetic poly (A)/transcriptional pause site, MCS, CMV
enhancer, hybrid promoter of U201 14 region (150 bp) of b-actin promoter and TATA
box region (130 bp) of CMV promoter, b-actin intron, luc+, SV40 late polyadenylation
signal, Amp (see FIG. 8)
[119]
[120] In order to examine the capability to induce the expression of a target protein, each of
the recombinant vectors described above was transformed into CHO cells, and then ex
pression levels of a luciferase gene inserted as a reporter gene were compared. A
pGL3-Basic vector, which has a basic structure of the vector used in the present
invention and dispenses with the promoter and enhancer sequences according to the
present invention, was used as a control group (see FIG. 1). As a result, the luciferase
expression level was remarkably increased in the recombinant vector having a hybrid
promoter of a CMV enhancer, 150 bp of a b-actin promoter, a CMV promoter and a b-
actin intron (pGL3-Ce /U/CTA/B of FIG. 8), compared to the recombinant vector
having a b-actin promoter (pGL3-BA of FIG. 3), the recombinant vector having a
hybrid promoter of 1.9 kb of a b-actin promoter and a TATA box region of a CMV
promoter, (pGL3-B/CTA of FIG. 5), the recombinant vector having a hybrid promoter
of 1.9 kb of a b-actin promoter, a TATA box region of a CMV promoter, and a b-actin
intron region (pGL3-B/C TA/B of FIG. 6), and the recombinant vector having a hybrid
promoter of 150 bp of a b-actin promoter, a TATA box region of a CMV promoter,
and a b-actin intron region (pGL3-U/C TA/B of FIG. 7) (see Tables 1 and 2, and FIG.
9).
[121] Therefore, it has been found that when a target protein-encoding gene, instead of the
luciferase gene, is inserted into the recombinant vector of the present invention, the
transcription and expression of the target gene are increased by the activity of the
hybrid promoter of the present invention, thereby mass-producing the target protein.
[122]
[123] In still another embodiment, the present invention provides a transformant that is
transformed with the recombinant vector.
[124] As used herein, the term "transformation" refers to introduction of a nucleic acid into
host cells. As a transformation method, any technique for introducing DNA into host
cells can be used, including various well-known techniques, such as electroporation,
calcium phosphate co-precipitation, retroviral infection, microinjection, DEAE-dextran
and cationic liposome, but is not limited thereto.
[125] As used herein, the term "transformant" refers to the whole or a part of an organism,
such as a cell, into which a foreign DNA is introduced by transformation. Examples of
a host cell may include prokaryotic cells, yeast, animal cells, plant cells, insect cells
and the like, preferably animal cells or animal cell-derived cells, and most preferably
Chinese Hamster Ovary (CHO) cells. Transformation of CHO cells with a target gene
along with an amplifiable gene such as dihydrofolate reductase (DHFR) or glutamine
synthetase (GS) offers effective platforms for expression of the required proteins. The
DHFR system is routinely used with CHO cells deficient in the DHFR activity (DHFR
). The target gene is delivered to the cells along with the DHFR marker gene, usually
on the same plasmid vector. Exposure of the transformed cells to the DHFR enzyme
inhibitor, methotrexate (MTX) promotes amplification of the DHFR and the cotransformed
target gene. MTX treatment enhances specific protein production
following an increased gene copy number.
[126]
[127] In still another embodiment, the present invention provides to a pharmaceutical com
position comprising the recombinant vector or the transformant as an effective in
gredient, and a pharmaceutically acceptable carrier. The composition of the present
invention is administered in a pharmaceutically effective amount.
[128] As used herein, the term "pharmaceutically effective amount" in the context of the
effective ingredient refers to an amount sufficient for exhibiting intended efficacy in a
reasonable benefit/risk ratio so as to be applicable to medical treatment.
[129] As used herein, the term "pharmaceutically acceptable carrier" refers to a material
which is used for production of a pharmaceutical agent or an agricultural chemical
(e.g., an animal drug), and has no adverse effect on effective ingredients. Any pharma
ceutically acceptable carrier known in the art may be used in the pharmaceutical com
position of the present invention.
[130] For oral administration, the pharmaceutically acceptable carrier may include a
binder, a lubricant, a disintegrator, an excipient, a solubilizer, a dispersing agent, a
stabilizer, a suspending agent, a coloring agent and a perfume. For injectable admin
istration, the pharmaceutically acceptable carrier may include a buffering agent, a
preserving agent, an analgesic, a solubilizer, an isotonic agent and a stabilizer. For
topical administration, the pharmaceutically acceptable carrier may include a base, an
excipient, a lubricant, and a preserving agent.
[131] The pharmaceutical composition of the present invention may be formulated into a
variety of dosage forms in combination with the aforementioned pharmaceutically ac
ceptable carriers. For example, for oral administration, the pharmaceutical composition
may be formulated into tablets, troches, capsules, elixirs, suspensions, syrups or
wafers. For injectable administration, the pharmaceutical composition may be
formulated into an ampule as a single-dose dosage form or a unit dosage form, such as
a multidose container. The pharmaceutical composition may be also formulated into
solutions, suspensions, tablets, pills, capsules and long-acting preparations.
[132] On the other hand, examples of the carrier, excipient and diluent suitable for the
pharmaceutical composition of the present invention may include lactose, dextrose,
sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate,
gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline
cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl
hydroxybenzoate, talc, magnesium stearate and mineral oils. In addition, the pharma
ceutical composition of the present invention may further include fillers, anticoagulating
agents, lubricants, humectants, perfumes and antiseptics.
[133] The pharmaceutical composition of the present invention may be administered via
any of the common routes, as long as it is able to reach a desired tissue. A variety of
modes of administration are contemplated, including intraperitoneally, intravenously,
intramuscularly, subcutaneously, intradermally, orally, topically, intranasally, intrapulmonarily
and intrarectally, but are not limited thereto.
[134] However, since peptides are digested upon oral administration, the effective in
gredient of the pharmaceutical composition for oral administration should be coated or
formulated for protection against degradation in the stomach. Preferably, the pharma
ceutical composition of the present invention may be administered in an injectable
form. In addition, the pharmaceutical composition of the present invention may be administered
using a certain apparatus capable of transporting the effective ingredient
into a target cell.
[135] The administration frequency and dose of the pharmaceutical composition of the
present invention can be determined by several related factors including the types of
diseases to be treated, administration routes, the patient's age, gender, weight and
severity of the illness, as well as by the types of the drug as an effective ingredient.
The composition of the present invention may be administered alone or in combination
with other therapeutic agent, and either sequentially or simultaneously, in a single dose
or multiple doses. Considering all of the above factors, a minimum amount to achieve
maximum efficacy without side effects can be readily determined by those skilled in
the art.
[136]
[137] In still another embodiment, the present invention provides a method for preparing a
target protein, comprising the steps of:
[138] 1) culturing the transformant of the present invention;
[139] 2) inducing the expression of a target protein from the transformant; and
[140] 3) harvesting the expressed target protein from the transformant or the culture
solution thereof.
[141] As used herein, the term "target protein" includes antibodies, enzymes, cytokines,
lymphokines, adhesion molecules, receptors and the derivatives or fragments thereof,
but is not limited thereto. Generally, all kinds of polypeptides which act as agonists or
antagonists and/or have therapeutic or diagnostic applications can be used as a target
protein. Other target proteins include, for example, anti-apoptotic proteins, chaperones,
metabolic enzymes, glycosylation enzymes and the derivatives or fragments thereof,
but are not limited thereto.
[142] As used herein, the term "polypeptide" refers to a polymer of amino acids without
regard to the length of the polymer; thus, peptides, oligopeptides, and proteins are
included within the definition of polypeptide. This term also does not specify or
exclude chemical or post-expression modifications of the polypeptides of the
invention, although chemical or post-expression modifications of these polypeptides
may be included or excluded as specific embodiments. Therefore, for example, modi
fications to polypeptides that include the covalent attachment of glycosyl groups,
acetyl groups, phosphate groups, lipid groups and the like are expressly encompassed
by the term. Modifications include acetylation, acylation, ADP-ribosylation,
amidation, covalent attachment of flavin, covalent attachment of a heme moiety,
covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a
lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization,
disulfide bond formation, demethylation, formation of cysteine, formation of
pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor
formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation,
proteolytic processing, phosphorylation, prenylation, racemization, selenoylation,
sulfation, transfer-RNA mediated addition of amino acids to proteins such
as arginylation, and ubiquitination (see, for instance Creighton, (1993), Posttranslational
Covalent Modification of Proteins, W.H. Freeman and Company, New York
B. C. Johnson, Ed., Academic Press, New York 1-12; Seifter, et al., (1990) Meth
Enzymol 182:626-646; Rattan et al., (1992) Ann N Y Acad Sci 663:48-62). Also
included within the definition are polypeptides which contain one or more analogs of
an amino acid (including, for example, non-naturally occurring amino acids, amino
acids which only occur naturally in an unrelated biological system, modified amino
acids from mammalian systems, etc.), polypeptides with substituted linkages, as well
as other modifications known in the art, both naturally occurring and non-naturally
occurring.
Examples of the target protein prepared according to the method of the present
invention may include, but are not limited to, human growth hormones, growth
hormone release hormones, growth hormone release peptides, interferons and in
terferon receptors (e.g., interferon-alpha, -beta and -gamma, Type I soluble interferon
receptor, etc.), granulocyte-colony stimulating factors (G-CSFs), granulocyte -
macrophage-colony stimulating factors (GM-CSFs), glucagons-like peptides (GLP-1,
etc.), G-protein-coupled receptors, interleukins (e.g., IL-1 receptor, IL-4 receptor, etc.),
enzymes (e.g., glucocerebrosidase, iduronate-2-sulfatase, alpha-galactosidase-A,
agalsidase alpha, beta- or alpha-L-iduronidase, butyrylcholinesterase, chitinase,
glutamate decarboxylase, imiglucerase, lipase, uricase, platelet-activating factor acetylhydrolase,
neutral endopeptidase, myeloperoxidase, etc.), interleukin- or cytokinebinding
proteins (e.g., IL-18bp, TNF-binding proteins, etc.), macrophage activating
factors, macrophage peptides, B cell factors, T cell factors, protein A, allergy in
hibitors, cell necrosis glycoprotein, immune toxins, lymph toxins, tumor necrosis
factors, tumor suppressing factors, transitional growth factors, alpha- 1 antitrypsin,
albumin, alpha-lactalbumin, apolipoprotein-E, erythropoietin, highly glycosylated ery
thropoietin, angiopoietin, hemoglobin, thrombin, thrombin receptor activating
peptides, thrombomodulin, blood factor VII, blood factor Vila, blood factor VIII,
blood factor IX, blood factor XIII, plasminogen activating factor, fibrin-binding
peptides, urokinases, streptokinases, hirudin, protein C, C-reactive proteins, rennin in
hibitors, collagenase inhibitors, superoxide dismutases, leptin, platelet-originated
growth factor, epithelial growth factor, epidermal growth factor, angiostatin, an
giotensin, myelopoiesis growth factor, myelopoiesis stimulating factor, calcitonin,
insulin, atriopeptin, cartilage inducer, elcatonin, joint tissue activating factor, tissue
factor pathway inhibitor, follicle stimulating hormone, progesterone forming hormone,
progesterone forming hormone releasing hormone, nerve growth factors (e.g., nerve
growth factor, cilliary neurotrophic factor, axogenesis factor- 1, brain-natriuretic
peptide, glial derived neurotrophic factor, netrin, neurophil inhibitor factor, neu
rotrophic factor, neuturin, etc.), parathormone, relaxin, cycretin, somatomedine,
insulin-like growth factor, adrenocortical hormones, glucagons, cholecystokynine,
pancreatic polypeptides, gastrin releasing peptide, corticotropin releasing factor,
thyroid stimulating hormone, autotaxin, lactoferrin, myostatin, receptors (e.g.,
TNFR(P75), TNFR(P55), IL-1 receptor, VEGF receptor, B cell activating factor
receptor, etc.), receptor antagonists (e.g., ILl-Ra, etc.), cell surface antigens (e.g., CD
2, 3, 4, 5, 7, 11a, lib, 18, 19, 20, 23, 25, 33, 38, 40, 45, 69, etc.), monoclonal an
tibodies, polyclonal antibodies, antibody fragments (e.g., scFv, Fab, Fab', F(ab') 2 and
Fd), virus -originated vaccine antigens. The antibody fragments include Fab, Fab',
F(ab') 2, Fd or scFv, which is capable of binding to a specific antigen, and preferably
Fab'.
Production systems for the target proteins described above may be in vitro or in vivo.
In vitro production systems may employ the use of eukaryotic or prokaryotic cells. For
example, the target protein can be obtained by culturing the transformant of the present
invention in vitro. The cultivation of the transformant may be performed according to
conventional methods in the art, and the conditions such as temperature, time and pH
of a medium may be suitably controlled. Culture media used for the cultivation need to
meet the requirements for growth of particular strains in an appropriate manner.
Culture media for various strains are disclosed in, for example, "Manual of Methods
for General Bacteriology" from American Society for Bacteriology (Washington D. C,
USA, 1981). A carbon source for the culture media may be sugar and carbohydrate
(e.g., glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose), oil
and fat (e.g., soybean oil, sunflower oil, peanut oil and coconut oil), a fatty acid (e.g.,
palmitic acid, stearic acid and linolenic acid), an alcohol (e.g., glycerol and ethanol),
and an organic acid (e.g., acetic acid). The carbon sources may be used alone or in a
mixture. A nitrogen source may also be a nitrogen-containing organic compound (e.g.,
peptone, yeast extract, meat extract, malt extract, corn steep liquor, soy meal and urea)
or an inorganic compound (e.g., ammonium sulfate, ammonium chloride, ammonium
phosphate, ammonium carbonate and ammonium nitrate). The nitrogen source may be
used alone or in a mixture. A phosphorous source may be potassium dihydrogen
phosphate, dipotassium hydrogen phosphate or its sodium salt thereof. In addition, the
culture media should contain a metal salt (e.g., magnesium sulfate or iron sulfate)
essential for growth. Finally, the culture medium may further include substances
essential for growth such as amino acids and vitamins in addition to the above
mentioned substances. Suitable precursors may be also added to the culture media.
Those components of culture media may be added to the culture media on a batch or
on a continuous basis during the cultivation.
[145] The pH of the culture medium may be adjusted with a basic compound (e.g., sodium
hydroxide, potassium hydroxide or ammonia), or an acidic compound (e.g., phosphoric
acid or sulfuric acid). A defoaming agent such as fatty acid poly glycol ester may be
added to prevent the formation of bubbles. An aerobic state may be maintained by
injecting oxygen or oxygen-containing gas (e.g., air) into the culture medium.
[146] For example, liquid culture media for animal cells may include DMEM, MEM,
RPM1 1640, IMDM, F10 medium, and F12 medium. The culture media may include
serum supplements such as fetal calf serum (FCS), or may be serum- free culture
media. Furthermore, a trans activator may be added to the media. The cultivation is
preferably performed at approximately pH 6.0 to 8.0. The cultivation is typically
carried out at approximately 30 to 40°C for approximately 15 to 200 hours. If required,
the medium may be changed, aerated or stirred.
[147] Since culture conditions vary depending on the cell type used, those skilled in the art
can appropriately determine suitable conditions. For example, CHO cells may be
cultured under a C0 2 atmosphere of 0 to 40%, preferably 2 to 10%, at a temperature of
30 to 39°C, preferably 37°C for 1 to 14 days.
[148] Various culture apparatuses can be used for animal cells, and exemplified by fer
mentation tank-type tank culture apparatuses, airlift-type culture apparatuses, culture
flask-type culture apparatuses, spinner flask-type culture apparatuses, microcarriertype
culture apparatuses, flow tank- type culture apparatuses, hollow fiber- type culture
apparatuses, roller bottle-type culture apparatuses, packed bed-type culture apparatuses
or the like.
[149] Meanwhile, in vivo production systems may include, for example, production
systems using animals or plants. A DNA of interest can be introduced into such an
animal or plant, and the polypeptide produced in the animal or plant in vivo can be
collected.
[150]
Mode for the Invention
[151] Hereinafter, the present invention will be described in more detail with reference to
Examples. However, these Examples are for illustrative purposes only, and the
invention is not intended to be limited by these Examples.
[152]
[153] Example 1: General molecular biological techniques
[154] Methods generally used in molecular biology, such as restriction enzyme treatment,
agarose gel electrophoresis, gel extraction, plasmid DNA purification, polymerase
chain reaction (PCR), DNA fragment ligation and E.coli transformation, were
performed according to the methods described in the literature with minor modi
fications (Sambrook J et al., 2001 Molecular cloning: A laboratory manual, 2nd
edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.).
[155]
[156] Example 2 : Preparation of plasmid vectors
[157] <2-l> Preparation of pGL3-BA vector
[158] PCR was performed using a total genomic DNA obtained from CHO cells as a
template and a primer pair of SEQ ID NOs: 1 and 2 to amplify a b-actin promoter
gene, and thus amplified PCR product was treated with restriction enzymes Nhel and
Hindlll. The resulting b-actin promoter DNA fragment (3.0 kb) was inserted into a
pGL3-Basic vector (Promega) treated with the same restriction enzymes, to thereby
prepare a pGL3-BA vector (FIG. 3). Here, PCR was performed under the following
conditions: initial denaturation at 94°C for 5 minutes; 25 cycles of denaturation at 94°C
for 1 minute, annealing at 55°C for 1 minute and polymerization at 72°C for 3.5
minutes; and final elongation at 72°C for 7 minutes.
[159] 5 -BA l_F(NheI): 5 -CAG CTA GCG GGA CCA AGA CAG AAC CAT AA-3'
(SEQ ID NO: 1)
[160] 3'-BA 4_R(HindIII): 5'-GTA AGC TTC GGC GAA CTA TAT CAG GGC A-3'
(SEQ ID NO: 2)
[161]
[162] As shown in FIG. 3, the prepared pGL3-BA vector includes f l ori, synthetic poly
(A)/transcriptional pause site, MCS, b-actin promoter, luc+, SV40 late polyadenylation
signal and Amp resistance gene.
[163]
[164] <2-2> Preparation of pGL3-B/C TA vector
[165] PCR was performed using a pcDNA3.1 vector (Invitrogen) as a template and a
primer pair of SEQ ID NOs: 4 and 5 to amplify a TATA box region (130 bp) of a
CMV promoter (FIG. 4), and thus amplified PCR product was treated with restriction
enzymes Sail and Hindlll. The resulting DNA fragment of the TATA box region was
inserted into the pGL3-BA vector treated with restriction enzymes Xhol and Hindlll,
to thereby prepare a pGL3-B/C TA vector (FIG. 5). The resulting vector pGL3-B/C TA
does not have Sail and Xhol restriction sites. Here, PCR was performed under the
following conditions: initial denaturation at 94°C for 5 minute; 25 cycles of de
naturation at 94°C for 1 minute, annealing at 55°C for 1 minute and polymerization at
72°C for 2.5 minute; and final elongation at 72°C for 7 minute.
[166] 5-CMV TA_F(SalI): 5'-CAG TCG ACT AGG CGT GTA CGG TGG GAG-3' (SEQ
ID NO: 4)
[167] 3'-BGH reverse priming site: 5'-TAG AAG GCA CAG TCG AGG-3' (SEQ ID NO:
5)
[168]
[169] As shown in FIG. 5, the prepared pGL3-B/C TA vector includes f l ori, synthetic poly
(A)/transcriptional pause site, MCS, a hybrid promoter of a b-actin promoter(1.9 kb)
and a TATA box region (130 bp) of a CMV promoter, luc+, SV40 late polyadenylation
signal and Amp resistance gene.
[170]
[171] <2-3> Preparation of pGL3-B/C TA/B vector
[172] PCR was performed using a pGL3-BA vector prepared in Example 2-1 as a template
and a primer pair of SEQ ID NOs: 6 and 2 to amplify a b-actin intron region, and thus
amplified PCR product was treated with restriction enzymes Sacl and Hindlll, to
thereby obtain a DNA fragment ( 1 kb) of b-actin intron. In addition, the pGL3-B/C TA
vector prepared in Example 2-2 was treated with restriction enzymes EcoRV and Sacl,
to thereby obtain a DNA fragment (370 bp) of TATA box region. After the pGL3-B/C
TA vector was treated with restriction enzymes EcoRV and Hindlll, the DNA fragment
( 1 kb) of b-actin intron and the DNA fragment (370 bp) of TATA box region were
inserted into the vector, to thereby prepare a pGL3-B/C TA/B vector (FIG. 6). The
resulting vector pGL3-B/C TA/B has two Sacl restriction sites. Here, PCR was
performed under the following conditions: initial denaturation at 94°C for 5 minute; 25
cycles of denaturation at 94°C for 1 minute, annealing at 55°C for 1 minute and poly
merization at 72°C for 3.5 minute; and final elongation at 72°C for 7 minute.
[173] 5'-BA-int(SacI): 5'-CAA GAG CTC TCT GGC TAA CTG AGC ACA GGC CTT
TC-3' (SEQ ID NO: 6)
[174] 3'-BA 4_R(HindIII): 5'-GTA AGC TTC GGC GAA CTA TAT CAG GGC A-3'
(SEQ ID NO: 2)
[175]
[176] As shown in FIG. 6, the prepared pGL3-B/C TA B vector includes f l ori, synthetic
poly (A)/transcriptional pause site, MCS, a hybrid promoter of a b-actin promoter (1.9
kb) and a TATA box region (130 bp) of a CMV promoter, a b-actin intron, luc+, SV40
late polyadenylation signal and Amp resistance gene.
[177]
[178] <2-4> Preparation of pGL3-U/C TA/B vector
[179] PCR was performed using the pGL3-B/C TA/B vector prepared in Example 2-3 as a
template and a primer pair of SEQ ID NOs: 3 and 2 to amplify a DNA fragment
covering a U201 14 region of a b-actin promoter, a TATA box region of a CMV
promoter and a b-actin intron, and thus amplified PCR product was treated with restriction
enzymes Nhel and Hindlll. The resulting DNA fragment was inserted into the
pGL3-Basic vector treated with the same restriction enzymes, to thereby prepare a
pGL3-U/CTA B vector (FIG. 7). Here, PCR was performed under the following
conditions: initial denaturation at 94°C for 5 minute; 25 cycles of denaturation at 94°C
for 1 minute, annealing at 55°C for 1 minute and polymerization at 72°C for 1.5
minute; and final elongation at 72°C for 7 minute.
[180] 5 -U201 14_F(NheI): 5 -CAC GCT AGC TCT CTC TTT TTT TTT TTT TAT-3'
(SEQ ID NO: 3)
[181] 3 -BA 4_R(HindIII): 5'-GTA AGC TTC GGC GAA CTA TAT CAG GGC A-3'
(SEQ ID NO: 2)
[182]
[183] As shown in FIG. 7, the prepared pGL3-U/CTA/B vector includes f l ori, synthetic
poly (A)/transcriptional pause site, MCS, a hybrid promoter of a U201 14 region of a b-
actin promoter and a TATA box region (130 bp) of a CMV promoter, a b-actin intron,
luc+, SV40 late polyadenylation signal and Amp resistance gene.
[184]
[185] <2-5> Preparation of pGL3-Ce U/CrA/B vector
[186] PCR was performed using a pcDNA3.1 vector (Invitrogen) as a template and a
primer pair of SEQ ID NOs: 7 and 8 to amplify an enhancer region of a CMV
promoter, and thus amplified PCR product was treated with restriction enzymes Mlul
and Nhel. The resulting DNA fragment of the CMV enhancer was inserted into the
pGL3-U/CTA B vector treated with the same restriction enzymes, to thereby prepare a
pGL3-Ce /U/CTA/B vector (FIG. 8). Here, PCR was performed under the following
conditions: initial denaturation at 94°C for 5 minute; 25 cycles of denaturation at 94°C
for 1 minute, annealing at 55°C for 1 minute and polymerization at 72°C for 1 minute;
and final elongation at 72°C for 7 minute.
[187] 5'-CMV En_F(MluI): 5'-CAG ACG CGT TGA CAT TGA TTA TTG ACT-3' (SEQ
ID NO: 7)
[188] 3'-CMV En_R(NheI): 5'-CAG GCT AGC AGT TGT TAC GAC ATT TTG-3' (SEQ
ID NO: 8)
[189]
[190] As shown in FIG. 8, the prepared pGL3-U/CTA/B vector includes f l ori, synthetic
poly (A)/transcriptional pause site, MCS, a hybrid promoter of a CMV enhancer, a
U201 14 region of a b-actin promoter and a TATA box region (130 bp) of a CMV
promoter, a b-actin intron, luc+, SV40 late polyadenylation signal and Amp resistance
gene.
[191]
[192] Example 3 : In vitro efficacy test of plasmid vectors
[193] Each of the recombinant vectors prepared in Example 2 was transformed into CHO
cells, and then luciferase expression levels were examined by ELISA.
[194] First, the recombinant vector was introduced into CHO cells using lipofectamine
(Invitrogen). Specifically, CHO cells were maintained in a DMEM medium
(Dulbecco's modified Eagles's medium, GIBCO-BRL) supplemented with heatinactivated
10% FBS (Fetal bovine serum, GIBCO-BRL). Each of the recombinant
vectors prepared in Example 2 and a pCHl 10 vector harboring b-gal were cotransformed
into the cultured CHO cells. One day before transformation, the CHO
cells were cultured in a 24-well plate (Falcon) at a density of 6xl0 4 cells per well.
[195] Meanwhile, Tube 1 (reaction amount for 1 well) containing each 500 ng of the re
combinant vectors prepared in Example 2, 150 ng of the pCHl 10 vector for b-gal
correction, 0.83 b of Plus Reagent, and 23.92 j i of Opti-MEM, and Tube 2 (reaction
amount for 1 well) containing 1.25 b of lipofectamine and 30 b of Opti-MEM were
left at room temperature for 15 minutes, respectively. Then, two tubes were mixed with
each other, followed by reacting at room temperature for further 15 minutes. The
medium of the well plate containing the cultured CHO cells was replaced with 200 b
of Opti-MEM, and then 60 b of the mixture was added to each well. The well plate
was then incubated at 5% C0 2, 37°C for 3 hours. After incubation, 260 b of DMEM
supplemented with 20% FBS was added to each well, and cultivation was performed
for further 2 days.
[196] After 2 days, the medium was removed from each well, and the well plate was
washed with 300 b of PBS. 100 b of 1 Reporter Lysis Buffer (Promega) was added
to each well, followed by freezing the well plate and thawing it at 37°C. The reaction
solution was gently shaken at room temperature, and then each 20 b was transferred
to an analysis plate to perform a luciferase assay and a b-gal assay. The b-gal assay
was performed so as to determine whether transformation had occurred uniformly, and
the luciferase assay results were corrected by the b-gal assay results.
[197] As shown in the following Tables 1 and 2, and FIG. 10, compared to the known
pGL3-BA vector, the pGL3-Ce /U/CTA B vector showed an increased luciferase ex
pression level, and the pGL3-B/C TA/B and pGL3-U/CTA/B vectors showed a similar
or slightly low luciferase expression level. In contrast, the pGL3-B/C TA vector showed
a lower luciferase expression level than the pGL3-BA vector. However, when the b-gal
assay was performed by co-transformation with the pCHl 10 vector for correction in
every experiment, it showed lower b-gal values (0.7-3), compared to the pGL3-Ce /
U/CTA/B vector (near 3) showing the high luciferase expression level.
[198]
[199] Table 1
[Table 1]
Table 2
[Table 2]
[201] *Luminescent: represents luminescence values expressed by luciferase gene inserted
in vector
[202] * ^1: represents galactosidase expression level
[203] * 1corrected: represents corrected values for comparison of galactosidase ex
pression level of each promoter
[204] *LUC average corrected: represents luciferase expression level corrected by average
[205] *Stdev: represents standard deviation of luciferase expression level
[206]
Industrial Applicability
[207] The present invention provides a novel promoter that is optimized for the production
of an antibody or a DNA vaccine. When a variety of target genes are inserted into a re
combinant vector including the hybrid promoter of the present invention, transcription
and expression of the target genes can be improved. Therefore, the recombinant vector
including the hybrid promoter of the present invention can be utilized for the de
velopment of antibody or DNA vaccine.

Claims
A hybrid promoter comprising a whole or a part of a CMV enhancer, a
whole or a part of a b-actin promoter, a whole or a part of a CMV
(Cytomegalovirus) promoter, and a whole or a part of a b-actin intron,
wherein the CMV enhancer, a b-actin promoter, CMV promoter and b-
actin intron are operably linked to each other.
The hybrid promoter according to claim 1, wherein the b-actin
promoter consists of one or more DNA fragments selected from the
group consisting of the following:
i) a DNA fragment having a nucleotide sequence represented by SEQ
ID NO: 9,
ii) a DNA fragment having a nucleotide sequence represented by SEQ
ID NO: 10, or
iii) a DNA fragment having a deletion, a substitution or an insertion of
one or more nucleotides in the nucleotide sequences of the DNA
fragments i) and ii), and having a promoter activity and an activity of
regulating the expression of a target gene operably linked downstream
of the promoter.
The hybrid promoter according to claim 1, wherein the b-actin
promoter is a b-actin promoter of CHO (Chinese Hamster Ovary) cells.
The hybrid promoter according to claim 1, wherein the CMV promoter
is a TATA box region.
The hybrid promoter according to claim 4, wherein the TATA box
region of the CMV promoter consists of a DNA fragment of the
following:
i) a DNA fragment having a nucleotide sequence represented by SEQ
ID NO: 11, or
ii) a DNA fragment having a deletion, a substitution or an insertion of
one or more nucleotides in the nucleotide sequence of the DNA
fragment i), and having a promoter activity and an activity of regulating
the expression of a target gene operably linked downstream of the
promoter.
The hybrid promoter according to claim 1, wherein the b-actin intron
consists of a DNA fragment of the following:
i) a DNA fragment having a nucleotide sequence represented by SEQ
ID NO: 12, or
ii) a DNA fragment having a deletion, a substitution or an insertion of

one or more nucleotides in the nucleotide sequence of the DNA
fragment i), and having a promoter activity and an activity of regulating
the expression of a target gene operably linked downstream of the
promoter.
The hybrid promoter according to claim 1, wherein the CMV enhancer
consists of a DNA fragment of the following:
i) a DNA fragment having a nucleotide sequence represented by SEQ
ID NO: 13, or
ii) a DNA fragment having a deletion, a substitution or an insertion of
one or more nucleotides in the nucleotide sequence of the DNA
fragment i), and having a promoter activity and an activity of regulating
the expression of a target gene operably linked downstream of the
promoter.
The hybrid promoter according to claim 1, which comprises the CMV
enhancer represented by SEQ ID NO: 13, the TATA box region of the
CMV promoter represented by SEQ ID NO: 11, the b-actin promoter
represented by SEQ ID NO: 9 and the b-actin intron region represented
by SEQ ID NO: 12.
The hybrid promoter according to claim 1, which comprises the CMV
enhancer represented by SEQ ID NO: 13, the TATA box region of
CMV promoter represented by SEQ ID NO: 11, the b-actin promoter
represented by SEQ ID NO: 10 and the b-actin intron region rep
resented by SEQ ID NO: 12.
A recombinant vector comprising the hybrid promoter of claim 1 and a
target protein-encoding gene operably linked thereto.
The recombinant vector according to claim 10, further comprising one
or more expression regulatory elements selected from the group
consisting of a replication origin, a selectable marker, a reporter gene, a
terminator and combination thereof.
The recombinant vector according to claim 11, wherein the selectable
marker is a drug resistance gene.
The recombinant vector according to claim 12, wherein the drug re
sistance gene is a gene resistant to antibiotics selected from the group
consisting of ampicillin, streptomycin, gentamicin, kanamycin, hygromycin,
tetracycline, chloramphenicol and neomycin.
The recombinant vector according to claim 11, wherein the reporter
gene is a gene encoding a protein selected from the group consisting of
green fluorescent protein (GFP), cyan fluorescent protein (CFP),

yellow fluorescent protein (YFP), red fluorescent protein (dsRFP), luciferase
(Luc), chloramphenicol acetyltransferase (CAT), b-
galactosidase (LacZ) and b-glucuronidase (Gus).
[Claim 15] A transformant in which the recombinant vector of claim 10 is in
troduced into a host cell.
[Claim 16] The transformant according to claim 15, wherein the host cell is an
animal cell or an animal cell-derived cell.
[Claim 17] The transformant according to claim 16, wherein the host cell is a CHO
(Chinese Hamster Ovary) cell.
[Claim 18] A pharmaceutical composition comprising the recombinant vector of
claim 10 or the transformant of claim 15 as an effective ingredient and
a pharmaceutically acceptable carrier.
[Claim 19] A method for preparing a target protein, comprising the steps of:
1) culturing the transformant of claim 15;
2) inducing the expression of a target protein from the transformant;
and
3) harvesting the expressed target protein from the transformant or the
culture solution thereof.
[Claim 20] The method according to claim 19, wherein the target protein is
selected from the group consisting of human growth hormones, growth
hormone release hormones, growth hormone release peptides, in
terferons, interferon receptors, colony stimulating factors, glucagons -
like peptides, G-protein-coupled receptors, interleukins, interleukin
receptors, enzymes, interleukin- or cytokine-binding proteins,
macrophage activating factors, macrophage peptides, B cell factors, T
cell factors, protein A, allergy inhibitors, cell necrosis glycoprotein,
immune toxins, lymph toxins, tumor necrosis factors, tumor sup
pressing factors, transitional growth factors, alpha- 1 antitrypsin,
albumin, alpha-lactalbumin, apolipoprotein-E, erythropoietin, highly
glycosylated erythropoietin, angiopoietin, hemoglobin, thrombin,
thrombin receptor activating peptides, thrombomodulin, blood factor
VII, Vila, VIII, IX, and XIII, plasminogen activating factor, fibrinbinding
peptides, urokinases, streptokinases, hirudin, protein C, Creactive
proteins, rennin inhibitors, collagenase inhibitors, superoxide
dismutases, leptin, platelet-originated growth factor, epithelial growth
factor, epidermal growth factor, angiostatin, angiotensin, myelopoiesis
growth factor, myelopoiesis stimulating factor, calcitonin, insulin,
atriopeptin, cartilage inducer, elcatonin, joint tissue activating factor,

tissue factor pathway inhibitor, follicle stimulating hormone, pro
gesterone forming hormone, progesterone forming hormone releasing
hormone, nerve growth factors, parathormone, relaxin, cycretin, somatomedine,
insulin-like growth factor, adrenocortical hormones,
glucagons, cholecystokynine, pancreatic polypeptides, gastrin releasing
peptide, corticotropin releasing factor, thyroid stimulating hormone,
autotaxin, lactoferrin, myostatin, receptors, receptor antagonists, cell
surface antigens, virus-originated vaccine antigens, monoclonal an
tibodies, polyclonal antibodies, and antibody fragments.