U.S. patent application number 10/828831 was filed with the patent office on 2006-01-19 for methods for modulating expression of exogenous genes in mammalian systems, and products related thereto.
Invention is credited to Ronald M. Evans, David No, Enrique Saez.
Application Number | 20060014711 10/828831 |
Document ID | / |
Family ID | 35600217 |
Filed Date | 2006-01-19 |
United States Patent
Application |
20060014711 |
Kind Code |
A1 |
Evans; Ronald M. ; et
al. |
January 19, 2006 |
Methods for modulating expression of exogenous genes in mammalian
systems, and products related thereto
Abstract
In accordance with the present invention, there are provided
various methods for modulating the expression of an exogenous gene
in a mammalian subject employing modified ecdysone receptors. Also
provided are modified ecdysone receptors, as well as homomeric and
heterodimeric receptors containing sane, nucleic acids encoding
invention modified ecdysone receptors, modified ecdysone response
elements, gene transfer vectors, recombinant cells, and transgenic
animals containing nucleic acids encoding invention modified
ecdysone receptor.
Inventors: |
Evans; Ronald M.; (La Jolla,
CA) ; No; David; (Irvine, CA) ; Saez;
Enrique; (San Diego, CA) |
Correspondence
Address: |
FOLEY & LARDNER LLP
P.O. BOX 80278
SAN DIEGO
CA
92138-0278
US
|
Family ID: |
35600217 |
Appl. No.: |
10/828831 |
Filed: |
April 20, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09042488 |
Mar 16, 1998 |
6723531 |
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10828831 |
Apr 20, 2004 |
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08974530 |
Nov 19, 1997 |
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10828831 |
Apr 20, 2004 |
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08628830 |
Apr 5, 1996 |
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08974530 |
Nov 19, 1997 |
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Current U.S.
Class: |
514/44R |
Current CPC
Class: |
C12N 2830/002 20130101;
A01K 67/0275 20130101; C12N 15/63 20130101; A61K 48/0058 20130101;
C12N 15/85 20130101; C07K 14/721 20130101; C12N 2830/15 20130101;
C12N 2830/75 20130101; A01K 2217/05 20130101; A61K 48/0066
20130101 |
Class at
Publication: |
514/044 |
International
Class: |
A61K 48/00 20060101
A61K048/00 |
Claims
1. A method for modulating the expression of an exogenous gene in a
mammalian subject containing: (i) a modified ecdysone receptor
which, in the presence of a ligand therefor, and optionally in the
further presence of a silent partner therefor, binds to a response
element, and wherein said modified ecdysone receptor comprises: (a)
a ligand binding domain that binds to an ecdysteroid, (b) a
DNA-binding domain obtained from a DNA-binding protein, which binds
to said response element; and (c) an activation domain of a
transcription factor, wherein at least one of said DNA-binding
domain or said activation domain is not obtained from a native
ecdysone receptor, with the proviso that when said activation
domain is derived from a glucocorticoid receptor, said DNA-binding
domain is not derived from a glucocorticoid receptor or an E. coli
LexA protein; and (ii) a DNA construct comprising said exogenous
gene under the control of said response element, wherein said
response element (a) has about 12-20 base pairs, (b) binds to said
modified ecdysone receptor, and (c) does not bind to farnesoid X
receptor (FXR); said method comprising providing to said subject an
effective amount of one or more ligands for said modified ecdysone
receptor; wherein said one or more ligands are not normally present
in said subject; and wherein said one or more ligands are not toxic
to said subject.
2. A method of inducing the expression of an exogenous gene in a
mammalian subject containing: (i) DNA encoding a modified ecdysone
receptor under the control of an inducible promoter, wherein said
modified ecdysone receptor, in the presence of a ligand therefor,
and optionally in the further presence of a silent partner
therefor, binds to a response element, and wherein said modified
ecdysone receptor comprises: (a) a ligand binding domain that binds
to an ecdysteroid, (b) a DNA-binding domain obtained from a
DNA-binding protein, which binds to said response element; and (c)
an activation domain of a transcription factor, wherein at least
one of said DNA-binding domain or said activation domain is not
obtained from a native ecdysone receptor, with the proviso that
when said activation domain is derived from a glucocorticoid
receptor, said DNA-binding domain is not derived from a
glucocorticoid receptor or an E. coli LexA protein; (ii) a DNA
construct comprising said exogenous gene under the control of said
response element, wherein said response element (a) has about 12-20
base pairs, (b) binds to said modified ecdysone receptor, and (c)
does not bind to famesoid X receptor (FXR); and (iii) one or more
ligands for said modified ecdysone receptor; said method comprising
subjecting said subject to conditions suitable to induce expression
of said modified ecdysone receptor.
3. A method of inducing expression of an exogenous gene in a
mammalian subject containing a DNA construct containing said
exogenous gene under the control of a response element, wherein
said response element (a) has about 12-20 base pairs, (b) binds to
a modified ecdysone receptor, and (c) does not bind to farnesoid X
receptor (FXR), said method comprising introducing into said
subject: a modified ecdysone receptor, wherein said modified
ecdysone receptor comprises: (a) a ligand binding domain that binds
to an ecdysteroid, (b) a DNA-binding domain obtained from a
DNA-binding protein, which binds to said response element; and (c)
an activation domain of a transcription factor, wherein at least
one of said DNA-binding domain or said activation domain is not
obtained from a native ecdysone receptor, with the proviso that
when said activation domain is derived from a glucocorticoid
receptor, said DNA-binding domain is not derived from a
glucocorticoid receptor or an E. coli LexA protein; and one or more
ligands for said modified ecdysone receptor, wherein said modified
ecdysone receptor, in combination with a ligand therefor, and
optionally in the further presence of a silent partner therefor,
binds to said response element, activating transcription
therefrom.
4. A method for modulating the expression of an exogenous gene in a
mammalian subject containing: (i) a DNA construct comprising said
exogenous gene under the control of an ecdysone response element;
and (ii) a modified receptor which, in the presence of a ligand
therefor, and optionally in the further presence of a silent
partner therefor, binds to said ecdysone response element, wherein
said modified receptor does not bind to endogenous response
elements, and wherein said modified receptor comprises: (a) a
ligand binding domain that binds to an ecdysteroid; (b) a
DNA-binding domain derived from a DNA-binding protein, wherein said
DNA-binding domain binds to said ecdysone response element but not
to endogenous response elements; and (c) an activation domain of a
transcription factor, with the proviso that when said activation
domain is derived from a glucocorticoid receptor, said DNA-binding
domain is not derived from a glucocorticoid receptor or an E. coli
LexA protein; said method comprising providing to said subject an
effective amount of one or more ligands for said modified receptor;
wherein said one or more ligands are not normally present in said
subject; and wherein said one or more ligands are not toxic to said
subject.
5. A method of inducing the expression of an exogenous gene in a
mammalian subject containing: (i) a DNA construct comprising an
exogenous gene under the control of an ecdysone response element,
(ii) DNA encoding a modified receptor under the control of an
inducible promoter, wherein said modified receptor, in the presence
of a ligand therefor, and optionally in the further presence of a
silent partner therefor, binds to said ecdysone response element,
wherein said modified receptor does not bind to endogenous response
elements, and wherein said modified receptor comprises: (a) a
ligand binding domain that binds to an ecdysteroid; (b) a
DNA-binding domain derived from a DNA-binding protein, wherein said
DNA-binding domain binds to said ecdysone response element but not
to endogenous response elements; and (c) an activation domain of a
transcription factor, with the proviso that when said activation
domain is derived from a glucocorticoid receptor, said DNA-binding
domain is not derived from a glucocorticoid receptor or an E. coli
LexA protein; (iii) one or more ligands for said modified receptor;
said method comprising subjecting said subject to conditions
suitable to induce expression of said modified receptor.
6. A method of inducing expression of an exogenous gene in a
mammalian subject containing a DNA construct containing said
exogenous gene under the control of an ecdysone response element,
said method comprising introducing into said subject: a modified
receptor, wherein said modified receptor does not bind to
endogenous response elements, and wherein said modified receptor
comprises: (a) a ligand binding domain that binds to an
ecdysteroid; (b) a DNA-binding domain derived from a DNA-binding
protein, wherein said DNA-binding domain binds to said ecdysone
response element but not to endogenous response elements; and (c)
an activation domain of a transcription factor, with the proviso
that when said activation domain is derived from a glucocorticoid
receptor, said DNA-binding domain is not derived from a
glucocorticoid receptor or an E. coli LexA protein; and one or more
ligands for said modified receptor, wherein said modified receptor,
in the presence of a ligand therefor, and optionally in the further
presence of a silent partner therefor, binds to said ecdysone
response element, activating transcription therefrom.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. Ser. No.
08/974,530, filed Nov. 19, 1997, now pending, which is, in turn, a
continuation-in-part of U.S. Ser. No. 08/628,830, filed Apr. 5,
1996, now pending, the entire contents of both of which are hereby
incorporated by reference herein.
FIELD OF THE INVENTION
[0002] The present invention relates to methods in the field of
recombinant DNA technology, and products related thereto. More
particularly, the invention relates to methods and products for
modulating the expression of exogenous genes in mammalian
systems.
BACKGROUND OF THE INVENTION
[0003] The steroid/thyroid hormone receptors comprise a superfamily
of ligand-dependent transcription factors that play a crucial role
in mediating changes in cell fate and function (Evans, R. M.,
Science 240:889-895 (1988)). The receptors transduce extracellular
hormonal signals to target genes that contain specific enhancer
sequences referred to as hormone response elements (HREs) Evans,
(1988); Green and Chambon, Trends Genet. 4:309-314 (1988);
Yamamoto, K. R., Annu. Rev. Genet. 19:209-252 (1985)). Each
receptor recognizes its own HRE, assuring that a distinct response
is triggered by each hormonal signal. Together the collection of
related transcription factors and their cognate response elements
provides a unique opportunity to control gene expression.
[0004] The DNA binding domain of each member of the steroid/thyroid
hormone superfamily of receptors has 66-68 amino acids. Twenty of
these, including nine cysteines, are conserved throughout the
family. The modular structure of members of this receptor
superfamily allows the exchange of homologous domains between
receptors to create functional chimeras. This strategy was used to
demonstrate that the DNA binding domain is solely responsible for
the specific recognition of the HRE in vivo (Green and Chambon,
Nature 325:75-78 (1987); Giguere et al., Nature 330:624-629 (1987);
Petkovich et al., Nature 330:444-450 (1987); Kumar et al., Cell
51:941-951 (1987); Umesono et al., Nature 336:262-265 (1988);
Thompson and Evans, Proc. Natl. Acad. Sci. U.S.A. 86:3494-3498
(1989) and in vitro (Kumar and Chambon, Cell 55:145-156 (1988)). By
analogy with the proposed structure for Xenopus transcription
factor IIIA (Miller et al., EMBO J. 4:1609-1614 (1985)), the
invariant cysteines are thought to form two "zinc fingers" that
mediate the DNA binding function (Hollenberg and Evans, Cell
55:899-906 (1988)). Involvement of these cysteines in Zn(II)
coordination is supported by extended X-ray absorption fine
structure (Freedman et al., Nature 334:543-546 (1988)), and DNA
binding by point mutagenesis experiments (Hollenberg and Evans,
(1988)); Severne et al., EMBO J. 7:2503-2508 (1988)).
[0005] The HREs are in fact structurally related but functionally
distinct. The glucocorticoid receptor response element (GRE),
estrogen receptor response element (ERE), and thyroid hormone
receptor response element (TRE) have been characterized in detail.
These particular response elements have been found to have a
palindromic pair of hexameric "half-sites" (Evans, (1988); Green
and Chambon, (1988)). With optimized pseudo- or consensus response
elements, only two nucleotides per half-site differ between GRE and
ERE (Klock et al., Nature 329:734-736 (1987)). On the other hand,
EREs and TREs have identical half-sites but the number of
nucleotide spacers between the two half sites is different (Glass
et al., Cell 54:313-323 (1988)).
[0006] In contrast to response elements having the palindromic
sequence motif, the following hormone receptors typically recognize
response elements having two half-sites in a direct-repeat (DR)
sequence motif: RXR, RAR, COUP-TF, PPAR, and the like (see, e.g.,
Mangelsdorf et al., The Retinoids: Biology, Chemistry, and
Medicine, 2nd Edition, Raven Press, Ltd., New York, 1994, Chapter
8). Thus at least three distinct means are used to achieve HRE
diversity: 1) binding site specificity for a particular half-site;
2) nucleotide spacing between the two half-sites; and 3) the
orientation of the half-sites to one another.
[0007] In insect systems, a pulse of the steroid hormone ecdysone
triggers metamorphosis in Drosophila melanogaster showing genomic
effects, such as chromosomal puffing, within minutes of hormone
addition. Mediating this response in insects is the functional
ecdysone receptor, a heterodimer of the ecdysone receptor (EcR) and
the product of the ultraspiracle gene (USP) (Yao et al. (1993)
Nature 366, 476-479; and Yao et al. (1992) Cell 71, 63-72).
Responsiveness to an insect ecdysteroid can be recreated in
cultured mammalian cells by co-transfection of EcR, USP, an
ecdysone responsive reporter, and treatment with ecdysone or the
synthetic analog muristerone A.
[0008] In the field of genetic engineering, precise control of gene
expression is an invaluable tool in studying, manipulating and
controlling development and other physiological processes. For
example applications for regulated gene expression in mammalian
systems include inducible gene targeting, overexpression of toxic
and teratogenic genes, anti-sense RNA expression, and gene therapy
(Jaenisch, R. (1988) Science 240, 1468-1474). For cultured cells,
glucocorticoids and other steroids have been used to induce the
expression of a desired gene.
[0009] As another means for controlling gene expression in a
mammalian system, an inducible tetracycline regulated system has
been devised and utilized in transgenic mice, whereby gene activity
is induced in the absence of the antibiotic and repressed in its
presence (see, e.g, Gossen et al. (1992) Proc. Natl. Acad. Sci. 89,
5547-5551; Gossen et al. (1993) TIBS 18, 471-475; Furth et al.
(1994) Proc. Natl. Acad. Sci. 91, 9302-9306; and Shockett et al.
(1995) Proc. Natl. Acad. Sci. 92, 6522-6526). However,
disadvantages of this system include the continuous treatment of
tetracycline to repress expression and the slow clearance of
antibiotic from bone which interferes with: quick and precise
induction. While this system has been improved by the recent
identification of a mutant tetracycline repressor which acts
conversely as an inducible activator, the pharmacokinetics of
tetracycline may hinder its use during development when a precise
and efficient "on-off" switch is essential (Gossen et al. (1995)
Science 268, 1766-1769).
[0010] Accordingly, there is a need in the art for improved methods
to precisely modulate the expression of exogenous genes in
mammalian subjects.
BRIEF DESCRIPTION OF THE INVENTION
[0011] In accordance with the present invention, there are provided
various methods for modulating the expression of an exogenous gene
in a mammalian subject. The invention method is useful in a wide
variety of applications where inducible in vivo expression of an
exogenous gene is desired, such as in vivo therapeutic methods for
delivering recombinant proteins into a variety of cells within a
patient.
[0012] Unlike prior art tetracycline based strategies, transferring
ecdysone responsiveness to mammalian cells takes advantage of a
naturally evolved steroid inducible system. Advantages of
ecdysteroid use include the lipophilic nature of the compounds
(which provides efficient penetrance thereof into all tissues,
including the brain), short half-lives (which allow for precise and
potent inductions), and favorable pharmacokinetics that prevent
storage and expedite clearance.
[0013] In accordance with another embodiment of the present
invention, there are provided modified ecdysone receptors, which
can be in the form of homodimeric species or heterodimeric species
comprising at least one silent partner of the steroid/thyroid
hormone superfamily of receptors, along with an invention modified
ecdysone receptor. Invention modified ecdysone receptors are
useful, for example, in methods for modulating expression of an
exogenous gene in a mammalian subject.
[0014] In accordance with additional embodiments of the present
invention, there are provided nucleic acids encoding invention
modified ecdysone receptors, modified ecdysone receptor response
elements, gene transfer vectors, recombinant cells, and transgenic
animals containing nucleic acid encoding invention modified
ecdysone receptor.
BRIEF DESCRIPTION OF THE FIGURES
[0015] FIGS. 1A-1D show the optimization of ecdysone responsiveness
using various combinations of USP or RXR with different modified
EcRs. In FIG. 1A, the numerical values on both sides of the figure
are on the same scale, with the GEcR/RXR value repeated for
clarity. Darkened and stripped bars represent reporter activity
with no hormone or 1 .mu.M muristerone A, respectively.
[0016] FIG. 1B shows FXR and VpEcR activity on ecdysone response
element (EcRE) and a hybrid ecdysone/glucocorticoid response
element (E/GRE) responsive reporters. VpEcR, VgEcR, and control
transfection without receptors were treated with 1 .mu.M
muristerone. FXR transfections were treated with 50 .mu.M Juvenile
Hormone III (Sigma). Darkened and stripped bars represent reporter
activity with no hormone or 1 .mu.M muristerone A/50 .mu.M Juvenile
Hormone III, respectively.
[0017] FIG. 1C shows that E/GRE and GRE are non-overlapping
response elements. Darkened and stripped bars represent reporter
activity with no hormone or 1 .mu.M muristerone A/1 .mu.M
dexamethasone, respectively.
[0018] FIG. 1D shows a schematic diagram of modified ecdysone
receptors. GEcR is a chimeric receptor containing the N-terminal
transactivation domain of GR and the DNA- and ligand-binding
domains of EcR. VpEcR is an N-terminal truncation of EcR, wherein
the activation domain of Vp16 is fused thereto at the amino
terminus thereof. VgEcR is identical to VpEcR except for the
following point mutations in the P box of the DNA binding domain:
E282G, G283S, and G286V. Vp16-EcR-B1 is a fusion of full length EcR
with the activation domain of Vp16, wherein the activation domain
of Vp16 is fused thereto at the carboxy terminus thereof. VgEcR-B1
is identical to Vp16-EcR-B1 except for the same point mutations in
the P box of the DNA binding domain as described above. In the
Figure, DBD=DNA binding domain and LBD=ligand binding domain.
[0019] FIG. 2 shows a schematic diagram of an invention ecdysone
inducible gene expression system. After expression of RXR and a
modified EcR, the two receptors can heterodimerize and
transactivate the ecdysone response element-containing promoter in
the presence of hormone. The ecdysone response elements are p laced
upstream of a minimal promoter (i.e., an enhancerless promoter)
which can drive the expression of any exogenous cDNA.
[0020] FIG. 3A shows a dose-dependent activation of N13 cells with
muristerone. N13 cells were grown with varying concentrations of
muristerone for 36 hours and then assayed for .beta.-galactosidase
activity (open squares) by standard ONPG assay or for luciferase
activity (closed circles). FIG. 3B shows the time-course of
luciferase activity of N13 cells treated with hormone. N13 cells
were grown in separate wells in the presence of 1 .mu.M
muristerone, harvested at varying times, and assayed for luciferase
activity as described in Example 3.
[0021] FIG. 4 shows muristerone activity in mice as described in
Example 4.
[0022] FIG. 5 compares the dose-dependent activation of N13 cells
with muristerone (X) and ponasterone A (open circles).
DETAILED DESCRIPTION OF THE INVENTION
[0023] In accordance with the present invention, there are provided
methods for modulating the expression of an exogenous gene in a
mammalian subject containing: [0024] (i) a DNA construct comprising
said exogenous gene under the control of an ecdysone response
element; and [0025] (ii) a modified ecdysone receptor which, in the
presence of a ligand therefor, and optionally in the further
presence of a receptor capable of acting as a silent partner
therefor, binds to said ecdysone response element; [0026] said
method comprising administering to said subject an effective amount
of a ligand for said modified ecdysone receptor; wherein said
ligand is not normally present in the cells of said subject; and
wherein said ligand is not toxic to said subject.
[0027] Thus, in accordance with the present invention the insect
molting hormone, ecdysone (as well as analogs and mimics thereof),
is advantageously employed as a regulated inducer of gene
expression in mammalian systems, i.e., background levels of
expression are substantially zero in the absence of conditions
required for induction. In a presently preferred aspect of the
invention, promoters containing a novel modified ecdysone response
element are employed in conjunction with an invention modified
ecdysone receptor (preferably having an altered DNA binding
specificity) to provide an extremely powerful and specific
inducible mammalian expression system. The low basal activity of
the invention expression system is advantageously suitable for the
expression of transcription factors and toxic genes. The excellent
dose response and induction rate characteristics of the invention
inducible expression system allow for precise control of both the
degree and duration of induction of a desired gene.
[0028] Since the invention method provides for regulated gene
expression by an exogenous non-mammalian inducer, it can be
advantageously employed in a variety of in vivo and in vitro
mammalian expression systems. For example, inducible expression of
cre recombinase in transgenic mammals, in accordance with invention
methods, would enable those of skill in the art to accomplish
temporally specific inducible gene targeting of the adult or the
developing embryo (O'Gorman et al. (1991) Science 251,
1351-1355).
[0029] As employed herein, the terms "modulate" and "modulating"
refer to the ability of a given ligand/receptor complex to effect
transactivation of transcription of an exogenous gene, relative to
such ability of said receptor in the absence of ligand. The actual
effect of complex formation on the transactivation activity of a
receptor will vary depending on the specific receptor species which
are part of the ligand/receptor-complex, and on the response
element with which the ligand/receptor complex interacts.
[0030] As used herein, when referring to genes, the phrase
"exogenous to said mammalian subject" or simply "exogenous" refers
to any gene wherein the gene product is not naturally expressed in
the particular cell where expression is desired. For example,
exogenous genes can be either natural or synthetic wild type genes
and therapeutic genes, which are introduced into the subject in the
form of DNA or RNA. The gene of interest can be introduced into
target cells (for in vitro applications), or the gene of interest
can be introduced directly into a subject, or indirectly introduced
by the transfer of transformed cells into a subject.
[0031] "Wild type" genes are those that are native to cells of a
particular type. Such genes may be undesirably overexpressed, or
may not be expressed in biologically significant levels. Thus, for
example, while a synthetic or natural gene coding for human insulin
would be exogenous genetic material to a yeast cell (since yeast
cells do not naturally contain insulin genes), a human insulin gene
inserted into a human skin fibroblast cell would be a wild type
gene with respect to that cell since human skin fibroblasts contain
genetic material encoding human insulin, although human skin
fibroblasts do not express human insulin in biologically
significant levels.
[0032] Wild type genes contemplated for use in the practice of the
present invention include genes which encode a gene product: [0033]
the substantial absence of which leads to the occurrence of a
non-normal state in said subject; or [0034] a substantial excess of
which leads to the occurrence of a non-normal state in said
subject; [0035] and the like.
[0036] As employed herein, the phrase "therapeutic gene" refers to
a gene which imparts a beneficial function to the host cell in
which such gene is expressed. Therapeutic genes are those that are
not naturally found in host cells. For example, a synthetic or
natural gene coding for wild type human insulin would be
therapeutic when inserted into a skin fibroblast cell so as to be
expressed in a human host, where the human host is not otherwise
capable of expressing functionally active human insulin in
biologically significant levels. In accordance with the methods
described herein, therapeutic genes are expressed at a level that
provides a therapeutically effective amount of the corresponding
therapeutic protein.
[0037] Therapeutic genes contemplated for use in the practice of
the present invention include genes which encode a gene product:
[0038] which is toxic to the cells in which it is expressed; or
[0039] which imparts a beneficial property to the host subject
(e.g., disease resistance, etc); [0040] and the like.
[0041] Numerous genomic and cDNA nucleic acid sequences coding for
a variety of proteins are well known in the art. Exogenous genetic
material useful in the practice of the present invention include
genes that encode biologically active proteins of interest, such
as, e.g., secretory proteins that can be released from said cell;
enzymes that can metabolize a substrate from a toxic substance to a
non-toxic substance, or from an inactive substance to a useful
substance; regulatory proteins; cell surface receptors; and the
like. Useful genes include genes that encode blood clotting factors
such as human factors VIII and IX; genes that encode hormones such
as insulin, parathyroid hormone, luteinizing hormone releasing
factor (LHRH), alpha and beta seminal inhibins, and human growth
hormone; genes that encode proteins such as enzymes, the absence of
which leads to the occurrence of an abnormal state; genes encoding
cytokines or lymphokines such as interferons, granulocytic
macrophage colony stimulating factor (GM-CSF), colony stimulating
factor-1 (CSF-1), tumor necrosis factor (TNF), and erythropoietin
(EPO); genes encoding inhibitor substances such as
alpha.sub.1-antitrypsin; genes encoding substances that function as
drugs, e.g., genes encoding the diphtheria and cholera toxins; and
the like.
[0042] Typically, nucleic acid sequence information for a desired
protein can be located in one of many public access databases,
e.g., GENBANK, EMBL, Swiss-Prot, and PIR, or in many biology
related journal publications. Thus, those of skill in the art have
access to nucleic acid sequence information for virtually all known
genes. Those of skill in the art can either obtain the
corresponding nucleic acid molecule directly from a public
depository or the institution that published the sequence.
Optionally, once the nucleic acid sequence encoding a desired
protein has been ascertained, the skilled artisan can employ
routine methods, e.g., polymerase chain reaction (PCR)
amplification, to isolate the desired nucleic acid molecule from
the appropriate nucleic acid library. Thus, all known nucleic acids
encoding proteins of interest are available for use in the methods
and products described herein.
[0043] As used herein, the terms "mammal" and "mammalian" refer to
humans; domesticated animals, e.g., rats, mice, rabbits, canines,
felines, and the like; farm animals, e.g., chickens, bovine,
porcine and ovine, and the like; and animals of zoological
interest, e.g., monkeys and baboons, and the like.
[0044] Modified ecdysone receptors contemplated for use in the
practice of the present invention comprise: [0045] a ligand binding
domain capable of binding an ecdysteroid; [0046] a DNA-binding
domain obtained from a DNA-binding protein; and [0047] an
activation domain of a transcription factor, [0048] wherein at
least one of said DNA-binding domain or said activation domain is
not obtained from a native ecdysone receptor, with the proviso that
when said activation domain is derived from a glucocorticoid
receptor, said DNA-binding domain is not derived from a
glucocorticoid receptor or an E. coli LexA protein. In accordance
with the present invention, modified ecdysone receptors function in
expression systems, preferably mammalian, to transactivate gene
expression from transcription regulatory regions having ecdysone
response elements. Preferably, in order to minimize induction of
undesired gene expression, modified ecdysone receptors of the
invention will have substantially no constitutive activity in
mammalian cells.
[0049] Ligand binding domains capable of binding an ecdysteroid, as
contemplated for use in the preparation of invention modified
ecdysone receptors are typically derived from the carboxy-terminal
portion of native ecdysone receptor and are able to bind
ecdysteroids (Koelle et al., Cell, 67:59-77, 1991; and
Christopherson et al., PNAS, USA, 89:6314-6318, 1992). Ligand
binding domains capable of binding an ecdysteroid can be
functionally located in either orientation and at various positions
within the modified ecdysone receptor of the invention. For
example, the ligand binding domain capable of binding an
ecdysteroid can be positioned at either the amino or carboxy
terminus of the modified receptor, or therebetween. In a preferred
embodiment of the present invention, the ligand binding domain
capable of binding an ecdysteroid is positioned at the carboxy
terminus of the modified receptor (see FIG. 1D).
[0050] DNA-binding domains contemplated for use in the preparation
of invention modified ecdysone receptors are typically obtained
from DNA-binding proteins (e.g., transcription factors). The term
"DNA-binding domain" is understood in the art to refer to an amino
acid sequence that is able to bind to DNA. As used herein, the term
"DNA-binding domain" encompasses a minimal peptide sequence of a
DNA-binding protein, up to the entire length of a DNA-binding
protein, so long as the DNA-binding domain functions to associate
with a particular response element.
[0051] Such DNA-binding domains are known to function
heterologously in combination with other functional protein domains
by maintaining the ability to bind the natural DNA recognition
sequence (see, e.g., Brent and Ptashne, 1985, Cell, 43:729-736).
For example, hormone receptors are known to have interchangeable
DNA-binding domains that function in chimeric proteins (see, e.g.,
U.S. Pat. No. 4,981,784; and Evans, R., 1988, Science,
240:889-895). Thus, similar to the ligand binding domain of
invention modified ecdysone receptor, the DNA-binding domain can be
positioned at either the carboxy terminus or the amino terminus, or
the DNA-binding domain can be positioned between the ligand binding
domain and the activation domain. In preferred embodiments of the
present invention, the DNA-binding domain is positioned internally
between the ligand binding domain and the activation domain.
[0052] "DNA-binding protein(s)" contemplated for use herein belong
to the well-known class of proteins that are able to directly bind
DNA and facilitate initiation or repression of transcription.
Exemplary DNA-binding proteins contemplated for use herein include
transcription control proteins (e.g., transcription factors and the
like; Conaway and Conaway, 1994, "Transcription Mechanisms and
Regulation", Raven Press Series on Molecular and Cellular Biology,
Vol. 3, Raven Press, Ltd., New York, N.Y.).
[0053] Transcription factors contemplated for use herein as a
source of such DNA binding domains include, e.g., homeobox
proteins, zinc finger proteins, hormone receptors, helix-turn-helix
proteins, helix-loop-helix proteins, basic-Zip proteins (bZip),
.beta.-ribbon factors, and the like. See, for example, Harrison,
S., "A Structural Taxonomy of DNA-binding Domains," Nature,
353:715-719. Homeobox DNA-binding proteins suitable for use herein
include, for example, HOX, STF-1 (Leonard et al., 1993, Mol. Endo.,
7:1275-1283), Antp, Mat .alpha.-2, INV, and the like. See, also,
Scott et al. (1989), Biochem. Biophys. Acta, 989:25-48. It has been
found that a fragment of 76 amino acids (corresponding to amino
acids 140-215 described in Leonard et al., 1993, Mol. Endo.,
7:1275-1283) containing the STF-1 homeodomain binds DNA as tightly
as wild-type STF-1. Suitable zinc finger DNA-binding proteins for
use herein include Zif268, GLI, XFin, and the like. See also, Klug
and Rhodes (1987), Trends Biochem. Sci., 12:464; Jacobs and
Michaels (1990), New Biol., 2:583; and Jacobs (1992), EMBO J.,
11:4507-4517.
[0054] Preferably, the DNA-binding domain used herein is obtained
from a member of the steroid/thyroid hormone superfamily of
receptors. As used herein, the phrase "member(s) of the
steroid/thyroid hormone superfamily of receptors" (also known as
"nuclear receptors" or "intracellular receptors") refers to hormone
binding proteins that operate as ligand-dependent transcription
factors, including identified members of the steroid/thyroid
hormone superfamily of receptors for which specific ligands have
not yet been identified (referred to hereinafter as "orphan
receptors").
[0055] Exemplary members of the steroid/thyroid hormone superfamily
of receptors (including the various isoforms thereof) include
steroid receptors such as glucocorticoid receptor (GR),
mineralocorticoid receptor (MR), estrogen receptor (ER),
progesterone receptor (PR), androgen receptor (AR), vitamin D.sub.3
receptor (VDR), and the like; plus retinoid receptors, such as the
various isoforms of retinoic acid receptor (e.g., RAR.alpha.,
RAR.beta., or RAR.gamma.), the various isoforms of retinoid X
receptor (e.g., RXR.alpha., RXR.beta., or RXR.gamma.), and the like
(see, e.g., U.S. Pat. Nos. 4,981,784; 5,171,671; and 5,071,773);
thyroid receptors (TR), such as TR.alpha., TR.beta., and the like;
insect derived receptors such as the ecdysone receptor, and the
like; as well as other gene products which, by their structure and
properties, are considered to be members of the superfamily, as
defined hereinabove, including the various isoforms thereof.
Examples of orphan receptors contemplated for use herein as a
source of DNA binding domain include HNF4 (see, for example, Sladek
et al., in Genes & Development 4: 2353-2365 (1990)), the COUP
family of receptors (see, for example, Miyajima et al., in Nucleic
Acids Research 16: 11057-11074 (1988), and Wang et al., in Nature
340: 163-166 (1989)), COUP-like receptors and COUP homologs, such
as those described by Mlodzik et al., in Cell 60: 211-224 (1990)
and Ladias et al., in Science 251: 561-565 (1991), various isoforms
of peroxisome proliferator-activated receptors (PPARs; see, for
example, Issemann and Green, supra), the insect derived knirps and
knirps-related receptors, and the like.
[0056] The DNA-binding domains of all members of the
steroid/thyroid hormone superfamily of receptors are related,
consisting of 66-68 amino acid residues, and possessing about 20
invariant amino acid residues, including nine cysteines. A member
of the superfamily can be characterized as a protein which contains
these 20 invariant amino acid residues. The highly conserved amino
acids of the DNA-binding domain of members of the superfamily are
as follows: TABLE-US-00001 (SEQ ID NO:1) Cys - X - X - Cys - X - X
- Asp* - X - Ala* - X - Gly* - X - Tyr* - X - X - X - X - Cys - X -
X - Cys - Lys* - X - Phe - Phe - X - Arg* - X - X - X - X - X - X -
X - X - X - (X - X -) Cys - X -X - X - X - X - (X - X - X -) Cys -
X -X - X - Lys - X - X - Arg - X - X - Cys - X - X - Cys - Arg* - X
- X - Lys* - Cys - X - X - X - Gly* - Met;
wherein X designates non-conserved amino acids within the
DNA-binding domain; an asterisk denotes the amino acid residues
which are almost universally conserved, but for which variations
have been found in some identified hormone receptors; and the
residues enclosed in parenthesis are optional residues (thus, the
DNA-binding domain is a minimum of 66 amino acids in length, but
can contain several additional residues).
[0057] Modification of existing DNA-binding domains to recognize
new target recognition sequences is also contemplated herein. For
example, in accordance with the present invention, it has been
found that the modification of the "P-box" sequence of DNA-binding
domains of members of the steroid/thyroid hormone superfamily of
receptors offers unique advantages not present in other chimeric
hormone receptors. For example, the modification of a P-box amino
acid sequence to preferentially bind to a different hormone
response element half-site than the naturally occurring P-box amino
acid sequence can reduce undesired background levels of gene
expression. Thus, invention receptors and methods provide the
advantage of increasing the selectivity of exogenous gene
expression in a particular subject.
[0058] As used herein, the phrase "P-box amino acid sequence"
refers to the proximal element region in a DNA-binding domain of a
hormone receptor that typically occurs at the junction of the first
zinc finger and the linker region, e.g., at about amino acids 19-23
of the DNA-binding domain (i.e., amino acids 19-23 of SEQ ID NO:1;
see, e.g., Umesono et al. (1989), Cell, 57:1139-1146, FIG. 2).
Umesono et al. (1989), supra, in Table 1, describe various
naturally occurring P-box amino acid sequences for a variety of
hormone receptor DNA-binding domains.
[0059] In one embodiment of the present invention, the P-box
sequence of a hormone receptor DNA-binding domain is modified to
have a P-box amino acid sequence that differs from the naturally
occurring P-box amino acid sequence. In a preferred embodiment of
the present invention, the modified P-box amino acid sequence
differs from the naturally occurring P-box amino acid sequence by 3
amino acids.
[0060] Preferably, the P-box amino acid sequence is modified so
that only the half-site nucleotide sequence recognized by the
DNA-binding domain is changed while not altering the spacing
between the two half-sites recognized by the DNA-binding domain.
For example, when the DNA-binding domain of the ecdysone receptor
is employed in an invention modified ecdysone receptor, the P-box
can be modified from the amino acid sequence EGCKG (SEQ ID NO:2;
which recognizes the half-site -AGGTCA-) to the amino acid sequence
GSCKV (SEQ ID NO:3; which recognizes the half-site seqeunce
-AGAACA-). In a presently preferred embodiment, when the
DNA-binding domain of invention modified ecdysone receptor is
derived from ecdysone receptor, the P-box amino acid sequence is
modified to GSCKV (SEQ ID NO:3).
[0061] It has also been found that in vitro evolution methods can
be applied to modify and improve existing DNA-binding domains (see,
e.g., Devlin et al., 1990, Science, 249:404-406; and Scott and
Smith, 1990, Science, 249:386-390).
[0062] Activation domains contemplated for use in the preparation
of invention modified ecdysone receptor are typically derived from
transcription factors and comprise a contiguous sequence of amino
acids that functions to activate gene expression when associated
with a suitable DNA-binding domain and a suitable ligand binding
domain. As with the ligand and DNA-binding domains employed for the
preparation of invention modified ecdysone receptors, the
activation domain can be positioned at the carboxy terminus, the
amino terminus or between the ligand binding domain and the DNA
binding domain. In preferred embodiments of present invention, the
activation domain is positioned at the amino terminus or the
carboxy terminus of the modified ecdysone receptor.
[0063] Suitable activation domains can be obtained from a variety
of sources, e.g., from the N-terminal region of a member of the
steroid/thyroid hormone superfamily of receptors, from a
transcription factor activation domain, such as, for example, VP16
or GAL4 activation domains, and the like. The presently most
preferred activation domain contemplated for use in the practice of
the present invention is obtained from the N-terminal region of the
VP16 protein.
[0064] The presently most preferred modified ecdysone receptors
contemplated for use herein are VgEcR (SEQ ID NO:5), VpEcR (SEQ ID
NO:7), GEcR (SEQ ID NO:9), Vp16-EcR-B1 or VgEcR-B1, with VgEcR (SEQ
ID NO:5) and VgEcR-B1 being especially preferred. The preparation
of several of these modified ecdysone receptors is set forth
hereinafter in Example 1. See also FIG. 1D. Those modified
receptors for which explicit methods of preparation is not provided
herein can readily be made using the methodology set forth herein
in combination with standard methodology well known to those of
skill in the art.
[0065] Invention modified ecdysone receptor proteins can be
produced by expressing nucleic acid constructs encoding the
chimeric proteins in suitable host cells as described in Example 1.
Recombinant methods of producing desired proteins by introducing an
expression construct into appropriate host cells are well-known in
the art. Modified ecdysone receptors of the invention can be
introduced into a particular subject by direct introduction of the
proteins themselves, by introducing DNA construct(s) encoding the
receptor into the subject, or into cells obtained from the subject
(wherein the cells are transformed and subsequently returned to the
subject).
[0066] In a preferred embodiment, invention modified ecdysone
receptors are expressed under the control of a tissue specific
promoter. As readily understood by those of skill in the art, the
term "tissue specific" refers to the substantially exclusive
initiation of transcription in the tissue from which a particular
promoter drives expression of a given gene.
[0067] In accordance with one aspect of the present invention,
invention modified ecdysone receptors are present in the form of
heterodimeric species comprising an invention modified ecdysone
receptor and at least one silent partner of the steroid/thyroid
hormone superfamily of receptors. Preferably, the silent partner is
a mammalian-derived receptor, with RXR being especially
preferred.
[0068] Silent partners contemplated herein are members of the
steroid/thyroid hormone superfamily of receptors which are capable
of forming heterodimeric species with the invention modified
ecdysone receptor, wherein the silent partner does not directly
participate in binding ligand (i.e., only the modified ecdysone
receptor co-partner of the heterodimer binds ligand). The silent
partner can either be endogenous to the cells of the subject or can
be provided to the subject by introducing DNA construct(s) encoding
receptor into the subject. A preferred silent partner for use
herein is RXR. In a particular embodiment of the invention methods,
exogenous RXR is provided to said mammalian subject.
[0069] The formation of heterodimeric receptor(s) can modulate the
ability of member(s) of the steroid/thyroid hormone superfamily of
receptors to trans-activate transcription of genes maintained under
expression control in the presence of ligand for said receptor. For
example, formation of a heterodimer of the modified ecdysone
receptor with another mammalian hormone receptor promotes the
ability of the modified ecdysone receptor to induce
trans-activation activity in the presence of an ecdysone response
element.
[0070] In accordance with another aspect of the present invention,
invention modified ecdysone receptors are present in the form of
homodimeric species comprising a plurality (i.e., at least two)
invention modified ecdysone receptors.
[0071] Ligands contemplated for use herein are compounds which,
inside a cell, bind to invention modified ecdysone receptors,
thereby creating a ligand/receptor complex, which in turn can bind
to an appropriate response element. The terms "ecdysone",
"ecdysteroid", "ecdysone-analogs", and "ecdysone mimics" as
interchangeably used herein, are employed herein in the generic
sense (in accordance with common usage in the art), referring to a
family of ligands with the appropriate binding and transactivation
activity (see, for example, Cherbas et al., in Biosynthesis,
metabolism and mode of action of invertebrate hormones (ed. J.
Hoffmann and M. Porchet), p. 305-322; Springer-Verlag, Berlin). An
ecdysone, therefore, is a steroid, steroid-like or non-steroidal
compound which acts to modulate gene transcription for a gene
maintained under the control of a suitable response-element, as
described herein.
[0072] 20-Hydroxy-ecdysone (also known as .beta.-ecdysone) is the
major naturally occurring ecdysone. Unsubstituted ecdysone (also
known as .alpha.-ecdysone) is converted in peripheral tissues to
.beta.-ecdysone. Analogs of the naturally occurring ecdysones are
also contemplated within the scope of the present invention.
Examples of such analogs, commonly referred to as ecdysteroids,
include ponasterone A, ponasterone B, ponasterone C, ponasterone D,
26-iodoponasterone A, muristerone A, inokosterone,
26-mesylinokosterone, sidasterone, buterosterone, ajugasterone,
makisterone, cyasterone, sengosterone, and the like. Since it has
been previously reported that the above-described ecdysones are
neither toxic, teratogenic, nor known to affect mammalian
physiology, they are ideal candidates for use as inducers in
cultured cells and transgenic mammals according to the invention
methods.
[0073] Additional compounds contemplated for use herein are mimics
of the naturally occurring ecdysones, i.e., synthetic organic
compounds which have binding and transactivation activities
characteristic of the naturally occurring ecdysones. Examples of
such compounds, referred to herein as ecdysone mimics, include
1,2-diacyl hydrazines (e.g., those described in U.S. Pat. Nos.
5,424,333 and 5,354,762, the entire contents of each of which are
hereby incorporated by reference herein),
N'-substituted-N,N'-di-substituted hydrazines (e.g., those
described in U.S. Pat. No. 5,117,057, the entire contents of which
are hereby incorporated by reference herein), dibenzoylalkyl
cyanohydrazines (e.g., those described in European Application No.
461,809, the entire contents of which are hereby incorporated by
reference herein), N-substituted-N-alkyl-N,N'-diaroyl hydrazines
(e.g., those described in U.S. Pat. No. 5,225,443, the entire
contents of which are hereby incorporated by reference herein),
N-substituted-N-acyl-N-alkyl, carbonyl hydrazines (e.g., those
described in European Application No. 234,944, the entire contents
of which are hereby incorporated by reference herein),
N-aroyl-N'-alkyl-N'-aroyl hydrazines (e.g., those described in U.S.
Pat. No. 4,985,461, the entire contents of which are hereby
incorporated by reference herein), and the like.
[0074] Compounds of specific interest are those having the formula:
##STR1## wherein: [0075] R.sup.1 is optionally hydrogen, lower
alkyl or substituted lower alkyl, alkenyl or substituted alkenyl,
alkynyl or substituted alkynyl, aryl or substituted aryl,
heteroaryl or substituted heteroaryl, and the like. R.sup.1 is not
present when X.sup.1 is part of a carbon-nitrogen double bond
linking R.sup.3 to the hydrazino group. [0076] R.sup.2 is
optionally hydrogen, alkyl or substituted alkyl, cyclohexyl or
substituted cyclohexyl, and the like. R.sup.2 is not present when
X.sup.2 is part of a carbon-nitrogen double bond linking R.sup.4 to
the hydrazino group. [0077] R.sup.3 and R.sup.4 are independently
part of an appropriately substituted carbon-nitrogen double bond
which links R.sup.3 and/or R.sup.4 to the hydrazino linkage, or
R.sup.3 and R.sup.4 are independently aryl or substituted aryl,
heteroaryl or substituted heteroaryl, provided, however, that when
two adjacent positions on the aryl or heteroaryl moieties are
substituted with alkoxy, thioalkyl, alkylamino, or dialkylamino
groups, these groups may be joined to form a 5- or 6-membered
heterocyclic ring system, or R.sup.3 and R.sup.4 are independently
heterocyclic or substituted heterocyclic, cycloalkyl or substituted
cycloalkyl, and the like. [0078] X.sup.1 and X.sup.2 are
independently --C(O)--, --C(S)--, --C(NR.sub.2)--,
--C(.dbd.CN)NH--, --C(O)O--, --C(O)NH--, --C(O)NHSO.sub.2--,
--CH.sub.2--, --SO.sub.2--, --P(O)CH.sub.3--, and the like, as well
as an appropriate substituted carbon-nitrogen double bond which
links R.sup.3 and/or R.sup.4 to the hydrazino linkage.
[0079] As employed herein, "alkyl" refers to alkyl groups having in
the range of 1 up to 8 carbon atoms; "lower alkyl" refers to alkyl
groups having in the range of 1 up to 4 carbon atoms; and
"substituted alkyl" or "substituted lower alkyl" comprises alkyl
(or lower alkyl) groups further bearing one or more substituents
selected from halogen, cyano, nitro, hydroxy, alkoxy (--OR),
thioalkyl (--SR), --NR.sub.2, --NRC(O)R, --OC(O)R, --C(O)OR,
--C(O)NR.sub.2, --C(O)R, wherein each R is independently hydrogen
or lower alkyl, and the like.
[0080] As employed herein, "cycloalkyl" refers to cyclic
ring-containing groups containing in the range of about 5 up to 8
carbon atoms, and "substituted cycloalkyl" refers to cycloalkyl
groups further bearing one or more substituents as set forth above,
as well as lower alkyl.
[0081] As employed herein, "heterocyclic" refers to cyclic (i.e.,
ring-containing) groups containing one or more (up to four)
heteroatoms (e.g., N, O, S, or the like) as part of the ring
structure, and having in the range of 2 up to 5 nuclear carbon
atoms and "substituted heterocyclic" refers to heterocyclic groups
further bearing one or more substituents as set forth above, as
well as lower alkyl.
[0082] As employed herein, "alkenyl" refers to straight or branched
chain hydrocarbyl groups having at least one carbon-carbon double
bond, and having in the range of about 2 up to 12 carbon atoms, and
"substituted alkenyl" refers to alkenyl groups further bearing one
or more substituents as set forth above.
[0083] As employed herein, "alkynyl" refers to straight or branched
chain hydrocarbyl groups having at least one carbon-carbon triple
bond, and having in the range of about 2 up to 12 carbon atoms, and
"substituted alkynyl" refers to alkynyl groups further bearing one
or more substituents as set forth above.
[0084] As employed herein, "aryl" refers to aromatic groups having
in the range of 6 up to 14 carbon atoms and "substituted aryl"
refers to aryl groups further bearing one or more substituents as
set forth above, as well as lower alkyl.
[0085] As employed herein, "heteroaryl" refers to aromatic groups
containing one or more heteroatoms (e.g., N, O, S, or the like) as
part of the ring structure, and having in the range of 3 up to 14
carbon atoms and "substituted heteroaryl" refers to heteroaryl
groups further bearing one or more substituents as set forth
above.
[0086] Presently preferred ecdysone mimics contemplated for use
herein include compounds wherein R.sup.1 is hydrogen; R.sup.2 is an
alkyl group possessing considerable bulk (such as, for example,
alkyl groups containing a tertiary carbon center, e.g.,
--C(R'').sub.3, wherein each R'' is methyl or greater). Examples of
alkyl groups having sufficient bulk for use herein include
tert-butyl, sec-butyl, isopropyl, isobutyl, cyclohexyl,
cyclopentyl, dicyclopropylmethyl, (cyclohexyl) ethyl, and the
like); X.sup.1 and X.sup.2 are both --C(O)--; R.sup.3 is phenyl,
substituted phenyl (with hydroxy, alkoxy, halo and/or substituted
amino substituents being preferred, with 3,4-disubstitution pattern
being especially preferred), heterocyclic (e.g., pyridyl or
pyrimidine) or substituted heterocyclic (with halo, alkyl,
thioalkyl, hydroxy, alkoxy, and/or amino substituents being
preferred); and R.sup.4 is phenyl or substituted phenyl, heteroaryl
or substituted heteroaryl or a bulky alkyl or cycloalkyl group.
[0087] Especially preferred ecdysone mimics contemplated for use
herein include
N'-(3,5-dimethylbenzoyl)-N-(4-ethylbenzoyl)-N'-(tert-butyl)
hydrazine, N,N'-dibenzoyl-N'-(tert-butyl) hydrazine,
N'-(3,5-dimethylbenzoyl)-N-(4-ethylbenzyl)-N'-(tert-butyl)
hydrazine,
N'-(3,5-dimethylbenzoyl)-N-(2-methyl-3,4-(ethylenedioxy)-benzoyl)-N'-(ter-
t-butyl) hydrazine,
3,5-di-tert-butyl-4-hydroxy-N-isobutyl-benzamide,
8-O-acetylharpagide, and the like.
[0088] Ligands contemplated for use in the practice of the present
invention are characterized as not normally being present in the
cells of the subject, meaning that the ligand is exogenous to the
subject. Ecdysteroids, for example, are not naturally present in
mammalian systems. Thus, in accordance with the invention method,
unless and until an ecdysteroid is administered to the subject,
substantially no expression of the desired gene occurs.
[0089] An effective amount of ligand contemplated for use in the
practice of the present invention is the amount of ligand (i.e.,
ecdysteroid) required to achieve the desired level of gene
expression product. Ligand can be administered in a variety of
ways, as are well-known in the art. For example, such ligands can
be administered topically, orally, intravenously,
intraperitoneally, intravascularly, and the like.
[0090] As readily recognized by those of skill in the art, it may
be desirable to be able to rapidly induce or rapidly turn off
expression by the invention expression system. This can readily be
accomplished by administration of a suitable ecdysone antagonist
before or after induction of the system (e.g., to prevent undesired
activation of the system, to promote rapid induction, to rapidly
terminate expression, and the like). Numerous ecdysone antagonists
are known in the art, e.g., ajugalactone.
[0091] In accordance with a particular embodiment of the present
invention, pharmaceutically acceptable formulations, and kits
thereof, comprising at least one ecdysteroid, and a
pharmaceutically acceptable carrier are contemplated. In accordance
with another aspect of the present invention, pharmaceutically
acceptable formulations consisting essentially of at least one
ecdysteroid and a pharmaceutically acceptable carrier, are
contemplated. Pharmaceutical formulations of the present invention
can be used in the form of a solid, a solution, an emulsion, a
dispersion, a micelle, a liposome, and the like, wherein the
resulting formulation contains one or more of the ecdysteroids of
the present invention, as an active ingredient, in admixture with
an organic or inorganic carrier or excipient suitable for enteral
or parenteral applications.
[0092] The active ingredient may be compounded, for example, with
the usual non-toxic, pharmaceutically acceptable carriers suitable
for oral, topical, nasal, transdermal, intravenous, subcutaneous,
intramuscular, intracutaneous, intraperitoneally, intravascular and
the like administration. Administration in the form of creams,
lotions, tablets, dispersible powders, granules, syrups, elixirs,
sterile aqueous or non-aqueous solutions, suspensions or emulsions,
and the like, is contemplated. Exemplary pharmaceutically
acceptable carriers include carriers for tablets, pellets,
capsules, suppositories, solutions, emulsions, suspensions, and any
other form suitable for use. Such carriers which can be used
include glucose, lactose, gum acacia, gelatin, mannitol, starch
paste, magnesium trisilicate, talc, corn starch, keratin, colloidal
silica, potato starch, urea, medium chain length triglycerides,
dextrans, and other carriers suitable for use in manufacturing
preparations, in solid, semisolid, or liquid form. In addition
auxiliary, stabilizing, thickening and coloring agents and perfumes
may be used. The active compound (i.e., ecdysteroid as described
herein) is included in the pharmaceutically acceptable formulation
in an amount sufficient to produce the desired effect upon the
process or condition of diseases.
[0093] Pharmaceutically acceptable formulations containing the
active ingredient may be in a form suitable for oral use, for
example, as aqueous or oily suspensions, syrups or elixirs,
tablets, troches, lozenges, dispersible powders or granules,
emulsions, or hard or soft capsules. For the preparation of oral
liquids, suitable carriers include emulsions, solutions,
suspensions, syrups, and the like, optionally containing additives
such as wetting agents, emulsifying and suspending agents,
dispersing agents, sweetening, flavoring, coloring, preserving and
perfuming agents, and the like. Formulations intended for oral use
may be prepared according to any method known to the art for the
manufacture of pharmaceutically acceptable formulations.
[0094] Tablets containing the active ingredient in admixture with
non-toxic pharmaceutically acceptable excipients may also be
manufactured by known methods. The excipients used may be, for
example, (1) inert diluents such as calcium carbonate, lactose,
calcium phosphate or sodium phosphate; (2) granulating and
disintegrating agents such as corn starch, potato starch or alginic
acid; (3) binding agents such as gum tragacanth, corn starch,
gelatin or acacia, and (4) lubricating agents such as magnesium
stearate, stearic acid or talc. The tablets may be uncoated or they
may be coated by known techniques to delay disintegration and
absorption in the gastrointestinal tract and thereby provide a
sustained action over a longer period. For example, a time delay
material such as glyceryl monostearate or glyceryl distearate may
be employed. They may also be coated by the techniques described in
the U.S. Pat. Nos. 4,256,108; 4,160,452; and 4,265,874, to form
osmotic therapeutic tablets for controlled release.
[0095] In some cases, formulations for oral use may be in the form
of hard gelatin capsules wherein the active ingredient is mixed
with an inert solid diluent, for example, calcium carbonate,
calcium phosphate or kaolin. They may also be in the form of soft
gelatin capsules wherein the active ingredient is mixed with water
or an oil medium, for example, peanut oil, liquid paraffin, or
olive oil.
[0096] The pharmaceutically acceptable formulations may be in the
form of a sterile injectable suspension. Suitable carriers include
non-toxic parenterally-acceptable sterile aqueous or non-aqueous
solutions, suspensions, or emulsions. This suspension may be
formulated according to known methods using suitable dispersing or
wetting agents and suspending agents. They can also be manufactured
in the form of sterile water, or some other sterile injectable
medium immediately before use. Sterile, fixed oils are
conventionally employed as a solvent or suspending medium. For this
purpose any bland fixed oil may be employed including synthetic
mono- or diglycerides, fatty acids (including oleic acid),
naturally occurring vegetable oils like sesame oil, coconut oil,
peanut oil, cottonseed oil, etc., or synthetic fatty vehicles like
ethyl oleate or the like. They may be sterilized, for example, by
filtration through a bacteria-retaining filter, by incorporating
sterilizing agents into the formulations, by irradiating the
formulations, or by heating the formulations. Sterile injectable
suspensions may also contain adjuvants such as preserving, wetting,
emulsifying, and dispersing agents. Buffers, preservatives,
antioxidants, and the like can be incorporated as required.
[0097] Compounds contemplated for use in the practice of the
present invention may also be administered in the form of
suppositories for rectal administration of the drug. These
formulations may be prepared by mixing the drug with a suitable
non-irritating excipient, such as cocoa butter, synthetic glyceride
esters of polyethylene glycols, which are solid at ordinary
temperatures, but liquefy and/or dissolve in the rectal cavity to
release the drug.
[0098] The pharmaceutically acceptable formulations are
administered in a manner compatible with the route of
administration, the dosage formulation, and in a therapeutically
effective amount. The required dosage will vary with the particular
treatment desired, the degree and duration of therapeutic effect
desired, the judgment of the practitioner, as well as properties
peculiar to each individual. Moreover, suitable dosage ranges for
systemic application depend on the route of administration. It is
anticipated that dosages between about 10 micrograms and about 1
milligram per kilogram of body weight per day will be used for
therapeutic treatment.
[0099] An effective amount of the pharmaceutically acceptable
formulation contemplated for use in the practice of the present
invention is the amount of the pharmaceutically acceptable
formulation (e.g., ecdysteroids(s)) required to achieve the desired
level of transcription and/or translation of exogenous nucleic
acid. A therapeutically effective amount is typically an amount of
a ligand or ligand precursor that, when administered in a
pharamceutically acceptable formulation, is sufficient to achieve a
plasma concentration of the transcribed or expressed nucleic acid
product from about 0.1 .mu.g/ml to about 100 .mu.g/ml, preferably
from about 1.0 .mu.g/ml to about 50 .mu.g/ml, more preferably at
least about 2 .mu.g/ml and usually 5 to 10 .mu.g/ml.
[0100] Pharmaceutically acceptable formulations containing suitable
ligand(s) are preferably administered intravenously, as by
injection of a unit dose, for example. The term "unit dose," when
used in reference to a pharmaceutically acceptable formulation of
the present invention, refers to a quantity of the pharmaceutical
formulation suitable as unitary dosage for the subject, each unit
containing a predetermined quantity of active material calculated
to produce the desired therapeutic effect in association with the
required diluent, i.e., carrier, or vehicle. It may be particularly
advantageous to administer such formulations in depot or
long-lasting form as discussed hereinafter.
[0101] Suitable regimes for initial administration and booster
shots are variable, but are typified by an initial administration
followed by repeated doses at one or more intervals by a subsequent
injection or other administration. Alternatively, continuous
intravenous infusion sufficient to maintain concentrations in the
blood in the ranges specified for in vivo therapies are
contemplated.
[0102] Ecdysone response elements contemplated for use in the
practice of the present invention (relating to modulation of the
expression of exogenous genes in a subject) include native, as well
as modified ecdysone response elements. Since invention modified
ecdysone receptors can function as either homodimers or as
heterodimers (with a silent partner therefor), any response element
that is responsive to an invention modified ecdysone receptor, in
the form of a homodimer or heterodimer, is contemplated for use in
the invention methods described herein. As is readily recognized by
those of skill in the art, modified receptors according to the
invention (whether in the form of a homodimer or a heterodimer) can
bind to either a response element having an inverted repeat motif
(i.e., two or more half sites in mirror image orientation with
respect to one another), or to a response element having a direct
repeat motif.
[0103] In a preferred embodiment of the invention, invention
modified ecdysone response elements are engineered so as to no
longer be capable of binding to a farnesoid hormone receptor (since
the mammalian farnesoid hormone receptor is able to bind to native
ecdysone receptor response element). Invention modified ecdysone
response elements provide low background expression levels of the
exogenous gene and increase the selectivity of the gene expression
system when used in mammalian systems.
[0104] Ecdysone response elements contemplated for use herein are
short cis-acting sequences (i.e., having about 12-20 bp) that are
required for activation of transcription in response to a suitable
ligand, such as ecdysone or muristerone A, associated with a
particular hormone receptor. The association of these response
elements with otherwise ecdysone-nonresponsive regulatory sequences
causes such regulatory sequences to become ecdysone responsive.
Ecdysone response element sequences function in a position- and
orientation-independent fashion.
[0105] The native ecdysone response element has been previously
described, see, e.g., Yao et al., Cell, 71:63-72, 1992. Modified
ecdysone response elements according to present invention comprise
two half-sites (in either direct repeat or inverted repeat
orientation to one another), separated by a spacer of 0-5
nucleotides. As used herein, the term "half-site" refers to a
contiguous 6 nucleotide sequence that is bound by a particular
member of the steroid/thyroid hormone superfamily of receptors.
Each half-site is typically separated by a spacer of 0 up to about
5 nucleotides. Typically, two half-sites with a corresponding
spacer make up a hormone response element. Hormone response
elements can be incorporated in multiple copies into various
transcription regulatory regions.
[0106] Preferred modified ecdysone response elements according to
the invention comprise, in any order, a first half-site and a
second half-site separated by a spacer of 0-5 nucleotides; [0107]
wherein the first and second half-sites are inverted with respect
to each other; [0108] wherein said first half-site has the
sequence: [0109] -RGBNNM-, (or complements thereof) wherein [0110]
each R is independently selected from A or G; [0111] each B is
independently selected from G, C, or T; [0112] each N is
independently selected from A, T, C, or G; and [0113] each M is
independently selected from A or C; with the proviso that at least
4 nucleotides of each -RGBNNM- group of nucleotides are identical
with the nucleotides at comparable positions of the sequence
-AGGTCA-; and [0114] said second half-site is obtained from a
glucocorticoid receptor subfamily response element.
[0115] The complement to the -RGBNNM- sequence set forth above is:
[0116] -YCVNNK-, wherein [0117] each Y is independently selected
from T or C; [0118] each V is independently selected from C, G, or
A; [0119] each N is independently selected from A, T, C, or G; and
[0120] each K is independently selected from T or G.
[0121] Exemplary first half-sites having the -RGBNNM- motif for use
in the invention modified ecdysone response element include, for
example, half-sites selected from -AGGGCA-, -AGTTCA-, -AGGTAA-,
-AGGTCA-, -GGTTCA-, -GGGTTA-, -GGGTGA-, -AGGTGA-, or -GGGTCA-. A
particularly preferred first half-site is -AGTGCA-.
[0122] Glucocorticoid receptor subfamily response elements
contemplated for use in the practice of the present invention are
response elements having half-sites that are typically bound by
glucocorticoid, mineralocorticoid, progesterone or androgen
receptors. Suitable half-sites from glucocorticoid receptor
subfamily response elements can be selected from the following
sequence (in either orientation): [0123] -RGNNCA- (or complements
thereof such as -YCNNGT-), wherein R, Y and N are as defined above.
Exemplary half-sites having the -RGNNCA- motif for use in the
invention modified ecdysone response element include -AGAACA-,
-GGAACA-, -AGTTCA-, -AGGTCA-, -GGAACA-, -GGTTCA-, -GGGTCA-,
-GGGTCA-, -AGGTGA-, -GGGTCA-, and the like, as well as complements
thereof. Particularly preferred half-sites having the -RGNNCA-
motif include -AGAACA- and -GGAACA-, with -AGAACA- being especially
preferred.
[0124] When the above-described modified ecdysone response elements
are employed to bind invention heterodimeric receptors, the second
half-site is inverted with respect to the first half-site. For
example, when describing a single-strand of an invention modified
ecdysone response element in the 5'-3' direction, the following
general motif can be employed: [0125] RGBNNM-(N).sub.x-TGNNCY (SEQ
ID NO:10), where x is an integer of 0 up to about 5, with x=1 being
especially preferred. As an alternative orientation to the above
described response element motif (SEQ ID NO:10), an invention
response element can be described in the 5'-3' direction as: [0126]
RGNNCA-(N).sub.x-KNNVCY (SEQ ID NO:11), where x is an integer of 0
up to about 5, with x=1 being especially preferred.
[0127] In preferred embodiments of the present invention, the first
half-site is obtained from an ecdysone response element and the
second half-site is obtained from a hormone response element
selected from a glucocorticoid response element, a
mineralocorticoid response element, a progesterone response element
or an androgen response element. In a particularly preferred
embodiment of the present invention, the first half-site is
obtained from an ecdysone response element and the second half-site
is obtained from a glucocorticoid response element.
[0128] In a particularly preferred embodiment of the invention
modified ecdysone response element, the first half-site is AGTGCA
and said second half-site is TGTTCT. The presently most preferred
modified-ecdysone response element for use in the invention methods
is: [0129] AGTGCA-N-TGTTCT (SEQ ID NO:12).
[0130] In another aspect of the invention, when modified ecdysone
receptors of the invention exist as homodimers, response elements
employed preferably have a direct repeat motif (instead of the
above-described inverted repeat motif), as follows: [0131]
RGBNNM-(N).sub.x'-RGBNNM (SEQ ID NO:13), where R, B, N and M are as
previously defined, and x' is an integer of 0 up to about 5, with
x'=3 being especially preferred.
[0132] Invention modified ecdysone response elements are
characterized as having substantially no constitutive activity,
which refers to the substantial absence of background levels of
gene expression initiated by invention modified ecdysone response
elements when introduced into mammalian expression systems. Since
it has been found that mammalian farnesoid hormone receptors are
able to bind to and transactivate gene expression from native
ecdysone response elements, in certain embodiments of the present
invention (e.g., where it is desired to avoid farnesoid-mediated
background expression), modified ecdysone response elements are
employed.
[0133] Presently preferred invention modified ecdysone response
elements are further characterized as having substantially no
binding affinity for farnesoid X receptor (FXR), i.e., invention
response elements are incapable of binding FXR (which would thereby
create undesired background levels of expression). Thus, presently
preferred invention modified ecdysone response elements preferably
induce basal levels of expression of substantially zero.
[0134] Response elements employed in the practice of the present
invention are operably linked to a suitable promoter for expression
of exogenous gene product(s). As used herein, the term "promoter"
refers to a specific nucleotide sequence recognized by RNA
polymerase, the enzyme that initiates RNA synthesis. This sequence
is the site at which transcription can be specifically initiated
under proper conditions. When exogenous genes, operatively linked
to a suitable promoter, are introduced into the cells of a suitable
host, expression of the exogenous genes is controlled by the
presence of ecdysteroid compounds, which are not normally present
in the host cells.
[0135] In accordance with another embodiment of the present
invention, there are provided methods of inducing the expression of
an exogenous gene in a mammalian subject containing: [0136] (i) a
DNA construct comprising an exogenous gene under the control of an
ecdysone response element, [0137] (ii) DNA encoding a modified
ecdysone receptor under the control of an inducible promoter;
wherein said modified ecdysone receptor, in the presence of a
ligand therefor, and optionally in the further presence of a
receptor capable of acting as a silent partner therefor, binds to
said ecdysone response element, and [0138] (iii) a ligand for said
modified ecdysone receptor; [0139] said method comprising
subjecting said subject to conditions suitable to induce expression
of said modified ecdysone receptor.
[0140] Inducible promoters contemplated for use in the practice of
the present invention are transcription regulatory regions that do
not function to transcribe mRNA unless inducing conditions are
present. Examples of suitable inducible promoters include DNA
sequences corresponding to: the E. coli lac operator responsive to
IPTG (see Nakamura et al., Cell, 18:1109-1117, 1979); the
metallothionein promoter metal-regulatory-elements responsive to
heavy-metal (e.g. zinc) induction (see Evans et. al, U.S. Pat. No.
4,870,009), the phage T7lac promoter responsive to IPTG (see
Studier et al., Meth. Enzymol., 185: 60-89, 1990; and U.S. Pat. No.
4,952,496), the heat-shock promoter, and the like.
[0141] In accordance with another embodiment of the present
invention, there are provided methods of inducing expression of an
exogenous gene in a mammalian subject containing a DNA construct
comprising said exogenous gene under the control of an ecdysone
response element, said method comprising introducing into said
subject: [0142] a modified ecdysone receptor; and [0143] a ligand
for said modified ecdysone receptor, [0144] wherein said receptor,
in combination with a ligand therefor, and optionally in the
further presence of a receptor capable of acting as a silent
partner therefor, binds to said ecdysone response element,
activating transcription therefrom.
[0145] In accordance with another embodiment of the present
invention, there are provided methods for the expression of
recombinant products detrimental to a host organism, said method
comprising: [0146] transforming suitable host cells with: [0147]
(i) a DNA construct encoding said recombinant product under the
control of an ecdysone response element, and [0148] (ii) DNA
encoding a modified ecdysone receptor; [0149] growing said host
cells in suitable media; and [0150] inducing expression of said
recombinant product by introducing into said host cells ligand(s)
for said modified ecdysone receptor, and optionally a receptor
capable of acting as a silent partner for said modified ecdysone
receptor.
[0151] Recombinant products detrimental to a host organism
contemplated for expression in accordance with the present
invention include any gene product that functions to confer a toxic
effect on the organism. For example, inducible expression of a
toxin such as the diptheroid toxin would allow for inducible tissue
specific ablation (Ross et al. (1993) Genes and Development 7,
1318-1324). Thus, the numerous gene products that are known to
induce apoptosis in cells expressing such products are contemplated
for use herein (see, e.g, Apoptosis, The Molecular Basis of Cell
Death, Current Communications In Cell & Molecular Biology, Cold
Spring Harbor Laboratory Press, 1991).
[0152] Suitable media contemplated for use in the practice of the
present invention include any growth and/or maintenance media, in
the substantial absence of ligand(s) which, in combination with an
invention modified ecdysone receptor, is (are) capable of binding
to an ecdysone response element.
[0153] In accordance with another embodiment of the present
invention, there are provided gene transfer vectors useful for the
introduction of invention constructs into suitable host cells. Such
gene transfer vectors comprise a transcription regulatory region
having a minimal promoter (i.e., a promoter region that does not
have an enhancer), and an invention modified ecdysone response
element, wherein said regulatory region is operatively associated
with DNA containing an exogenous gene, and wherein said modified
ecdysone response element is present in multiple copies. The number
of copies of response elements can readily be varied by those of
skill in the art. For example, transcription regulatory regions can
contain from 1 up to about 50 copies of a particular response
element, preferably 2 up to about 25 copies, more preferably 3 up
to about 10-15 copies, with about 4-6 copies being especially
preferred.
[0154] Gene transfer vectors (also referred to as "expression
vectors") contemplated for use herein are recombinant nucleic acid
molecules that are used to transport exogenous nucleic acid into
cells for expression and/or replication thereof. Expression vectors
may be either circular or linear, and are capable of incorporating
a variety of nucleic acid constructs therein. Expression vectors
typically come in the form of a plasmid that, upon introduction
into an appropriate host cell, results in expression of the
inserted DNA.
[0155] As used herein, the phrase "transcription regulatory region"
refers to the region of a gene or expression construct that
controls the initiation of mRNA transcription. Regulatory regions
contemplated for use herein typically comprise at least a minimal
promoter in combination with an ecdysone response element. A
minimal promoter, when combined with an enhancer region (e.g., a
hormone response element), functions to initiate mRNA transcription
in response to a ligand/receptor complex. However, transcription
will not occur unless the required inducer (ligand) is present.
[0156] As used herein, the phrase "operatively associated with"
refers to the functional relationship of DNA with regulatory and
effector sequences of nucleotides, such as promoters, enhancers,
transcriptional and translational stop sites, and other signal
sequences. For example, operative linkage of DNA to a promoter
refers to the physical and functional relationship between the DNA
and promoter such that the transcription of such DNA is initiated
from the promoter by an RNA polymerase that specifically
recognizes, binds to and transcribes the DNA.
[0157] Preferably, the transcription regulatory region further
comprises a binding site for an ubiquitous transcription factor.
Such a binding site is preferably positioned between the promoter
and modified ecdysone response element of the invention. Suitable
ubiquitous transcription factors for use herein are well-known in
the art and include, for example, Sp1.
[0158] Expression vectors suitable for use in the practice of the
present invention are well known to those of skill in the art and
include those that are replicable in eukaryotic cells and/or
prokaryotic cells as well as those that remain episomal and those
that integrate into the host cell genome. Expression vectors
typically further contain other functionally important nucleic acid
sequences, such as expression constructs encoding antibiotic
resistance proteins, and the like.
[0159] Exemplary eukaryotic expression vectors include eukaryotic
constructs, such as the pSV-2 gpt system (Mulligan et al., Nature,
1979, 277:108-114); pBlueSkript (Stratagene, La Jolla, Calif.), the
expression cloning vector described by Genetics Institute (Science,
1985, 228:810-815), and the like. Each of these plasmid vectors are
capable of promoting expression of the invention chimeric protein
of interest.
[0160] Promoters, depending upon the nature of the regulation, may
be constitutively or inducibly regulated, or may be tissue-specific
(e.g., expressed only in T-cells, endothelial cels, smooth muscle
cells, and the like). Exemplary promoters contemplated for use in
the practice of the present invention include the SV40 early
promoter, the cytomegalovirus (CMV) promoter, the mouse mammary
tumor virus (MMTV) steroid-inducible promoter, Moloney murine
leukemia virus (MMLV) promoter, elongation factor 1.alpha.
(EF1.alpha.) promoter, albumin promoter, APO A1 promoter, cyclic
AMP dependent kinase II (CaMKII) promoter, keratin promoter, CD3
promoter, immunoglobulin light or heavy chain promoters,
neurofiliment promoter, neuron specific enolase promoter, L7
promoter, CD2 promoter, myosin light chain kinase promoter, HOX
gene promoter, thymidine kinase (TK) promoter, RNA Pol II promoter,
MYOD promoter, MYF5 promoter, phophoglycerokinase (PGK) promoter,
Stf1 promoter, Low Density Lipoprotein (LDL) promoter, and the
like.
[0161] Suitable means for introducing (transducing) expression
vectors containing nucleic acid constructs according to the
invention into host cells to produce transduced recombinant cells
(i.e., cells containing recombinant heterologous nucleic acid) are
well-known in the art (see, for review, Friedmann, 1989, Science,
244:1275-1281; Mulligan, 1993, Science, 260:926-932, each of which
are incorporated herein by reference in their entirety). Exemplary
methods of transduction include, e.g., infection employing viral
vectors (see, e.g., U.S. Pat. Nos. 4,405,712 and 4,650,764),
calcium phosphate transfection (U.S. Pat. Nos. 4,399,216 and
4,634,665), dextran sulfate transfection, electroporation,
lipofection (see, e.g., U.S. Pat. Nos. 4,394,448 and 4,619,794),
cytofection, particle bead bombardment, and the like. The
heterologous nucleic acid can optionally include sequences which
allow for its extrachromosomal (i.e., episomal) maintenance, or the
heterologous nucleic acid can be donor nucleic acid that integrates
into the genome of the host.
[0162] In a specific embodiment, said gene transfer vector is a
viral vector, preferably a retroviral vector. Retroviral vectors
are gene transfer plasmids that have an expression construct
encoding an heterologous gene residing between two retroviral LTRs.
Retroviral vectors typically contain appropriate packaging signals
that enable the retroviral vector, or RNA transcribed using the
retroviral vector as a template, to be packaged into a viral virion
in an appropriate packaging cell line (see, e.g., U.S. Pat. No.
4,650,764).
[0163] Suitable retroviral vectors for use herein are described,
for example, in U.S. Pat. Nos. 5,399,346 and 5,252,479; and in WIPO
publications WO 92/07573, WO 90/06997, WO 89/05345, WO 92/05266 and
WO 92/14829, incorporated herein by reference, which provide a
description of methods for efficiently introducing nucleic acids
into human cells using such retroviral vectors. Other retroviral
vectors include, for example, mouse mammary tumor virus vectors
(e.g., Shackleford et al., 1988, PNAS, USA, 85:9655-9659), and the
like.
[0164] Various procedures are also well-known in the art for
providing helper cells which produce retroviral vector particles
which are essentially free of replicating virus. See, for example,
U.S. Pat. No. 4,650,764; Miller, Human Gene Therapy, 1:5-14 (1990);
Markowitz, et. al., Journal of Virology, 61(4):1120-1124 (1988);
Watanabe, et al., Molecular and Cellular Biology, 3(12):2241-2249
(1983); Danos, et al., Proc. Natl. Acad. Sci., 85:6460-6464 (1988);
and Bosselman, et al., Molecular and Cellular Biology,
7(5):1797-1806 (1987), which disclose procedures for producing
viral vectors and helper cells which minimize the chances for
producing a viral vector which includes a replicating virus.
[0165] Recombinant retroviruses suitable for carrying out the
invention methods are produced employing well-known methods for
producing retroviral virions. See, for example, U.S. Pat. No.
4,650,764; Miller, Human Gene Therapy, 1:5-14 (1990); Markowitz, et
al., Journal of Virology, 61(4):1120-1124 (1988); Watanabe, et al.,
Molecular and Cellular Biology, 3(12):2241-2249 (1983); Danos, et
al., Proc. Natl. Acad. Sci., 85:6460-6464 (1988); and Bosselman, et
al., Molecular and Cellular Biology, 7(5):1797-1806 (1987).
[0166] In accordance with another embodiment of the present
invention, there are provided recombinant cells containing a
nucleic acid encoding modified ecdysone receptor(s) according to
the invention. Exemplary eukaryotic cells for introducing
expression vectors according to the invention include, e.g., CV-1
cells, P19 cells and NT2/D1 cells (which are derived from human
embryo carcinomas), COS cells, mouse L cells, Chinese hamster ovary
(CHO) cells, primary human fibroblast cells, human embryonic kidney
cells, African green monkey cells, HEK 293 (ATCC accession #CRL
1573; U.S. Pat. No. 5,024,939), Ltk cells (ATCC accession #CCL1.3),
COS-7 cells (ATCC under accession #CRL 1651), DG44 cells
(dhfr.sup.- CHO cells; see, e.g., Urlaub et al. (1986) Cell. Molec.
Genet. 12: 555), cultured primary tissues, cultured tumor cells,
and the like. Presently preferred cells include CV-1 and 293
cells.
[0167] In accordance with another embodiment of the present
invention, there is provided a transgenic mammal containing a
nucleic acid encoding an invention modified ecdysone receptor. As
used herein, the phrase "transgenic mammal" refers to a mammal that
contains one or more inheritable expression constructs containing a
recombinant modified ecdysone receptor transgene and/or an
exogenous gene under the transcription control of an invention
modified ecdysone response element. Preferably, an invention
transgenic mammal also contains one or more inheritable expression
constructs containing a member of the steroid/thyroid hormone
superfamily of receptors that functions as a silent partner for
modified ecdysone receptor (e.g., RXR).
[0168] Methods of making transgenic mammals using a articular
nucleic acid construct are well-known in the art. When preparing
invention transgenic animals, it is referred that two transgenic
lines are generated. The first line will express, for example, RXR
and a modified EcR (e.g., VpEcR). Tissue specificity is conferred
by the selection of tissue-specific promoters (e.g., T-cell
specific) that will then direct the expression of the receptors. A
second line contains an ecdysone responsive promoter controlling
the expression of an exogenous cDNA.
[0169] In a preferred embodiment of the present invention, an
invention transgenic mammal contains one or more expression
constructs containing nucleic acid encoding a modified ecdysone
receptor, exogenous RXR, and an exogenous gene under the
transcription control of an invention modified ecdysone response
element. It has been found that in transgenic mice containing an
ecdysone inducible promoter (i.e., an invention modified ecdysone
response element) and expressing a modified ecdysone receptor and
RXR, muristerone treatment can activate gene expression. Thus, with
tissue specific expression of the modified ecdysone receptor and
RXR and timely hormone treatment, inducible gene expression can be
achieved with spatial, dosage, and temporal specificity.
[0170] In accordance with another embodiment of the present
invention, there are provided methods for inducing expression of an
exogenous gene in a transgenic mammal containing a modified
ecdysone receptor according to the invention, said method
comprising: [0171] introducing into said mammal a DNA construct
encoding an exogenous gene under the transcription control of an
ecdysone response element responsive to said modified ecdysone
receptor; and [0172] administering to said mammal an amount of
ligand for said modified ecdysone receptor effective to induce
expression of said exogenous gene. As discussed hereinbefore, the
modified ecdysone receptor forms a homodimer, or optionally a
heterodimer in the presence of a silent partner of the
steroid/thyroid hormone superfamily of receptors, and functions to
activate transcription from an expression vector having a response
element responsive to the particular homodimer or heterodimer
formed.
[0173] In accordance with another embodiment of the present
invention, there are provided methods for the induction of two
different genes in a mammalian subject comprising: activating a
first exogenous gene employing the invention ecdysone inducible
system; and activating a second gene using a tetracycline inducible
system. The invention method for inducing two different genes is
particularly advantagous because it permits the temporal, spatial,
and dosage specific control of two exogenous genes.
[0174] The tetracycline inducible system is well-known in the art
(see, e.g, Gossen et al. (1992) Proc. Natl. Acad. Sci. 89,
5547-5551; Gossen et al. (1993) TIBS 18, 471-475; Furth et al.
(1994) Proc. Natl. Acad. Sci. 91, 9302-9306; and Shockett et al.
(1995) Proc. Natl. Acad. Sci. 92, 6522-6526).
[0175] All U.S. and Foreign Patent publications, textbooks, and
journal publications referred to herein are hereby expressly
incorporated by reference in their entirety. The invention will now
be described in greater detail by reference to the following
non-limiting examples.
EXAMPLE 1
Preparation of Modified Ecdysone Receptors
Plasmid Preparation:
[0176] The plasmids CMX-EcR, CMX-USP, CMX-FXR, CMX-hRXRa,
EcREx5-.DELTA.MTV-Luc, CMX-GEcR, MMTV-luc, and CMX-GR have been
previously described (Yao, et al., Nature 366:476-479 (1993) and
Forman, et al. Cell 81:687-693 (1995)).
[0177] The plasmid CMX-VpEcR was constructed by ligation of an
EcoRI fragment of psk-EcR and CMX-Vp16.
[0178] The plasmid CMX-VgEcR was generated by site-directed
mutagenesis of CMX-VpEcR using the Transformer Mutagenesis Kit
(Clontech) and the mutagenic oligonucleotide (SEQ ID NO:14): [0179]
5'-TACAACGCCCTCACCTGTGGATCCTGCAAGGTGTTTCTTTCGACGCAGC-3'.
Mutagenesis of VpEcR to VgEcR altered the P-box region of the DNA
binding domain of ecdysone receptor to resemble that of GR (Umesono
and Evans, Cell 57:1139-1146 (1989)). The following amino acids in
the DNA-binding domain of the ecdysone receptor were altered:
E282G, G283S, and G286V (E=glutamate, G=glycine, S=serine,
V=valine).
[0180] The reporter construct EcREx4-.DELTA.HSP-.beta.-gal was
constructed by oligomerizing two annealed oligonucleotides
containing the HSP-EcRE (Yao, et al., Nature 366:476-479
(1993)).
[0181] EcREx4-Sp1x3-.DELTA.HSP-.beta.gal was constructed by
ligating the following annealed oligonucleotides into the Asp718
site of EcREx4-HSP-.beta.-gal (SEQ ID NO:15): [0182]
5'-GTACTCCCGGGGCGGGGCTATGCGGGGCGGGGCTAATCGCTAGGGGCGGGGCA-3' and
(SEQ ID NO:16): [0183]
5'-GTACTGCCCCGCCCCTAGCGATTAGCCCCGCCCCGCATAGCCCCGCCC CGGGA-3'.
.DELTA.HSP is a minimal promoter derived from the Drosophila heat
shock promoter with its enhancers deleted.
[0184] To generate the construct E/GREx4-.DELTA.MTV-Luc, the
following oligonucleotides (SEQ ID NO:17): [0185]
5'-AGCTCGATGGACAAGTGCATTGTTCTTTGCTGAA-3'; and (SEQ ID NO:18):
[0186] 5'-AGCTTTCAGCAAGAGAACAATGCACTTGTCCATCG-3', were annealed,
multimerized, and ligated into the HindIII site of .DELTA.MTV-Luc.
The resulting reporter contained 4 copies of the invention modified
ecdysone response element E/GRE.
[0187] To produce the plasmid pRC-ESH.beta., a BglII/(XhoI)
fragment containing EcREx4-Sp1x3-.DELTA.HSP-.beta.-gal was
subcloned into BglII/(NotI) digested pRC-CMV (Invitrogen, San
Diego, Calif.), which contains a neomycin resistance gene.
Cell Culture and Transient Transfections:
[0188] CV-1 cells were maintained in DMEM supplemented with 10%
Fetal Bovine Serum. Transient transfections were performed using
DOTAP transfection reagent (Boehringer-Mannheim). Transfections
using .beta.-galactosidase as the reporter were assayed either by
Galactolight luminescent assay (Tropix, Bedford, Mass.) or by
standard liquid ONPG assay (Sigma, St. Louis, Mo.). The values were
normalized by co-transfection of CMX-luciferase. Transfections
using luciferase as the reporter were assayed by standard
techniques using luciferin and ATP. These values were normalized by
co-transfection of CMX-.beta.-galactosidase. Hormone treated cells
were treated with ethanol, 50 .mu.M Juvenile Hormone III (Sigma), 1
.mu.M muristerone A (Zambon, Bresso, IT), or 1 .mu.M dexamethasone
(Sigma) unless otherwise noted.
[0189] To maximize the sensitivity of the invention ecdysone
inducible system, modifications of the ecdysone receptor were made.
The N-terminal transactivation domain of the ecdysone receptor was
replaced by the corresponding domain of the glucocorticoid receptor
(GR), resulting in the modified ecdysone receptor GEcR (See FIG.
1D). CV-1 cells were transfected with the plasmid CMX-GEcR encoding
the modified ecdysone receptor as discussed above. After
transfection, cells were either treated with ethanol or 1 .mu.M
muristerone A. This hybrid modified ecdysone receptor boosted
muristerone responsiveness from 3- to 1'-fold in a transient
transfection assay (FIG. 1A). Replacement of the natural
heterodimeric partner for the ecdysone receptor, USP, by its
mammalian homologue, the retinoid X receptor (RXR), produced a more
potent ligand dependent heterodimer, providing a 34 fold induction
(FIG. 1A).
[0190] A more potent heterodimer, however, was obained by combining
RXR and VpEcR, an N-terminal truncation of the ecdysone receptor
attached to the VP16 activation domain, resulting in a 212 fold
induction (FIGS. 1A and 1D). Different from most nuclear
receptor/VP16 fusion proteins which exhibit high constitutive
activity, VpEcR generates ligand dependent superinduction while
maintaining a very low basal activity (Underhill et al., Mol.
Encod. 8:274-285 (1994) and Perlmann et al., Genes & Devel.
7:1411-1422 (1993)).
[0191] In addition, the reporter vector was also modified by
inserting consensus binding sites for the ubiquitous transcription
factor Sp1 between the minimal promoter and the ecdysone response
elements (Kamine et al., Proc. Natl. Acad. Sci. 88:8510-8514 (1991)
and Strahle te al., EMBO 7:3389-3395 (1988)). The addition of Sp1
sites to the ecdysone responsive promoter increases the absolute
activity 5-fold (FIG. 1A).
EXAMPLE 2
Construction of a Novel Ecdysone Response Element
[0192] Although no mammalian transcription factors have been shown
to have a natural enhancer element like the ecdysone response
element, which is composed of two inverted half-sites of the
sequence AGGTCA spaced by one nucleotide, it is difficult to
preclude such a possibility. The recently cloned farnesoid X
receptor (FXR) can very weakly activate certain synthetic promoters
containing an ecdysone response element in response to extremely
high concentrations of farnesoids (Forman et al., Cell 81:687-693
(1995)).
[0193] In FXR containing cells and in transgenic mice, activation
of gene expression by endogenous receptors would create undesirable
background levels of reporter protein. To circumvent this potential
problem, the DNA binding specificity of VpEcR was altered to mimic
that of GR, which binds as a homodimer to an inverted repeat of the
sequence AGAACA, spaced by three nucleotides. This altered binding
specificity was achieved by mutating three amino acid residues of
VpEcR in the P-box of the DNA binding domain, a region previously
shown to be essential for DNA sequence recognition (Umesono and
Evans, Cell 57:1139-1146 (1989)). This new hybrid modified ecdysone
receptor is referred herein as VgEcR and is responsive to a new
hybrid respone element referred to herein as the E/GRE (SEQ ID
NO:12), which contains two different half-site motifs, RGBNNM and
RGNNCA, spaced by one nucleotide (FIG. 1B). This new response
element is a hybrid between the glucocorticoid response element
(GRE) and that of type II nuclear receptors like RXR, EcR, retinoic
acid receptor (RAR), thyroid hormone receptor (T3R), etc. Although
FXR can activate a promoter containing the wild type ecdysone
response element, it cannot activate one containing the E/GRE (FIG.
1B; note log scale). The E/GRE reporter is not activated by GR nor
does VgEcR activate a dexamethasone responsive promoter (FIG.
1C).
EXAMPLE 3
Assay for Ecdysone Responsiveness in Stable Cell Lines
[0194] Stable cell lines were generated containing the modified
ecdysone receptor VpEcR, a heterodimeric partner (RXR), and an
ecdysone inducible reporter (FIG. 2). 293 cells were transfected
with the following linearized plasmids, pRC-ESH.beta.,
EcREx5-.DELTA.MTV-Luc, CMX-VpEcR, and CMX-hRXRa. The following day,
the cells were split 1:10 and were allowed to recover one day prior
to selection with 1 mg/ml G418 (GIBCO). After 14 days of selection,
14 individual clones were isolated and grown separately in the
presence of 0.5 mg/ml G418. Of 14 G418 resistant clones, 10
demonstrated differing degrees of muristerone responsiveness.
[0195] One of these cell lines, N13, was grown in the presence or
absence of 1 .mu.M muristerone for 20 hours. Cell lysates were then
assayed for .beta.-galactosidase and luciferase activities as
described in Example 1. X-gal staining was performed on the stable
cell lines. Cells were fixed briefly with 10% formaldehyde in PBS
and then stained with X-Gal (Molecular Probes, Eugene, Oreg.) for 2
to 6 hours. After 24 hours of treatment with 1 .mu.M muristerone,
100% of the cells turned dark blue after 3 hours of staining. Thus,
mammalian cells containing the modified ecdysone receptor VpEcR, a
heterodimeric partner (RXR), and a reporter gene construct
regulated by a modified ecdysone response element, function to
efficiently express an exogenous gene in response to a ligand,
e.g., ecdysone.
[0196] A dose-response assay was conducted by growing N13 cells
with varying concentrations of muristerone for 36 hours and then
assaying for .beta.-galactosidase activity (using the well-known
ONPG assay), or the cells were assayed for luciferase activity.
Dose response curves of stably integrated .beta.-galactosidase and
luciferase reporters in N13 cells revealed that inducibility
approaching 3 orders of magnitude can be achieved at a final
concentration 10 .mu.M muristerone (FIG. 3A). One-hundred fold
induction was achieved by muristerone concentrations as low as 100
nM (FIG. 3A).
[0197] Finally, the kinetics of muristerone mediated induction was
measured. N13 cells were grown in separate wells in the presence of
1 .mu.M muristerone, harvested at varying times, and assayed for
luciferase activity. Inductions of 100-fold in 3 hrs., 1000 fold in
8 hrs., and maximal effects of 20,000 fold after 20 hours of
treatment were observed (FIG. 3B). Similar results were observed in
stable lines containing CMX-VgEcR and the E/GRE reporters.
EXAMPLE 4
Bioavailability and Activity of Muristerone
[0198] In order to use muristerone as a potential hormone in mice,
its toxicity and bioavailability was examined. For toxicity
studies, adult mice were injected intraperitonealy with 20 mg of
muristerone A suspended in sesame oil. The mice were then observed
for approximately two months. For teratogenic studies, pregnant
mice were injected with 20 mg of muristerone A suspended in sesame
oil and both the mother and pups were observed for three months.
The results indicate that muristerone maintains its activity when
injected into mice, and that it is neither toxic, teratogenic, nor
inactivated by serum binding proteins. In addition to the inert
qualities of muristerone (an ecdysone), overexpression of VpEcR and
RXR appears not to be toxic.
[0199] For muristerone bioavailability studies, adult mice were
injected intraperitoneally with sesame oil with or without 10 mg of
muristerone, and were subsequently sacrificed for serum collection.
After twelve hours, blood was drawn from the mice, and the serum
was isolated by brief centrifugation of the whole blood. In order
to conduct transfection assays to test for muristerone activity,
serum from sesame oil injected mice was divided, and half was
supplemented with muristerone to a final concentration of 10 .mu.M.
The three batches of mouse serum were diluted 1:10 in DMEM and
placed onto CV-1 cells transfected with CMX-GEcR, CMX-hRXRa, and
EcREx5-DMTV-Luc.
[0200] The results are shown in FIG. 4 and indicate that serum from
muristerone treated mice yielded a luciferase activity comparable
to that seen from untreated mouse serum supplemented with 1 .mu.M
muristerone. The results indicate that single-site injected
material should be widely circulated, and that there is little or
no blunting of activity due to association with serum proteins.
EXAMPLE 5
Muristerone Dependent Gene Expression in Transgenic Mice
[0201] To produce transgenic mice, the following DNA constructs
were prepared and subsequently injected into fertilized eggs:
CD3-VpEcR, CD3-RXR, ESH.beta. (Lee et al., J. Exp. Med.
175:1013-1025 (1992)). Two separate lines of transgenic mice were
generated harboring either an ecdysone inducible reporter,
ESH.beta., or a T-cell specific expression construct of VpEcR and
RXR, respectively. The former are referred to as reporter mice, the
latter are referred to as receptor mice, and double transgenic mice
are referred to as receptor/reporter mice. Constructs CD3-VpEcR and
CD3-RXR were mixed and coinjected, while ESH.beta. was injected
alone. Primary genotyping was performed by Southern blot analysis
and the transmission of transgenic mice was monitored by dot blot
analysis. Receptor mice were analyzed for VpEcR and RXR expression
by Northern blot analysis of RNA collected from these mice. For
Northern blot analysis, 15 .mu.g of total RNA obtained from the
thymus, and various tissues as a control, was run on a denaturing
gel and blotted onto a nitrocellulose membrane. The blot was probed
with a radiolabeled .beta.-gal-specific probe and exposed on film
for 2 days. These receptor mice were healthy, fertile, and appeared
normal by visual inspection.
[0202] In addition, the transgene was transferred to the offspring
as expected by Mendelian genetics. This data suggests that
overexpression of VpEcR and RXR in T-cells is not toxic.
[0203] Receptor expressing mice were bred with reporter mice
(containing ESH.beta.) to produce double transgenic
receptor/reporter mice. Adult receptor/reporter transgenic mice
(genotype=CD3-VpEcR; CD3-RXR; and ESH.beta.) were injected
intraperitonealy with sesame oil with or without 10 mg of
muristerone. Subsequently, a Northern blot analysis was performed
on the double transgenic lines using RNA isolated 48 hours after
treatment from various tissues including the thymus, brain and
liver, to test for the specific induction of an ecdysone inducible
promoter. The probe used was specific to the activity of the
ecdysone inducible promoter. The autoradiograph was exposed for 36
hrs. The results of the Northern analysis indicate that muristerone
treatment of the transgenic mouse containing a T-cell specific
expression construct of VpEcR and RXR, and the ecdysone inducible
reporter ESH.beta., caused a significant induction from an ecdysone
inducible promoter in the thymus, while low basal activity is
observed in its absence.
EXAMPLE 6
Assay for Ponasterone Responsiveness
[0204] A dose-response assay was conducted as described in Example
3, by growing N13 cells with varying concentrations of muristerone
or ponasterone A for 36 hours and then assaying for
.beta.-galactosidase activity (using the well-known ONPG assay), or
the cells were assayed for luciferase activity. Dose response
curves of stably integrated .beta.-galactosidase and luciferase
reporters in N13 cells revealed that inducibility exceeding 3
orders of magnitude can be achieved with both ligands at final
concentrations of about 10.sup.-4 (see FIG. 5).
[0205] While the invention has been described in detail with
reference to certain preferred embodiments thereof, it will be
understood that modifications and variations are within the spirit
and scope of that which is described and claimed.
Sequence CWU 1
1
18 1 71 PRT Artificial Sequence Description of Artificial Sequence
Consensus peptide sequence 1 Cys Xaa Xaa Cys Xaa Xaa Asp Xaa Ala
Xaa Gly Xaa Tyr Xaa Xaa Xaa 1 5 10 15 Xaa Cys Xaa Xaa Cys Lys Xaa
Phe Phe Xaa Arg Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa
Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 35 40 45 Xaa Xaa Xaa
Lys Xaa Xaa Arg Xaa Xaa Cys Xaa Xaa Cys Arg Xaa Xaa 50 55 60 Lys
Cys Xaa Xaa Xaa Gly Met 65 70 2 5 PRT Artificial Sequence
Description of Artificial Sequence Synthetic peptide 2 Glu Gly Cys
Lys Gly 1 5 3 5 PRT Artificial Sequence Description of Artificial
Sequence Synthetic peptide 3 Gly Ser Cys Lys Val 1 5 4 2241 DNA
Artificial Sequence Description of Artificial Sequence Recombinant
VgEcR 4 atg gcc ccc ccg acc gat gtc agc ctg ggg gac gag ctc cac tta
gac 48 Met Ala Pro Pro Thr Asp Val Ser Leu Gly Asp Glu Leu His Leu
Asp 1 5 10 15 ggc gag gac gtg gcg atg gcg cat gcc gac gcg cta gac
gat ttc gat 96 Gly Glu Asp Val Ala Met Ala His Ala Asp Ala Leu Asp
Asp Phe Asp 20 25 30 ctg gac atg ttg ggg gac ggg gat tcc ccg ggt
ccg gga ttt acc ccc 144 Leu Asp Met Leu Gly Asp Gly Asp Ser Pro Gly
Pro Gly Phe Thr Pro 35 40 45 cac gac tcc gcc ccc tac ggc gct ctg
gat atg gcc gac ttc gag ttt 192 His Asp Ser Ala Pro Tyr Gly Ala Leu
Asp Met Ala Asp Phe Glu Phe 50 55 60 gag cag atg ttt acc gat gcc
ctt gga att gac gag tac ggt ggg aag 240 Glu Gln Met Phe Thr Asp Ala
Leu Gly Ile Asp Glu Tyr Gly Gly Lys 65 70 75 80 ctt cta ggt acc tct
aga agg ata tcg aat tct ata tct tca ggt cgc 288 Leu Leu Gly Thr Ser
Arg Arg Ile Ser Asn Ser Ile Ser Ser Gly Arg 85 90 95 gat gat ctc
tcg cct tcg agc agc ttg aac gga tac tcg gcg aac gaa 336 Asp Asp Leu
Ser Pro Ser Ser Ser Leu Asn Gly Tyr Ser Ala Asn Glu 100 105 110 agc
tgc gat gcg aag aag agc aag aag gga cct gcg cca cgg gtg caa 384 Ser
Cys Asp Ala Lys Lys Ser Lys Lys Gly Pro Ala Pro Arg Val Gln 115 120
125 gag gag ctg tgc ctg gtt tgc ggc gac agg gcc tcc ggc tac cac tac
432 Glu Glu Leu Cys Leu Val Cys Gly Asp Arg Ala Ser Gly Tyr His Tyr
130 135 140 aac gcc ctc acc tgt gga tcc tgc aag gtg ttc ttt cga cgc
agc gtt 480 Asn Ala Leu Thr Cys Gly Ser Cys Lys Val Phe Phe Arg Arg
Ser Val 145 150 155 160 acg aag agc gcc gtc tac tgc tgc aag ttc ggg
cgc gcc tgc gaa atg 528 Thr Lys Ser Ala Val Tyr Cys Cys Lys Phe Gly
Arg Ala Cys Glu Met 165 170 175 gac atg tac atg agg cga aag tgt cag
gag tgc cgc ctg aaa aag tgc 576 Asp Met Tyr Met Arg Arg Lys Cys Gln
Glu Cys Arg Leu Lys Lys Cys 180 185 190 ctg gcc gtg ggt atg cgg ccg
gaa tgc gtc gtc ccg gag aac caa tgt 624 Leu Ala Val Gly Met Arg Pro
Glu Cys Val Val Pro Glu Asn Gln Cys 195 200 205 gcg atg aag cgg cgc
gaa aag aag gcc cag aag gag aag gac aaa atg 672 Ala Met Lys Arg Arg
Glu Lys Lys Ala Gln Lys Glu Lys Asp Lys Met 210 215 220 acc act tcg
ccg agc tct cag cat ggc ggc aat ggc agc ttg gcc tct 720 Thr Thr Ser
Pro Ser Ser Gln His Gly Gly Asn Gly Ser Leu Ala Ser 225 230 235 240
ggt ggc ggc caa gac ttt gtt aag aag gag att ctt gac ctt atg aca 768
Gly Gly Gly Gln Asp Phe Val Lys Lys Glu Ile Leu Asp Leu Met Thr 245
250 255 tgc gag ccg ccc cag cat gcc act att ccg cta cta cct gat gaa
ata 816 Cys Glu Pro Pro Gln His Ala Thr Ile Pro Leu Leu Pro Asp Glu
Ile 260 265 270 ttg gcc aag tgt caa gcg cgc aat ata cct tcc tta acg
tac aat cag 864 Leu Ala Lys Cys Gln Ala Arg Asn Ile Pro Ser Leu Thr
Tyr Asn Gln 275 280 285 ttg gcc gtt ata tac aag tta att tgg tac cag
gat ggc tat gag cag 912 Leu Ala Val Ile Tyr Lys Leu Ile Trp Tyr Gln
Asp Gly Tyr Glu Gln 290 295 300 cca tct gaa gag gat ctc agg cgt ata
atg agt caa ccc gat gag aac 960 Pro Ser Glu Glu Asp Leu Arg Arg Ile
Met Ser Gln Pro Asp Glu Asn 305 310 315 320 gag agc caa acg gac gtc
agc ttt cgg cat ata acc gag ata acc ata 1008 Glu Ser Gln Thr Asp
Val Ser Phe Arg His Ile Thr Glu Ile Thr Ile 325 330 335 ctc acg gtc
cag ttg att gtt gag ttt gct aaa ggt cta cca gcg ttt 1056 Leu Thr
Val Gln Leu Ile Val Glu Phe Ala Lys Gly Leu Pro Ala Phe 340 345 350
aca aag ata ccc cag gag gac cag atc acg tta cta aag gcc tgc tcg
1104 Thr Lys Ile Pro Gln Glu Asp Gln Ile Thr Leu Leu Lys Ala Cys
Ser 355 360 365 tcg gag gtg atg atg ctg cgt atg gca cga cgc tat gac
cac agc tcg 1152 Ser Glu Val Met Met Leu Arg Met Ala Arg Arg Tyr
Asp His Ser Ser 370 375 380 gac tca ata ttc ttc gcg aat aat aga tca
tat acg cgg gat tct tac 1200 Asp Ser Ile Phe Phe Ala Asn Asn Arg
Ser Tyr Thr Arg Asp Ser Tyr 385 390 395 400 aaa atg gcc gga atg gct
gat aac att gaa gac ctg ctg cat ttc tgc 1248 Lys Met Ala Gly Met
Ala Asp Asn Ile Glu Asp Leu Leu His Phe Cys 405 410 415 cgc caa atg
ttc tcg atg aag gtg gac aac gtc gaa tac gcg ctt ctc 1296 Arg Gln
Met Phe Ser Met Lys Val Asp Asn Val Glu Tyr Ala Leu Leu 420 425 430
act gcc att gtg atc ttc tcg gac cgg ccg ggc ctg gag aag gcc caa
1344 Thr Ala Ile Val Ile Phe Ser Asp Arg Pro Gly Leu Glu Lys Ala
Gln 435 440 445 cta gtc gaa gcg atc cag agc tac tac atc gac acg cta
cgc att tat 1392 Leu Val Glu Ala Ile Gln Ser Tyr Tyr Ile Asp Thr
Leu Arg Ile Tyr 450 455 460 ata ctc aac cgc cac tgc ggc gac tca atg
agc ctc gtc ttc tac gca 1440 Ile Leu Asn Arg His Cys Gly Asp Ser
Met Ser Leu Val Phe Tyr Ala 465 470 475 480 aag ctg ctc tcg atc ctc
acc gag ctg cgt acg ctg ggc aac cag aac 1488 Lys Leu Leu Ser Ile
Leu Thr Glu Leu Arg Thr Leu Gly Asn Gln Asn 485 490 495 gcc gag atg
tgt ttc tca cta aag ctc aaa aac cgc aaa ctg ccc aag 1536 Ala Glu
Met Cys Phe Ser Leu Lys Leu Lys Asn Arg Lys Leu Pro Lys 500 505 510
ttc ctc gag gag atc tgg gac gtt cat gcc atc ccg cca tcg gtc cag
1584 Phe Leu Glu Glu Ile Trp Asp Val His Ala Ile Pro Pro Ser Val
Gln 515 520 525 tcg cac ctt cag att acc cag gag gag aac gag cgt ctc
gag cgg gct 1632 Ser His Leu Gln Ile Thr Gln Glu Glu Asn Glu Arg
Leu Glu Arg Ala 530 535 540 gag cgt atg cgg gca tcg gtt ggg ggc gcc
att acc gcc ggc att gat 1680 Glu Arg Met Arg Ala Ser Val Gly Gly
Ala Ile Thr Ala Gly Ile Asp 545 550 555 560 tgc gac tct gcc tcc act
tcg gcg gcg gca gcc gcg gcc cag cat cag 1728 Cys Asp Ser Ala Ser
Thr Ser Ala Ala Ala Ala Ala Ala Gln His Gln 565 570 575 cct cag cct
cag ccc cag ccc caa ccc tcc tcc ctg acc cag aac gat 1776 Pro Gln
Pro Gln Pro Gln Pro Gln Pro Ser Ser Leu Thr Gln Asn Asp 580 585 590
tcc cag cac cag aca cag ccg cag cta caa cct cag cta cca cct cag
1824 Ser Gln His Gln Thr Gln Pro Gln Leu Gln Pro Gln Leu Pro Pro
Gln 595 600 605 ctg caa ggt caa ctg caa ccc cag ctc caa cca cag ctt
cag acg caa 1872 Leu Gln Gly Gln Leu Gln Pro Gln Leu Gln Pro Gln
Leu Gln Thr Gln 610 615 620 ctc cag cca cag att caa cca cag cca cag
ctc ctt ccc gtc tcc gct 1920 Leu Gln Pro Gln Ile Gln Pro Gln Pro
Gln Leu Leu Pro Val Ser Ala 625 630 635 640 ccc gtg ccc gcc tcc gta
acc gca cct ggt tcc ttg tcc gcg gtc agt 1968 Pro Val Pro Ala Ser
Val Thr Ala Pro Gly Ser Leu Ser Ala Val Ser 645 650 655 acg agc agc
gaa tac atg ggc gga agt gcg gcc ata gga ccc atc acg 2016 Thr Ser
Ser Glu Tyr Met Gly Gly Ser Ala Ala Ile Gly Pro Ile Thr 660 665 670
ccg gca acc acc agc agt atc acg gct gcc gtt acc gct agc tcc acc
2064 Pro Ala Thr Thr Ser Ser Ile Thr Ala Ala Val Thr Ala Ser Ser
Thr 675 680 685 aca tca gcg gta ccg atg ggc aac gga gtt gga gtc ggt
gtt ggg gtg 2112 Thr Ser Ala Val Pro Met Gly Asn Gly Val Gly Val
Gly Val Gly Val 690 695 700 ggc ggc aac gtc agc atg tat gcg aac gcc
cag acg gcg atg gcc ttg 2160 Gly Gly Asn Val Ser Met Tyr Ala Asn
Ala Gln Thr Ala Met Ala Leu 705 710 715 720 atg ggt gta gcc ctg cat
tcg cac caa gag cag ctt atc ggg gga gtg 2208 Met Gly Val Ala Leu
His Ser His Gln Glu Gln Leu Ile Gly Gly Val 725 730 735 gcg gtt aag
tcg gag cac tcg acg act gca tag 2241 Ala Val Lys Ser Glu His Ser
Thr Thr Ala 740 745 5 746 PRT Artificial Sequence Description of
Artificial Sequence Recombinant VgEcR 5 Met Ala Pro Pro Thr Asp Val
Ser Leu Gly Asp Glu Leu His Leu Asp 1 5 10 15 Gly Glu Asp Val Ala
Met Ala His Ala Asp Ala Leu Asp Asp Phe Asp 20 25 30 Leu Asp Met
Leu Gly Asp Gly Asp Ser Pro Gly Pro Gly Phe Thr Pro 35 40 45 His
Asp Ser Ala Pro Tyr Gly Ala Leu Asp Met Ala Asp Phe Glu Phe 50 55
60 Glu Gln Met Phe Thr Asp Ala Leu Gly Ile Asp Glu Tyr Gly Gly Lys
65 70 75 80 Leu Leu Gly Thr Ser Arg Arg Ile Ser Asn Ser Ile Ser Ser
Gly Arg 85 90 95 Asp Asp Leu Ser Pro Ser Ser Ser Leu Asn Gly Tyr
Ser Ala Asn Glu 100 105 110 Ser Cys Asp Ala Lys Lys Ser Lys Lys Gly
Pro Ala Pro Arg Val Gln 115 120 125 Glu Glu Leu Cys Leu Val Cys Gly
Asp Arg Ala Ser Gly Tyr His Tyr 130 135 140 Asn Ala Leu Thr Cys Gly
Ser Cys Lys Val Phe Phe Arg Arg Ser Val 145 150 155 160 Thr Lys Ser
Ala Val Tyr Cys Cys Lys Phe Gly Arg Ala Cys Glu Met 165 170 175 Asp
Met Tyr Met Arg Arg Lys Cys Gln Glu Cys Arg Leu Lys Lys Cys 180 185
190 Leu Ala Val Gly Met Arg Pro Glu Cys Val Val Pro Glu Asn Gln Cys
195 200 205 Ala Met Lys Arg Arg Glu Lys Lys Ala Gln Lys Glu Lys Asp
Lys Met 210 215 220 Thr Thr Ser Pro Ser Ser Gln His Gly Gly Asn Gly
Ser Leu Ala Ser 225 230 235 240 Gly Gly Gly Gln Asp Phe Val Lys Lys
Glu Ile Leu Asp Leu Met Thr 245 250 255 Cys Glu Pro Pro Gln His Ala
Thr Ile Pro Leu Leu Pro Asp Glu Ile 260 265 270 Leu Ala Lys Cys Gln
Ala Arg Asn Ile Pro Ser Leu Thr Tyr Asn Gln 275 280 285 Leu Ala Val
Ile Tyr Lys Leu Ile Trp Tyr Gln Asp Gly Tyr Glu Gln 290 295 300 Pro
Ser Glu Glu Asp Leu Arg Arg Ile Met Ser Gln Pro Asp Glu Asn 305 310
315 320 Glu Ser Gln Thr Asp Val Ser Phe Arg His Ile Thr Glu Ile Thr
Ile 325 330 335 Leu Thr Val Gln Leu Ile Val Glu Phe Ala Lys Gly Leu
Pro Ala Phe 340 345 350 Thr Lys Ile Pro Gln Glu Asp Gln Ile Thr Leu
Leu Lys Ala Cys Ser 355 360 365 Ser Glu Val Met Met Leu Arg Met Ala
Arg Arg Tyr Asp His Ser Ser 370 375 380 Asp Ser Ile Phe Phe Ala Asn
Asn Arg Ser Tyr Thr Arg Asp Ser Tyr 385 390 395 400 Lys Met Ala Gly
Met Ala Asp Asn Ile Glu Asp Leu Leu His Phe Cys 405 410 415 Arg Gln
Met Phe Ser Met Lys Val Asp Asn Val Glu Tyr Ala Leu Leu 420 425 430
Thr Ala Ile Val Ile Phe Ser Asp Arg Pro Gly Leu Glu Lys Ala Gln 435
440 445 Leu Val Glu Ala Ile Gln Ser Tyr Tyr Ile Asp Thr Leu Arg Ile
Tyr 450 455 460 Ile Leu Asn Arg His Cys Gly Asp Ser Met Ser Leu Val
Phe Tyr Ala 465 470 475 480 Lys Leu Leu Ser Ile Leu Thr Glu Leu Arg
Thr Leu Gly Asn Gln Asn 485 490 495 Ala Glu Met Cys Phe Ser Leu Lys
Leu Lys Asn Arg Lys Leu Pro Lys 500 505 510 Phe Leu Glu Glu Ile Trp
Asp Val His Ala Ile Pro Pro Ser Val Gln 515 520 525 Ser His Leu Gln
Ile Thr Gln Glu Glu Asn Glu Arg Leu Glu Arg Ala 530 535 540 Glu Arg
Met Arg Ala Ser Val Gly Gly Ala Ile Thr Ala Gly Ile Asp 545 550 555
560 Cys Asp Ser Ala Ser Thr Ser Ala Ala Ala Ala Ala Ala Gln His Gln
565 570 575 Pro Gln Pro Gln Pro Gln Pro Gln Pro Ser Ser Leu Thr Gln
Asn Asp 580 585 590 Ser Gln His Gln Thr Gln Pro Gln Leu Gln Pro Gln
Leu Pro Pro Gln 595 600 605 Leu Gln Gly Gln Leu Gln Pro Gln Leu Gln
Pro Gln Leu Gln Thr Gln 610 615 620 Leu Gln Pro Gln Ile Gln Pro Gln
Pro Gln Leu Leu Pro Val Ser Ala 625 630 635 640 Pro Val Pro Ala Ser
Val Thr Ala Pro Gly Ser Leu Ser Ala Val Ser 645 650 655 Thr Ser Ser
Glu Tyr Met Gly Gly Ser Ala Ala Ile Gly Pro Ile Thr 660 665 670 Pro
Ala Thr Thr Ser Ser Ile Thr Ala Ala Val Thr Ala Ser Ser Thr 675 680
685 Thr Ser Ala Val Pro Met Gly Asn Gly Val Gly Val Gly Val Gly Val
690 695 700 Gly Gly Asn Val Ser Met Tyr Ala Asn Ala Gln Thr Ala Met
Ala Leu 705 710 715 720 Met Gly Val Ala Leu His Ser His Gln Glu Gln
Leu Ile Gly Gly Val 725 730 735 Ala Val Lys Ser Glu His Ser Thr Thr
Ala 740 745 6 2241 DNA Artificial Sequence Description of
Artificial Sequence Recombinant VpEcR 6 atg gcc ccc ccg acc gat gtc
agc ctg ggg gac gag ctc cac tta gac 48 Met Ala Pro Pro Thr Asp Val
Ser Leu Gly Asp Glu Leu His Leu Asp 1 5 10 15 ggc gag gac gtg gcg
atg gcg cat gcc gac gcg cta gac gat ttc gat 96 Gly Glu Asp Val Ala
Met Ala His Ala Asp Ala Leu Asp Asp Phe Asp 20 25 30 ctg gac atg
ttg ggg gac ggg gat tcc ccg ggt ccg gga ttt acc ccc 144 Leu Asp Met
Leu Gly Asp Gly Asp Ser Pro Gly Pro Gly Phe Thr Pro 35 40 45 cac
gac tcc gcc ccc tac ggc gct ctg gat atg gcc gac ttc gag ttt 192 His
Asp Ser Ala Pro Tyr Gly Ala Leu Asp Met Ala Asp Phe Glu Phe 50 55
60 gag cag atg ttt acc gat gcc ctt gga att gac gag tac ggt ggg aag
240 Glu Gln Met Phe Thr Asp Ala Leu Gly Ile Asp Glu Tyr Gly Gly Lys
65 70 75 80 ctt cta ggt acc tct aga agg ata tcg aat tct ata tct tca
ggt cgc 288 Leu Leu Gly Thr Ser Arg Arg Ile Ser Asn Ser Ile Ser Ser
Gly Arg 85 90 95 gat gat ctc tcg cct tcg agc agc ttg aac gga tac
tcg gcg aac gaa 336 Asp Asp Leu Ser Pro Ser Ser Ser Leu Asn Gly Tyr
Ser Ala Asn Glu 100 105 110 agc tgc gat gcg aag aag agc aag aag gga
cct gcg cca cgg gtg caa 384 Ser Cys Asp Ala Lys Lys Ser Lys Lys Gly
Pro Ala Pro Arg Val Gln 115 120 125 gag gag ctg tgc ctg gtt tgc ggc
gac agg gcc tcc ggc tac cac tac 432 Glu Glu Leu Cys Leu Val Cys Gly
Asp Arg Ala Ser Gly Tyr His Tyr 130 135 140 aac gcc ctc acc tgt gag
ggc tgc aag ggg ttc ttt cga cgc agc gtt 480 Asn Ala Leu Thr Cys Glu
Gly Cys Lys Gly Phe Phe Arg Arg Ser Val 145 150 155 160 acg aag agc
gcc gtc tac tgc tgc aag ttc ggg cgc gcc tgc gaa atg 528 Thr Lys Ser
Ala Val Tyr Cys Cys Lys Phe Gly Arg Ala Cys Glu Met 165 170 175 gac
atg tac atg agg cga aag tgt cag gag tgc cgc ctg aaa aag tgc 576 Asp
Met Tyr Met Arg Arg Lys Cys Gln Glu Cys Arg Leu Lys Lys Cys 180 185
190 ctg gcc gtg ggt atg cgg ccg gaa tgc gtc gtc ccg gag aac caa tgt
624 Leu Ala Val Gly Met Arg Pro Glu Cys Val Val Pro Glu Asn Gln Cys
195 200 205 gcg atg aag
cgg cgc gaa aag aag gcc cag aag gag aag gac aaa atg 672 Ala Met Lys
Arg Arg Glu Lys Lys Ala Gln Lys Glu Lys Asp Lys Met 210 215 220 acc
act tcg ccg agc tct cag cat ggc ggc aat ggc agc ttg gcc tct 720 Thr
Thr Ser Pro Ser Ser Gln His Gly Gly Asn Gly Ser Leu Ala Ser 225 230
235 240 ggt ggc ggc caa gac ttt gtt aag aag gag att ctt gac ctt atg
aca 768 Gly Gly Gly Gln Asp Phe Val Lys Lys Glu Ile Leu Asp Leu Met
Thr 245 250 255 tgc gag ccg ccc cag cat gcc act att ccg cta cta cct
gat gaa ata 816 Cys Glu Pro Pro Gln His Ala Thr Ile Pro Leu Leu Pro
Asp Glu Ile 260 265 270 ttg gcc aag tgt caa gcg cgc aat ata cct tcc
tta acg tac aat cag 864 Leu Ala Lys Cys Gln Ala Arg Asn Ile Pro Ser
Leu Thr Tyr Asn Gln 275 280 285 ttg gcc gtt ata tac aag tta att tgg
tac cag gat ggc tat gag cag 912 Leu Ala Val Ile Tyr Lys Leu Ile Trp
Tyr Gln Asp Gly Tyr Glu Gln 290 295 300 cca tct gaa gag gat ctc agg
cgt ata atg agt caa ccc gat gag aac 960 Pro Ser Glu Glu Asp Leu Arg
Arg Ile Met Ser Gln Pro Asp Glu Asn 305 310 315 320 gag agc caa acg
gac gtc agc ttt cgg cat ata acc gag ata acc ata 1008 Glu Ser Gln
Thr Asp Val Ser Phe Arg His Ile Thr Glu Ile Thr Ile 325 330 335 ctc
acg gtc cag ttg att gtt gag ttt gct aaa ggt cta cca gcg ttt 1056
Leu Thr Val Gln Leu Ile Val Glu Phe Ala Lys Gly Leu Pro Ala Phe 340
345 350 aca aag ata ccc cag gag gac cag atc acg tta cta aag gcc tgc
tcg 1104 Thr Lys Ile Pro Gln Glu Asp Gln Ile Thr Leu Leu Lys Ala
Cys Ser 355 360 365 tcg gag gtg atg atg ctg cgt atg gca cga cgc tat
gac cac agc tcg 1152 Ser Glu Val Met Met Leu Arg Met Ala Arg Arg
Tyr Asp His Ser Ser 370 375 380 gac tca ata ttc ttc gcg aat aat aga
tca tat acg cgg gat tct tac 1200 Asp Ser Ile Phe Phe Ala Asn Asn
Arg Ser Tyr Thr Arg Asp Ser Tyr 385 390 395 400 aaa atg gcc gga atg
gct gat aac att gaa gac ctg ctg cat ttc tgc 1248 Lys Met Ala Gly
Met Ala Asp Asn Ile Glu Asp Leu Leu His Phe Cys 405 410 415 cgc caa
atg ttc tcg atg aag gtg gac aac gtc gaa tac gcg ctt ctc 1296 Arg
Gln Met Phe Ser Met Lys Val Asp Asn Val Glu Tyr Ala Leu Leu 420 425
430 act gcc att gtg atc ttc tcg gac cgg ccg ggc ctg gag aag gcc caa
1344 Thr Ala Ile Val Ile Phe Ser Asp Arg Pro Gly Leu Glu Lys Ala
Gln 435 440 445 cta gtc gaa gcg atc cag agc tac tac atc gac acg cta
cgc att tat 1392 Leu Val Glu Ala Ile Gln Ser Tyr Tyr Ile Asp Thr
Leu Arg Ile Tyr 450 455 460 ata ctc aac cgc cac tgc ggc gac tca atg
agc ctc gtc ttc tac gca 1440 Ile Leu Asn Arg His Cys Gly Asp Ser
Met Ser Leu Val Phe Tyr Ala 465 470 475 480 aag ctg ctc tcg atc ctc
acc gag ctg cgt acg ctg ggc aac cag aac 1488 Lys Leu Leu Ser Ile
Leu Thr Glu Leu Arg Thr Leu Gly Asn Gln Asn 485 490 495 gcc gag atg
tgt ttc tca cta aag ctc aaa aac cgc aaa ctg ccc aag 1536 Ala Glu
Met Cys Phe Ser Leu Lys Leu Lys Asn Arg Lys Leu Pro Lys 500 505 510
ttc ctc gag gag atc tgg gac gtt cat gcc atc ccg cca tcg gtc cag
1584 Phe Leu Glu Glu Ile Trp Asp Val His Ala Ile Pro Pro Ser Val
Gln 515 520 525 tcg cac ctt cag att acc cag gag gag aac gag cgt ctc
gag cgg gct 1632 Ser His Leu Gln Ile Thr Gln Glu Glu Asn Glu Arg
Leu Glu Arg Ala 530 535 540 gag cgt atg cgg gca tcg gtt ggg ggc gcc
att acc gcc ggc att gat 1680 Glu Arg Met Arg Ala Ser Val Gly Gly
Ala Ile Thr Ala Gly Ile Asp 545 550 555 560 tgc gac tct gcc tcc act
tcg gcg gcg gca gcc gcg gcc cag cat cag 1728 Cys Asp Ser Ala Ser
Thr Ser Ala Ala Ala Ala Ala Ala Gln His Gln 565 570 575 cct cag cct
cag ccc cag ccc caa ccc tcc tcc ctg acc cag aac gat 1776 Pro Gln
Pro Gln Pro Gln Pro Gln Pro Ser Ser Leu Thr Gln Asn Asp 580 585 590
tcc cag cac cag aca cag ccg cag cta caa cct cag cta cca cct cag
1824 Ser Gln His Gln Thr Gln Pro Gln Leu Gln Pro Gln Leu Pro Pro
Gln 595 600 605 ctg caa ggt caa ctg caa ccc cag ctc caa cca cag ctt
cag acg caa 1872 Leu Gln Gly Gln Leu Gln Pro Gln Leu Gln Pro Gln
Leu Gln Thr Gln 610 615 620 ctc cag cca cag att caa cca cag cca cag
ctc ctt ccc gtc tcc gct 1920 Leu Gln Pro Gln Ile Gln Pro Gln Pro
Gln Leu Leu Pro Val Ser Ala 625 630 635 640 ccc gtg ccc gcc tcc gta
acc gca cct ggt tcc ttg tcc gcg gtc agt 1968 Pro Val Pro Ala Ser
Val Thr Ala Pro Gly Ser Leu Ser Ala Val Ser 645 650 655 acg agc agc
gaa tac atg ggc gga agt gcg gcc ata gga ccc atc acg 2016 Thr Ser
Ser Glu Tyr Met Gly Gly Ser Ala Ala Ile Gly Pro Ile Thr 660 665 670
ccg gca acc acc agc agt atc acg gct gcc gtt acc gct agc tcc acc
2064 Pro Ala Thr Thr Ser Ser Ile Thr Ala Ala Val Thr Ala Ser Ser
Thr 675 680 685 aca tca gcg gta ccg atg ggc aac gga gtt gga gtc ggt
gtt ggg gtg 2112 Thr Ser Ala Val Pro Met Gly Asn Gly Val Gly Val
Gly Val Gly Val 690 695 700 ggc ggc aac gtc agc atg tat gcg aac gcc
cag acg gcg atg gcc ttg 2160 Gly Gly Asn Val Ser Met Tyr Ala Asn
Ala Gln Thr Ala Met Ala Leu 705 710 715 720 atg ggt gta gcc ctg cat
tcg cac caa gag cag ctt atc ggg gga gtg 2208 Met Gly Val Ala Leu
His Ser His Gln Glu Gln Leu Ile Gly Gly Val 725 730 735 gcg gtt aag
tcg gag cac tcg acg act gca tag 2241 Ala Val Lys Ser Glu His Ser
Thr Thr Ala 740 745 7 746 PRT Artificial Sequence Description of
Artificial Sequence Recombinant VpEcR 7 Met Ala Pro Pro Thr Asp Val
Ser Leu Gly Asp Glu Leu His Leu Asp 1 5 10 15 Gly Glu Asp Val Ala
Met Ala His Ala Asp Ala Leu Asp Asp Phe Asp 20 25 30 Leu Asp Met
Leu Gly Asp Gly Asp Ser Pro Gly Pro Gly Phe Thr Pro 35 40 45 His
Asp Ser Ala Pro Tyr Gly Ala Leu Asp Met Ala Asp Phe Glu Phe 50 55
60 Glu Gln Met Phe Thr Asp Ala Leu Gly Ile Asp Glu Tyr Gly Gly Lys
65 70 75 80 Leu Leu Gly Thr Ser Arg Arg Ile Ser Asn Ser Ile Ser Ser
Gly Arg 85 90 95 Asp Asp Leu Ser Pro Ser Ser Ser Leu Asn Gly Tyr
Ser Ala Asn Glu 100 105 110 Ser Cys Asp Ala Lys Lys Ser Lys Lys Gly
Pro Ala Pro Arg Val Gln 115 120 125 Glu Glu Leu Cys Leu Val Cys Gly
Asp Arg Ala Ser Gly Tyr His Tyr 130 135 140 Asn Ala Leu Thr Cys Glu
Gly Cys Lys Gly Phe Phe Arg Arg Ser Val 145 150 155 160 Thr Lys Ser
Ala Val Tyr Cys Cys Lys Phe Gly Arg Ala Cys Glu Met 165 170 175 Asp
Met Tyr Met Arg Arg Lys Cys Gln Glu Cys Arg Leu Lys Lys Cys 180 185
190 Leu Ala Val Gly Met Arg Pro Glu Cys Val Val Pro Glu Asn Gln Cys
195 200 205 Ala Met Lys Arg Arg Glu Lys Lys Ala Gln Lys Glu Lys Asp
Lys Met 210 215 220 Thr Thr Ser Pro Ser Ser Gln His Gly Gly Asn Gly
Ser Leu Ala Ser 225 230 235 240 Gly Gly Gly Gln Asp Phe Val Lys Lys
Glu Ile Leu Asp Leu Met Thr 245 250 255 Cys Glu Pro Pro Gln His Ala
Thr Ile Pro Leu Leu Pro Asp Glu Ile 260 265 270 Leu Ala Lys Cys Gln
Ala Arg Asn Ile Pro Ser Leu Thr Tyr Asn Gln 275 280 285 Leu Ala Val
Ile Tyr Lys Leu Ile Trp Tyr Gln Asp Gly Tyr Glu Gln 290 295 300 Pro
Ser Glu Glu Asp Leu Arg Arg Ile Met Ser Gln Pro Asp Glu Asn 305 310
315 320 Glu Ser Gln Thr Asp Val Ser Phe Arg His Ile Thr Glu Ile Thr
Ile 325 330 335 Leu Thr Val Gln Leu Ile Val Glu Phe Ala Lys Gly Leu
Pro Ala Phe 340 345 350 Thr Lys Ile Pro Gln Glu Asp Gln Ile Thr Leu
Leu Lys Ala Cys Ser 355 360 365 Ser Glu Val Met Met Leu Arg Met Ala
Arg Arg Tyr Asp His Ser Ser 370 375 380 Asp Ser Ile Phe Phe Ala Asn
Asn Arg Ser Tyr Thr Arg Asp Ser Tyr 385 390 395 400 Lys Met Ala Gly
Met Ala Asp Asn Ile Glu Asp Leu Leu His Phe Cys 405 410 415 Arg Gln
Met Phe Ser Met Lys Val Asp Asn Val Glu Tyr Ala Leu Leu 420 425 430
Thr Ala Ile Val Ile Phe Ser Asp Arg Pro Gly Leu Glu Lys Ala Gln 435
440 445 Leu Val Glu Ala Ile Gln Ser Tyr Tyr Ile Asp Thr Leu Arg Ile
Tyr 450 455 460 Ile Leu Asn Arg His Cys Gly Asp Ser Met Ser Leu Val
Phe Tyr Ala 465 470 475 480 Lys Leu Leu Ser Ile Leu Thr Glu Leu Arg
Thr Leu Gly Asn Gln Asn 485 490 495 Ala Glu Met Cys Phe Ser Leu Lys
Leu Lys Asn Arg Lys Leu Pro Lys 500 505 510 Phe Leu Glu Glu Ile Trp
Asp Val His Ala Ile Pro Pro Ser Val Gln 515 520 525 Ser His Leu Gln
Ile Thr Gln Glu Glu Asn Glu Arg Leu Glu Arg Ala 530 535 540 Glu Arg
Met Arg Ala Ser Val Gly Gly Ala Ile Thr Ala Gly Ile Asp 545 550 555
560 Cys Asp Ser Ala Ser Thr Ser Ala Ala Ala Ala Ala Ala Gln His Gln
565 570 575 Pro Gln Pro Gln Pro Gln Pro Gln Pro Ser Ser Leu Thr Gln
Asn Asp 580 585 590 Ser Gln His Gln Thr Gln Pro Gln Leu Gln Pro Gln
Leu Pro Pro Gln 595 600 605 Leu Gln Gly Gln Leu Gln Pro Gln Leu Gln
Pro Gln Leu Gln Thr Gln 610 615 620 Leu Gln Pro Gln Ile Gln Pro Gln
Pro Gln Leu Leu Pro Val Ser Ala 625 630 635 640 Pro Val Pro Ala Ser
Val Thr Ala Pro Gly Ser Leu Ser Ala Val Ser 645 650 655 Thr Ser Ser
Glu Tyr Met Gly Gly Ser Ala Ala Ile Gly Pro Ile Thr 660 665 670 Pro
Ala Thr Thr Ser Ser Ile Thr Ala Ala Val Thr Ala Ser Ser Thr 675 680
685 Thr Ser Ala Val Pro Met Gly Asn Gly Val Gly Val Gly Val Gly Val
690 695 700 Gly Gly Asn Val Ser Met Tyr Ala Asn Ala Gln Thr Ala Met
Ala Leu 705 710 715 720 Met Gly Val Ala Leu His Ser His Gln Glu Gln
Leu Ile Gly Gly Val 725 730 735 Ala Val Lys Ser Glu His Ser Thr Thr
Ala 740 745 8 3126 DNA Artificial Sequence Description of
Artificial Sequence Recombinant GEcR 8 atg gac tcc aaa gaa tca tta
act cct ggt aga gaa gaa aac ccc agc 48 Met Asp Ser Lys Glu Ser Leu
Thr Pro Gly Arg Glu Glu Asn Pro Ser 1 5 10 15 agt gtg ctt gct cag
gag agg gga gat gtg atg gac ttc tat aaa acc 96 Ser Val Leu Ala Gln
Glu Arg Gly Asp Val Met Asp Phe Tyr Lys Thr 20 25 30 cta aga gga
gga gct act gtg aag gtt tct gcg tct tca ccc tca ctg 144 Leu Arg Gly
Gly Ala Thr Val Lys Val Ser Ala Ser Ser Pro Ser Leu 35 40 45 gct
gtc gct tct caa tca gac tcc aag cag cga aga ctt ttg gtt gat 192 Ala
Val Ala Ser Gln Ser Asp Ser Lys Gln Arg Arg Leu Leu Val Asp 50 55
60 ttt cca aaa ggc tca gta agc aat gcg cag cag cca gat ctg tcc aaa
240 Phe Pro Lys Gly Ser Val Ser Asn Ala Gln Gln Pro Asp Leu Ser Lys
65 70 75 80 gca gtt tca ctc tca atg gga ctg tat atg gga gag aca gaa
aca aaa 288 Ala Val Ser Leu Ser Met Gly Leu Tyr Met Gly Glu Thr Glu
Thr Lys 85 90 95 gtg atg gga aat gac ctg gga ttc cca cag cag ggc
caa atc agc ctt 336 Val Met Gly Asn Asp Leu Gly Phe Pro Gln Gln Gly
Gln Ile Ser Leu 100 105 110 tcc tcg ggg gaa aca gac tta aag ctt ttg
gaa gaa agc att gca aac 384 Ser Ser Gly Glu Thr Asp Leu Lys Leu Leu
Glu Glu Ser Ile Ala Asn 115 120 125 ctc aat agg tcg acc agt gtt cca
gag aac ccc aag agt tca gca tcc 432 Leu Asn Arg Ser Thr Ser Val Pro
Glu Asn Pro Lys Ser Ser Ala Ser 130 135 140 act gct gtg tct gct gcc
ccc aca gag aag gag ttt cca aaa act cac 480 Thr Ala Val Ser Ala Ala
Pro Thr Glu Lys Glu Phe Pro Lys Thr His 145 150 155 160 tct gat gta
tct tca gaa cag caa cat ttg aag ggc cag act ggc acc 528 Ser Asp Val
Ser Ser Glu Gln Gln His Leu Lys Gly Gln Thr Gly Thr 165 170 175 aac
ggt ggc aat gtg aaa ttg tat acc aca gac caa agc acc ttt gac 576 Asn
Gly Gly Asn Val Lys Leu Tyr Thr Thr Asp Gln Ser Thr Phe Asp 180 185
190 att ttg cag gat ttg gag ttt tct tct ggg tcc cca ggt aaa gag acg
624 Ile Leu Gln Asp Leu Glu Phe Ser Ser Gly Ser Pro Gly Lys Glu Thr
195 200 205 aat gag agt cct tgg aga tca gac ctg ttg ata gat gaa aac
tgt ttg 672 Asn Glu Ser Pro Trp Arg Ser Asp Leu Leu Ile Asp Glu Asn
Cys Leu 210 215 220 ctt tct cct ctg gcg gga gaa gac gat tca ttc ctt
ttg gaa gga aac 720 Leu Ser Pro Leu Ala Gly Glu Asp Asp Ser Phe Leu
Leu Glu Gly Asn 225 230 235 240 tcg aat gag gac tgc aag cct ctc att
tta ccg gac act aaa ccc aaa 768 Ser Asn Glu Asp Cys Lys Pro Leu Ile
Leu Pro Asp Thr Lys Pro Lys 245 250 255 att aag gat aat gga gat ctg
gtt ttg tca agc ccc agt aat gta aca 816 Ile Lys Asp Asn Gly Asp Leu
Val Leu Ser Ser Pro Ser Asn Val Thr 260 265 270 ctg ccc caa gtg aaa
aca gaa aaa gaa gat ttc atc gaa ctc tgc acc 864 Leu Pro Gln Val Lys
Thr Glu Lys Glu Asp Phe Ile Glu Leu Cys Thr 275 280 285 cct ggg gta
att aag caa gag aaa ctg ggc aca gtt tac tgt cag gca 912 Pro Gly Val
Ile Lys Gln Glu Lys Leu Gly Thr Val Tyr Cys Gln Ala 290 295 300 agc
ttt cct gga gca aat ata att ggt aat aaa atg tct gcc att tct 960 Ser
Phe Pro Gly Ala Asn Ile Ile Gly Asn Lys Met Ser Ala Ile Ser 305 310
315 320 gtt cat ggt gtg agt acc tct gga gga cag atg tac cac tat gac
atg 1008 Val His Gly Val Ser Thr Ser Gly Gly Gln Met Tyr His Tyr
Asp Met 325 330 335 aat aca gca tcc ctt tct caa cag cag gat cag aag
cct att ttt aat 1056 Asn Thr Ala Ser Leu Ser Gln Gln Gln Asp Gln
Lys Pro Ile Phe Asn 340 345 350 gtc att cca cca att ccc gtt ggt tcc
gaa aat tgg aat agg tgc caa 1104 Val Ile Pro Pro Ile Pro Val Gly
Ser Glu Asn Trp Asn Arg Cys Gln 355 360 365 gga tct gga gat gac aac
ttg act tct ctg ggg act ctg aac ttc cct 1152 Gly Ser Gly Asp Asp
Asn Leu Thr Ser Leu Gly Thr Leu Asn Phe Pro 370 375 380 ggt cga aca
gtt ttt tct aat ggc tat tca agc ccc agc atg aga cca 1200 Gly Arg
Thr Val Phe Ser Asn Gly Tyr Ser Ser Pro Ser Met Arg Pro 385 390 395
400 gat gta agc tct cct cca tcc agc tcc tca aca gca aca aca gga cca
1248 Asp Val Ser Ser Pro Pro Ser Ser Ser Ser Thr Ala Thr Thr Gly
Pro 405 410 415 cct ccc agc ggc cgc gtg caa gag gag ctg tgc ctg gtt
tgc ggc gac 1296 Pro Pro Ser Gly Arg Val Gln Glu Glu Leu Cys Leu
Val Cys Gly Asp 420 425 430 agg gcc tcc ggc tac cac tac aac gcc ctc
acc tgt gga tcc tgc aag 1344 Arg Ala Ser Gly Tyr His Tyr Asn Ala
Leu Thr Cys Gly Ser Cys Lys 435 440 445 gtg ttc ttt cga cgc agc gtt
acg aag agc gcc gtc tac tgc tgc aag 1392 Val Phe Phe Arg Arg Ser
Val Thr Lys Ser Ala Val Tyr Cys Cys Lys 450 455 460 ttc ggg cgc gcc
tgc gaa atg gac atg tac atg agg cga aag tgt cag 1440 Phe Gly Arg
Ala Cys Glu Met Asp Met Tyr Met Arg Arg Lys Cys Gln 465 470 475 480
gag tgc cgc ctg aaa aag tgc ctg gcc gtg ggt atg cgg ccg gaa tgc
1488 Glu Cys Arg Leu Lys Lys Cys Leu Ala Val Gly Met Arg Pro Glu
Cys 485 490 495 gtc gtc ccg gag aac caa tgt gcg atg aag cgg cgc gaa
aag aag gcc
1536 Val Val Pro Glu Asn Gln Cys Ala Met Lys Arg Arg Glu Lys Lys
Ala 500 505 510 cag aag gag aag gac aaa atg acc act tcg ccg agc tct
cag cat ggc 1584 Gln Lys Glu Lys Asp Lys Met Thr Thr Ser Pro Ser
Ser Gln His Gly 515 520 525 ggc aat ggc agc ttg gcc tct ggt ggc ggc
caa gac ttt gtt aag aag 1632 Gly Asn Gly Ser Leu Ala Ser Gly Gly
Gly Gln Asp Phe Val Lys Lys 530 535 540 gag att ctt gac ctt atg aca
tgc gag ccg ccc cag cat gcc act att 1680 Glu Ile Leu Asp Leu Met
Thr Cys Glu Pro Pro Gln His Ala Thr Ile 545 550 555 560 ccg cta cta
cct gat gaa ata ttg gcc aag tgt caa gcg cgc aat ata 1728 Pro Leu
Leu Pro Asp Glu Ile Leu Ala Lys Cys Gln Ala Arg Asn Ile 565 570 575
cct tcc tta acg tac aat cag ttg gcc gtt ata tac aag tta att tgg
1776 Pro Ser Leu Thr Tyr Asn Gln Leu Ala Val Ile Tyr Lys Leu Ile
Trp 580 585 590 tac cag gat ggc tat gag cag cca tct gaa gag gat ctc
agg cgt ata 1824 Tyr Gln Asp Gly Tyr Glu Gln Pro Ser Glu Glu Asp
Leu Arg Arg Ile 595 600 605 atg agt caa ccc gat gag aac gag agc caa
acg gac gtc agc ttt cgg 1872 Met Ser Gln Pro Asp Glu Asn Glu Ser
Gln Thr Asp Val Ser Phe Arg 610 615 620 cat ata acc gag ata acc ata
ctc acg gtc cag ttg att gtt gag ttt 1920 His Ile Thr Glu Ile Thr
Ile Leu Thr Val Gln Leu Ile Val Glu Phe 625 630 635 640 gct aaa ggt
cta cca gcg ttt aca aag ata ccc cag gag gac cag atc 1968 Ala Lys
Gly Leu Pro Ala Phe Thr Lys Ile Pro Gln Glu Asp Gln Ile 645 650 655
acg tta cta aag gcc tgc tcg tcg gag gtg atg atg ctg cgt atg gca
2016 Thr Leu Leu Lys Ala Cys Ser Ser Glu Val Met Met Leu Arg Met
Ala 660 665 670 cga cgc tat gac cac agc tcg gac tca ata ttc ttc gcg
aat aat aga 2064 Arg Arg Tyr Asp His Ser Ser Asp Ser Ile Phe Phe
Ala Asn Asn Arg 675 680 685 tca tat acg cgg gat tct tac aaa atg gcc
gga atg gct gat aac att 2112 Ser Tyr Thr Arg Asp Ser Tyr Lys Met
Ala Gly Met Ala Asp Asn Ile 690 695 700 gaa gac ctg ctg cat ttc tgc
cgc caa atg ttc tcg atg aag gtg gac 2160 Glu Asp Leu Leu His Phe
Cys Arg Gln Met Phe Ser Met Lys Val Asp 705 710 715 720 aac gtc gaa
tac gcg ctt ctc act gcc att gtg atc ttc tcg gac cgg 2208 Asn Val
Glu Tyr Ala Leu Leu Thr Ala Ile Val Ile Phe Ser Asp Arg 725 730 735
ccg ggc ctg gag aag gcc caa cta gtc gaa gcg atc cag agc tac tac
2256 Pro Gly Leu Glu Lys Ala Gln Leu Val Glu Ala Ile Gln Ser Tyr
Tyr 740 745 750 atc gac acg cta cgc att tat ata ctc aac cgc cac tgc
ggc gac tca 2304 Ile Asp Thr Leu Arg Ile Tyr Ile Leu Asn Arg His
Cys Gly Asp Ser 755 760 765 atg agc ctc gtc ttc tac gca aag ctg ctc
tcg atc ctc acc gag ctg 2352 Met Ser Leu Val Phe Tyr Ala Lys Leu
Leu Ser Ile Leu Thr Glu Leu 770 775 780 cgt acg ctg ggc aac cag aac
gcc gag atg tgt ttc tca cta aag ctc 2400 Arg Thr Leu Gly Asn Gln
Asn Ala Glu Met Cys Phe Ser Leu Lys Leu 785 790 795 800 aaa aac cgc
aaa ctg ccc aag ttc ctc gag gag atc tgg gac gtt cat 2448 Lys Asn
Arg Lys Leu Pro Lys Phe Leu Glu Glu Ile Trp Asp Val His 805 810 815
gcc atc ccg cca tcg gtc cag tcg cac ctt cag att acc cag gag gag
2496 Ala Ile Pro Pro Ser Val Gln Ser His Leu Gln Ile Thr Gln Glu
Glu 820 825 830 aac gag cgt ctc gag cgg gct gag cgt atg cgg gca tcg
gtt ggg ggc 2544 Asn Glu Arg Leu Glu Arg Ala Glu Arg Met Arg Ala
Ser Val Gly Gly 835 840 845 gcc att acc gcc ggc att gat tgc gac tct
gcc tcc act tcg gcg gcg 2592 Ala Ile Thr Ala Gly Ile Asp Cys Asp
Ser Ala Ser Thr Ser Ala Ala 850 855 860 gca gcc gcg gcc cag cat cag
cct cag cct cag ccc cag ccc caa ccc 2640 Ala Ala Ala Ala Gln His
Gln Pro Gln Pro Gln Pro Gln Pro Gln Pro 865 870 875 880 tcc tcc ctg
acc cag aac gat tcc cag cac cag aca cag ccg cag cta 2688 Ser Ser
Leu Thr Gln Asn Asp Ser Gln His Gln Thr Gln Pro Gln Leu 885 890 895
caa cct cag cta cca cct cag ctg caa ggt caa ctg caa ccc cag ctc
2736 Gln Pro Gln Leu Pro Pro Gln Leu Gln Gly Gln Leu Gln Pro Gln
Leu 900 905 910 caa cca cag ctt cag acg caa ctc cag cca cag att caa
cca cag cca 2784 Gln Pro Gln Leu Gln Thr Gln Leu Gln Pro Gln Ile
Gln Pro Gln Pro 915 920 925 cag ctc ctt ccc gtc tcc gct ccc gtg ccc
gcc tcc gta acc gca cct 2832 Gln Leu Leu Pro Val Ser Ala Pro Val
Pro Ala Ser Val Thr Ala Pro 930 935 940 ggt tcc ttg tcc gcg gtc agt
acg agc agc gaa tac atg ggc gga agt 2880 Gly Ser Leu Ser Ala Val
Ser Thr Ser Ser Glu Tyr Met Gly Gly Ser 945 950 955 960 gcg gcc ata
gga ccc atc acg ccg gca acc acc agc agt atc acg gct 2928 Ala Ala
Ile Gly Pro Ile Thr Pro Ala Thr Thr Ser Ser Ile Thr Ala 965 970 975
gcc gtt acc gct agc tcc acc aca tca gcg gta ccg atg ggc aac gga
2976 Ala Val Thr Ala Ser Ser Thr Thr Ser Ala Val Pro Met Gly Asn
Gly 980 985 990 gtt gga gtc ggt gtt ggg gtg ggc ggc aac gtc agc atg
tat gcg aac 3024 Val Gly Val Gly Val Gly Val Gly Gly Asn Val Ser
Met Tyr Ala Asn 995 1000 1005 gcc cag acg gcg atg gcc ttg atg ggt
gta gcc ctg cat tcg cac caa 3072 Ala Gln Thr Ala Met Ala Leu Met
Gly Val Ala Leu His Ser His Gln 1010 1015 1020 gag cag ctt atc ggg
gga gtg gcg gtt aag tcg gag cac tcg acg act 3120 Glu Gln Leu Ile
Gly Gly Val Ala Val Lys Ser Glu His Ser Thr Thr 1025 1030 1035 1040
gca tag 3126 Ala 9 1041 PRT Artificial Sequence Description of
Artificial Sequence Recombinant GEcR 9 Met Asp Ser Lys Glu Ser Leu
Thr Pro Gly Arg Glu Glu Asn Pro Ser 1 5 10 15 Ser Val Leu Ala Gln
Glu Arg Gly Asp Val Met Asp Phe Tyr Lys Thr 20 25 30 Leu Arg Gly
Gly Ala Thr Val Lys Val Ser Ala Ser Ser Pro Ser Leu 35 40 45 Ala
Val Ala Ser Gln Ser Asp Ser Lys Gln Arg Arg Leu Leu Val Asp 50 55
60 Phe Pro Lys Gly Ser Val Ser Asn Ala Gln Gln Pro Asp Leu Ser Lys
65 70 75 80 Ala Val Ser Leu Ser Met Gly Leu Tyr Met Gly Glu Thr Glu
Thr Lys 85 90 95 Val Met Gly Asn Asp Leu Gly Phe Pro Gln Gln Gly
Gln Ile Ser Leu 100 105 110 Ser Ser Gly Glu Thr Asp Leu Lys Leu Leu
Glu Glu Ser Ile Ala Asn 115 120 125 Leu Asn Arg Ser Thr Ser Val Pro
Glu Asn Pro Lys Ser Ser Ala Ser 130 135 140 Thr Ala Val Ser Ala Ala
Pro Thr Glu Lys Glu Phe Pro Lys Thr His 145 150 155 160 Ser Asp Val
Ser Ser Glu Gln Gln His Leu Lys Gly Gln Thr Gly Thr 165 170 175 Asn
Gly Gly Asn Val Lys Leu Tyr Thr Thr Asp Gln Ser Thr Phe Asp 180 185
190 Ile Leu Gln Asp Leu Glu Phe Ser Ser Gly Ser Pro Gly Lys Glu Thr
195 200 205 Asn Glu Ser Pro Trp Arg Ser Asp Leu Leu Ile Asp Glu Asn
Cys Leu 210 215 220 Leu Ser Pro Leu Ala Gly Glu Asp Asp Ser Phe Leu
Leu Glu Gly Asn 225 230 235 240 Ser Asn Glu Asp Cys Lys Pro Leu Ile
Leu Pro Asp Thr Lys Pro Lys 245 250 255 Ile Lys Asp Asn Gly Asp Leu
Val Leu Ser Ser Pro Ser Asn Val Thr 260 265 270 Leu Pro Gln Val Lys
Thr Glu Lys Glu Asp Phe Ile Glu Leu Cys Thr 275 280 285 Pro Gly Val
Ile Lys Gln Glu Lys Leu Gly Thr Val Tyr Cys Gln Ala 290 295 300 Ser
Phe Pro Gly Ala Asn Ile Ile Gly Asn Lys Met Ser Ala Ile Ser 305 310
315 320 Val His Gly Val Ser Thr Ser Gly Gly Gln Met Tyr His Tyr Asp
Met 325 330 335 Asn Thr Ala Ser Leu Ser Gln Gln Gln Asp Gln Lys Pro
Ile Phe Asn 340 345 350 Val Ile Pro Pro Ile Pro Val Gly Ser Glu Asn
Trp Asn Arg Cys Gln 355 360 365 Gly Ser Gly Asp Asp Asn Leu Thr Ser
Leu Gly Thr Leu Asn Phe Pro 370 375 380 Gly Arg Thr Val Phe Ser Asn
Gly Tyr Ser Ser Pro Ser Met Arg Pro 385 390 395 400 Asp Val Ser Ser
Pro Pro Ser Ser Ser Ser Thr Ala Thr Thr Gly Pro 405 410 415 Pro Pro
Ser Gly Arg Val Gln Glu Glu Leu Cys Leu Val Cys Gly Asp 420 425 430
Arg Ala Ser Gly Tyr His Tyr Asn Ala Leu Thr Cys Gly Ser Cys Lys 435
440 445 Val Phe Phe Arg Arg Ser Val Thr Lys Ser Ala Val Tyr Cys Cys
Lys 450 455 460 Phe Gly Arg Ala Cys Glu Met Asp Met Tyr Met Arg Arg
Lys Cys Gln 465 470 475 480 Glu Cys Arg Leu Lys Lys Cys Leu Ala Val
Gly Met Arg Pro Glu Cys 485 490 495 Val Val Pro Glu Asn Gln Cys Ala
Met Lys Arg Arg Glu Lys Lys Ala 500 505 510 Gln Lys Glu Lys Asp Lys
Met Thr Thr Ser Pro Ser Ser Gln His Gly 515 520 525 Gly Asn Gly Ser
Leu Ala Ser Gly Gly Gly Gln Asp Phe Val Lys Lys 530 535 540 Glu Ile
Leu Asp Leu Met Thr Cys Glu Pro Pro Gln His Ala Thr Ile 545 550 555
560 Pro Leu Leu Pro Asp Glu Ile Leu Ala Lys Cys Gln Ala Arg Asn Ile
565 570 575 Pro Ser Leu Thr Tyr Asn Gln Leu Ala Val Ile Tyr Lys Leu
Ile Trp 580 585 590 Tyr Gln Asp Gly Tyr Glu Gln Pro Ser Glu Glu Asp
Leu Arg Arg Ile 595 600 605 Met Ser Gln Pro Asp Glu Asn Glu Ser Gln
Thr Asp Val Ser Phe Arg 610 615 620 His Ile Thr Glu Ile Thr Ile Leu
Thr Val Gln Leu Ile Val Glu Phe 625 630 635 640 Ala Lys Gly Leu Pro
Ala Phe Thr Lys Ile Pro Gln Glu Asp Gln Ile 645 650 655 Thr Leu Leu
Lys Ala Cys Ser Ser Glu Val Met Met Leu Arg Met Ala 660 665 670 Arg
Arg Tyr Asp His Ser Ser Asp Ser Ile Phe Phe Ala Asn Asn Arg 675 680
685 Ser Tyr Thr Arg Asp Ser Tyr Lys Met Ala Gly Met Ala Asp Asn Ile
690 695 700 Glu Asp Leu Leu His Phe Cys Arg Gln Met Phe Ser Met Lys
Val Asp 705 710 715 720 Asn Val Glu Tyr Ala Leu Leu Thr Ala Ile Val
Ile Phe Ser Asp Arg 725 730 735 Pro Gly Leu Glu Lys Ala Gln Leu Val
Glu Ala Ile Gln Ser Tyr Tyr 740 745 750 Ile Asp Thr Leu Arg Ile Tyr
Ile Leu Asn Arg His Cys Gly Asp Ser 755 760 765 Met Ser Leu Val Phe
Tyr Ala Lys Leu Leu Ser Ile Leu Thr Glu Leu 770 775 780 Arg Thr Leu
Gly Asn Gln Asn Ala Glu Met Cys Phe Ser Leu Lys Leu 785 790 795 800
Lys Asn Arg Lys Leu Pro Lys Phe Leu Glu Glu Ile Trp Asp Val His 805
810 815 Ala Ile Pro Pro Ser Val Gln Ser His Leu Gln Ile Thr Gln Glu
Glu 820 825 830 Asn Glu Arg Leu Glu Arg Ala Glu Arg Met Arg Ala Ser
Val Gly Gly 835 840 845 Ala Ile Thr Ala Gly Ile Asp Cys Asp Ser Ala
Ser Thr Ser Ala Ala 850 855 860 Ala Ala Ala Ala Gln His Gln Pro Gln
Pro Gln Pro Gln Pro Gln Pro 865 870 875 880 Ser Ser Leu Thr Gln Asn
Asp Ser Gln His Gln Thr Gln Pro Gln Leu 885 890 895 Gln Pro Gln Leu
Pro Pro Gln Leu Gln Gly Gln Leu Gln Pro Gln Leu 900 905 910 Gln Pro
Gln Leu Gln Thr Gln Leu Gln Pro Gln Ile Gln Pro Gln Pro 915 920 925
Gln Leu Leu Pro Val Ser Ala Pro Val Pro Ala Ser Val Thr Ala Pro 930
935 940 Gly Ser Leu Ser Ala Val Ser Thr Ser Ser Glu Tyr Met Gly Gly
Ser 945 950 955 960 Ala Ala Ile Gly Pro Ile Thr Pro Ala Thr Thr Ser
Ser Ile Thr Ala 965 970 975 Ala Val Thr Ala Ser Ser Thr Thr Ser Ala
Val Pro Met Gly Asn Gly 980 985 990 Val Gly Val Gly Val Gly Val Gly
Gly Asn Val Ser Met Tyr Ala Asn 995 1000 1005 Ala Gln Thr Ala Met
Ala Leu Met Gly Val Ala Leu His Ser His Gln 1010 1015 1020 Glu Gln
Leu Ile Gly Gly Val Ala Val Lys Ser Glu His Ser Thr Thr 1025 1030
1035 1040 Ala 10 17 DNA Artificial Sequence Description of
Artificial Sequence Modified ecdysone response element 10
rgbnnmnnnn ntgnncy 17 11 17 DNA Artificial Sequence Description of
Artificial Sequence Modified ecdysone response element 11
rgnncannnn nknnvcy 17 12 13 DNA Artificial Sequence Description of
Artificial Sequence Modified ecdysone response element 12
agtgcantgt tct 13 13 17 DNA Artificial Sequence Description of
Artificial Sequence Modified ecdysone response element 13
rgbnnmnnnn nrgbnnm 17 14 49 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 14 tacaacgccc
tcacctgtgg atcctgcaag gtgtttcttt cgacgcagc 49 15 53 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 15 gtactcccgg ggcggggcta tgcggggcgg ggctaatcgc
taggggcggg gca 53 16 53 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 16 gtactgcccc
gcccctagcg attagccccg ccccgcatag ccccgccccg gga 53 17 34 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 17 agctcgatgg acaagtgcat tgttctttgc tgaa 34 18 35
DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 18 agctttcagc aagagaacaa tgcacttgtc catcg
35
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