U.S. patent application number 11/941876 was filed with the patent office on 2008-08-14 for codon optimized synthetic plasmids.
This patent application is currently assigned to VGX PHARMACEUTICLAS, INC.. Invention is credited to Ronald V. Abruzzese, Ruxandra Draghia-Akli, Douglas R. Kern.
Application Number | 20080194511 11/941876 |
Document ID | / |
Family ID | 30115994 |
Filed Date | 2008-08-14 |
United States Patent
Application |
20080194511 |
Kind Code |
A1 |
Draghia-Akli; Ruxandra ; et
al. |
August 14, 2008 |
CODON OPTIMIZED SYNTHETIC PLASMIDS
Abstract
One aspect of the current invention is an optimized synthetic
mammalian expression plasmid (e.g. pAV0201). This new plasmid
comprise a therapeutic element, and a replication element. The
therapeutic element of the new plasmid comprises a eukaryotic
promoter; a 5' untranslated region ("UTR"); a
codon-optimized-eukaryotic therapeutic gene sequence; and a poly
adenylation signal. The therapeutic elements of this plasmid are
operatively linked and located in a first operatively-linked
arrangement. Additionally, the optimized synthetic mammalian
expression plasmid comprises replication elements, wherein the
replication elements are operatively linked and located in a second
operatively-linked arrangement. The replication elements comprise a
selectable marker gene promoter, a ribosomal binding site, and an
origin of replication. The first-operatively-linked arrangement and
the second-operatively-linked arrangement comprise a circular
structure of the codon optimized synthetic mammalian expression
plasmid.
Inventors: |
Draghia-Akli; Ruxandra;
(Houston, TX) ; Abruzzese; Ronald V.; (Leander,
TX) ; Kern; Douglas R.; (The Woodlands, TX) |
Correspondence
Address: |
Pepper Hamilton LLP
400 Berwyn Park, 899 Cassatt Road
Berwyn
PA
19312-1183
US
|
Assignee: |
VGX PHARMACEUTICLAS, INC.
Blue Bell
PA
|
Family ID: |
30115994 |
Appl. No.: |
11/941876 |
Filed: |
November 16, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10619939 |
Jul 15, 2003 |
7316925 |
|
|
11941876 |
|
|
|
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60396247 |
Jul 16, 2002 |
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Current U.S.
Class: |
514/44R |
Current CPC
Class: |
A61K 48/00 20130101;
C12N 15/85 20130101; C12N 2840/203 20130101; C12N 15/67 20130101;
C07K 14/60 20130101; C12N 2830/15 20130101; A61P 5/06 20180101;
A61P 5/02 20180101; C12N 2840/20 20130101; C12N 2830/008
20130101 |
Class at
Publication: |
514/44 |
International
Class: |
A61K 31/711 20060101
A61K031/711 |
Claims
1.-31. (canceled)
32. A method for plasmid mediated gene supplementation comprising:
delivering into a subject a codon optimized synthetic mammalian
expression plasmid; wherein the codon optimized synthetic mammalian
expression plasmid encodes a growth hormone releasing hormone
("GHRH") or functional biological equivalent in the subject.
33. The method of claim 32, wherein delivering into the cells of
the subject the codon optimized synthetic mammalian expression
plasmid is via electroporation.
34. The method of claim 32, wherein the cells of the subject are
somatic cells, stem cells, or germ cells.
35. The method of claim 32, wherein the codon optimized synthetic
mammalian expression plasmid is selected from the group consisting
of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, and SEQ
ID NO:21.
36. The method of claim 32, wherein the encoded GHRH is a
biologically active polypeptide; and the encoded functional
biological equivalent of GHRH is a polypeptide that has been
engineered to contain a distinct amino acid sequence while
simultaneously having similar or improved biologically activity
when compared to the GHRH polypeptide.
37. The method of claim 32, wherein the encoded GHRH or functional
biological equivalent thereof facilitates growth hormone ("GH")
secretion in the subject.
38. The method of claim 32, wherein the codon optimized synthetic
mammalian expression plasmid encodes a protein sequence chosen from
SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID
NO:8.
39. The method of claim 32, wherein the codon optimized synthetic
mammalian expression plasmid comprises a 5' untranslated region
(UTR).
40. The method of claim 39, wherein the 5' UTR comprises a portion
of a human growth hormone 5' UTR.
41. The method of claim 32, wherein the codon optimized synthetic
mammalian expression plasmid comprises an origin of replication
comprising SEQ ID NO:12.
42. The method of claim 32, wherein the codon optimized synthetic
mammalian expression plasmid comprises a prokaryotic ribosomal
binding site ("RBS").
43. The method of claim 32, wherein the codon optimized synthetic
mammalian expression plasmid comprises a eukaryotic poly A
signal.
44. The method of claim 39, wherein the 5' UTR comprises a portion
of a eukaryotic 5' UTR.
45. The method of claim 32, wherein the codon optimized synthetic
mammalian expression plasmid comprises a prokaryotic promoter that
comprises PNEO and a transposon fragment ("Tn5").
46. The method of claim 32, wherein the codon optimized synthetic
mammalian expression plasmid comprises a selectable marker
gene.
47. The method of claim 32, wherein the codon optimized synthetic
mammalian expression plasmid comprises SEQ ID NO: 2.
48. The method of claim 32, wherein the delivering into a subject
is accomplished by administration selected from the group
consisting of: electroporation, a gene gun, and applying large
volumes of liquid.
Description
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent
Application Ser. No. 60/396,247, entitled "Codon Optimized
Synthetic Plasmids," filed on Jul. 16, 2002, the entire content of
which is hereby incorporated by reference.
BACKGROUND
[0002] One aspect of the current invention is an optimized nucleic
acid delivery vehicle, or synthetic expression plasmid. The
synthetic expression plasmid of this invention has reduced
components, and has been optimized to increase efficacy, and reduce
adverse reactions in vivo. In addition to a mammalian gene of
interest, a typical nucleic acid delivery vehicle or synthetic
expression plasmid contains many structural elements necessary for
the in vitro amplification of the plasmid in a bacterial host.
Consequently, some of the inherent bacterial nucleic acid sequences
can cause adverse effects when the amplified plasmid is introduced
into a mammalian host. For example, the presence of CpG sequences
are known to cause both gene silencing and initiate an immune
response in mammals. By utilizing codon optimization, essential
bacterial structural elements (e.g. bacterial antibiotic resistant
genes) are synthetically constructed and used to replace codons
that contained detrimental sequences, but do not effect the final
gene product. The current invention involves a "synthetic plasmid
backbone" (pAV0201) that provides a clean lineage, which is useful
for plasmid supplementation therapy in mammals.
[0003] A plasmid based mammalian expression system is minimally
composed of a plasmid backbone, a synthetic delivery promoter in
addition to the nucleic acid encoding a therapeutic expression
product. A plasmid backbone typically contains a bacterial origin
of replication, and a bacterial antibiotic selection gene, which
are necessary for the specific growth of only the bacteria that are
transformed with the proper plasmid. However, there are plasmids,
called mini-circles, that lack both the antibiotic resistance gene
and the origin of replication (Darquet et al., 1997; Darquet et
al., 1999; Soubrier et al., 1999). The use of in vitro amplified
expression plasmid DNA (i.e. non-viral expression systems) avoids
the risks associated with viral vectors. The non-viral expression
systems products generally have low toxicity due to the use of
"species-specific" components for gene delivery, which minimizes
the risks of immunogenicity generally associated with viral
vectors. One aspect of the current invention is a new, versatile,
and codon optimized plasmid based mammalian expression system that
will reduce the adverse effects associated with prokaryotic nucleic
acid sequences in mammalian hosts. In addition, this new plasmid
will constitute the base of a species-specific library of plasmids
for expression of hormones or other proteins for agricultural and
companion animal applications.
[0004] Codon optimization: Expression of eukaryotic gene products
in prokaryotes is sometimes limited by the presence of codons that
are infrequently used in E. coli. Expression of such genes can be
enhanced by systematic substitution of the endogenous codons with
codons over represented in highly expressed prokaryotic genes.
Although not wanting to be bound by theory, it is commonly thought
that rare codons cause pausing of the ribosome. Pausing of the
ribosome can lead to a failure to complete the nascent polypeptide
chain and a uncoupling of transcription and translation.
Additionally, pausing of the ribosome is thought to expose the 3'
end of the mRNA to cellular ribonucleases. An invention thought to
circumvented such problems for prokaryotic expression of eukaryotic
genes was discussed in U.S. Pat. No. 6,114,148 issued on Sep. 5,
2000 and titled "High level expression of proteins" with Seed, et
al., listed as inventors ("the Seed '148 patent"). The Seed '148
patent features a synthetic gene that encodes a protein normally
expressed in a mammalian cell wherein a non-preferred codon in the
natural gene encoding the protein has been replaced by a preferred
codon encoding the same amino acid. In contrast, the use of
prokaryotic codons in mammalian systems can lead to detrimental
effects (e.g. increased immune response). Furthermore, there are
species specific differences with codons that are preferred, or
less-preferred among species of a genus (Narum et al., 2001). One
aspect of the current invention is the codon optimization of
modified mammalian gene sequences. Publicly available databases for
optimized codons have been referenced in the following articles:
Nagata T, Uchijima M, Yoshida A, Kawashima M, Koide Y. Biochem
Biophys Res Commun 261:445-51 (1999). Codon optimization effect on
translational efficiency of DNA vaccine in mammalian cells:
analysis of plasmid DNA encoding a CTL epitope derived from
microorganisms; Uchijima, M, Yoshida, A, Nagata, T, Koide, Y. J
Immunol 161:5594-9 (1998). Optimization of codon usage of plasmid
DNA vaccine is required for the effective MHC class I-restricted T
cell responses against an intracellular bacterium; Meetei, A R and
Rao, M R. Protein Expr Purif 13:184-90 (1998). Hyperexpression of
rat spermatidal protein TP2 in Escherichia coli by codon
optimization and engineering the vector-encoded 5' UTR; Andre, S,
Seed, B, Eberle, J, Schraut, W, Bultmann, A, Haas, J. J Virol
72:1497-503 (1998). Increased immune response elicited by DNA
vaccination with a synthetic gp120 sequence with optimized codon
usage; Hale, R S and Thompson, G. Protein Expr Purif 12:185-8
(1998). Codon optimization of the gene encoding a domain from human
type 1 neurofibromin protein results in a threefold improvement in
expression level in Escherichia coli; Hubatsch, I, Ridderstrom, M,
Mannervik, B. Biochem J 330:175-9 (1998). Human glutathione
transferase A4-4: an alpha class enzyme with high catalytic
efficiency in the conjugation of 4-hydroxynonenal and other
genotoxic products of lipid peroxidation; Kim, C H, Oh, Y, Lee, T
H. Gene 199:293-301 (1997). Codon optimization for high-level
expression of human erythropoietin (EPO) in mammalian cells; Deng,
T. FEBS Lett 409(2):269-72 (1997). Bacterial expression and
purification of biologically active mouse c-Fos proteins by
selective codon optimization; Cormack, B P, Bertram, G, Egerton, M,
Gow, N A, Falkow, S, Brown, A J. Microbiology 143:303-11 (1997).
Yeast-enhanced green fluorescent protein, a reporter of gene
expression in Candida albicans; Prapunwattana, P, Sirawarapom, W,
Yuthavong, Y, Santi, D V. Mol Biochem Parasitol 83:93-106 (1996)
Chemical synthesis of the Plasmodium falciparum dihydrofolate
reductase-thymidylate synthase gene; Pikaart, M J and Felsenfeld,
G. Protein Expr Purif 8:469-75 (1996). Expression and codon usage
optimization of the erythroid-specific transcription factor cGATA-1
in baculoviral and bacterial systems; Yang, T T, Cheng, L, Kain, S
R. Nucleic Acids Res 24:4592-3 (1996). Optimized codon usage and
chromophore mutations provide enhanced sensitivity with the green
fluorescent protein; Gouka, R J, Punt, P J, Hessing, J G, van den
Hondel, Calif. Appl Environ Microbiol 62:1951-7 (1996). Analysis of
heterologous protein production in defined recombinant Aspergillus
awamori strains; Altmann, S W, Timans, J C, Rock, F L, Bazan, J F,
Kastelein, R A. Protein Expr Purif 6:722-6 (1995). Expression and
purification of a synthetic human obese gene product; Kane, J.,
Current Opinion in Biotechnology 6:494-500 (1995). Effects of rare
codon clusters on gene expression in Escherichia coli; Airenne, K
J, Sarkkinen, P, Punnonen, E L, Kulomaa, M S. Gene, 144:75-80
(1994). Production of recombinant avidin in Escherichia coli; Wang,
B Q, Lei, L, Burton, Z F. Protein Expr Purif 5:476-485 (1994).
Importance of codon preference for production of human RAP74 and
reconstitution of the RAP30/74 complex; Gerchman, S E, Graziano, V,
Ramakrishnan, V. Protein Expr Purif 5:242-51 (1994). Expression of
chicken linker histones in E. coli: sources of problems and methods
for overcoming some of the difficulties; Dittrich, W, Williams, K
L, Slade, M B. Bio/Technology 12:614-8 (1994). Production and
Secretion of Recombinant Proteins in Dictyostelium discoideum;
Holler, T P, Foltin, S K, Ye, Q Z, Hupe, D J. Gene 136:323-8
(1993). HIV1 integrase expressed in Escherichia coli from a
synthetic gene; Kane, J F, Violand, B N, Curran, D F, Staten, N R,
Duffin, K L, Bogosian, G. Nucleic Acids Res 20:6707-12 (1992).
Novel in-frame two codon translational hop during synthesis of
bovine placental lactogen in a recombinant strain of Escherichia
coli; Kotula, L and Curtis, P J. Biotechnology (NY) 9:1386-9
(1991). Evaluation of foreign gene codon optimization in yeast:
expression of a mouse IG kappa chain; Makoff, A J, Oxer, M D,
Romanos, M A, Fairweather, N F, Ballantine, S, Nucleic Acids Res.
17:10191-10202 (1989). Expression of tetanus toxin fragment C in E.
coli: high level expression by removing rare codons; Misra, R and
Reeves, P. Eur J Biochem 152:151-5 (1985). Intermediates in the
synthesis of TolC protein include an incomplete peptide stalled at
a rare Arg codon; Robinson, M, Lilley, R, Little, S, Emtage, J S,
Yarranton, G, Stephens, P, Millican, A, Eaton, M, Humphreys, G.
Nucleic Acids Res 12:6663-71 (1984). Codon usage can affect
efficiency of translation of genes in Escherichia coli; Pedersen,
S. EMBO J 3:2895-8 (1984). Escherichia coli ribosomes translate in
vivo with variable rate.
[0005] As mentioned above, a plasmid backbone typically contains a
bacterial origin of replication, and a bacterial antibiotic
selection gene, which are necessary for the specific growth of only
the bacteria that are transformed with the proper plasmid. However,
the nucleotide sequence of the bacterial gene products can
adversely affect a mammalian host receiving plasmid DNA. For
example, it was desirable to avoid CpG sequences, as these
sequences have been shown to cause a recipient host to have an
immune response (Manders and Thomas, 2000; Scheule, 2000) to
plasmids as well as possible gene silencing (Shi et al., 2002;
Shiraishi et al., 2002). Thus, the DNA coding regions of any
expressed genes avoid the "cg" sequence, without changing the amino
acid sequence. Another aspect of the current invention involves the
removal of unnecessary DNA sequences that were left over from prior
cloning procedures. As a result of codon optimization, and removal
of unnecessary DNA sequences, a synthetically generated plasmid
backbone ("pAV0201") with a unique cloning site that was
constructed to generate a clean lineage of plasmid, which will be
useful for plasmid mediated gene supplementation.
[0006] Growth Hormone ("GH") and Immune Function: Another aspect of
the current invention is utilizing the synthetically generated
plasmid backbone pAV0201 for plasmid mediated gene supplementation.
The central role of growth hormone ("GH") is controlling somatic
growth in humans and other vertebrates, and the physiologically
relevant pathways regulating GH secretion from the pituitary is
well known (Berneis and Keller, 1996). The GH production pathway is
composed of a series of interdependent genes whose products are
required for normal growth (Cuttler, 1996). The GH pathway genes
include: (1) ligands, such as GH and insulin-like growth factor-I
("IGF-I"); (2) transcription factors such as prophet of pit 1, or
prop 1, and pit 1: (3) agonists and antagonists, such as growth
hormone releasing hormone ("GHRH") and somatostatin ("SS"),
respectively; and (4) receptors, such as GHRH receptor ("GHRH-R")
and the GH receptor ("GH-R"). These genes are expressed in
different organs and tissues, including the hypothalamus,
pituitary, liver, and bone. Effective and regulated expression of
the GH pathway is essential for optimal linear growth, as well as
homeostasis of carbohydrate, protein, and fat metabolism GH
synthesis and secretion from the anterior pituitary is stimulated
by GHRH and inhibited by somatostatin, both hypothalamic hormones
(Frohman et al., 1992). GH increases production of IGF-J, primarily
in the liver, and other target organs. IGF-I and GH, in turn,
feedback on the hypothalamus and pituitary to inhibit GHRH and GH
release. GH elicits both direct and indirect actions on peripheral
tissues, the indirect effects being mediated mainly by IGF-I.
[0007] The immune function is modulated by IGF-I (Geffner, 1997;
LeRoith et al., 1996), which has two major effects on B cell
development: potentiation and maturation, and as a B-cell
proliferation cofactor that works together with interlukin-7
("IL-7"). These activities were identified through the use of
anti-IGF-I antibodies, antisense sequences to IGF-I, and the use of
recombinant IGF-I to substitute for the activity. There is evidence
that macrophages are a rich source of IGF-I. The treatment of mice
with recombinant IGF-I confirmed these observations as it increased
the number of pre-B and mature B cells in bone marrow. The mature B
cell remained sensitive to IGF-I as immunoglobulin production was
also stimulated by IGF-I in vitro and in vivo.
[0008] The production of recombinant proteins in the last 2 decades
provided a useful tool for the treatment of many diverse
conditions. For example, GH-deficiencies in short stature children,
anabolic agent in burn, sepsis, and AIDS patients (Carrel and
Allen, 2000; Hart et al., 2001; Lal et al., 2000; Mulligan et al.,
1999). However, resistance to GH action has been reported in
malnutrition and infection (Kotzmann et al., 2001). Long-term
studies on transgenic animals and in patients undergoing GH
therapies have shown no correlation in between GH or IGF-I therapy
and cancer development. GH replacement therapy is widely used
clinically, with beneficial effects, but therapy is associated with
several disadvantages (Blethen, 1995): GH must be administered
subcutaneously or intramuscularly once a day to three times a week
for months, or usually years; insulin resistance and impaired
glucose tolerance (Burgert et al., 2002); accelerated bone
epiphysis growth and closure in pediatric patients (Blethen and
Rundle, 1996).
[0009] In contrast, essentially no side effects have been reported
for recombinant GHRH therapies. Extracranially secreted GHRH, as
mature peptide or truncated molecules (as seen with pancreatic
islet cell tumors and variously located carcinoids) are often
biologically active and can even produce acromegaly (Faglia et al.,
1992; Melmed, 1991). Administration of recombinant GHRH to
GH-deficient children or adult humans augments IGF-I levels,
increases GH secretion proportionally to the GHRH dose, yet still
invokes a response to bolus doses of recombinant GHRH (Bercu et
al., 1997). Thus, GHRH administration represents a more
physiological alternative of increasing subnormal GH and IGF-I
levels (Corpas et al., 1993b).
[0010] GH is released in a distinctive pulsatile pattern that has
profound importance for its biological activity. Secretion of GH is
stimulated by the GHRH, and inhibited by somatostatin, and both
hypothalamic hormones. GH pulses are a result of GHRH secretion
that is associated with a diminution or withdrawal of somatostatin
secretion. In addition, the pulse generator mechanism is timed by
GH-negative feedback. The endogenous rhythm of GH secretion becomes
entrained to the imposed rhythm of exogenous GH administration.
Effective and regulated expression of the GH and insulin-like
growth factor-I ("IGF-I") pathway is essential for optimal linear
growth, homeostasis of carbohydrate, protein, and fat metabolism,
and for providing a positive nitrogen balance. Numerous studies in
humans, sheep or pigs showed that continuous infusion with
recombinant GHRH protein restores the normal GH pattern without
desensitizing GHRH receptors or depleting GH supplies as this
system is capable of feed-back regulation, which is abolished in
the GH therapies. Although recombinant GHRH protein therapy
entrains and stimulates normal cyclical GH secretion with virtually
no side effects (Duck et al., 1992), the short half-life of GHRH in
vivo requires frequent (one to three times a day) intravenous,
subcutaneous or intranasal (requiring 300-fold higher dose)
administration (Evans et al., 1985). Thus, as a chronic treatment,
GHRH administration is not practical.
[0011] Wild type GHRH has a relatively short half-life in the
circulatory system, both in humans and in farm animals (Frohman et
al., 1986). After 60 minutes of incubation in plasma 95% of the
GHRH(1-44)NH.sub.2 is degraded, while incubation of the shorter
(1-40)OH form of the hormone, under similar conditions, shows only
a 77% degradation of the peptide after 60 minutes of incubation
(Frohman et al., 1989a). Incorporation of cDNA coding for a
particular protease-resistant GHRH analog in a gene therapy vector
results in a molecule with a longer half-life in serum
(Draghia-Akli et al., 1999), increased potency, and provides
greater GH release in plasmid-injected animals as described in U.S.
Pat. No. 6,551,996 that was issued on Apr. 23, 2003 titled "Super
Active Porcine Growth Hormone Releasing Hormone Analog" with
Schwartz, et al., listed as inventors, ("the Schwartz '996
patent"), the entire content is herein incorporated by reference.
The Schwartz '996 patent teaches that an application of a GHRH
analog containing mutations that improve the ability to elicit the
release of growth hormone. In addition, the Schwartz '996 patent
relates to the treatment of growth deficiencies; the improvement of
growth performance; the stimulation of production of growth hormone
in an animal at a greater level than that associated with normal
growth; and the enhancement of growth utilizing the administration
of growth hormone releasing hormone analog and is herein
incorporated by reference. Mutagenesis via amino acid replacement
of protease sensitive amino acids prolongs the serum half-life of
the GHRH molecule. Furthermore, the enhancement of biological
activity of GHRH is achieved by using super-active analogs that may
increase its binding affinity to specific receptors as described in
the Schwartz '996 patent.
[0012] Extracranially secreted GHRH, as processed protein species
GHRH(1-40) hydroxy or GHRH(1-44) amide or even as shorter truncated
molecules, are biological active. It has been reported that a low
level of GHRH (100 pg/ml) in the blood supply stimulates GH
secretion (Corpas et al., 1993a). Direct plasmid DNA gene transfer
is currently the basis of many emerging therapeutic strategies and
thus does not require viral genes or lipid particles (Aihara and
Miyazaki, 1998; Lesbordes et al., 2002). Skeletal muscle is a
target tissue because muscle fiber has a long life span and can be
transduced by circular DNA plasmids that express over months or
years in an immunocompetent host (Danko and Wolff, 1994; Wolff et
al., 1992). Previous reports demonstrated that human GHRH cDNA
could be delivered to muscle by an injectable myogenic expression
vector in mice where it transiently stimulated GH secretion over a
period of two weeks in immunocompetent mice (Draghia-Akli et al.,
1997), and for 5 month in immunodeficient mice (Draghia-Akli et
al., 2002)(human hormones are immunogenic in normal immunocompetent
rodents, and transgene expression is transitory in these
cases).
[0013] U.S. Pat. No. 5,061,690 issued on Oct. 29, 1991 and titled
"Method for increasing milk production in mammals and/or increasing
the birth weight of their newborn and improving postnatal growth
"with Kann, et al., listed as inventors, ("the Kann '690 patent").
The Kann '690 patent is directed toward increasing both birth
weight and milk production by supplying to pregnant female mammals
an effective amount of human GHRH or one of it analogs for 10-20
days. Application of the analogs lasts only throughout the
lactation period. However, multiple administrations are presented,
and there is no teachings regarding administration of the growth
hormone releasing hormone a nucleic acid delivery vehicle or a
codon optimized synthetic mammalian expression plasmid.
[0014] U.S. Pat. No. 5,134,120 issued on Jul. 28, 1992 and titled
"Use of growth hormone to enhance porcine weight gain" with Boyd,
et al., listed as inventors, ("the Boyd '120 patent"); and U.S.
Pat. No. 5,292,721 issued on Mar. 8, 1994 and titled "Use of growth
hormone to enhance porcine fetal energy and sow lactation
performance" with Boyd, et al., listed as inventors, ("the Boyd
'721 patent"). Both the Boyd '120, and Boyd 721 patent teach that
by deliberately increasing growth hormone in swine during the last
2 weeks of pregnancy through a 3 week lactation resulted in the
newborn piglets having marked enhancement of the ability to
maintain plasma concentrations of glucose and free fatty acids when
fasted after birth. In addition, the Boyd '120 and Boyd '721
patents teach that treatment of the sow during lactation results in
increased milk fat in the colostrum and an increased milk yield.
These effects are important in enhancing survivability of newborn
pigs and weight gain prior to weaning. However Boyd '120 and Boyd
'721 patents provide no teachings regarding administration of the
growth hormone releasing hormone a nucleic acid delivery vehicle or
a codon optimized synthetic mammalian expression plasmid.
[0015] In summary, previous studies have shown that it is possible
to treat various disease conditions in a limited capacity utilizing
recombinant protein technology, but these treatments have some
significant drawbacks. It has also been taught that nucleic acid
expression plasmids that encode recombinant proteins are viable
solutions to the problems of frequent injections and high cost of
traditional recombinant therapy. However, the nucleic acid
expression plasmids also have some drawbacks when injected into a
mammalian host. The synthetic plasmids of this invention have
reduced components, and have been codon optimized to increase
efficacy, and reduce adverse reactions in vivo. The introduction of
point mutations in to the encoded recombinant proteins was a
significant step forward in producing proteins that are more stable
in vivo than the wild type counterparts. Since there is a need in
the art to expanded treatments for subjects with a disease by
utilizing nucleic acid expression constructs that are delivered
into a subject and express stable therapeutic proteins in vivo, the
combination of codon optimization of an encoded therapeutic
mammalian gene in an optimized plasmid backbone will further
enhance the art of plasmid mediated gene supplementation.
SUMMARY
[0016] One aspect of the current invention is an optimized
synthetic mammalian expression plasmid (e.g. pAV0201). This new
plasmid comprises a therapeutic element, and a replication element.
The therapeutic element of the new plasmid comprises a eukaryotic
promoter; a 5' untranslated region ("UTR"); a
codon-optimized-eukaryotic therapeutic gene sequence; and a
polyadenylation signal. The therapeutic elements of this plasmid
are operatively linked and located in a first operatively-linked
arrangement. Additionally, the optimized synthetic mammalian
expression plasmid comprises replication elements, wherein the
replication elements are operatively linked and located in a second
operatively-linked arrangement. The replication elements comprise a
selectable marker gene promoter, a ribosomal binding site, a
optimized selectable marker gene sequence, and an origin of
replication. The first-operatively-linked arrangement and the
second-operatively-linked arrangement comprise a circular structure
of the codon optimized synthetic mammalian expression plasmid.
[0017] In preferred embodiments, the synthetic mammalian expression
plasmid comprises a pUC-18 prokaryotic origin of replication
sequence. However, the origin of replication may also comprise an
autonomously replication sequence ("ARS"). In a preferred
embodiment, the optimized prokaryotic antibiotic resistant gene
comprises kanamycin. In another preferred embodiment, the poly
adenylation signal ("PolyA") comprises a human growth hormone
("hGH") poly A signal, and a hGH 5' untranslated region ("5'UTR").
The codon optimized mammalian therapeutic gene sequence comprises a
sequence that encodes a modified species specific growth hormone
releasing hormone ("GHRH"). In preferred embodiments, the codon
optimized sequence comprises porcine, mouse, rat, bovine, ovine,
and chicken GHRH (e.g. Seq ID#4, Seq ID#5; Seq ID#6; Seq ID#7; Seq
ID#8; and Seq ID#9). Similarly, species specific, and codon
optimized plasmids are disclosed (e.g. SEQ ID#17; SEQ ID#18; SEQ
ID#19; SEQ ID#20; and SEQ ID#21).
[0018] Another aspect of the current invention is a method for
plasmid mediated gene supplementation that comprises delivering a
codon optimized synthetic mammalian expression plasmid into a
subject. The codon optimized synthetic mammalian expression plasmid
encodes a growth hormone releasing hormone ("GHRH") or functional
biological equivalent in the subject. The method of delivering the
codon optimized synthetic mammalian expression plasmid into the
cells of the subject is via electroporation. In a preferred
embodiment, the cells of the subject can be somatic cells, stem
cells, or germ cells. The codon optimized synthetic mammalian
expression plasmids consisting of Seq ID#17, Seq ID#18, Seq ID#19,
Seq ID#20, and Seq ID#21 have been contemplated by the inventors.
The encoded GHRH is a biologically active polypeptide; and the
encoded functional biological equivalent of GHRH is a polypeptide
that has been engineered to contain a distinct amino acid sequence
while simultaneously having similar or improved biologically
activity when compared to the GHRH polypeptide. One result of
expressing the encoded GHRH or functional biological equivalent
thereof in a subject is the facilitation of growth hormone ("GH")
secretion in the subject.
BRIEF DESCRIPTION OF FIGURES
[0019] FIG. 1 shows a general map of a plasmid construct (pAV0125,
this plasmid contains the porcine modified HV-GHRH sequence) used
prior construction of an optimized synthetic plasmid of the current
invention;
[0020] FIG. 2 shows a general map of a synthetic plasmid construct
(pAV0201, this construct contains the porcine modified GHRH called
HV-GHRH) of the current invention, which contains codon
optimization;
[0021] FIG. 3 shows the optimized nucleic acid sequence for the
kanamycin gene and the corresponding translated amino acid
sequence;
[0022] FIG. 4 shows a schematic map of a 228 bp synthetic nucleic
acid sequence for mouse GHRH ("mGHRH");
[0023] FIG. 5 shows the optimized nucleic acid sequence for the
mGHRH gene and the corresponding translated amino acid
sequence;
[0024] FIG. 6 shows the optimized nucleic acid sequence for the
original mGHRH gene ("GHRH-m-ori"), and the optimized mGHRH gene
("GHRH-m-opt") after removing some CpG islands and other motifs
that can decrease protein expression, the changes did not effect
the amino acid sequence;
[0025] FIG. 7 shows a comparison of the translated amino acid
sequence from the original ("GHRH-m-Ori") and optimized nucleic
acid sequence for the mouse GHRH gene ("GHRH-m-Opti");
[0026] FIG. 8 shows a schematic map of a 231 bp synthetic nucleic
acid sequence for rat GHRH ("rGHRH");
[0027] FIG. 9 shows the optimized nucleic acid sequence for the
rGHRH gene and the corresponding translated amino acid
sequence;
[0028] FIG. 10 shows the optimized nucleic acid sequence for the
original rGHRH gene ("GHRH-R-ori"), and the optimized rGHRH gene
("GHRH-R-opt") after removing some CpG islands and other motifs
that can decrease protein expression, the changes did not effect
the amino acid sequence;
[0029] FIG. 11 shows a comparison of the translated amino acid
sequence from the original ("GHRH-R-Ori") and optimized nucleic
acid sequence for the rat GHRH gene ("GHRH-R-Opti");
[0030] FIG. 12 shows a schematic map of a 222 bp synthetic nucleic
acid sequence for bovine GHRH ("bGHRH");
[0031] FIG. 13 shows the optimized nucleic acid sequence for the
bGHRH gene and the corresponding translated amino acid
sequence;
[0032] FIG. 14 shows the optimized nucleic acid sequence for the
original bGHRH gene ("GHRH-B-ori"), and the optimized bGHRH gene
("GHRH-B-opt") after removing some CpG islands and other motifs
that can decrease protein expression, the changes did not effect
the amino acid sequence;
[0033] FIG. 15 shows a comparison of the translated amino acid
sequence from the original ("GHRH-B-Ori") and optimized nucleic
acid sequence for the bovine GHRH gene ("GHRH-B-Opti");
[0034] FIG. 16 shows a schematic map of a 222 bp synthetic nucleic
acid sequence for ovine GHRH ("oGHRH");
[0035] FIG. 17 shows the optimized nucleic acid sequence for the
oGHRH gene and the corresponding translated amino acid
sequence;
[0036] FIG. 18 shows the optimized nucleic acid sequence for the
original oGHRH gene ("GHRH--O-ori"), and the optimized oGHRH gene
("GHRH--O-opt") after removing some CpG islands and other motifs
that can decrease protein expression, the changes did not effect
the amino acid sequence;
[0037] FIG. 19 shows a comparison of the translated amino acid
sequence from the original ("GHRH-O-Ori") and optimized nucleic
acid sequence for the ovine GHRH gene ("GHRH--O-Opti");
[0038] FIG. 20 shows a schematic map of a 234 bp synthetic nucleic
acid sequence for chicken GHRH ("cGHRH");
[0039] FIG. 21 shows the optimized nucleic acid sequence for the
cGHRH gene and the corresponding translated amino acid
sequence;
[0040] FIG. 22 shows the optimized nucleic acid sequence for the
original cGHRH gene ("GHRH-Chi-ori"), and the optimized cGHRH gene
("GHRH-Chi-opt") after removing some CpG islands and other motifs
that can decrease protein expression, the changes did not effect
the amino acid sequence;
[0041] FIG. 23 shows a comparison of the translated amino acid
sequence from the original ("GHRH-Chi-Ori") and optimized nucleic
acid sequence for the chicken GHRH gene ("GHRH-Chi-Opti");
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0042] Terms:
[0043] The term "coding region" as used herein refers to any
portion of the DNA sequence that is transcribed into messenger RNA
(mRNA) and then translated into a sequence of amino acids
characteristic of a specific polypeptide.
[0044] The term "analog" as used herein includes any mutant of
GHRH, or synthetic or naturally occurring peptide fragments of
GHRH.
[0045] The term "codon" as used herein refers to any group of three
consecutive nucleotide bases in a given messenger RNA molecule, or
coding strand of DNA that specifies a particular amino-acid, a
starting or stopping signal for translation. The term codon also
refers to base triplets in a DNA strand.
[0046] The term "delivery" as used herein is defined as a means of
introducing a material into a subject, a cell or any recipient, by
means of chemical or biological process, injection, mixing,
electroporation, sonoporation, orcombinationthereof, either under
orwithout pressure.
[0047] The term "encoded GHRH" as used herein is a biologically
active polypeptide.
[0048] The term "functional biological equivalent" of GHRH as used
herein is a polypeptide that has been engineered to contain a
distinct amino acid sequence while simultaneously having similar or
improved biologically activity when compared to the GHRH
polypeptide.
[0049] The term "heterologous nucleic acid sequence" as used herein
is defined as a DNA sequence consisting of differing regulatory and
expression elements.
[0050] The term "growth hormone" ("GH") as used herein is defined
as a hormone that relates to growth and acts as a chemical
messenger to exert its action on a target cell.
[0051] The term "growth hormone releasing hormone" ("GHRH") as used
herein is defined as a hormone that facilitates or stimulates
release of growth hormone, and in a lesser extent other pituitary
hormones, as prolactin.
[0052] The term "non-optimized codon" as used herein refers to a
codon that does not have a match codon frequencies in target and
host organisms. The non-optimized codons of this invention were
determined using Aptagen's Gene Forge.RTM. codon optimization and
custom gene synthesis platform (Aptagen, Inc., 2190 Fox Mill Rd.
Suite 300, Herndon, Va. 20171). Other publicly available databases
for optimized codons are available and will work equally as
well.
[0053] The term "nucleic acid expression construct" as used herein
refers to any type of genetic construct comprising a nucleic acid
coding for a RNA capable of being transcribed. The term "expression
vector" or "expression plasmid" can also be used
interchangeably.
[0054] The term "operatively linked" as used herein refers to
elements or structures in a nucleic acid sequence that are linked
by operative ability and not physical location. The elements or
structures are capable of, or characterized by accomplishing a
desired operation. It is recognized by one of ordinary skill in the
art that it is not necessary for elements or structures in a
nucleic acid sequence to be in a tandem or adjacent order to be
operatively linked.
[0055] The term "optimized codon" as used herein refers to a codon
that has a match codon frequencies in target and host organisms,
but does not alter the amino acid sequence of the original
translated protein. The optimized codons of this invention were
determined using Aptagen's Gene Forge.RTM. codon optimization and
custom gene synthesis platform (Aptagen, Inc., 2190 Fox Mill Rd.
Suite 300, Herndon, Va. 20171). Other publicly available databases
for optimized codons are available and will work equally as
well.
[0056] The term "optimized nucleic acid delivery vehicle" as used
herein refers to any vector that delivers a nucleic acid into a
cell or organism wherein at least one of the codons has been
optimized for expression in a host organism. The term "synthetic
expression plasmid" can also be used interchangeably with the term
optimized nucleic acid delivery vehicle.
[0057] The term "promoter" as used herein refers to a sequence of
DNA that directs the transcription of a gene. A promoter may direct
the transcription of a prokaryotic or eukaryotic gene. A promoter
may be "inducible", initiating transcription in response to an
inducing agent or, in contrast, a promoter may be "constitutive",
whereby an inducing agent does not regulate the rate of
transcription. A promoter may be regulated in a tissue-specific or
tissue-preferred manner, such that it is only active in
transcribing the operable linked coding region in a specific tissue
type or types.
[0058] The term "replication element" as used herein comprises
nucleic acid sequences that will lead to replication of a plasmid
in a specified host. One skilled in the art of molecular biology
will recognize that the replication element may include, but is not
limited to a selectable marker gene promoter, a ribosomal binding
site, a selectable marker gene sequence, and a origin of
replication.
[0059] The term "subject" as used herein refers to any species of
the animal kingdom. In preferred embodiments it refers more
specifically to humans and animals used for: pets (e.g. cats, dogs,
etc.); work (e.g. horses, cows, etc.); food (chicken, fish, lambs,
pigs, etc); and all others known in the art.
[0060] The term "therapeutic element" as used herein comprises
nucleic acid sequences that will lead to an in vivo expression of
an encoded gene product. One skilled in the art of molecular
biology will recognize that the therapeutic element may include,
but is not limited to a promoter sequence, a poly A sequence, or a
3' or 5' UTR.
[0061] The term "vector" as used herein refers to any vehicle that
delivers a nucleic acid into a cell or organism. Examples include
plasmid vectors, viral vectors, liposomes, or cationic lipids.
[0062] The new synthetic constructs of the current invention are
injected intramuscularly into a correspondent species. For example,
the bovine GHRH ("bGHRH") construct is utilized in cows, and ovine
GHRH ("oGHRH") construct is utilized in sheep. Although not wanting
to be bound by theory, the ovine GHRH will be produced by the sheep
muscle fibers, and then delivered into the circulatory system. The
circulating hormone will enhance the synthesis and secretion of
ovine growth hormone in the anterior pituitary. The new synthetic
constructs can promote long-term expression because the new plasmid
backbone lacks CpG islands and other bacterial components that
alert the immune system of the presence of a foreign antigen. By
decreasing the immune response against the plasmid fragment and its
products can function in the muscle cells for longer durations of
time, which lowers cost of treatment by decreasing the number of
treatments. Furthermore, the usage of species-specific transgene
will ensure long term expression by the lack of neutralizing
antibodies against a foreign GHRH.
[0063] Plasmid mediated gene supplementation. The delivery of
specific genes to somatic tissue in a manner that can correct
inborn or acquired deficiencies and imbalances has been
demonstrated in prior art. Plasmid mediated gene supplementation
offers a number of advantages over the administration of
recombinant proteins. These advantages include the conservation of
native protein structure, improved biological activity, avoidance
of systemic toxicities, and avoidance of infectious and toxic
impurities. In addition, plasmid mediated gene supplementation
allows for prolonged exposure to the protein in the therapeutic
range, because the newly secreted protein is present continuously
in the blood circulation.
[0064] Although not wanting to be bound by theory, the primary
limitation of using a recombinant protein is the limited
availability of protein after each administration. Plasmid mediated
gene supplementation using injectable DNA plasmid expression
vectors overcomes this drawback, because a single injection into
the subject's skeletal muscle permits physiologic expression for
extensive periods of time. Injection of the vectors can promote the
production of enzymes and hormones in animals in a manner that more
closely mimics the natural process. Furthermore, among the
non-viral techniques for gene transfer in vivo, the direct
injection of plasmid DNA into muscle tissue is simple, inexpensive,
and safe.
[0065] In a plasmid based expression system, a non-viral gene
vector may be composed of a synthetic gene delivery system in
addition to the nucleic acid encoding a therapeutic gene product.
In this way, the risks associated with the use of most viral
vectors can be avoided. Additionally, no integration of plasmid
sequences into host chromosomes has been reported in vivo to date,
so that this type of gene transfer should neither activate
oncogenes nor inactivate tumor suppressor genes. As episomal
systems residing outside the chromosomes, plasmids have defined
pharmacokinetics and elimination profiles, leading to a finite
duration of gene expression in target tissues.
[0066] One aspect of the current invention is a new, versatile, and
codon optimized plasmid based mammalian expression system that will
reduce the adverse effects associated with prokaryotic nucleic acid
sequences in mammalian hosts. In addition, this new plasmid will
constitute the base of a species-specific library of plasmids for
expression of hormones or other proteins for agricultural and
companion animal applications. The synthetic expression plasmid of
this invention has reduced components, and has been optimized to
increase efficacy, and reduce adverse reactions in vivo. In
addition to a mammalian gene of interest, a typical nucleic acid
delivery vehicle or synthetic expression plasmid contains many
structural elements useful for the in vitro amplification of the
plasmid in a bacterial host. Consequently, some of the inherent
bacterial nucleic acid sequences can cause adverse effects when the
amplified plasmid is introduced into a mammalian host. For example,
the presence of CpG sequences are known to cause both gene
silencing and initiate an immune response in mammals. By utilizing
codon optimization, essential bacterial structural elements (e.g.
bacterial antibiotic resistant genes) are synthetically constructed
and used to replace codons that contained detrimental sequences,
but do not effect the final gene product. The current invention
involves a "synthetic plasmid backbone" (pAV0201) that provides a
clean lineage, which is useful for plasmid supplementation therapy
in mammals.
[0067] A plasmid based mammalian expression system is minimally
composed of a plasmid backbone, a synthetic delivery promoter in
addition to the nucleic acid encoding a therapeutic expression
product. A plasmid backbone typically contains a bacterial origin
of replication, and a bacterial antibiotic selection gene, which
are necessary for the specific growth of only the bacteria that are
transformed with the proper plasmid. However, there are plasmids
that lack both the antibiotic resistance gene and the origin of
replication, such plasmids are called mini-circles (Darquet et al.,
1997; Darquet et al., 1999; Soubrier et al., 1999). The use of in
vitro amplified expression plasmid DNA (i.e. non-viral expression
systems) avoids the risks associated with viral vectors. The
non-viral expression systems products generally have low toxicity
due to the use of "species-specific" components for gene delivery,
which minimizes the risks of immunogenicity generally associated
with viral vectors. One aspect of the current invention is a new,
versatile, and codon optimized plasmid based mammalian expression
system, which will constitute the base of a species-specific
library of plasmids for expression of hormones or other proteins
for agricultural and companion animal applications. For example,
optimized synthetic sequences can be produced such that codon
frequencies are matched in target and host organisms to ensure
proper folding. A bias of GC content can be used to increase mRNA
stability or reduce secondary structures. Tandem repeat codons or
base runs that may impair the gene can be minimized with codon
optimization. Modification of ribosome binding sites and mRNA
degradation sites can be utilized. Optimization can also reduce or
eliminate problem secondary structures within the transcribed
mRNA.
[0068] Vectors. One skilled in the art recognizes that expression
vectors derived from various bacterial plasmids may be used for
delivery of nucleotide sequences to a targeted organ, tissue or
cell population. Methods which are well known to those skilled in
the art can be used to construct recombinant vectors that will
express a gene of interest or a gene encoding a growth hormone
releasing hormone analog. Transient expression may last for a month
or more with a non-replicating vector and even longer if
appropriate replication elements are a part of the vector system,
wherein the term "vector" is used to refer to a carrier nucleic
acid molecule into which a nucleic acid sequence can be inserted
for introduction into a cell where the vector can be replicated and
the nucleic acid sequence can be expressed. The term vector can
also be referred to as a nucleic acid construct. A nucleic acid
sequence can be "exogenous," which means that it is foreign to the
cell into which the vector is being introduced or that the sequence
is homologous to a sequence in the cell but in a position within
the host cell nucleic acid in which the sequence is ordinarily not
found. Vectors include plasmids, cosmids, and artificial
chromosomes (e.g., YACs). One of skill in the art would be well
equipped to construct a vector through standard recombinant
techniques, which are described in Maniatis et al., 1988 the
entirety is incorporated herein by reference. The selected
expressed nucleic acid sequences of a constructed vector could then
be codon optimized as described below.
[0069] The term "expression vector" refers to a vector or nucleic
acid expression construct containing a nucleic acid sequence coding
for at least part of a gene product capable of being transcribed.
In a specific embodiment the nucleic acid sequence encodes part or
all of GHRH. In some cases, RNA molecules are then translated into
a protein, polypeptide, or peptide. In other cases, these sequences
are not translated, for example, in the production of antisense
molecules or ribozymes. Expression vectors can contain a variety of
"control sequences," which refer to nucleic acid sequences
necessary for the transcription and possibly translation of an
operably linked coding sequence in a particular host organism. In
addition to control sequences that govern transcription and
translation, vectors and expression vectors may contain nucleic
acid sequences that serve other functions as well and are described
infra.
[0070] In a preferred embodiment, the nucleic acid construction
construct or vector of the present invention is a plasmid which
comprises a synthetic myogenic (muscle-specific) promoter, a
synthetic nucleotide sequence encoding a growth hormone releasing
hormone or its analog, and a 3 untranslated region. In other
alternative embodiments, optimized porcine growth hormone,
optimized human growth hormone, optimized mouse growth hormone,
optimized rat growth hormone, optimized bovine growth hormone,
optimized ovine growth hormone, optimized chicken growth hormone,
or skeletal alpha actin 3 untranslated regions are utilized in the
vector.
[0071] Promoters and Enhancers. A "promoter" is a control sequence
that is a region of a nucleic acid sequence at which initiation and
rate of transcription are controlled. It may contain genetic
elements at which regulatory proteins and molecules may bind such
as RNA polymerase and other transcription factors. The phrases
"operatively positioned," "operatively linked," "under control,"
and "under transcriptional control" mean that a promoter is in a
correct functional location and/or orientation in relation to a
nucleic acid sequence to control transcriptional initiation and/or
expression of that sequence. A promoter may or may not be used in
conjunction with an "enhancer," which refers to a cis-acting
regulatory sequence involved in the transcriptional activation of a
nucleic acid sequence.
[0072] A promoter may be one of naturally-coding sequences located
upstream of the coding segment and/or exon. Such a promoter can be
referred to as "endogenous." Similarly, an enhancer may be one
naturally associated with a nucleic acid sequence, located either
downstream or upstream of that sequence. Alternatively, certain
advantages will be gained by positioning the coding nucleic acid
segment under the control of a recombinant or heterologous
promoter, which refers to a promoter that is not normally
associated with a nucleic acid sequence in its natural environment.
A recombinant or heterologous enhancer refers also to an enhancer
not normally associated with a nucleic acid sequence in its natural
environment. Such promoters or enhancers may include promoters or
enhancers of other genes, and promoters or enhancers isolated from
any other prokaryotic, viral, or eukaryotic cell, and promoters or
enhancers not "naturally occurring," i.e., containing different
elements of different transcriptional regulatory regions, and/or
mutations that alter expression. In addition to producing nucleic
acid sequences of promoters and enhancers synthetically, sequences
may be produced using recombinant cloning and/or nucleic acid
amplification technology, including PCR.TM.. Furthermore, it is
contemplated the control sequences that direct transcription and/or
expression of sequences within non-nuclear organelles such as
mitochondria, chloroplasts, and the like, can be employed as
well.
[0073] Naturally, it will be important to employ a promoter and/or
enhancer that effectively directs the expression of the DNA segment
in the cell type, organelle, and organism chosen for expression.
Those of skill in the art of molecular biology generally know the
use of promoters, enhancers, and cell type combinations for protein
expression. The promoters employed may be constitutive,
tissue-specific, inducible, and/or useful under the appropriate
conditions to direct high level expression of the introduced DNA
segment, such as is advantageous in the large-scale production of
recombinant proteins and/or peptides. The promoter may be
heterologous or endogenous. In a specific embodiment the promoter
is a synthetic myogenic promoter.
[0074] The identity of tissue-specific promoters or elements, as
well as assays to characterize their activity, is well known to
those of skill in the art. Examples of such regions include the
human LIMK2 gene, the somatostatin receptor 2 gene, murine
epididymal retinoic acid-binding gene, human CD4, mouse alpha2 (XI)
collagen, DIA dopamine receptor gene, insulin-like growth factor
II, human platelet endothelial cell adhesion molecule-1.
[0075] Initiation Signals and Internal Ribosome Binding Sites. A
specific initiation signal also may be required for efficient
translation of coding sequences. These signals include the ATG
initiation codon or adjacent sequences. Exogenous translational
control signals, including the ATG initiation codon, may need to be
provided. One of ordinary skill in the art would readily be capable
of determining this and providing the necessary signals. It is well
known that the initiation codon must be "in-frame" with the reading
frame of the desired coding sequence to ensure translation of the
entire insert. The exogenous translational control signals and
initiation codons can be either natural or synthetic. The
efficiency of expression may be enhanced by the inclusion of
appropriate transcription enhancer elements.
[0076] In certain embodiments of the invention, the use of internal
ribosome entry sites (IRES) elements are used to create multigene,
or polycistronic, messages. IRES elements are able to bypass the
ribosome scanning model of 5' methylated Cap dependent translation
and begin translation at internal sites. IRES elements from two
members of the picornavirus family (polio and encephalomyocarditis)
have been described, as well an IRES from a mammalian message. IRES
elements can be linked to heterologous open reading frames.
Multiple open reading frames can be transcribed together, each
separated by an IRES, creating polycistronic messages. By virtue of
the IRES element, each open reading frame is accessible to
ribosomes for efficient translation. Multiple genes can be
efficiently expressed using a single promoter/enhancer to
transcribe a single message.
[0077] Multiple Cloning Sites. Vectors can include a multiple
cloning site ("MCS"), which is a nucleic acid region that contains
multiple restriction enzyme sites, any of which can be used in
conjunction with standard recombinant technology to digest the
vector. "Restriction enzyme digestion" refers to catalytic cleavage
of a nucleic acid molecule with an enzyme that functions only at
specific locations in a nucleic acid molecule. Many of these
restriction enzymes are commercially available. Use of such enzymes
is widely understood by those of skill in the art. Frequently, a
vector is linearized or fragmented using a restriction enzyme that
cuts within the MCS to enable exogenous sequences to be ligated to
the vector. "Ligation" refers to the process of forming
phosphodiester bonds between two nucleic acid fragments, which may
or may not be contiguous with each other. Techniques involving
restriction enzymes and ligation reactions are well known to those
of skill in the art of recombinant technology.
[0078] Splicing Sites. Most transcribed eukaryotic RNA molecules
will undergo RNA splicing to remove introns from the primary
transcripts. Vectors containing genomic eukaryotic sequences may
require donor and/or acceptor splicing sites to ensure proper
processing of the transcript for protein expression.
[0079] Polyadenylation Signals. In expression, one will typically
include a polyadenylation signal to effect proper polyadenylation
of the transcript. The nature of the polyadenylation signal is not
believed to be crucial to the successful practice of the invention,
and/or any such sequence may be employed. Preferred embodiments
include the bovine or human growth hormone polyadenylation signal,
convenient and/or known to function well in various target cells.
Also contemplated as an element of the expression cassette is a
transcriptional termination site. These elements can serve to
enhance message levels and/or to minimize read through from the
cassette into other sequences.
[0080] Origins of Replication. In order to propagate a vector in a
host cell, it may contain one or more origins of replication sites
(often termed "ori"), which is a specific nucleic acid sequence at
which replication is initiated. Alternatively an autonomously
replicating sequence (ARS) can be employed if the host cell is
yeast.
[0081] Selectable and Screenable Markers. In certain embodiments of
the invention, the cells that contain the nucleic acid construct of
the present invention may be identified in vitro or in vivo by
including a marker in the expression vector. Such markers would
confer an identifiable change to the cell permitting easy
identification of cells containing the expression vector.
Generally, a selectable marker is one that confers a property that
allows for selection. A positive selectable marker is one in which
the presence of the marker allows for its selection, while a
negative selectable marker is one in which its presence prevents
its selection. An example of a positive selectable marker is a drug
resistance marker, such as the antibiotic resistance gene on the
plasmid constructs (such as kanamycin, ampicylin, gentamycin,
tetracycline, or chloramphenicol).
[0082] Usually the inclusion of a drug selection marker aids in the
cloning and identification of transformants, for example, genes
that confer resistance to neomycin, puromycin, hygromycin, DHFR,
GPT, zeocin and histidinol are useful selectable markers. In
addition to markers conferring a phenotype that allows for the
discrimination of transformants based on the implementation of
conditions, other types of markers including screenable markers
such as GFP, whose basis is calorimetric analysis, are also
contemplated. Alternatively, screenable enzymes may be utilized.
One of skill in the art would also know how to employ immunologic
markers, possibly in conjunction with FACS analysis. The marker
used is not believed to be important, so long as it is capable of
being expressed simultaneously with the nucleic acid encoding a
gene product. Further examples of selectable and screenable markers
are well known to one of skill in the art.
[0083] The invention may be better understood with reference to the
following examples, which are representative of some of the
embodiments of the invention, and are not intended to limit the
invention.
EXAMPLE 1
[0084] Optimized Plasmid Backbone. One aspect of the current
invention is the optimized plasmid backbone. The new synthetic
plasmids presented below contain eukaryotic sequences that are
synthetically optimized for species specific mammalian
transcription. An existing pSP-HV-GHRH plasmid ("pAV0125") (Seq
ID#1), as shown in FIG. 1 was synthetically optimized to form a new
plasmid ("pAV0201")(Seq ID#2). The plasmid pAV0125 was described in
U.S. Pat. No. 6,551,996 that was issued on Apr. 23, 2003 titled
"Super Active Porcine Growth Hormone Releasing Hormone Analog" with
Schwartz, et al., listed as inventors, ("the Schwartz '996
patent"). This 3,534 bp plasmid pAV0125 (Seq ID #1) contains a
plasmid backbone with various component from different commercially
available plasmids, for example, a synthetic promoter SPc5-12 (Seq
ID #15), a modified porcine GHRH sequence (Seq ID #4), and a 3' end
of human growth hormone (Seq ID #10). The new optimized synthetic
expression vector (Seq ID #2) contains 2,739 bp and is shown in
FIG. 2. The therapeutic encoded gene for the optimized plasmid in
FIG. 2 may also include optimized nucleic acid sequences that
encode the following modified GHRH molecules.
TABLE-US-00001 ENCODED GHRH AMINO ACID SEQUENCE wt-GHRH
YADAIFTNSYRKVLGQLSARKLLQDIMSRQQGERNQE QGA-OH HV-GHRH
HVDAIFTNSYRKVLAQLSARKLLQDILNRQQGERNQEQ GA-OH TI-GHRH
YIDAIFTNSYRKVLAQLSARKLLQDILNRQQGERNQEQ GA-OH TV-GHRH
YVDAIFTNSYRKVLAQLSARKLLQDILNRQQGERNQEQ GA-OH 15/27/28-GHRH
YADAIFTNSYRKVLAQLSARKLLQDILNRQQGERNQE QGA-OH
[0085] In general, the encoded GHRH or functional biological
equivalent thereof is of formula:
-A.sub.-1-A.sub.2-DAIFTNSYRKVL-A.sub.3-QLSARKLLQDI-A.sub.4-A.sub.5-RQQGE-
RNQEQGA-OH
wherein: A.sub.1 is a D- or L-isomer of an amino acid selected from
the group consisting of tyrosine ("Y"), or histidine ("H"); A.sub.2
is a D- or L-isomer of an amino acid selected from the group
consisting of alanine ("A"), valine ("V"), or isoleucine ("I");
A.sub.3 is a D- or L-isomer of an amino acid selected from the
group consisting of alanine ("A") or glycine ("G"); A.sub.4 is a D-
or L-isomer of an amino acid selected from the group consisting of
methionein ("M"), or leucine ("L"); A.sub.5 is a D- or L-isomer of
an amino acid selected from the group consisting of serine ("S") or
asparagines ("N").
[0086] An example of this new optimized synthetic expression vector
was denoted as pAV0201 (Seq ID#2). In order to construct pAV0201
(Seq ID#2), the unwanted sequences from the pAV0125 (Seq ID#1) were
initially removed. A software program called Vector NTI (version
7.0) was used to generate and match sequences that could be
compared and were known to be extraneous (e.g. LacZ promoter).
There are many programs such as Vector NTI (version 7.0) that are
known in the art and could have been used with similar results to
compare and identify specific nucleic acid sequences. Once the
extraneous DNA sequences were identified in the pAV0125 plasmid,
they were removed by from the plasmid creating a truncated-pAV0125
plasmid. The Gene Forge.RTM. optimized synthetic sequences were
used to produced codon frequencies that were matched in target and
host organisms to ensure proper folding. Gene Forge.RTM. was also
used to identify and correct a number of deleterious structural
elements in the relevant nucleic acid sequences. For example, a
bias of GC content can be used to increase mRNA stability or reduce
secondary structures; tandem repeat codons or base runs that may
impair the gene can be minimized with codon optimization;
modification of ribosome binding sites and mRNA degradation sites
can be utilized; codon optimization can also reduce or eliminate
problem secondary structures within the transcribed mRNA. Although
Gene Forge.RTM. is a proprietary product of Aptagen that speeds
codon optimization analysis, publicly available databases are
available that allow a person with average skill in the art to
replicate codon optimization protocol.
[0087] The pAV0125 plasmid contained a human Growth Hormone poly
adenylation region that was approximately 618 bp. The original 618
bp region contained multiple poly adenylation sites and was reduced
to only one. As a result over 400 bp were removed to an optimized
length of 190 bp (Seq ID #10). Another 210 bp poly A site is Seq ID
#16. The origin of replication (Seq ID #12) was not altered.
[0088] A summary of the changes made to the pAV0125 plasmid
backbone changes are as follows:
[0089] 1. Although not wanting to be bound by theory, CpG islands
are known to enhance immune responses, and are used to boost immune
responses in vaccines (Manders and Thomas, 2000; McCluskie et al.,
2000; Scheule, 2000)), the Gene Forge.RTM. system can identified
and removed as many CpG island as possible without changing the
translated amino acid sequence. Additionally, a Nco I site was
removed from the Kanamycin sequence without altering the amino acid
sequence. Currently the NcoI is an unique site, which makes the
plasmid backbone more versatile.
[0090] 2. The lacZ promoter region that was located downstream of
the hGH polyA site was determined to be unnecessary, and it was
subsequently removed.
[0091] 3. A portion of the hGH polyA region was removed to produce
a more compact plasmid that is able to accommodate longer DNA
fragments or transgenes.
[0092] 4. A 118 bp portion of the lacZ coding sequence that was
located between the KanR gene and the C5-12 synthetic promoter was
determined to be unnecessary, and it was subsequently removed.
[0093] As a result of the above modifications to the plasmid
backbone, a new synthetic plasmid as shown in FIG. 2 was
constructed. The pAV0201 optimized plasmid comprises a 2,739 bp
circular plasmid (Seq ID#2). The pAV0201 plasmid contains at least
one eukaryotic coding region, and at least one prokaryotic coding
sequence, wherein it has been contemplated that the eukaryotic
coding region contains a modified growth hormone releasing hormone
("GHRH"). The pAV0201 plasmid also contains a poly A signal,
wherein the human growth hormone poly A has been utilized. The
pAV0201 plasmid also contains a eukaryotic promoter, and it has
been contemplated that the c5-12 synthetic eukaryotic promoter of
skeletal actin will be used, although other may be equally useful.
The pAV0201 also contains a prokaryotic promoter. The prokaryotic
promoter is PNEO, and a 19-47 bp sequence of transposon fragment
("Tn5") with accession number V00618. Additionally one NEO ribosome
binding site ("RBS") is present in the pAV0201 plasmid. A
complementary origin of replication sequence ("pUC ori") from the
pUC18 plasmid (e.g. 685-1466 bp of pUC18). A 5' untranslated region
("5'UTR") was inserted into the pAV0201 plasmid. The 5' UTR is from
human growth hormone hGH 5' UTR (i.e. 504-557 bp) accession number
M13438.
EXAMPLE 2
[0094] Optimized Synthetic GHRH sequences. Another aspect of the
current invention is to utilize the above optimized plasmid
backbone (pAV0201) and insert codon optimized species specific
eukaryotic nucleic acid expression sequences. Although not wanting
to limit the scope of the invention, five novel species of
optimized GHRH nucleic acid sequences have been inserted into the
pAV0201 plasmid backbone using the Nco I and Hind III restriction
sites. Each sequence was codon optimized for expression in the
corresponding species. The corresponding species in the below
examples are as follows: mouse; rat; bovine; ovine; and chicken.
The selection of these 5 species is not intended to limit the scope
of species specific GHRH insertions into the pAV0201 plasmid
backbone. In addition the structural features of pAV0201, each
eukaryotic expression sequence also contains a signal peptide
sequence for the purpose of making a signal peptide upstream from
the mature peptide. Each signal peptide sequence has been
contemplated to be the appropriate for the specific species of
interest. However, in one example below (e.g. the chicken GHRH
sequence), the rat GHRH signal peptide has been utilized. While the
natural cDNA sequences (Baird et al., 1986) are known for mouse
(Frohman et al., 1989b), rat (Bohlen et al., 1984; Mayo et al.,
1985), ovine (Brazeau et al., 1984), bovine (Esch et al., 1983),
porcine (Bohlen et al., 1983), chicken (McRory et al., 1997), the
codon optimization expression sequences in conjunction with the
pAV0201 based plasmid backbone make each of the constructs entirely
unique.
[0095] One aspect of the current invention is the insertion of the
codon optimized nucleic acid expression sequence for mouse GHRH
("mGHRH") (Seq ID#5) into the pAV0201 plasmid backbone to give
pAV0202 (Seq ID#17). A schematic representation of the optimized
nucleic acid expression sequence for mGHRH is shown in FIG. 4. The
optimized 228 bp mGHRH fragment was sub-cloned into the pAV0201
vector using the Nco I and Hind III restriction enzyme cut sites,
and standard methods known to one with ordinary skill in the art of
molecular biology. FIG. 5 shows a detailed nucleic acid and amino
acid sequence of the mGHRH motif, wherein all changes to the
nucleic acid expression sequences are labeled in bold. The nucleic
acid alignment between the original sequence (GHRH-M Ori) and Gene
Forge optimized sequence (GHRH-M Opti) are shown in FIG. 6, changes
are labeled in bold. FIG. 7 shows a comparison to indicate that the
amino acid sequence has not changed due to codon optimization.
[0096] Another aspect of the current invention is the insertion of
the codon optimized nucleic acid expression sequence for rat GHRH
("rGHRH") (Seq ID#6) into the pAV0201 plasmid backbone to give
pAV0203 (Seq ID#18). A schematic representation of the optimized
nucleic acid expression sequence for rGHRH is shown in FIG. 8. The
optimized 231 bp rGHRH fragment was sub-cloned into the pAV0201
vector using the Nco I and Hind III restriction enzyme cut sites,
and standard methods known to one with ordinary skill in the art of
molecular biology. FIG. 9 shows a detailed nucleic acid and amino
acid sequence of the rGHRH motif, wherein all changes to the
nucleic acid expression sequences are labeled in bold. The nucleic
acid alignment between the original sequence (GHRH-R Ori) and Gene
Forge optimized sequence (GHRH-R Opti) are shown in FIG. 10,
changes are labeled in bold. FIG. 11 shows a comparison to indicate
that the amino acid sequence has not changed due to codon
optimization.
[0097] Another aspect of the current invention is the insertion of
the codon optimized nucleic acid expression sequence for bovine
GHRH ("bGHRH") (Seq ID#7) into the pAV0201 plasmid backbone to give
pAV0204 (Seq ID#19). A schematic representation of the optimized
nucleic acid expression sequence for bGHRH is shown in FIG. 12. The
optimized 222 bp bGHRH fragment was sub-cloned into the pAV0201
vector using the Nco I and Hind III restriction enzyme cut sites,
and standard methods known to one with ordinary skill in the art of
molecular biology. FIG. 13 shows a detailed nucleic acid and amino
acid sequence of the bGHRH motif, wherein all changes to the
nucleic acid expression sequences are labeled in bold. The nucleic
acid alignment between the original sequence (GHRH-B Ori) and Gene
Forge optimized sequence (GHRH-B Opti) are shown in FIG. 14,
changes are labeled in bold. FIG. 15 shows a comparison to indicate
that the amino acid sequence has not changed due to codon
optimization.
[0098] Another aspect of the current invention is the insertion of
the codon optimized nucleic acid expression sequence for ovine GHRH
("oGHRH") (Seq ID#8) into the pAV0201 plasmid backbone to give
pAV0205 (Seq ID#20). A schematic representation of the optimized
nucleic acid expression sequence for oGHRH is shown in FIG. 16. The
optimized 222 bp oGHRH fragment was sub-cloned into the pAV0201
vector using the NcoI and Hind III restriction enzyme cut sites,
and standard methods known to one with ordinary skill in the art of
molecular biology. FIG. 17 shows a detailed nucleic acid and amino
acid sequence of the oGHRH motif, wherein all changes to the
nucleic acid expression sequences are labeled in bold. The nucleic
acid alignment between the original sequence (GHRH-O Ori) and Gene
Forge optimized sequence (GHRH-O Opti) are shown in FIG. 18,
changes are labeled in bold. FIG. 19 shows a comparison to indicate
that the amino acid sequence has not changed due to codon
optimization.
[0099] Another aspect of the current invention is the insertion of
the codon optimized nucleic acid expression sequence for chicken
GHRH ("cGHRH") (Seq ID#9) into the pAV0201 plasmid backbone to give
pAV0206 (Seq ID#21). A schematic representation of the optimized
nucleic acid expression sequence for cGHRH is shown in FIG. 20. The
optimized 234 bp cGHRH fragment was sub-cloned into the pAV0201
vector using the Nco I and Hind III restriction enzyme cut sites,
and standard methods known to one with ordinary skill in the art of
molecular biology. FIG. 21 shows a detailed nucleic acid and amino
acid sequence of the cGHRH motif, wherein all changes to the
nucleic acid expression sequences are labeled in bold. The nucleic
acid alignment between the original sequence (GHRH-C Ori) and Gene
Forge optimized sequence (GHRH-C Opti) are shown in FIG. 22,
changes are labeled in bold. FIG. 23 shows a comparison to indicate
that the amino acid sequence has not changed due to codon
optimization. For this particular sequence, the chicken pre-pro
hormone signal was replaced with the more compact, shorter rat
pre-pro sequence.
[0100] The above optimized plasmid constructs can be administered
to a mammalian host for various therapeutic effects. One skilled in
the art recognizes that different methods of delivery may be
utilized to administer an optimized synthetic expression vector
into a cell. Examples include: (1) methods utilizing physical
means, such as electroporation (electricity), a gene gun (physical
force) or applying large volumes of a liquid (pressure); and (2)
methods wherein said vector is complexed to another entity, such as
a liposome or transporter molecule.
[0101] Accordingly, the present invention provides a method of
transferring a therapeutic gene to a host, which comprises
administering the vector of the present invention, preferably as
part of a composition, using any of the aforementioned routes of
administration or alternative routes known to those skilled in the
art and appropriate for a particular application. Effective gene
transfer of a vector to a host cell in accordance with the present
invention to a host cell can be monitored in terms of a therapeutic
effect (e.g. alleviation of some symptom associated with the
particular disease being treated) or, further, by evidence of the
transferred gene or expression of the gene within the host (e.g.,
using the polymerase chain reaction in conjunction with sequencing,
Northern or Southern hybridizations, or transcription assays to
detect the nucleic acid in host cells, or using immunoblot
analysis, antibody-mediated detection, mRNA or protein half-life
studies, or particularized assays to detect protein or polypeptide
encoded by the transferred nucleic acid, or impacted in level or
function due to such transfer).
[0102] These compositions and methods described herein are by no
means all-inclusive, and further methods to suit the specific
application will be apparent to the ordinary skilled artisan.
Moreover, the effective amount of the compositions can be further
approximated through analogy to compounds known to exert the
desired effect.
Sequence CWU 1
1
4313534DNAartificial sequencePlasmid vector having an analog GHRH
sequence. 1gttgtaaaac gacggccagt gaattgtaat acgactcact atagggcgaa
ttggagctcc 60accgcggtgg cggccgtccg ccctcggcac catcctcacg acacccaaat
atggcgacgg 120gtgaggaatg gtggggagtt atttttagag cggtgaggaa
ggtgggcagg cagcaggtgt 180tggcgctcta aaaataactc ccgggagtta
tttttagagc ggaggaatgg tggacaccca 240aatatggcga cggttcctca
cccgtcgcca tatttgggtg tccgccctcg gccggggccg 300cattcctggg
ggccgggcgg tgctcccgcc cgcctcgata aaaggctccg gggccggcgg
360cggcccacga gctacccgga ggagcgggag gcgccaagct ctagaactag
tggatcccaa 420ggcccaactc cccgaaccac tcagggtcct gtggacagct
cacctagctg ccatggtgct 480ctgggtgttc ttctttgtga tcctcaccct
cagcaacagc tcccactgct ccccacctcc 540ccctttgacc ctcaggatgc
ggcggcacgt agatgccatc ttcaccaaca gctaccggaa 600ggtgctggcc
cagctgtccg cccgcaagct gctccaggac atcctgaaca ggcagcaggg
660agagaggaac caagagcaag gagcataatg actgcaggaa ttcgatatca
agcttatcgg 720ggtggcatcc ctgtgacccc tccccagtgc ctctcctggc
cctggaagtt gccactccag 780tgcccaccag ccttgtccta ataaaattaa
gttgcatcat tttgtctgac taggtgtcct 840tctataatat tatggggtgg
aggggggtgg tatggagcaa ggggcaagtt gggaagacaa 900cctgtagggc
ctgcggggtc tattgggaac caagctggag tgcagtggca caatcttggc
960tcactgcaat ctccgcctcc tgggttcaag cgattctcct gcctcagcct
cccgagttgt 1020tgggattcca ggcatgcatg accaggctca gctaattttt
gtttttttgg tagagacggg 1080gtttcaccat attggccagg ctggtctcca
actcctaatc tcaggtgatc tacccacctt 1140ggcctcccaa attgctggga
ttacaggcgt gaaccactgc tcccttccct gtccttctga 1200ttttaaaata
actataccag caggaggacg tccagacaca gcataggcta cctggccatg
1260cccaaccggt gggacatttg agttgcttgc ttggcactgt cctctcatgc
gttgggtcca 1320ctcagtagat gcctgttgaa ttcgataccg tcgacctcga
gggggggccc ggtaccagct 1380tttgttccct ttagtgaggg ttaatttcga
gcttggcgta atcatggtca tagctgtttc 1440ctgtgtgaaa ttgttatccg
ctcacaattc cacacaacat acgagccgga agcataaagt 1500gtaaagcctg
gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
1560ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
caacgcgcgg 1620ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
gctcactgac tcgctgcgct 1680cggtcgttcg gctgcggcga gcggtatcag
ctcactcaaa ggcggtaata cggttatcca 1740cagaatcagg ggataacgca
ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga 1800accgtaaaaa
ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
1860acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
agataccagg 1920cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
gaccctgccg cttaccggat 1980acctgtccgc ctttctccct tcgggaagcg
tggcgctttc tcatagctca cgctgtaggt 2040atctcagttc ggtgtaggtc
gttcgctcca agctgggctg tgtgcacgaa ccccccgttc 2100agcccgaccg
ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
2160acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
tatgtaggcg 2220gtgctacaga gttcttgaag tggtggccta actacggcta
cactagaaga acagtatttg 2280gtatctgcgc tctgctgaag ccagttacct
tcggaaaaag agttggtagc tcttgatccg 2340gcaaacaaac caccgctggt
agcggtggtt tttttgtttg caagcagcag attacgcgca 2400gaaaaaaagg
atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagaaga
2460actcgtcaag aaggcgatag aaggcgatgc gctgcgaatc gggagcggcg
ataccgtaaa 2520gcacgaggaa gcggtcagcc cattcgccgc caagctcttc
agcaatatca cgggtagcca 2580acgctatgtc ctgatagcgg tccgccacac
ccagccggcc acagtcgatg aatccagaaa 2640agcggccatt ttccaccatg
atattcggca agcaggcatc gccatgggtc acgacgagat 2700cctcgccgtc
gggcatgcgc gccttgagcc tggcgaacag ttcggctggc gcgagcccct
2760gatgctcttc gtccagatca tcctgatcga caagaccggc ttccatccga
gtacgtgctc 2820gctcgatgcg atgtttcgct tggtggtcga atgggcaggt
agccggatca agcgtatgca 2880gccgccgcat tgcatcagcc atgatggata
ctttctcggc aggagcaagg tgagatgaca 2940ggagatcctg ccccggcact
tcgcccaata gcagccagtc ccttcccgct tcagtgacaa 3000cgtcgagcac
agctgcgcaa ggaacgcccg tcgtggccag ccacgatagc cgcgctgcct
3060cgtcctgcag ttcattcagg gcaccggaca ggtcggtctt gacaaaaaga
accgggcgcc 3120cctgcgctga cagccggaac acggcggcat cagagcagcc
gattgtctgt tgtgcccagt 3180catagccgaa tagcctctcc acccaagcgg
ccggagaacc tgcgtgcaat ccatcttgtt 3240caatcatgcg aaacgatcct
catcctgtct cttgatcaga tcttgatccc ctgcgccatc 3300agatccttgg
cggcaagaaa gccatccagt ttactttgca gggcttccca accttaccag
3360agggcgcccc agctggcaat tccggttcgc ttgctgtcca taaaaccgcc
cagtctagca 3420actgttggga agggcgatcg gtgcgggcct cttcgctatt
acgccagctg gcgaaagggg 3480gatgtgctgc aaggcgatta agttgggtaa
cgccagggtt ttcccagtca cgac 353422739DNAartificial sequenceOptimized
vector having an analog GHRH sequence. 2ccaccgcggt ggcggccgtc
cgccctcggc accatcctca cgacacccaa atatggcgac 60gggtgaggaa tggtggggag
ttatttttag agcggtgagg aaggtgggca ggcagcaggt 120gttggcgctc
taaaaataac tcccgggagt tatttttaga gcggaggaat ggtggacacc
180caaatatggc gacggttcct cacccgtcgc catatttggg tgtccgccct
cggccggggc 240cgcattcctg ggggccgggc ggtgctcccg cccgcctcga
taaaaggctc cggggccggc 300ggcggcccac gagctacccg gaggagcggg
aggcgccaag cggatcccaa ggcccaactc 360cccgaaccac tcagggtcct
gtggacagct cacctagctg ccatggtgct ctgggtgttc 420ttctttgtga
tcctcaccct cagcaacagc tcccactgct ccccacctcc ccctttgacc
480ctcaggatgc ggcggtatgc agatgccatc ttcaccaaca gctaccggaa
ggtgctgggc 540cagctgtccg cccgcaagct gctccaggac atcatgagca
ggcagcaggg agagaggaac 600caagagcaag gagcataatg actgcaggaa
ttcgatatca agcttatcgg ggtggcatcc 660ctgtgacccc tccccagtgc
ctctcctggc cctggaagtt gccactccag tgcccaccag 720ccttgtccta
ataaaattaa gttgcatcat tttgtctgac taggtgtcct tctataatat
780tatggggtgg aggggggtgg tatggagcaa ggggcaagtt gggaagacaa
cctgtagggc 840tcgagggggg gcccggtacc agcttttgtt ccctttagtg
agggttaatt tcgagcttgg 900tcttccgctt cctcgctcac tgactcgctg
cgctcggtcg ttcggctgcg gcgagcggta 960tcagctcact caaaggcggt
aatacggtta tccacagaat caggggataa cgcaggaaag 1020aacatgtgag
caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
1080tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
aagtcagagg 1140tggcgaaacc cgacaggact ataaagatac caggcgtttc
cccctggaag ctccctcgtg 1200cgctctcctg ttccgaccct gccgcttacc
ggatacctgt ccgcctttct cccttcggga 1260agcgtggcgc tttctcatag
ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc 1320tccaagctgg
gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
1380aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
agcagccact 1440ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
cagagttctt gaagtggtgg 1500cctaactacg gctacactag aagaacagta
tttggtatct gcgctctgct gaagccagtt 1560accttcggaa aaagagttgg
tagctcttga tccggcaaac aaaccaccgc tggtagcggt 1620ggtttttttg
tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
1680ttgatctttt ctacggggtc tgacgctcag ctagcgctca gaagaactcg
tcaagaaggc 1740gatagaaggc gatgcgctgc gaatcgggag cggcgatacc
gtaaagcacg aggaagcggt 1800cagcccattc gccgccaagc tcttcagcaa
tatcacgggt agccaacgct atgtcctgat 1860agcggtccgc cacacccagc
cggccacagt cgatgaatcc agaaaagcgg ccattttcca 1920ccatgatatt
cggcaagcag gcatcgccat gagtcacgac gagatcctcg ccgtcgggca
1980tgcgcgcctt gagcctggcg aacagttcgg ctggcgcgag cccctgatgc
tcttcgtcca 2040gatcatcctg atcgacaaga ccggcttcca tccgagtacg
tgctcgctcg atgcgatgtt 2100tcgcttggtg gtcgaatggg caggtagccg
gatcaagcgt atgcagccgc cgcattgcat 2160cagccatgat ggatactttc
tcggcaggag caaggtgaga tgacaggaga tcctgccccg 2220gcacttcgcc
caatagcagc cagtcccttc ccgcttcagt gacaacgtcg agcacagctg
2280cgcaaggaac gcccgtcgtg gccagccacg atagccgcgc tgcctcgtcc
tgcagttcat 2340tcagggcacc ggacaggtcg gtcttgacaa aaagaaccgg
gcgcccctgc gctgacagcc 2400ggaacacggc ggcatcagag cagccgattg
tctgttgtgc ccagtcatag ccgaatagcc 2460tctccaccca agcggccgga
gaacctgcgt gcaatccatc ttgttcaatc atgcgaaacg 2520atcctcatcc
tgtctcttga tcagatcttg atcccctgcg ccatcagatc cttggcggca
2580agaaagccat ccagtttact ttgcagggct tcccaacctt accagagggc
gccccagctg 2640gcaattccgg ttcgcttgct gtccataaaa ccgcccagtc
tagcaactgt tgggaagggc 2700gatcgtgtaa tacgactcac tatagggcga
attggagct 27393795DNAartificial sequenceNucleic acid sequence for
the antibiotic resistance gene kanamycin. 3atgattgaac aagatggatt
gcacgcaggt tctccggccg cttgggtgga gaggctattc 60ggctatgact gggcacaaca
gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca 120gcgcaggggc
gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
180caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg
cgcagctgtg 240ctcgacgttg tcactgaagc gggaagggac tggctgctat
tgggcgaagt gccggggcag 300gatctcctgt catctcacct tgctcctgcc
gagaaagtat ccatcatggc tgatgcaatg 360cggcggctgc atacgcttga
tccggctacc tgcccattcg accaccaagc gaaacatcgc 420atcgagcgag
cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
480gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg
catgcccgac 540ggcgaggatc tcgtcgtgac tcatggcgat gcctgcttgc
cgaatatcat ggtggaaaat 600ggccgctttt ctggattcat cgactgtggc
cggctgggtg tggcggaccg ctatcaggac 660atagcgttgg ctacccgtga
tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc 720ctcgtgcttt
acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
780gacgagttct tctga 7954219DNAartificial sequenceSequence for an
analog porcine GHRH sequence. 4atggtgctct gggtgttctt ctttgtgatc
ctcaccctca gcaacagctc ccactgctcc 60ccacctcccc ctttgaccct caggatgcgg
cggcacgtag atgccatctt caccaacagc 120taccggaagg tgctggccca
gctgtccgcc cgcaagctgc tccaggacat cctgaacagg 180cagcagggag
agaggaacca agagcaagga gcataatga 2195246DNAartificial
sequenceSequence for an analog mouse GHRH sequence. 5gccatggtgc
tctgggtgct ctttgtgatc ctcatcctca ccagcggcag ccactgcagc 60ctgcctccca
gccctccctt caggatgcag aggcacgtgg acgccatctt caccaccaac
120tacaggaagc tgctgagcca gctgtacgcc aggaaggtga tccaggacat
catgaacaag 180cagggcgaga ggatccagga gcagagggcc aggctgagct
gataagcttg cgatgagttc 240ttctaa 2466234DNAartificial
sequenceSequence for an analog rat GHRH sequence. 6gccatggccc
tgtgggtgtt cttcgtgctg ctgaccctga ccagcggaag ccactgcagc 60ctgcctccca
gccctccctt cagggtgcgc cggcacgccg acgccatctt caccagcagc
120tacaggagga tcctgggcca gctgtacgct aggaagctcc tgcacgagat
catgaacagg 180cagcagggcg agaggaacca ggagcagagg agcaggttca
actgataagc ttgc 2347225DNAartificial sequenceSequence for an analog
bovine GHRH sequence. 7gccatggtgc tgtgggtgtt cttcctggtg accctgaccc
tgagcagcgg ctcccacggc 60tccctgccct cccagcctct gcgcatccct cgctacgccg
acgccatctt caccaacagc 120taccgcaagg tgctcggcca gctcagcgcc
cgcaagctcc tgcaggacat catgaaccgg 180cagcagggcg agcgcaacca
ggagcaggga gcctgataag cttgc 2258225DNAartificial sequenceSequence
for an analog ovine GHRH sequence. 8gccatggtgc tgtgggtgtt
cttcctggtg accctgaccc tgagcagcgg aagccacggc 60agcctgccca gccagcccct
gaggatccct aggtacgccg acgccatctt caccaacagc 120tacaggaaga
tcctgggcca gctgagcgct aggaagctcc tgcaggacat catgaacagg
180cagcagggcg agaggaacca ggagcagggc gcctgataag cttgc
2259246DNAartificial sequenceSequence for an analog chicken GHRH
sequence. 9gccatggtgc tctgggtgct ctttgtgatc ctcatcctca ccagcggcag
ccactgcagc 60ctgcctccca gccctccctt caggatgcag aggcacgtgg acgccatctt
caccaccaac 120tacaggaagc tgctgagcca gctgtacgcc aggaaggtga
tccaggacat catgaacaag 180cagggcgaga ggatccagga gcagagggcc
aggctgagct gataagcttg cgatgagttc 240ttctaa 24610190DNAartificial
sequenceNucleic acid sequence of human growth hormone poly A tail.
10gggtggcatc cctgtgaccc ctccccagtg cctctcctgg ccctggaagt tgccactcca
60gtgcccacca gccttgtcct aataaaatta agttgcatca ttttgtctga ctaggtgtcc
120ttctataata ttatggggtg gaggggggtg gtatggagca aggggcaagt
tgggaagaca 180acctgtaggg 1901155DNAartificial sequenceNucleic acid
sequence of human growth hormone 5' untranslated region
11caaggcccaa ctccccgaac cactcagggt cctgtggaca gctcacctag ctgcc
5512782DNAartificial sequenceNucleic acid sequence of a plasmid
pUC-18 origin of replicaiton 12tcttccgctt cctcgctcac tgactcgctg
cgctcggtcg ttcggctgcg gcgagcggta 60tcagctcact caaaggcggt aatacggtta
tccacagaat caggggataa cgcaggaaag 120aacatgtgag caaaaggcca
gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg 180tttttccata
ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
240tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
ctccctcgtg 300cgctctcctg ttccgaccct gccgcttacc ggatacctgt
ccgcctttct cccttcggga 360agcgtggcgc tttctcatag ctcacgctgt
aggtatctca gttcggtgta ggtcgttcgc 420tccaagctgg gctgtgtgca
cgaacccccc gttcagcccg accgctgcgc cttatccggt 480aactatcgtc
ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
540ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
gaagtggtgg 600cctaactacg gctacactag aaggacagta tttggtatct
gcgctctgct gaagccagtt 660accttcggaa aaagagttgg tagctcttga
tccggcaaac aaaccaccgc tggtagcggt 720ggtttttttg tttgcaagca
gcagattacg cgcagaaaaa aaggatctca agaagatcct 780tt
782135DNAartificial sequenceThis is a NEO ribosomal binding site
13tcctc 51429DNAartificial sequenceNucleic acid sequence of a
prokaryotic PNEO promoter. 14accttaccag agggcgcccc agctggcaa
2915323DNAartificial sequenceNucleic acid sequence of a eukaryotic
promoter c5-12. 15cggccgtccg ccctcggcac catcctcacg acacccaaat
atggcgacgg gtgaggaatg 60gtggggagtt atttttagag cggtgaggaa ggtgggcagg
cagcaggtgt tggcgctcta 120aaaataactc ccgggagtta tttttagagc
ggaggaatgg tggacaccca aatatggcga 180cggttcctca cccgtcgcca
tatttgggtg tccgccctcg gccggggccg cattcctggg 240ggccgggcgg
tgctcccgcc cgcctcgata aaaggctccg gggccggcgg cggcccacga
300gctacccgga ggagcgggag gcg 32316210DNAartificial
sequenceOptimized nucleic acid sequence of a human growth hormone
poly Atail 16ttatcggggt ggcatccctg tgacccctcc ccagtgcctc tcctggccct
ggaagttgcc 60actccagtgc ccaccagcct tgtcctaata aaattaagtt gcatcatttt
gtctgactag 120gtgtccttct ataatattat ggggtggagg ggggtggtat
ggagcaaggg gcaagttggg 180aagacaacct gtagggctcg agggggggcc
210172722DNAartificial sequencePlasmid vector having a codon
optimized mouse GHRH sequence 17ccaccgcggt ggcggccgtc cgccctcggc
accatcctca cgacacccaa atatggcgac 60gggtgaggaa tggtggggag ttatttttag
agcggtgagg aaggtgggca ggcagcaggt 120gttggcgctc taaaaataac
tcccgggagt tatttttaga gcggaggaat ggtggacacc 180caaatatggc
gacggttcct cacccgtcgc catatttggg tgtccgccct cggccggggc
240cgcattcctg ggggccgggc ggtgctcccg cccgcctcga taaaaggctc
cggggccggc 300ggcggcccac gagctacccg gaggagcggg aggcgccaag
cggatcccaa ggcccaactc 360cccgaaccac tcagggtcct gtggacagct
cacctagctg ccatggtgct ctgggtgctc 420tttgtgatcc tcatcctcac
cagcggcagc cactgcagcc tgcctcccag ccctcccttc 480aggatgcaga
ggcacgtgga cgccatcttc accaccaact acaggaagct gctgagccag
540ctgtacgcca ggaaggtgat ccaggacatc atgaacaagc agggcgagag
gatccaggag 600cagagggcca ggctgagctg ataagcttat cggggtggca
tccctgtgac ccctccccag 660tgcctctcct ggccctggaa gttgccactc
cagtgcccac cagccttgtc ctaataaaat 720taagttgcat cattttgtct
gactaggtgt ccttctataa tattatgggg tggagggggg 780tggtatggag
caaggggcaa gttgggaaga caacctgtag ggctcgaggg ggggcccggt
840accagctttt gttcccttta gtgagggtta atttcgagct tggtcttccg
cttcctcgct 900cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
gtatcagctc actcaaaggc 960ggtaatacgg ttatccacag aatcagggga
taacgcagga aagaacatgt gagcaaaagg 1020ccagcaaaag gccaggaacc
gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg 1080cccccctgac
gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
1140actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
ctgttccgac 1200cctgccgctt accggatacc tgtccgcctt tctcccttcg
ggaagcgtgg cgctttctca 1260tagctcacgc tgtaggtatc tcagttcggt
gtaggtcgtt cgctccaagc tgggctgtgt 1320gcacgaaccc cccgttcagc
ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc 1380caacccggta
agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
1440agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
acggctacac 1500tagaagaaca gtatttggta tctgcgctct gctgaagcca
gttaccttcg gaaaaagagt 1560tggtagctct tgatccggca aacaaaccac
cgctggtagc ggtggttttt ttgtttgcaa 1620gcagcagatt acgcgcagaa
aaaaaggatc tcaagaagat cctttgatct tttctacggg 1680gtctgacgct
cagctagcgc tcagaagaac tcgtcaagaa ggcgatagaa ggcgatgcgc
1740tgcgaatcgg gagcggcgat accgtaaagc acgaggaagc ggtcagccca
ttcgccgcca 1800agctcttcag caatatcacg ggtagccaac gctatgtcct
gatagcggtc cgccacaccc 1860agccggccac agtcgatgaa tccagaaaag
cggccatttt ccaccatgat attcggcaag 1920caggcatcgc catgagtcac
gacgagatcc tcgccgtcgg gcatgcgcgc cttgagcctg 1980gcgaacagtt
cggctggcgc gagcccctga tgctcttcgt ccagatcatc ctgatcgaca
2040agaccggctt ccatccgagt acgtgctcgc tcgatgcgat gtttcgcttg
gtggtcgaat 2100gggcaggtag ccggatcaag cgtatgcagc cgccgcattg
catcagccat gatggatact 2160ttctcggcag gagcaaggtg agatgacagg
agatcctgcc ccggcacttc gcccaatagc 2220agccagtccc ttcccgcttc
agtgacaacg tcgagcacag ctgcgcaagg aacgcccgtc 2280gtggccagcc
acgatagccg cgctgcctcg tcctgcagtt cattcagggc accggacagg
2340tcggtcttga caaaaagaac cgggcgcccc tgcgctgaca gccggaacac
ggcggcatca 2400gagcagccga ttgtctgttg tgcccagtca tagccgaata
gcctctccac ccaagcggcc 2460ggagaacctg cgtgcaatcc atcttgttca
atcatgcgaa acgatcctca tcctgtctct 2520tgatcagatc ttgatcccct
gcgccatcag atccttggcg gcaagaaagc catccagttt 2580actttgcagg
gcttcccaac cttaccagag ggcgccccag ctggcaattc cggttcgctt
2640gctgtccata aaaccgccca gtctagcaac tgttgggaag ggcgatcgtg
taatacgact 2700cactataggg cgaattggag ct 2722182725DNAartificial
sequencePlasmid vector having a codon optimized rat GHRH sequence
18ccaccgcggt ggcggccgtc cgccctcggc accatcctca cgacacccaa atatggcgac
60gggtgaggaa tggtggggag ttatttttag agcggtgagg aaggtgggca ggcagcaggt
120gttggcgctc taaaaataac tcccgggagt tatttttaga gcggaggaat
ggtggacacc 180caaatatggc gacggttcct cacccgtcgc catatttggg
tgtccgccct cggccggggc 240cgcattcctg ggggccgggc ggtgctcccg
cccgcctcga taaaaggctc cggggccggc 300ggcggcccac gagctacccg
gaggagcggg aggcgccaag cggatcccaa ggcccaactc 360cccgaaccac
tcagggtcct gtggacagct cacctagctg
ccatggccct gtgggtgttc 420ttcgtgctgc tgaccctgac cagcggaagc
cactgcagcc tgcctcccag ccctcccttc 480agggtgcgcc ggcacgccga
cgccatcttc accagcagct acaggaggat cctgggccag 540ctgtacgcta
ggaagctcct gcacgagatc atgaacaggc agcagggcga gaggaaccag
600gagcagagga gcaggttcaa ctgataagct tatcggggtg gcatccctgt
gacccctccc 660cagtgcctct cctggccctg gaagttgcca ctccagtgcc
caccagcctt gtcctaataa 720aattaagttg catcattttg tctgactagg
tgtccttcta taatattatg gggtggaggg 780gggtggtatg gagcaagggg
caagttggga agacaacctg tagggctcga gggggggccc 840ggtaccagct
tttgttccct ttagtgaggg ttaatttcga gcttggtctt ccgcttcctc
900gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
ctcactcaaa 960ggcggtaata cggttatcca cagaatcagg ggataacgca
ggaaagaaca tgtgagcaaa 1020aggccagcaa aaggccagga accgtaaaaa
ggccgcgttg ctggcgtttt tccataggct 1080ccgcccccct gacgagcatc
acaaaaatcg acgctcaagt cagaggtggc gaaacccgac 1140aggactataa
agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
1200gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
tggcgctttc 1260tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
gttcgctcca agctgggctg 1320tgtgcacgaa ccccccgttc agcccgaccg
ctgcgcctta tccggtaact atcgtcttga 1380gtccaacccg gtaagacacg
acttatcgcc actggcagca gccactggta acaggattag 1440cagagcgagg
tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
1500cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
tcggaaaaag 1560agttggtagc tcttgatccg gcaaacaaac caccgctggt
agcggtggtt tttttgtttg 1620caagcagcag attacgcgca gaaaaaaagg
atctcaagaa gatcctttga tcttttctac 1680ggggtctgac gctcagctag
cgctcagaag aactcgtcaa gaaggcgata gaaggcgatg 1740cgctgcgaat
cgggagcggc gataccgtaa agcacgagga agcggtcagc ccattcgccg
1800ccaagctctt cagcaatatc acgggtagcc aacgctatgt cctgatagcg
gtccgccaca 1860cccagccggc cacagtcgat gaatccagaa aagcggccat
tttccaccat gatattcggc 1920aagcaggcat cgccatgagt cacgacgaga
tcctcgccgt cgggcatgcg cgccttgagc 1980ctggcgaaca gttcggctgg
cgcgagcccc tgatgctctt cgtccagatc atcctgatcg 2040acaagaccgg
cttccatccg agtacgtgct cgctcgatgc gatgtttcgc ttggtggtcg
2100aatgggcagg tagccggatc aagcgtatgc agccgccgca ttgcatcagc
catgatggat 2160actttctcgg caggagcaag gtgagatgac aggagatcct
gccccggcac ttcgcccaat 2220agcagccagt cccttcccgc ttcagtgaca
acgtcgagca cagctgcgca aggaacgccc 2280gtcgtggcca gccacgatag
ccgcgctgcc tcgtcctgca gttcattcag ggcaccggac 2340aggtcggtct
tgacaaaaag aaccgggcgc ccctgcgctg acagccggaa cacggcggca
2400tcagagcagc cgattgtctg ttgtgcccag tcatagccga atagcctctc
cacccaagcg 2460gccggagaac ctgcgtgcaa tccatcttgt tcaatcatgc
gaaacgatcc tcatcctgtc 2520tcttgatcag atcttgatcc cctgcgccat
cagatccttg gcggcaagaa agccatccag 2580tttactttgc agggcttccc
aaccttacca gagggcgccc cagctggcaa ttccggttcg 2640cttgctgtcc
ataaaaccgc ccagtctagc aactgttggg aagggcgatc gtgtaatacg
2700actcactata gggcgaattg gagct 2725192716DNAartificial
sequencePlasmid vector having a codon optimized bovine GHRH
sequence 19ccaccgcggt ggcggccgtc cgccctcggc accatcctca cgacacccaa
atatggcgac 60gggtgaggaa tggtggggag ttatttttag agcggtgagg aaggtgggca
ggcagcaggt 120gttggcgctc taaaaataac tcccgggagt tatttttaga
gcggaggaat ggtggacacc 180caaatatggc gacggttcct cacccgtcgc
catatttggg tgtccgccct cggccggggc 240cgcattcctg ggggccgggc
ggtgctcccg cccgcctcga taaaaggctc cggggccggc 300ggcggcccac
gagctacccg gaggagcggg aggcgccaag cggatcccaa ggcccaactc
360cccgaaccac tcagggtcct gtggacagct cacctagctg ccatggtgct
gtgggtgttc 420ttcctggtga ccctgaccct gagcagcggc tcccacggct
ccctgccctc ccagcctctg 480cgcatccctc gctacgccga cgccatcttc
accaacagct accgcaaggt gctcggccag 540ctcagcgccc gcaagctcct
gcaggacatc atgaaccggc agcagggcga gcgcaaccag 600gagcagggag
cctgataagc ttatcggggt ggcatccctg tgacccctcc ccagtgcctc
660tcctggccct ggaagttgcc actccagtgc ccaccagcct tgtcctaata
aaattaagtt 720gcatcatttt gtctgactag gtgtccttct ataatattat
ggggtggagg ggggtggtat 780ggagcaaggg gcaagttggg aagacaacct
gtagggctcg agggggggcc cggtaccagc 840ttttgttccc tttagtgagg
gttaatttcg agcttggtct tccgcttcct cgctcactga 900ctcgctgcgc
tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
960acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
aaggccagca 1020aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
ttccataggc tccgcccccc 1080tgacgagcat cacaaaaatc gacgctcaag
tcagaggtgg cgaaacccga caggactata 1140aagataccag gcgtttcccc
ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 1200gcttaccgga
tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
1260acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
gtgtgcacga 1320accccccgtt cagcccgacc gctgcgcctt atccggtaac
tatcgtcttg agtccaaccc 1380ggtaagacac gacttatcgc cactggcagc
agccactggt aacaggatta gcagagcgag 1440gtatgtaggc ggtgctacag
agttcttgaa gtggtggcct aactacggct acactagaag 1500aacagtattt
ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
1560ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
gcaagcagca 1620gattacgcgc agaaaaaaag gatctcaaga agatcctttg
atcttttcta cggggtctga 1680cgctcagcta gcgctcagaa gaactcgtca
agaaggcgat agaaggcgat gcgctgcgaa 1740tcgggagcgg cgataccgta
aagcacgagg aagcggtcag cccattcgcc gccaagctct 1800tcagcaatat
cacgggtagc caacgctatg tcctgatagc ggtccgccac acccagccgg
1860ccacagtcga tgaatccaga aaagcggcca ttttccacca tgatattcgg
caagcaggca 1920tcgccatgag tcacgacgag atcctcgccg tcgggcatgc
gcgccttgag cctggcgaac 1980agttcggctg gcgcgagccc ctgatgctct
tcgtccagat catcctgatc gacaagaccg 2040gcttccatcc gagtacgtgc
tcgctcgatg cgatgtttcg cttggtggtc gaatgggcag 2100gtagccggat
caagcgtatg cagccgccgc attgcatcag ccatgatgga tactttctcg
2160gcaggagcaa ggtgagatga caggagatcc tgccccggca cttcgcccaa
tagcagccag 2220tcccttcccg cttcagtgac aacgtcgagc acagctgcgc
aaggaacgcc cgtcgtggcc 2280agccacgata gccgcgctgc ctcgtcctgc
agttcattca gggcaccgga caggtcggtc 2340ttgacaaaaa gaaccgggcg
cccctgcgct gacagccgga acacggcggc atcagagcag 2400ccgattgtct
gttgtgccca gtcatagccg aatagcctct ccacccaagc ggccggagaa
2460cctgcgtgca atccatcttg ttcaatcatg cgaaacgatc ctcatcctgt
ctcttgatca 2520gatcttgatc ccctgcgcca tcagatcctt ggcggcaaga
aagccatcca gtttactttg 2580cagggcttcc caaccttacc agagggcgcc
ccagctggca attccggttc gcttgctgtc 2640cataaaaccg cccagtctag
caactgttgg gaagggcgat cgtgtaatac gactcactat 2700agggcgaatt ggagct
2716202716DNAartificial sequencePlasmid vector having a codon
optimized ovine GHRH sequence 20ccaccgcggt ggcggccgtc cgccctcggc
accatcctca cgacacccaa atatggcgac 60gggtgaggaa tggtggggag ttatttttag
agcggtgagg aaggtgggca ggcagcaggt 120gttggcgctc taaaaataac
tcccgggagt tatttttaga gcggaggaat ggtggacacc 180caaatatggc
gacggttcct cacccgtcgc catatttggg tgtccgccct cggccggggc
240cgcattcctg ggggccgggc ggtgctcccg cccgcctcga taaaaggctc
cggggccggc 300ggcggcccac gagctacccg gaggagcggg aggcgccaag
cggatcccaa ggcccaactc 360cccgaaccac tcagggtcct gtggacagct
cacctagctg ccatggtgct gtgggtgttc 420ttcctggtga ccctgaccct
gagcagcgga agccacggca gcctgcccag ccagcccctg 480aggatcccta
ggtacgccga cgccatcttc accaacagct acaggaagat cctgggccag
540ctgagcgcta ggaagctcct gcaggacatc atgaacaggc agcagggcga
gaggaaccag 600gagcagggcg cctgataagc ttatcggggt ggcatccctg
tgacccctcc ccagtgcctc 660tcctggccct ggaagttgcc actccagtgc
ccaccagcct tgtcctaata aaattaagtt 720gcatcatttt gtctgactag
gtgtccttct ataatattat ggggtggagg ggggtggtat 780ggagcaaggg
gcaagttggg aagacaacct gtagggctcg agggggggcc cggtaccagc
840ttttgttccc tttagtgagg gttaatttcg agcttggtct tccgcttcct
cgctcactga 900ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
gctcactcaa aggcggtaat 960acggttatcc acagaatcag gggataacgc
aggaaagaac atgtgagcaa aaggccagca 1020aaaggccagg aaccgtaaaa
aggccgcgtt gctggcgttt ttccataggc tccgcccccc 1080tgacgagcat
cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
1140aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
cgaccctgcc 1200gcttaccgga tacctgtccg cctttctccc ttcgggaagc
gtggcgcttt ctcatagctc 1260acgctgtagg tatctcagtt cggtgtaggt
cgttcgctcc aagctgggct gtgtgcacga 1320accccccgtt cagcccgacc
gctgcgcctt atccggtaac tatcgtcttg agtccaaccc 1380ggtaagacac
gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
1440gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
acactagaag 1500aacagtattt ggtatctgcg ctctgctgaa gccagttacc
ttcggaaaaa gagttggtag 1560ctcttgatcc ggcaaacaaa ccaccgctgg
tagcggtggt ttttttgttt gcaagcagca 1620gattacgcgc agaaaaaaag
gatctcaaga agatcctttg atcttttcta cggggtctga 1680cgctcagcta
gcgctcagaa gaactcgtca agaaggcgat agaaggcgat gcgctgcgaa
1740tcgggagcgg cgataccgta aagcacgagg aagcggtcag cccattcgcc
gccaagctct 1800tcagcaatat cacgggtagc caacgctatg tcctgatagc
ggtccgccac acccagccgg 1860ccacagtcga tgaatccaga aaagcggcca
ttttccacca tgatattcgg caagcaggca 1920tcgccatgag tcacgacgag
atcctcgccg tcgggcatgc gcgccttgag cctggcgaac 1980agttcggctg
gcgcgagccc ctgatgctct tcgtccagat catcctgatc gacaagaccg
2040gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg cttggtggtc
gaatgggcag 2100gtagccggat caagcgtatg cagccgccgc attgcatcag
ccatgatgga tactttctcg 2160gcaggagcaa ggtgagatga caggagatcc
tgccccggca cttcgcccaa tagcagccag 2220tcccttcccg cttcagtgac
aacgtcgagc acagctgcgc aaggaacgcc cgtcgtggcc 2280agccacgata
gccgcgctgc ctcgtcctgc agttcattca gggcaccgga caggtcggtc
2340ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga acacggcggc
atcagagcag 2400ccgattgtct gttgtgccca gtcatagccg aatagcctct
ccacccaagc ggccggagaa 2460cctgcgtgca atccatcttg ttcaatcatg
cgaaacgatc ctcatcctgt ctcttgatca 2520gatcttgatc ccctgcgcca
tcagatcctt ggcggcaaga aagccatcca gtttactttg 2580cagggcttcc
caaccttacc agagggcgcc ccagctggca attccggttc gcttgctgtc
2640cataaaaccg cccagtctag caactgttgg gaagggcgat cgtgtaatac
gactcactat 2700agggcgaatt ggagct 2716212725DNAartificial
sequencePlasmid vector having a codon optimized chicken GHRH
sequence 21ccaccgcggt ggcggccgtc cgccctcggc accatcctca cgacacccaa
atatggcgac 60gggtgaggaa tggtggggag ttatttttag agcggtgagg aaggtgggca
ggcagcaggt 120gttggcgctc taaaaataac tcccgggagt tatttttaga
gcggaggaat ggtggacacc 180caaatatggc gacggttcct cacccgtcgc
catatttggg tgtccgccct cggccggggc 240cgcattcctg ggggccgggc
ggtgctcccg cccgcctcga taaaaggctc cggggccggc 300ggcggcccac
gagctacccg gaggagcggg aggcgccaag cggatcccaa ggcccaactc
360cccgaaccac tcagggtcct gtggacagct cacctagctg ccatggccct
gtgggtgttc 420tttgtgctgc tgaccctgac ctccggaagc cactgcagcc
tgccacccag cccacccttc 480cgcgtcaggc gccacgccga cggcatcttc
agcaaggcct accgcaagct cctgggccag 540ctgagcgcac gcaactacct
gcacagcctg atggccaagc gcgtgggcag cggactggga 600gacgaggccg
agcccctgag ctgataagct tatcggggtg gcatccctgt gacccctccc
660cagtgcctct cctggccctg gaagttgcca ctccagtgcc caccagcctt
gtcctaataa 720aattaagttg catcattttg tctgactagg tgtccttcta
taatattatg gggtggaggg 780gggtggtatg gagcaagggg caagttggga
agacaacctg tagggctcga gggggggccc 840ggtaccagct tttgttccct
ttagtgaggg ttaatttcga gcttggtctt ccgcttcctc 900gctcactgac
tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
960ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
tgtgagcaaa 1020aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
ctggcgtttt tccataggct 1080ccgcccccct gacgagcatc acaaaaatcg
acgctcaagt cagaggtggc gaaacccgac 1140aggactataa agataccagg
cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc 1200gaccctgccg
cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
1260tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
agctgggctg 1320tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
tccggtaact atcgtcttga 1380gtccaacccg gtaagacacg acttatcgcc
actggcagca gccactggta acaggattag 1440cagagcgagg tatgtaggcg
gtgctacaga gttcttgaag tggtggccta actacggcta 1500cactagaaga
acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
1560agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
tttttgtttg 1620caagcagcag attacgcgca gaaaaaaagg atctcaagaa
gatcctttga tcttttctac 1680ggggtctgac gctcagctag cgctcagaag
aactcgtcaa gaaggcgata gaaggcgatg 1740cgctgcgaat cgggagcggc
gataccgtaa agcacgagga agcggtcagc ccattcgccg 1800ccaagctctt
cagcaatatc acgggtagcc aacgctatgt cctgatagcg gtccgccaca
1860cccagccggc cacagtcgat gaatccagaa aagcggccat tttccaccat
gatattcggc 1920aagcaggcat cgccatgagt cacgacgaga tcctcgccgt
cgggcatgcg cgccttgagc 1980ctggcgaaca gttcggctgg cgcgagcccc
tgatgctctt cgtccagatc atcctgatcg 2040acaagaccgg cttccatccg
agtacgtgct cgctcgatgc gatgtttcgc ttggtggtcg 2100aatgggcagg
tagccggatc aagcgtatgc agccgccgca ttgcatcagc catgatggat
2160actttctcgg caggagcaag gtgagatgac aggagatcct gccccggcac
ttcgcccaat 2220agcagccagt cccttcccgc ttcagtgaca acgtcgagca
cagctgcgca aggaacgccc 2280gtcgtggcca gccacgatag ccgcgctgcc
tcgtcctgca gttcattcag ggcaccggac 2340aggtcggtct tgacaaaaag
aaccgggcgc ccctgcgctg acagccggaa cacggcggca 2400tcagagcagc
cgattgtctg ttgtgcccag tcatagccga atagcctctc cacccaagcg
2460gccggagaac ctgcgtgcaa tccatcttgt tcaatcatgc gaaacgatcc
tcatcctgtc 2520tcttgatcag atcttgatcc cctgcgccat cagatccttg
gcggcaagaa agccatccag 2580tttactttgc agggcttccc aaccttacca
gagggcgccc cagctggcaa ttccggttcg 2640cttgctgtcc ataaaaccgc
ccagtctagc aactgttggg aagggcgatc gtgtaatacg 2700actcactata
gggcgaattg gagct 272522264PRTartificial sequenceAmino acid sequence
for a coding sequence having an antibiotic resistance gene
kanamycin 22Met Ile Glu Gln Asp Gly Leu His Ala Gly Ser Pro Ala Ala
Trp Val1 5 10 15Glu Arg Leu Phe Gly Tyr Asp Trp Ala Gln Gln Thr Ile
Gly Cys Ser20 25 30Asp Ala Ala Val Phe Arg Leu Ser Ala Gln Gly Arg
Pro Val Leu Phe35 40 45Val Lys Thr Asp Leu Ser Gly Ala Leu Asn Glu
Leu Gln Asp Glu Ala50 55 60Ala Arg Leu Ser Trp Leu Ala Thr Thr Gly
Val Pro Cys Ala Ala Val65 70 75 80Leu Asp Val Val Thr Glu Ala Gly
Arg Asp Trp Leu Leu Leu Gly Glu85 90 95Val Pro Gly Gln Asp Leu Leu
Ser Ser His Leu Ala Pro Ala Glu Lys100 105 110Val Ser Ile Met Ala
Asp Ala Met Arg Arg Leu His Thr Leu Asp Pro115 120 125Ala Thr Cys
Pro Phe Asp His Gln Ala Lys His Arg Ile Glu Arg Ala130 135 140Arg
Thr Arg Met Glu Ala Gly Leu Val Asp Gln Asp Asp Leu Asp Glu145 150
155 160Glu His Gln Gly Leu Ala Pro Ala Glu Leu Phe Ala Arg Leu Lys
Ala165 170 175Arg Met Pro Asp Gly Glu Asp Leu Val Val Thr His Gly
Asp Ala Cys180 185 190Leu Pro Asn Ile Met Val Glu Asn Gly Arg Phe
Ser Gly Phe Ile Asp195 200 205Cys Gly Arg Leu Gly Val Ala Asp Arg
Tyr Gln Asp Ile Ala Leu Ala210 215 220Thr Arg Asp Ile Ala Glu Glu
Leu Gly Gly Glu Trp Ala Asp Arg Phe225 230 235 240Leu Val Leu Tyr
Gly Ile Ala Ala Pro Asp Ser Gln Arg Ile Ala Phe245 250 255Tyr Arg
Leu Leu Asp Glu Phe Phe2602375PRTartificial sequenceAmino acid
sequence for an analog mouse GHRH sequence 23Ala Met Val Leu Trp
Val Leu Phe Val Ile Leu Ile Leu Thr Ser Gly1 5 10 15Ser His Cys Ser
Leu Pro Pro Ser Pro Pro Phe Arg Met Gln Arg His20 25 30Val Asp Ala
Ile Phe Thr Thr Asn Tyr Arg Lys Leu Leu Ser Gln Leu35 40 45Tyr Ala
Arg Lys Val Ile Gln Asp Ile Met Asn Lys Gln Gly Glu Arg50 55 60Ile
Gln Glu Gln Arg Ala Arg Leu Ser Ala Cys65 70 7524231DNAartificial
sequenceNucleic acid sequence for an original mouse GHRH sequence
24gccatggtgc tctgggtgct ctttgtgatc ctcatcctca ccagcggcag ccactgcagc
60ctgcctccca gccctccctt caggatgcag aggcacgtgg acgccatctt caccaccaac
120tacaggaagc tgctgagcca gctgtacgcc aggaaggtga tccaggacat
catgaacaag 180cagggcgaga ggatccagga gcagagggcc aggctgagct
gataagcttg c 2312573PRTartificial sequenceAmino acid sequence for
an original mouse GHRH sequence 25Met Val Leu Trp Val Leu Phe Val
Ile Leu Ile Leu Thr Ser Gly Ser1 5 10 15His Cys Ser Leu Pro Pro Ser
Pro Pro Phe Arg Met Gln Arg His Val20 25 30Asp Ala Ile Phe Thr Thr
Asn Tyr Arg Lys Leu Leu Ser Gln Leu Tyr35 40 45Ala Arg Lys Val Ile
Gln Asp Ile Met Asn Lys Gln Gly Glu Arg Ile50 55 60Gln Glu Gln Arg
Ala Arg Leu Ser Ala65 702676PRTartificial sequenceAmino acid
sequence for an analog rat GHRH sequence 26Ala Met Ala Leu Trp Val
Phe Phe Val Leu Leu Thr Leu Thr Ser Gly1 5 10 15Ser His Cys Ser Leu
Pro Pro Ser Pro Pro Phe Arg Val Arg Arg His20 25 30Ala Asp Ala Ile
Phe Thr Ser Ser Tyr Arg Arg Ile Leu Gly Gln Leu35 40 45Tyr Ala Arg
Lys Leu Leu His Glu Ile Met Asn Arg Gln Gln Gly Glu50 55 60Arg Asn
Gln Glu Gln Arg Ser Arg Phe Asn Ala Cys65 70 7527234DNAartificial
sequenceNucleic acid sequence for an original rat GHRH sequence
27gccatggcac tctgggtgtt ctttgtgctc ctcaccctca ccagtggctc ccactgctca
60ctgcccccct cacctccctt cagggtgcgg cggcacgccg acgccatctt caccagcagc
120tacaggagaa tcctgggcca gctgtacgcc aggaaactgc tgcacgagat
catgaacagg 180cagcagggcg agaggaacca ggagcagagg tccaggttca
actgataagc ttgc 2342874PRTartificial sequenceAmino acid sequence
for an original rat GHRH sequence 28Met Ala Leu Trp Val Phe Phe Val
Leu Leu Thr Leu Thr Ser Gly Ser1 5 10 15His Cys Ser Leu Pro Pro Ser
Pro Pro Phe Arg Val Arg Arg His Ala20 25 30Asp Ala Ile Phe Thr Ser
Ser Tyr Arg Arg Ile Leu Gly Gln Leu Tyr35 40 45Ala Arg Lys Leu Leu
His Glu Ile Met Asn Arg Gln Gln Gly Glu Arg50
55 60Asn Gln Glu Gln Arg Ser Arg Phe Asn Ala65 702973PRTartificial
sequenceAmino acid sequence for an analog bovine GHRH sequence
29Ala Met Val Leu Trp Val Phe Phe Leu Val Thr Leu Thr Leu Ser Ser1
5 10 15Gly Ser His Gly Ser Leu Pro Ser Gln Pro Leu Arg Ile Pro Arg
Tyr20 25 30Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly
Gln Leu35 40 45Ser Ala Arg Lys Leu Leu Gln Asp Ile Met Asn Arg Gln
Gln Gly Glu50 55 60Arg Asn Gln Glu Gln Gly Ala Ala Cys65
7030222DNAartificial sequenceNucleic acid sequence for an original
bovine GHRH sequence 30ccatggtgct ctgggtgttc ttcctcgtga ccctcaccct
cagcagcggc tcccacggtt 60ccctgccttc ccagcctctc aggattccac ggtacgccga
cgccatcttc accaacagct 120accggaaggt gctgggccag ctgtccgccc
ggaagctgct gcaggacatc atgaacaggc 180agcagggcga gagaaaccag
gagcagggcg cctgataagc tt 2223171PRTartificial sequenceAmino acid
sequence for an original bovine GHRH sequence 31Met Val Leu Trp Val
Phe Phe Leu Val Thr Leu Thr Leu Ser Ser Gly1 5 10 15Ser His Gly Ser
Leu Pro Ser Gln Pro Leu Arg Ile Pro Arg Tyr Ala20 25 30Asp Ala Ile
Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln Leu Ser35 40 45Ala Arg
Lys Leu Leu Gln Asp Ile Met Asn Arg Gln Gln Gly Glu Arg50 55 60Asn
Gln Glu Gln Gly Ala Ala65 703273PRTartificial sequenceAmino acid
sequence for an analog ovine GHRH sequence 32Ala Met Val Leu Trp
Val Phe Phe Leu Val Thr Leu Thr Leu Ser Ser1 5 10 15Gly Ser His Gly
Ser Leu Pro Ser Gln Pro Leu Arg Ile Pro Arg Tyr20 25 30Ala Asp Ala
Ile Phe Thr Asn Ser Tyr Arg Lys Ile Leu Gly Gln Leu35 40 45Ser Ala
Arg Lys Leu Leu Gln Asp Ile Met Asn Arg Gln Gln Gly Glu50 55 60Arg
Asn Gln Glu Gln Gly Ala Ala Cys65 7033222DNAartificial
sequenceNucleic acid sequence for an original ovine GHRH sequence
33ccatggtgct ctgggtgttc ttcctcgtga ccctcaccct cagcagcggc tcccacggtt
60ccctgccttc ccagcctctc aggattccac ggtacgccga cgccatcttc accaacagct
120accggaagat cctgggccag ctgtccgccc ggaagctgct gcaggacatc
atgaacaggc 180agcagggcga gagaaaccag gagcagggcg cctgataagc tt
2223471PRTartificial sequenceAmino acid sequence for an original
ovine GHRH sequence 34Met Val Leu Trp Val Phe Phe Leu Val Thr Leu
Thr Leu Ser Ser Gly1 5 10 15Ser His Gly Ser Leu Pro Ser Gln Pro Leu
Arg Ile Pro Arg Tyr Ala20 25 30Asp Ala Ile Phe Thr Asn Ser Tyr Arg
Lys Ile Leu Gly Gln Leu Ser35 40 45Ala Arg Lys Leu Leu Gln Asp Ile
Met Asn Arg Gln Gln Gly Glu Arg50 55 60Asn Gln Glu Gln Gly Ala
Ala65 7035234DNAartificial sequenceNucleic acid sequence for an
analog chicken GHRH sequence 35gccatggccc tgtgggtgtt ctttgtgctg
ctgaccctga cctccggaag ccactgcagc 60ctgccaccca gcccaccctt ccgcgtcagg
cgccacgccg acggcatctt cagcaaggcc 120taccgcaagc tcctgggcca
gctgagcgca cgcaactacc tgcacagcct gatggccaag 180cgcgtgggca
gcggactggg agacgaggcc gagcccctga gctgataagc ttgc
2343676PRTartificial sequenceAmino acid sequence for an analog
chicken GHRH sequence 36Ala Met Ala Leu Trp Val Phe Phe Val Leu Leu
Thr Leu Thr Ser Gly1 5 10 15Ser His Cys Ser Leu Pro Pro Ser Pro Pro
Phe Arg Val Arg Arg His20 25 30Ala Asp Gly Ile Phe Ser Lys Ala Tyr
Arg Lys Leu Leu Gly Gln Leu35 40 45Ser Ala Arg Asn Tyr Leu His Ser
Leu Met Ala Lys Arg Val Gly Ser50 55 60Gly Leu Gly Asp Glu Ala Glu
Pro Leu Ser Ala Cys65 70 7537231DNAartificial sequenceNucleic acid
sequence for an original chicken GHRH sequence 37ccatggcact
ctgggtgttc tttgtgctcc tcaccctcac cagtggctcc cactgctcac 60tgcccccctc
acctcccttc agggtgcggc ggcacgccga tgggatcttc agcaaagcct
120acaggaaact cctgggccag ctgtccgcaa gaaattacct gcactccctg
atggccaagc 180gggtcggcag cggcctgggg gacgaggcgg aaccgctcag
ctgataagct t 2313874PRTartificial sequenceAmino acid sequence for
an original chicken GHRH sequence 38Met Ala Leu Trp Val Phe Phe Val
Leu Leu Thr Leu Thr Ser Gly Ser1 5 10 15His Cys Ser Leu Pro Pro Ser
Pro Pro Phe Arg Val Arg Arg His Ala20 25 30Asp Gly Ile Phe Ser Lys
Ala Tyr Arg Lys Leu Leu Gly Gln Leu Ser35 40 45Ala Arg Asn Tyr Leu
His Ser Leu Met Ala Lys Arg Val Gly Ser Gly50 55 60Leu Gly Asp Glu
Ala Glu Pro Leu Ser Ala65 703940PRTartificial sequenceAmino acid
sequence for GHRH sequence wt-GHRH 39Tyr Ala Asp Ala Ile Phe Thr
Asn Ser Tyr Arg Lys Val Leu Gly Gln1 5 10 15Leu Ser Ala Arg Lys Leu
Leu Gln Asp Ile Met Ser Arg Gln Gln Gly20 25 30Glu Arg Asn Gln Glu
Gln Gly Ala35 404040PRTartificial sequenceAmino acid sequence for
GHRH sequence HV-GHRH 40His Val Asp Ala Ile Phe Thr Asn Ser Tyr Arg
Lys Val Leu Ala Gln1 5 10 15Leu Ser Ala Arg Lys Leu Leu Gln Asp Ile
Leu Asn Arg Gln Gln Gly20 25 30Glu Arg Asn Gln Glu Gln Gly Ala35
404140PRTartificial sequenceAmino acid sequence for GHRH sequence
TI-GHRH 41Tyr Ile Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu
Ala Gln1 5 10 15Leu Ser Ala Arg Lys Leu Leu Gln Asp Ile Leu Asn Arg
Gln Gln Gly20 25 30Glu Arg Asn Gln Glu Gln Gly Ala35
404240PRTartificial sequenceAmino acid sequence for GHRH sequence
TV-GHRH 42Tyr Val Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu
Ala Gln1 5 10 15Leu Ser Ala Arg Lys Leu Leu Gln Asp Ile Leu Asn Arg
Gln Gln Gly20 25 30Glu Arg Asn Gln Glu Gln Gly Ala35
404340PRTartificial sequenceAmino acid sequence for GHRH sequence
15/27/28-GHRH 43Tyr Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val
Leu Ala Gln1 5 10 15Leu Ser Ala Arg Lys Leu Leu Gln Asp Ile Leu Asn
Arg Gln Gln Gly20 25 30Glu Arg Asn Gln Glu Gln Gly Ala35 40
* * * * *